Antibody or antigen binding fragment thereof, composition and use of an antibody or antigen binding

专利摘要:
endoglobin antibodies. the present application relates to compositions of humanized and humanized / deimunized anti-endoglobin antibodies and antigen-binding fragments thereof. one aspect concerns antibodies that have one or more modifications to at least one amino acid residue from at least one of the structure regions of the variable heavy chain, the light chain variable, or both. another aspect refers to antibodies that bind to endoglina and inhibit angiogenesis. another aspect refers to the deimmunization of humanized antibodies to reduce immunogenicity. another aspect relates to the use of humanized and humanized / deimmunized antibodies that bind endoglobin for the detection, diagnosis or treatment of a disease or condition associated with endoglobin, angiogenesis or a combination of these.
公开号:BR112012007318B1
申请号:R112012007318-4
申请日:2010-09-29
公开日:2020-03-10
发明作者:Charles Theuer；Maximiliano Vasquez
申请人:Tracon Pharmaceuticals, Inc.；
IPC主号:

专利说明:

ANTIGEN OR ANTIGEN BINDING FRAGMENT OF THE SAME, COMPOSITION AND USE OF AN ANTIBODY OR FRAGMENT OF
ANTIGEN BINDING
PREVIOUS REFERENCE
This patent application claims the right to US provisional application No. 61 / 247,290, filed on September 30, 2009 and US non-provisional application No. 12 / 751,907, filed on March 31, 2010, the disclosures being all of these. orders incorporated herein by reference in their entirety.
BACKGROUND OF THE TECHNIQUE Endoglobin, also known as, inter alia, CD105 or edg-1, is a homodimeric type I membrane glycoprotein that is expressed at high levels in the proliferation of vascular endothelial cells (Burrows et al., 1995, Res. Cancer Clin. 1: 1623-1634). Thus, endoglobin is mainly a marker associated with the proliferation of endothelial cells submitted to active angiogenesis. However, there is some expression of endoglobin by the vascular endothelium of normal tissues (Burrows et al, supra; Wang et al, 1993, Int. J. Cancer 54: 363-370). Human endogline is known to bind specifically to the transforming growth factor-β (TGF-β), and the amino acid sequence deduced from endogline has great homology with β-glycan, a type of TGF-β receptor. Endoglobin (EDG) has been the target of antibodies based on methods of reducing tumor vasculature, as EDG is an antigen associated with the proliferation of endothelial cells and leukemia. Its expression is overregulated in tumors associated with the vascular endothelium, and EDG is essential for angiogenesis. Angiogenesis includes the formation of new blood capillary vessels that lead to neovascularization as well as the maintenance of the existing vasculature. It is a complex process that includes a series of sequential steps, including degradation mediated by basal membrane endothelial cells and vascular interstitial matrix, migration of endothelial cells, proliferation of endothelial cells and capillary formation by endothelial cells. Various anti-globin antibodies, in particular anti-globin monoclonal antibodies ("mAb"), have been described. MAb SN6 is an antibody generated from immunization of mice with mixtures of glycoproteins from cell membranes of human leukemia cells (Haruta and Seon, 1986, Proc. Natl. Acad. Sci. 83: 7898-7902). SN6 is a mAb murine that recognizes human endogline. MAb 44G4 is an antibody generated from the immunization of mice with whole cell suspensions of human pre-B leukemic cells (Gougos and Letarte, 1988, J. Immunol 141: 1925-1933; 1990, J. Biol Chem. 265: 8361 -8364). 44G4 is also a murine mAb that recognizes human endogline. MAb MJ7 / 18 is an antibody produced from the immunization of rats with inflamed mouse skins (Ge and Butcher, 1994, supra). MJ7 / 18 is a mAb that recognizes murine endoglin. MAb Tec-11 is an antibody generated from the immunization of mice with human umbilical vein endothelial cells (Burrows et al., 1995, Clin. Cancer Res. 1: 1623-1634). Tec-11 is a mAb murine with limited reactivity to human endogline. Endoglobin antibodies represent an important area for the development of therapies for the treatment of a variety of diseases and conditions that involve, being influenced by, or affected by angiogenesis.
Angiogenesis is a physiological process by which new blood vessels develop from pre-existing vessels (Varner, et al., Cell Adh. Commun. 1995, 3: 367-374; Blood, et al., Biochim. Biophys. Acta 1990, 1032: 89-118; Weidner, et al., J. Natl. Cancer Inst. 1992, 84: 18751887). Angiogenesis has been suggested to play a role in both normal and pathological processes. For example, angiogenic processes are involved in the development of vascular systems in animal organs and tissues. These processes are also involved in the transient stages of angiogenesis, for example, during the menstrual cycle, in pregnancy and in the regeneration of wounds. On the other hand, several diseases are associated with unregulated angiogenesis.
In certain pathological conditions, angiogenesis is stimulated as a means to provide blood and adequate nutrients to cells within the affected tissue. Many of these pathological conditions involve abnormal cell proliferation and / or regulation. Therefore, inhibition of angiogenesis is a potentially useful approach for the treatment of diseases that are characterized by the development of new blood vessels. For example, angiogenesis is involved in pathological conditions, including: various forms of ocular and non-ocular diseases characterized by angiogenesis / neovascularization (eg, macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (eg, IHD), rheumatoid arthritis, osteoarthritis, and various forms of cancer, solid tumors, metastases and the like.
SUMMARY OF THE INVENTION
Here, humanized antibodies or their antigen binding fragments are provided, which bind to endogline. Such antibodies have in vitro and in vivo purification, detection, diagnosis and therapeutic uses. Humanized antibodies or antigen-binding fragments thereof are also provided here that bind to one or more species or variants of endogline and inhibit angiogenesis. Also provided herein are methods of treating diseases associated with angiogenesis with humanized antibodies or antigen binding fragments thereof that bind to endoglobin.
The humanized antibodies and antigen binding fragments that bind to endogline and are described herein can be used to treat or prevent macular degeneration, CNV, diabetic retinopathy, or proliferative vitreoretinopathy. Described herein are methods of treating or preventing macular degeneration, CNV, diabetic retinopathy, or proliferative vitreoretinopathy by administering the antibodies and antigen binding fragments described herein. Humanized antibodies and antigen-binding fragments that bind to endogline and which are described here can also decrease blood vessels, inhibit the proliferation of endothelial cells associated with eye disease, evident bleeding symptoms, treat blurred vision, provide stasis of loss of vision, and / or preventing loss of blood vessels. The humanized antibodies and antigen binding fragments described herein can also be used in medications for the treatment of macular degeneration, NVC, diabetic retinopathy or proliferative vitreoretinopathy. The humanized antibodies and antigen binding fragments described herein can also be used in medicines for the treatment of cancer.
Here antibodies, or antigen binding fragments thereof, are provided, having a heavy chain variable region having an amino acid sequence defined as SEQ ID NO: 41 and a light chain variable region having an amino acid sequence defined as SEQ ID NO. : 3.
Here antibodies, or antigen binding fragments thereof, are provided that bind to the endoglobin, comprising a light chain variable region that has an amino acid sequence defined as SEQ ID NO: 3 and a heavy chain variable region having a set of amino acid sequence defined as SEQ ID NO: 41, wherein: said heavy chain variable region further comprises one or more modifications selected from the group consisting of a substitution of glycine (G) for alanine (A) at position 49 ; a replacement of asparagine (N) with serine (S) at position 76; a replacement of threonine (T) with arginine (R) at position 77; a replacement of leucine (L) with valine (V) at position 78; a replacement of asparagine (N) with isoleucine (I) at position 82a; a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89; a replacement of threonine (T) with arginine (R) or glycine (G) at position 94; a replacement of leucine (L) with threonine (T) at position 108; a substitution of valine (V) with leucine (L) at position 109, and a substitution of serine (S) with alanine (A) at position 113, using the Kabat numbering system; and the light chain variable region further comprises one or more modifications selected from the group consisting of a replacement of aspartic acid (D) with glutamine (Q) in position 1; a substitution of glutamine (Q) for valine (V) in position 3; a replacement of methionine (M) with leucine (L) in position 4; a replacement of threonine (T) by serine (S) at position 5; a substitution of tyrosine (Y) for phenylalanine (F) at position 36; a replacement of leucine (L) with proline (P) at position 46; a replacement of leucine (L) with tryptophan (W) at position 47; a replacement of serine (S) by valine (V) or alanine (A) at position 60; a replacement of aspartic acid (D) with serine (S) at position 70; a substitution of phenylalanine (F) for tyrosine (Y) at position 71; a replacement of glutamine (G) with alanine (A) at position 100, and a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system. Antibodies or antigen-binding fragments thereof are provided herein which bind to endoglobin, having a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises: (i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQ ID NO: 68; (ii) an FR1 heavy chain having the amino acid sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 44 except for one or more conservative substitutions; (iii) an FR2 heavy chain having the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence of SEQ ID NO: 45 except for the replacement of glycine (G) by alanine (A) at position 49 using the numbering system Kabat; and (iv) an FR3 heavy chain having the amino acid sequence of SEQ ID NO: 47 or the amino acid sequence of SEQ ID NO: 47 except for one or more substitutions of the group consisting of: (a) an asparagine substitution (N ) by serine (S) at position 76; (b) a replacement of threonine (T) with arginine (R) at position 77; (c) a replacement of leucine (L) with valine (V) at position 78; (d) a replacement of asparagine (N) with isoleucine (I) at position 82a; (e) a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89; and (f) a replacement of threonine (T) with arginine (R) or glycine (G) at position 94 using the Kabat numbering system; and (v) an FR4 heavy chain having the amino acid sequence of SEQ ID NO: 56 or the amino acid sequence of SEQ ID NO: 56 except for one or more substitutions selected from the group consisting of: (a) a leucine substitution ( L) by threonine (T) at position 108; (b) a substitution of valine (V) for leucine (L) at position 109; and (c) a replacement of serine (S) by alanine (A) at position 113 using the Kabat numbering system; and said light chain variable region comprises: (i) a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, a CDR3 of SEQ ID NO: 65; (ii) an FR1 light chain having the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 except for one or more substitutions selected from the group consisting of: (a) an aspartic acid substitution ( D) by glutamine (Q) in position 1; (b) a substitution of glutamine (Q) for valine (V) in position 3; (c) a substitution of methionine (M) for leucine (L) at position 4; and (d) a substitution of threonine (T) for serine (S) in position 5; using the Kabat numbering system; and (iii) an FR2 light chain having the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence of SEQ ID NO: 20 except for one or more substitutions selected from the group consisting of: (a) a tyrosine substitution ( Y) by phenylalanine (F) at position 36; (b) a substitution of leucine (L) for proline (P) at position 46; and (c) a replacement of leucine (L) with tryptophan (W) at position 47 using the Kabat numbering system; and (iv) an FR3 light chain having the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence of SEQ ID NO: 28 except for one or more substitutions selected from the group consisting of: (a) a serine substitution ( S) by valine (V) or alanine (A) at position 60; (b) a replacement of aspartic acid (D) with serine (S) at position 70; and (b) a substitution of phenylalanine (F) for tyrosine (Y) at position 71 using the Kabat numbering system; and (v) an FR4 light chain having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35 except for one or more substitutions selected from the group consisting of: (a) a glycine substitution ( G) by alanine (A) at position 100; and (b) a replacement of isoleucine (I) with leucine (L) at position 106 using the Kabat numbering system.
An antibody, or antigen binding fragment thereof, is provided herein, comprising a heavy chain variable region having an amino acid sequence defined as SEQ ID NO: 42 and a light chain variable region having an amino acid sequence defined as SEQ ID NO: 4.
An antibody, or antigen binding fragment thereof, is provided herein which binds to the endogline, comprising a light chain variable region having an amino acid sequence defined as SEQ ID NO: 4 and a heavy chain variable region having a sequence amino acid defined as SEQ ID NO: 42, wherein: said heavy chain variable region further comprises one or more modifications selected from the group consisting of a substitution of glycine (G) for alanine (A) at position 49; a replacement of asparagine (N) with serine (S) at position 76; a replacement of threonine (T) with arginine (R) at position 77; a replacement of leucine (L) with valine (V) at position 7 8; a replacement of asparagine (N) with isoleucine (I) at position 82a; a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89; a replacement of arginine (R) with threonine (T) or glycine (G) at position 94; a replacement of leucine (L) with threonine (T) at position 108; a substitution of valine (V) for leucine (L) in position 109, and a substitution of serine (S) for alanine (A) in position 113, using the numbering system of Kabat and the light chain variable region also comprises a or more modifications selected from the group consisting of a replacement of aspartic acid (D) with glutamine (Q) in position 1; a substitution of glutamine (Q) for valine (V) in position 3; a replacement of methionine (M) with leucine (L) in position 4; a replacement of threonine (T) by serine (S) at position 5; a substitution of tyrosine (Y) for phenylalanine (F) at position 36; a substitution of proline (P) for leucine (L) at position 46; a substitution of tryptophan (W) for leucine (L) at position 47; a replacement of serine (S) by valine (V) or alanine (A) at position 60; a replacement of aspartic acid (D) with serine (S) at position 70; a substitution of tyrosine (Y) for phenylalanine (F) at position 71; a replacement of glutamine (G) with alanine (A) at position 100, and a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system.
Here an antibody, or antigen binding fragment thereof, is provided, which binds to the endoglobin, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises: (i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQ ID NO: 68; (ii) an FR1 heavy chain having the amino acid sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 44, except for one or more conservative substitutions; (iii) an FR2 heavy chain having the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence of SEQ ID NO: 45, except for a substitution of glycine (G) with alanine (A) at position 49, using the Kabat numbering system; and (iv) an FR3 heavy chain having the amino acid sequence of SEQ ID NO: 47 or the amino acid sequence of SEQ ID NO: 47, except for one or more substitutions selected from the group consisting of: (a) the substitution an asparagine (N) by serine (S) at position 76; (b) a replacement of threonine (T) with arginine (R) at position 77; (c) a replacement of leucine (L) with valine (V) at position 78; (d) a replacement of asparagine (N) with isoleucine at position 82a; (e) a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89, and (f) a replacement of arginine (R) with threonine (T) or glycine (G) at position 94, using the Kabat numbering system and (v) a heavy chain FR4 having the amino acid sequence of SEQ ID NO: 56 or the amino acid sequence of SEQ ID NO: 56, except for one or more substitutions selected from the group consisting of in: (a) a replacement of leucine (L) with threonine (T) in position 108; (b) a substitution of valine (V) with leucine (L) at position 109, and (c) a substitution of serine (S) with alanine (A) at position 113, using the Kabat numbering system; and said light chain variable region comprises: (i) a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65; (ii) an FR1 light chain having the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6, except for one or more substitutions selected from the group consisting of: (a) a substitution of aspartic acid (D) by glutamine (Q) in position 1; (b) a substitution of glutamine (Q) for valine (V) in position 3; (c) a replacement of methionine (M) with leucine (L) at position 4; and (d) a replacement of threonine (T) by serine (S) in position 5; using the Kabat numbering system and (iii) an FR2 light chain having the amino acid sequence of SEQ ID NO: 21 or the amino acid sequence of SEQ ID NO: 20, except for one or more substitutions selected from the group consisting of in: (a) a substitution of tyrosine (Y) for phenylalanine (F) at position 36; (b) a substitution of proline (P) with leucine (L) at position 46, and (c) a substitution of tryptophan (W) with leucine (L) at position 47, using the Kabat numbering system and (iv) a FR3 light chain having the amino acid sequence of SEQ ID NO: 29 or the amino acid sequence of SEQ ID NO: 28, except for one or more substitutions selected from the group consisting of: (a) a serine substitution (S ) by valine (V) or alanine (A) at position 60; (b) a substitution of aspartic acid (D) with serine (S) at position 70, and (b) a substitution of tyrosine (Y) with phenylalanine (F) at position 71, using the Kabat numbering system and (v ) a FR4 light chain having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35, except for one or more substitutions selected from the group consisting of: (a) a glycine substitution ( G) with alanine (A) at position 100, and (b) a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system.
An antibody, or antigen binding fragment thereof, is provided herein, comprising a light chain variable region having an amino acid sequence defined as SEQ ID NO: 93 (VK1AA) and a heavy chain variable region having a sequence set amino acid defined as SEQ ID NO: 89 (VH1A2).
An antibody, antigen-binding fragment thereof, which binds to the endogline, is provided here, comprising a heavy chain variable region having an amino acid sequence defined as SEQ ID NO: 89 and a light chain variable region having a sequence of amino acids defined as SEQ ID NO: 93, where: (i) the heavy chain variable region further comprises one or more modifications selected from the group consisting of a substitution of glycine (G) for alanine (A) or serine ( S) in position 49; a replacement of alanine (A) with isoleucine (I) at position 51; a replacement of lysine (K) with arginine (R) or asparagine (Q) at position 52b; a substitution of leucine (L) for valine (V) in 78 position, using the Kabat numbering system and (ii) the light chain variable region also comprises one or more modifications selected from the group consisting of a substitution methionine (M) by leucine (L) at position 4; a substitution of alanine (A) for valine (V) in position 19; a replacement of threonine (T) by serine (S) at position 22; a replacement of alanine (A) with isoleucine (I) at position 48, and a replacement of threonine (T) with serine (S) at position 51 using the Kabat numbering system.
An antibody, or antigen binding fragment thereof, of claim 2 is provided herein comprising a heavy chain variable region having an amino acid sequence defined as SEQ ID NO: 88, 89, 90, 91 or 92, and a variable region light chain having an amino acid sequence defined as SEQ ID NO: 93, 94, 95, 96, 97, 100, 102, or 103.
In one aspect, the antibodies and antigen binding fragment described herein are humanized and can be of any isotype. Also covered here are AVIMERs, diabody and heavy chain dimers (including camelid and shark heavy chain constructions).
The terms "antigen binding portion of an antibody", "antigen binding fragment", "antigen binding domain", "antibody fragment" or "functional antibody fragment" are used interchangeably to refer to to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. Non-limiting examples of antibody fragments included within such terms include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, Cl and Ch1 domains, (ii) an F (ab 'fragment) ) 2, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fd fragment consisting of the Vh and Chi domains, (iv) an Fv fragment containing the Vl and Vh domains of a single region of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341: 544-546.), which contains a Vh domain and (vi) an isolated CDR. In addition, "one half" of antibodies comprising a single heavy chain and a single light chain are included in this definition. Other forms of single chain antibodies, such as diabody, are also included in this document.
An antigen binding fragment can be any of those described herein, including, but not limited to, a Fab fragment, a Fab ', an F (ab') 2 fragment, an Fv fragment (including Covalently unbound and ligated Fv fragments covalently), an scFv fragment, a single chain polypeptide bond, an Fd fragment, an Fv fragment or a dAb fragment. In a non-limiting form, the antigen binding fragment is a scFv that can optionally be further fused to an Fc portion of a human antibody.
In a form of non-limiting modality, the antibody fragment, or antigen-binding fragment thereof that binds to the endoglobin comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 41, 42, or 43, and a light chain variable region that has an amino acid sequence defined in SEQ ID NO: 3, 4 or 5.
In another form of non-limiting modality, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 41 and a light chain variable region having a set of amino acid sequence defined in SEQ ID NO: 3.
In another form of non-limiting modality, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 41 and a light chain variable region having a sequence set amino acid defined in SEQ ID NO: 4.
In another form of non-limiting modality, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 41 and a light chain variable region having a sequence of amino acid defined in SEQ ID NO: 5.
In another form of non-limiting modality, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 42 and a light chain variable region having a set of amino acid sequence defined in SEQ ID NO: 3.
In another form of non-limiting modality, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 42 and a light chain variable region having a sequence set amino acid defined in SEQ ID NO: 4.
In another form of non-limiting modality, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 42 and a light chain variable region having a sequence set amino acid defined in SEQ ID NO: 5.
In another form of non-limiting modality, the antibody, or antigen binding fragment comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence set defined in SEQ ID NO: 3.
In another form of non-limiting modality, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence defined in SEQ ID NO: 4.
In another form of non-limiting modality, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence defined in SEQ ID NO: 43 and a light chain variable region having an amino acid sequence defined in SEQ ID NO: 5.
In yet another form of non-limiting modality, the antibody, or antigen binding of the same variable region of the heavy chain, further comprises one or more modifications selected from the group consisting of: a replacement of asparagine (N) by serine (S ) at position 76; a replacement of threonine (T) with arginine (R) at position 77; a replacement of asparagine (N) with isoleucine (I) at position 82a; a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89; a replacement of threonine (T) with glycine (G) at position 94; a replacement of leucine (L) with threonine (T) at position 108; a substitution of valine (V) with leucine (L) at position 109, and a substitution of serine (S) with alanine (A) at position 113; and said light chain variable region further comprises one or more modifications selected from the group consisting of: a replacement of aspartic acid (D) with glutamine (Q) in position 1; a substitution of glutamine (Q) for valine (V) in position 3; a replacement of threonine (T) by serine (S) at position 5; a substitution of tyrosine (Y) with phenylalanine (F) at position 36; a replacement of serine (S) by valine (V) or alanine (A) at position 60; a replacement of aspartic acid (D) with serine (S) at position 70; a substitution of glycine (G) for alanine (A) at position 100, and a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system.
In one aspect, the antibodies and antigen binding fragments described herein can be modified. For example, in a form of embodiment, the compound can be modified to alter a pharmacokinetic property of the compound, such as, for example, in vivo stability, solubility, bioavailability, or half-life. Such modifications include, but are not limited to, PEGylation and / or glycosylation.
The antibodies and antigen binding fragments described herein can be formulated for rapid or prolonged release using conventional means. In a non-limiting form, a quick release is made, for example, by intravenous injection. In another form of non-limiting modality, prolonged release is done, for example, by deposition by the subcutaneous route. In another form of non-limiting modality, delivery is achieved through aerosol administration.
Here, compositions of the antibodies and antigen binding fragments described herein and an acceptable carrier or excipient are provided.
Here, polynucleotides (nucleic acids) are provided comprising a nucleotide sequence that encodes the antibodies or antigen binding fragments described herein.
Antibodies and antigen binding fragments thereof as described herein can be used to treat various diseases and conditions associated with angiogenesis, for example, various forms of eye diseases characterized by angiogenesis / neovascularization (for example, macular degeneration, diabetic retinopathy), nephropathy diabetic, chronic inflammatory diseases (eg, IHD), rheumatoid arthritis, osteoarthritis, and various forms of cancer, solid tumors, and metastases. In addition, these antibodies and antigen-binding fragments thereof described herein can be used in the formulation of a medicament for the prophylaxis, diagnosis or treatment of diseases and conditions associated with angiogenesis, for example, various forms of ocular and non-ocular diseases characterized by angiogenesis / neovascularization (eg, macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (eg, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancer, solid tumors, and metastases.
Here, a method is provided to induce a host immune response in a patient against endoglobin by administering a composition to the patient, wherein the composition comprises a humanized anti-globin antibody or antigen binding fragments thereof that induce an effective immune response. of the host against the epitope recognized specifically by said antibody or fragment thereof. The host's immune response can be a humoral immune response or a cell-mediated immune response. If the immune response is a humoral immune response, it may be a protective antibody response that inhibits angiogenesis, an angiogenesis-dependent disease, or an angiogenesis-dependent disorder. Immune responses also include inducing or blocking cell signaling pathways (for example, Smad signaling). Angiogenesis-dependent disease or disorder can be, for example, various forms of ocular and non-ocular diseases characterized by angiogenesis / neovascularization (eg macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (eg IHD), rheumatoid arthritis, osteoarthritis, various forms of cancer (primary tumors and metastases) and the like. In a modality form, the protective antibody response inhibits angiogenesis.
Here is provided a method of affecting the cell signaling pathways associated with endoglobin and angiogenesis. Angiogenic cells can be contacted (in vitro, in vivo or ex vivo) with an antibody or antigen-binding fragment thereof described in an amount sufficient to alter cell signaling pathways. In a non-limiting example, in response to antibody binding, Smad 1.5 and / or 8 signaling is inhibited about 1.5 times or more in angiogenic cells. In another non-limiting example, Smad 3 levels increase by 1.5 times or more, indicating that the cells are returning to a quiescent state.
Here is provided a method for inhibiting angiogenesis or an angiogenesis-dependent disease or disorder in an individual by administering a composition provided here to a patient. Angiogenesis-dependent disease or disorder can be one of the following several forms of ocular and non-ocular diseases characterized by angiogenesis / neovascularization (for example, macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (for example IHD), rheumatoid arthritis , osteoarthritis, and various forms of cancer, solid tumors and metastases. In a form of modality, inhibiting angiogenesis or an angiogenesis-dependent disease or disorder alleviates the symptoms associated with the disease or disorder. In another form of modality, inhibition of angiogenesis or an angiogenesis-dependent disease or disorder results in decreasing tumor size, preventing tumor progression, decreasing cell proliferation, increasing apoptosis, or increasing a patient's survival . Inhibition of angiogenesis can result in a decrease in tumor size or prevent tumor progression. The method may also include surgical removal of cancer, and / or administration of one or more additional anti-cancer agents or treatments to a cancer patient.
Here a method of preventing or treating cancer or metastasis in an individual is provided by administering a composition provided herein. In a form of embodiment, administration of the pharmaceutical composition prolongs the life of the individual to be treated. A cancer / tumor to be treated includes a solid tumor; a tumor can be a primary tumor or a metastatic tumor. Examples of solid tumors that are from a tissue or organ selected from skin, lung, melanoma, pancreas, breast, ovary, colon, rectum, stomach, thyroid, ovary, larynx, prostate, colorectal, head, neck, eyes, mouth , throat, esophagus, chest, bone, testicular, marrow, lymphoid, bone, sarcoma, kidney, sweat glands, liver, kidney, brain, for example, glioblastoma multiforme and similar tissues. In a non-limiting example, a solid tumor is a colon tumor, a breast tumor, a kidney tumor, a lung tumor, a prostate tumor, an ovarian tumor, or metastasis of any such tumor. The method may also include surgical removal of the cancer and / or administration of one or more anti-cancer agents. An anticancer agent can be administered before, concomitantly, or subsequently the administration of the pharmaceutical composition. An anti-cancer agent can be administered within one week before the pharmaceutical composition, within one week after the pharmaceutical composition, or the anti-cancer agent can be administered the same day as the pharmaceutical composition. If an anticancer agent is administered on the same day as the pharmaceutical composition, administration may be concomitant.
Here a method is provided for the prevention or treatment of a cancer or a metastasis by surgical removal of the cancer / tumor and concomitant administration of an anti-cancer agent or treatment and a composition provided herein to an individual.
Here, a method of inhibiting angiogenesis is provided, by contacting a cell or tissue with a therapeutically effective amount of an antibody or antigen binding fragment thereof as described herein sufficient to inhibit angiogenesis.
Here, a method of inhibiting the growth of cancer cells is provided, by contacting a therapeutically effective amount of an antibody or antigen binding fragment thereof as described herein sufficient to inhibit the growth of cancer cells or to cause apoptosis of the cancer cell.
Here a method is provided, comprising contacting a tissue with an antibody or antigen binding fragment thereof as described herein, wherein the contact inhibits angiogenesis. The tissue may be tissue grown from a biopsy sample or may be present in an individual.
Provided herein is a method of preventing or treating a proliferative (for example, angiogenic) cell disorder by administration to an individual who has or is at risk of having a cell proliferative disorder of an effective amount of a composition provided herein effective to treat the condition. proliferative cell disorder. The proliferative cell disorder may, for example, be a solid or non-solid benign or malignant tumor and the tumor may be metastatic or non-metastatic. Treatment can result in improvement of the individual's condition and can be assessed by determining whether one or more of the following factors has occurred: decreased cell proliferation, decreased number of cells, increased apoptosis, or decreased survival of at least a portion of cells comprising the proliferative cell disorder. One or more of these events can, in some cases, result in the partial or total elimination of the cancer and prolong the patient's survival. Optionally, the method may also include administering an anti-cancer or treatment agent to the individual.
Here a method is provided for treating diabetic retinopathy, macular degeneration, choroidal neovascularization or neovascular glaucoma in a patient, by administering to the patient a therapeutically effective amount of a composition provided herein. Treatment can result in improvement of the individual's condition and can be assessed by determining whether one or more of the following factors has occurred: decreased macular edema, decreased NVC areas, or increased visual acuity.
In the methods provided herein, the individual can be a human or a non-human subject. Compositions and the anticancer agent or treatments provided herein can be administered once or several times, depending on the patient's health, the progression or condition of the disease, and the effectiveness of the treatment. Adjustments to therapy and treatments can be made throughout the course of treatment.
Compositions can be administered locally, regionally or systemically, such as, for example, administration by subcutaneous, subcutaneous, intravitreous, intradermal, intravenous, intra-arterial, intraperitoneal or intramuscular injection.
In addition, the humanized antibodies and antigen binding fragments described herein can also be used in combination with known therapies and / or compounds for the treatment of macular degeneration, NVC, diabetic retinopathy or proliferative vitreoretinopathy. Examples of such compounds include, but are not limited to, bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), or Macugen. In addition to the routes of administration described herein, the humanized antiendoglina antibodies and antigen binding fragments can be administered via intravitreal routes. Non-limiting examples of modes of administration include intravitreal intravitreal injection and the use of intravitreal implants.
Another aspect is the treatment of a chronic inflammatory disease in an individual by administering a composition of an antibody or antigen-binding fragment described herein. Non-limiting examples of chronic inflammatory diseases include Crohn's disease and ulcerative colitis.
Another aspect is the treatment of rheumatoid arthritis in an individual by administering a composition of an antibody or antigen-binding fragment described herein.
Another aspect is the treatment of osteoarthritis in a subject by administering an antibody composition or antigen binding fragment described herein. The treatment of a subject with rheumatoid arthritis and / or osteoarthritis can be assessed by several means, including improvement in the appropriate categories of ACR scores measured according to published guidelines.
Here is provided a method of monitoring the effectiveness of one or more of any of the methods provided herein. Increased levels of soluble endogline were correlated with decreased survival in cancer patients. Thus, in one aspect, levels of soluble endogline can be monitored before and during therapy. A decrease in the levels of soluble endogline may therefore be an indication that a therapeutic regimen is effective in treating the patient.
One form of embodiment of the present invention contemplates the use of any of the compositions of the present invention to formulate a medicament for the treatment of a disorder of the present invention. Medicines can be formulated based on the physical characteristics of the patient / individual in need of treatment, and can be formulated in single or multiple formulations based on the stage of the cancerous tissue. The medicaments of the present invention can be packaged in a suitable pharmaceutical package with labels appropriate for distribution to hospitals and clinics where the package insert is for indicating the treatment of a disorder as described herein in a subject. Medicines can be packaged as single or multiple units. Instructions for the dose and administration of the pharmaceutical compositions of the present invention can be included with the pharmaceutical product packages.
Here a diagnostic method is provided to provide a sample of cancer cells from a solid tumor or plasma of a patient to be tested, to detect in the sample the expression of at least one gene or gene product chosen from a panel of genes or gene products whose expression has been correlated with the sensitivity or resistance to an angiogenesis inhibitor, in which at least one gene or gene product is chosen from one or more genes or gene products selected from the group consisting of VEGF, VEGF receptor, HIF-Ια, placental growth factor receptor, and CD105, and comparing the expression level of at least one gene or gene product detected in the patient sample to an expression level of at least a gene or gene product that has been correlated with sensitivity or resistance to the angiogenesis inhibitor. In one embodiment, the angiogenesis inhibitor is chosen from VEGF receptor inhibitors, VEGF inhibitors, and endoglobin inhibitors.
Here a kit is provided for detecting levels of gene expression that have been correlated with the sensitivity or resistance to an angiogenesis inhibitor in a sample of cancer cells or human plasma. In one form of modality, one or more genes are selected from among VEGF, VEGF receptor, HIF-Ια, placental growth factor receptor, and endogline.
INCORPORATION AS A REFERENCE
All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety, unless specifically noted otherwise. . This application contains references to amino acid sequences that have been presented simultaneously in the attachment as the text file sequence list "35882-706-202-SeqList.txt", file size 67,843 kilobytes (KB), created on March 30, 2010. The aforementioned sequence listing is incorporated by reference in its entirety in accordance with 37 CFR § 1.52 (e) (5).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides a humanized variable light chain (Vl) O2-Vk1-39 having the chimeric murine monoclonal Vl (underlined) TRC105 CDRs grafted between the structure regions (FRs) 1-3 of the human sequence O2-Vk1-39 and a region of structure 4 from the human sequence Jk4 (SEQ ID NO: 4) (all in bold). Variations that can be made to human FRs are indicated in positions 1, 3, 4, 5, 36, 46, 47, 60, 70, 71, 100, and 106 of the sequence (sequence revealed in SEQ ID NO: 86) using the Kabat numbering system (shown in italics below the humanized sequence).
Figure 2 provides a VH3-15 humanized variable heavy chain (Vh) having the chimeric murine monoclonal antibody TRC105 Vh CDRs (underlined) grafted between the VH3-15 human sequence regions (FRs) 1-3 and a region of structure 4 from the human JH4 sequence (SEQ ID NO: 42) (all in bold). One or more variations that can be made to human FRs are indicated at positions 49, 76, 77, 78, 82a, 89, 94, 108, 109, and 113 of the sequence (sequence disclosed in SEQ ID NO: 87) using the system of Kabat numbering (shown in italics below the humanized sequence).
Figure 3 provides the diagram of the TGF-p / ALK5 signaling path. The TGF-p / ALK5 (A) pathway leads to the inhibition of cell proliferation and migration. The TGF-p / ALK1 (B) pathway induces endothelial cell proliferation and migration and requires CD105 (endoglobin) for ALK1 signaling. Dotted lines indicate inactive or blocked roads. The bold arrow indicates the stimulation of a signaling path.
Figure 4 provides a sequence alignment of exemplary mouse amino acids and humanized VK chains (SEQ ID NOS 1-5, respectively, in order of appearance) and Vh chains (SEQ ID NOS 39-43, respectively, in order of appearance) produced according to the invention described herein.
Figure 5 shows an alignment of amino acid sequences of the exemplary mouse and super-humanized VK chains (SEQ ID NOS 1 and 69-72, respectively, in order of appearance) and Vh chains (SEQ ID 39 and 73-75, respectively, in order of appearance) produced in accordance with the invention described herein.
Figure 6 provides amino acid sequence alignment and comparison of exemplary mouse and humanized and superhumanized VK chains (SEQ ID NOS 1, 3 and 70, respectively, in order of appearance) and VH chains (SEQ ID NOS 39, 41 and 74, respectively, in order of appearance) produced according to the invention described herein.
Figure 7 illustrates the binding of the humanized variant constructed for endoglobin in an ELISA competition assay.
Figure 8 ELISA competition with humanized and humanized / de-immunized anti-CD105 antibodies. Variable concentrations of each antibody were mixed with a fixed concentration of biotinylated anti-CD105 reference antibody (6.25ng / ml) and bound to CD105 (100ng / ml) captured on a Nunc Maxisorp plate. The binding was detected using streptavidin-HRP and TMB substrate. Absorbance (OD) at 450nm was measured on a plate reader and this was plotted against the test antibody concentration.
Figure 9 illustrates the binding assay data for variant VK1AAVH1A plus VK2 containing controls.
Figure 10 illustrates the binding assay data for chimeric compared to VK1AAVH1A2 and VK2AAVH1A2.
Figure 11 illustrates the binding assay data for chimeric compared to VK2AAVH1Q.
Figure 12 illustrates the binding assay data for chimeric compared to VK2AAVH1R.
Figure 13 illustrates the binding assay data for chimeric compared to VK1AAVH1A2.
Figure 14 illustrates the binding assay data for chimeric compared to VK1AAVH1Q.
Figure 15 illustrates the binding assay data for chimeric compared to VK1AAVH1R.
Figure 16 illustrates the binding assay data for chimeric compared to VK2AAVH1A2.
Figure 17 illustrates the first humanized heavy chain variable region de-immunized with bold and underlined CDRs (sequence disclosed in SEQ ID NO: 89).
Variations that can be made are indicated in the identified positions of the sequence using the Kabat numbering system (sequence revealed in SEQ ID NO: 116) (shown in italics below the humanized sequence). Variations can be made as a single mutation or as more than one mutation, and variations can be made with mutations in any combination.
Figure 18 illustrates the first humanized variable region light chain de-immunized with bold and underlined CDRs (sequence disclosed in SEQ ID NO: 93). Variations that can be made are indicated in the identified positions of the sequence using the Kabat numbering system (sequence disclosed in SEQ ID NO: 117) (shown in italics below the humanized sequence). Variations can be made as a single mutation or as more than one mutation, and variations can be made with mutations in any combination.
Figure 19 illustrates the analysis of the heavy chain of regions of (SEQ ID NO: 41) using iTope ™. Peptides spanning the entire sequence were tested as 9mer peptides in increments of one amino acid. The prediction of binding of each residue as a p1 anchor of a 9mer MHC class II allele peptide is indicated by an "O" if the binding resulted in 0.55-0.6 and an "X" if the connection result was> 0.6. Regions containing potentially immunogenic peptides are indicated in the "iTope" column, dark gray indicates high affinity promiscuous MHC class II binding peptides, light gray indicates moderate affinity promiscuous MHC class II binding peptides. The numbers of MHC class II alleles predicted for binding are shown in the columns: "total" and "high affinity". Potential anchorage residues p1 identified as germline sequences are shown in reverse type in the "Sequence" column.
Figure 20 illustrates the analysis of variant regions of heavy chain variants (SEQ ID NO: 118) using iTope ™. Peptides spanning the entire sequence were tested as 9mer peptides in increments of one amino acid. The prediction of binding of each residue as a p1 anchor of a 9mer MHC class II allele peptide is indicated by an "O" if the binding resulted in 0.55-0.6 and an "X" if the connection result was> 0.6. Regions containing potentially immunogenic peptides are indicated in the "iTope" column, dark gray indicates high affinity promiscuous MHC class II binding peptides, light gray indicates moderate affinity promiscuous MHC class II binding peptides. The numbers of MHC class II alleles predicted for binding are shown in the columns: "total" and "high affinity" and the binding difference is shown. Potential p1 anchorage residues identified as germline sequences are shown in the reverse type in the column " Sequence "and the amino acid differences in the variants are framed.
Figure 21 illustrates the analysis of the light chain regions (SEQ ID NO: 3) using iTope ™. Peptides spanning the entire sequence were tested as 9mer peptides in increments of one amino acid. The prediction of binding of each residue as a p1 anchor of a 9mer MHC class II allele peptide is indicated by an "O" if the binding result was 0.55-0.6 and an "X" if the connection result was> 0.6. Regions containing potentially immunogenic peptides are indicated in the "iTope" column, dark gray indicates high affinity promiscuous MHC class II binding peptides, light gray indicates moderate affinity promiscuous MHC class II binding peptides. The numbers of MHC class II alleles predicted for binding are shown in the columns: "total" and "high affinity." Potential anchor residues p1 identified as germline sequences are shown in reverse type in the "Sequence" column.
Figure 22 illustrates the analysis of the variant regions of the light chain (SEQ ID NO: 101) using iTope ™. Peptides spanning the entire sequence were tested as 9mer peptides in increments of one amino acid. The prediction of binding of each residue as a p1 anchor of a 9mer MHC class II allele core peptide is indicated by an "O" if the binding resulted in 0.55-0.6 and an "X" if the result of connection was> 0.6. Regions containing potentially immunogenic peptides are indicated in the "iTope" column, dark gray indicates high affinity promiscuous MHC class II binding peptides, light gray indicates moderate affinity promiscuous MHC class II binding peptides. The numbers of MHC class II alleles predicted for binding are shown in the "total" and "high affinity" columns and the binding difference is shown. Potential p1 anchorage residues identified as germline sequences are shown in the reverse type in the "Sequence" and the amino acid differences in the variants are framed.
Figure 23 illustrates the frequency of MHC Class II allotypes in the world population and the study population.
Figure 24 The chimeric anti-gland antibody, the humanized anti-gland antibody VK1VH1 and the humanized / de-immunized anti-gland antibody VK1AAVH1A2 were tested in EpiScreen ™ for duration of T cell assays using 20-donor CMSP. Bulk cultures of PBMC incubated with test antibodies were shown on days 5, 6, 7 and 8, and pulsed with 3 H-thymidine. The cells were harvested and the incorporation of radioactivity was measured by scintillation counting. The results for each triplicate sample were calculated and normalized by converting the stimulation index (SI). The SI for each time point with each donor is shown above for the chimeric antibody TRC105 (Figure 24A), VK1VH1 (Figure 24B) and VK1AAVH1A2 (Figure 24C). The cut-off point for determining positive responses with an SIh2 is highlighted by the dotted line and significant responses (p <0.05 in a Student's t test) are indicated (*).
DETAILED DESCRIPTION OF THE INVENTION
It should be understood that this modality is not limited to particular formulations or process parameters, since these can, of course, vary. It is also to be understood that the terminology used here is for the purpose of describing only particular forms of modality, and is not intended to be limiting. In addition, it is understood that a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
In accordance with the present application, conventional molecular biology, microbiology, and recombinant DNA techniques can be employed within the art of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al, "Molecular Cloning: A Labocamundongory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Labocamundongory Handbook" Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984), each of which is specifically incorporated herein by reference in its entirety.
Murine monoclonal antibodies (mAbs) have been elevated against endoglobin which modulates endoglobin activity and thus inhibits angiogenesis and / or inhibits vasodilation of small blood vessels. These murine antibodies are described in U.S. patents 5,928,641, 6,200,566, 6,190,660, and 7,097,836 which are incorporated herein in their entirety. In addition, the ex vivo and in vivo in terms of the efficiency of a number of these antibodies has been demonstrated; these monoclonal antibodies that bind to endoglobin are of interest as endoglobin modulating compounds. The therapeutic use of these murine antibodies is not feasible, however, as their administration has a number of limitations, including immunogenicity, for example, in the form of human anti-mouse antibodies (HAMA). Humanized antibodies are made to treat these reactions.
The humanized antibodies that bind to endogline, which are described herein, exhibit reduced immunogenicity while maintaining and / or improving their specificity. In addition, to solve the problems associated with murine antibodies, humanized antibodies that bind to endogline decrease and / or inhibit angiogenesis that are described herein exhibit reduced immunogenicity while maintaining and / or improving their specificity. These humanized endoglobin antibodies are useful for the diagnosis and treatment of various conditions and diseases, as well as for the purification and detection of endoglobin.
I- ANTI-ENDOGLINE ANTIBODIES
Provided here are humanized antibodies, and antigen-binding fragments that bind to endoglobin. Endoglobin can be found in cells that understand and support the existing vasculature, as well as cells that are promoting the growth of, and becoming part of, new vascularization. These antibodies and antigen-binding fragments can bind to endogline and thus inhibit angiogenesis, inhibit the existing vasculature or the maintenance of the existing vasculature, and / or inhibit the dilation of small vessels. Hereinafter, a reference to the terms "antibody" or "antibodies" is to be considered an integral part of any of the antigen binding fragments described herein and the terms are to be interchangeable, where applicable. In addition to their use for the purification of endoglobin, these antibodies are useful for detection, purification and diagnostic purposes, as well as therapeutic purposes. The antibodies provided herein can be used for formulating medicaments for the treatment of a variety of conditions and diseases, methods for treating such conditions and diseases, and methods of detection or diagnosis. As used herein, angiogenesis is inclusive of the growth and / or development of new blood vessels (also referred to as neovascularization), excessive small vessel dilation, or prolonged vascular growth, and maintenance of the existing vasculature. Angiogenesis conditions and diseases refer to those related diseases and conditions, caused by, or associated with angiogenesis. Non-limiting examples of such diseases include, for example, various forms of eye diseases characterized by angiogenesis / neovascularization (eg, macular degeneration, NVC, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (eg, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancer (primary tumor and metastases).
A. ANTIBODY TERMINOLOGY
As used herein, "antibody" refers to an immunoglobulin (Ig) naturally occurring or partially or synthetically produced. The term also encompasses any polypeptide or protein that has a binding domain that is, or is homologous to, an antigen binding domain. The term further includes "antigen binding fragments" and similar interchangeable terms for binding fragments such as those described below. Complementarity determining regions (CDR) of grafted antibodies and other humanized antibodies (including CDR modifications and structural region modifications) are also contemplated by this term.
Native antibodies and native immunoglobulins are generally heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is normally linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain ("Vh") at one end followed by a number of constant domains ("CH"). Each light chain has a variable domain at one end ("VL") and a constant domain ("CL") at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
The terms "synthetic polynucleotide", "synthetic gene" or "synthetic polypeptide," as used herein, mean that the corresponding polynucleotide sequence or part thereof, or amino acid sequence or part thereof, is derived from a sequence that it has been designed, or synthesized again, or modified, compared to a naturally occurring equivalent sequence. Synthetic polynucleotides (antibodies or antigen binding fragments) or synthetic genes can be prepared by methods known in the art, including, but not limited to, chemical synthesis of nucleic acid or amino acid sequences. Synthetic genes are typically different from naturally occurring genes, either at the amino acid, or polynucleotide (or both) levels, and are typically located within the context of synthetic expression control sequences. For example, sequences of synthetic genes may include the amino acid, or polynucleotide, sequences that have been altered, for example, by substitution, deletion or addition, of one or more, amino acids, or nucleotides, thus providing an antibody sequence of amino acids, or a polynucleotide encoding sequence that is different from the source sequence. Polynucleotide sequences of synthetic genes may not necessarily encode proteins with different amino acids, compared to the natural gene, for example, they may also encompass synthetic polynucleotide sequences that incorporate different codons, but that encode the same amino acid (ie, nucleotide changes represent silent mutations at the amino acid level).
With regard to antibodies, "variable domain" refers to the variable domains of antibodies that are used in the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed across antibody variable domains. Rather, it is concentrated in three segments called hypervariable regions (also known as CDRs) in both light and heavy chain variable domains. More highly conserved portions of variable domains are called "framework regions" or "FRs." The unmodified variable domains of heavy and light chains each contain four FRs (FR1, FR2, FR3 and FR4), largely adopting a β-leaf configuration interspersed with three CDRs, which form loop connections, in some cases, with a part of the β-leaf structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs in the other chain, contribute to the formation of the antibody antigen binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669).
The terms "hypervariable region" and "CDR" when used herein, refer to the amino acid residues of an antibody that are responsible for antigen binding. CDRs comprise amino acid residues from three regions of sequences that bind complementarily to an antigen and are known as CDR1, CDR2 and CDR3 for each of the Vh and Vl chains. In the light chain variable domain, CDRs typically correspond to about 24-34 residues (CDRL1) and 50-56 (CDRL2) and 89-97 (CDRL3), and in the heavy chain variable domain CDRs typically correspond to about 31-35 residues (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). It is understood that CDRs of different antibodies may contain inserts, thus, the number of amino acids may be different. The Kabat numbering system accounts for such inserts with a numbering scheme that uses letters linked to specific residues (for example, 27A, 27B, 27C, 27D, 27E and 27F of CDRL1 in the light chain) to reflect any insertions in the numbers between different antibodies. Alternatively, in the light chain variable domain, CDRs typically correspond to about 26-32 residues (CDRL1), 50-52 (CDRL2) and 91-96 (CDRL3), and in the heavy chain variable domain, CDRs typically correspond to to about 26-32 residues (CDRH1), 53-55 (CDRH2) and 96-101 (CDRH3) according to Chothia and Lesk, J. Mol. Biol, 196: 901-917 (1987)).
As used herein, "structural region" or "FR" refers to structural amino acid residues that form a part of the antigen receptacle or binding socket. In some embodiments, the structural residues form a closed circuit that is a part of the antigen binding receptacle or socket and the amino acid residues in the loop may or may not be in contact with the antigen. Structure regions generally comprise the regions between CDRs. In the light chain variable domain, FRs typically correspond to about 0-23 residues (FRL1), 35-49 (FRL2) and 57-88 (FRL3), and 98-109 and in the FRs heavy chain variable domain typically correspond to about 0-30 residues (FRH1), 36-49 (FRH2) and 66-94 (FRH3), and 103-133 according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). As discussed above, with the Kabat numbering for the light chain, the heavy chain is also responsible for insertions in a similar way (for example, 35A, 35B of CDRH1 in the heavy chain). Alternatively, in the light chain variable domain, FRs typically correspond to about 025 residues (FRL1) and 33-49 (FRL2) 53-90 (FRL3), and 97-109 (FRL4), and, in the variable domain of heavy chain, FRs typically correspond to about 0-25 residues (FRH1) and 33-52 (FRH2) and 56-95 (FRH3), and 102-113 (FRH4) according to Chothia and Lesk, J. Mol. Biol, 196: 901-917 (1987)).
The amino acids of an FR loop can be evaluated and determined by inspecting the three-dimensional structure of an antibody heavy chain and / or antibody light chain. The three-dimensional structure can be analyzed by positions accessible to the amino acid solvent as such positions are likely to form a loop and / or provide contact with the antigen in a variable domain of the antibody. Some of the positions accessible to the solvent can tolerate sequence diversity of amino acids and others (for example, the structural positions) are generally less diverse. The three-dimensional structure of the variable domain of antibodies can be derived from a crystal structure or protein modeling.
Antibody constant (Fc) domains are not directly involved in binding an antibody to an antigen, but instead exhibit various effector functions, such as participation of the antibody in antibody-dependent cell toxicity through interactions with, for example , Fc receptors (FCR). Fc domains can also increase the bioavailability of an circulating antibody after administration to a patient.
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains (Fc) that correspond to the different classes of immunoglobulins that are denominated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.
The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be attributed to one of two clearly distinct types, called kappa or ("κ" or "K") and lambda or ("λ"), based on amino acid sequences of their constant domains.
The terms "antigen binding portion of an antibody", "antigen binding fragments", "antigen binding domain", "antibody fragment" or "functional antibody fragment" are used interchangeably here to refer to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. Non-limiting examples of antibody fragments included within such terms include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the V1, Vh, Cl and Ch1 domains, (ii) an F (ab 'fragment) ) 2, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fd fragment consisting of the Vh and Chi domains, (iv) an Fv fragment containing the Vl and Vh domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544 546), which contains an isolated Vh domain and (vi) a CDR. In addition, included in this definition are "a medium" single chain antibodies comprising a single heavy chain and a single light chain. Other forms of single chain antibodies, such as diabody, are also included in this document.
The "F (ab ') 2" and "Fab'" moieties can be produced by treating an Ig with a protease, such as pepsin and papain, and include antibody fragments generated by digestion of immunoglobulin near the disulfide bonds that exist between the hinge regions in each of the two heavy chains. For example, papain cleaves IgG upstream of the disulfide bonds that exist between the hinge regions on each of the two heavy chains to generate two homologous antibody fragments in which a light chain of VL and CL compound (constant region of the chain light), and a fragment heavy chain composed of Vh and Chyi (γ1) a region of the constant region of the heavy chain) are linked at their C-terminal regions via a disulfide bond. Each of these two fragments of homologous antibodies is called Fab '. Pepsin also cleaves downstream of IgG from the disulfide bonds between the hinge regions in each of the two heavy chains to generate an antibody fragment slightly larger than the fragment to which the above two Fab 'are linked in the hinge region . This antibody fragment is called F (ab ') 2. The Fab fragment also contains the light chain constant domain and the first heavy chain constant domain (Ch1). Fab 'fragments differ from Fab fragments by adding some residues at the carboxylic terminus of the CHI domain of the heavy chain including one or more cysteine (s) from the hinge region of the antibody. Fab'-SH is the designation here for Fab 'where the cysteine residue (s) from the constant domains support a free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. "Fv" refers to an antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain and a variable domain of light chain in association, non-covalent or covalent bond (Fv-linked disulfide have been described in the art, Reiter et al. (1996) Nature Biotechnology 14: 1239-1245) . It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, a combination of one or more of the CDRs of each of the VH and VL chains gives antigen binding specificity to the antibody. For example, it would be understood that, for example, CDRH3 and CDRL3 could be sufficient to confer antigen binding specificity of an antibody when transferred to the VH and VL chains of an antibody receptor or an antigen binding fragment thereof and this combination of CDRs can be tested for affinity, binding etc. using any of the techniques described here. Even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind the antigen, although probably less affinity than when combined with a second variable domain. In addition, although the two domains of an Fv fragment (VL and VH) are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to be made as a single protein chain in which the regions Vl and Vh form monovalent molecules (known as single-chain Fv (scFv) ;. Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Osbourn et al (1998) Nat. Biotechnol. 16: 778). Such scFvs are also intended to fall within the scope of the term "antigen binding portion" of an antibody. Any specific scFv Vh and Vl sequences can be linked to an Fc region of the cDNA or genomic sequences in order to generate expression vectors encoding complete Ig (e.g., IgG), molecules or other isotypes. Vh and Vl can also be used in the generation of Fab, Fv or other fragments of Igs using protein chemistry or recombinant DNA technology.
Antibody fragments "single chain Fv" or "sfv" comprise the Vh and Vl domains of an antibody, where these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the Vh and Vl domains that allows the sFv to form the desired structure for antigen binding. For a review of sFvs see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). The term "AVIMER ™" refers to a class of therapeutic proteins of human origin, which are not related to antibodies and antibody fragments, and are composed of several modular and reusable binding domains, referred to as A domains (also referred to as class A module, complementary repetition type, or class A domain LDL receiver). They were developed from human receptors of extracellular domains by in vitro exon and phage disposition (Silverman et al, 2005, Nat Biotechnol 23: 1493-1494; Silverman et al, 2006, Nat Biotechnol 24: 220). The resulting proteins can contain multiple, independent binding domains that can exhibit improved affinity (in some cases, subnanomolar) and specificity compared to a single epitope of binding proteins. See, for example, U.S. Patent Application. Nos. 2005 / 0.221.384, 2005 / 0.164.301,2005 / 0.053.973 and 2005 / 0.089.932, 2005 / 0.048.512, and 2004 / 0.175.756, each of which is incorporated herein by reference into its entirety.
Each of the 217 known human A domains comprises ~ 35 amino acids (~ 4 kDa), and these domains are separated by bonds of an average of five amino acids in length. Native A domains bend quickly and efficiently to form a uniform and stable structure mediated primarily through calcium binding and disulfide formation. A conserved structure motif of only 12 amino acids is necessary for this common structure. The end result is a single protein chain containing multiple domains, each of which represents a separate function. Each domain of proteins is linked independently and the energy contributions of each domain are additive. These proteins were called "avimers ™" of avidity multimers. The term "diabody" refers to small antibody fragments with two antigen binding sites, where fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (Vh -Vl). When using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444 6448 (1993).
Antigen-binding polypeptides also include heavy chain dimers, such as, for example, camelid and shark antibodies. Camel and shark antibodies comprise a homodimeric pair of two chains of type-V and type-C domains (neither has a light chain). Since the Vh region of an IgG heavy chain dimer on a camelid does not have to do hydrophobic interactions with a light chain, the region on the heavy chain that normally contacts a light chain is changed to hydrophilic amino acid residues in a camelid. Vhh dimer IgGs heavy chain domains are called shark Ig-NARS Vhh domains that comprise a homodimer of a variable domain (called a V-NAR domain) and five constant type-C domains (C-NAR domains). In camelids, the diversity of antibody repertoire is determined by CDRs 1, 2 and 3 in the Vh or Vhh regions. The CDR3 of the camel's Vhh region is characterized by its relatively long length, with an average of 16 amino acids (Muyldermans et al, 1994, Protein Engineering 7 (9): 1129). This is in contrast to the CDR3 antibody regions of many other species. For example, the mouse Vh CDR3 has an average of 9 amino acids. Libraries of camelids derived from variable regions of antibodies, which maintain the in vivo diversity of variable regions of a camelid, can be made, for example, the methods disclosed in the Patent Application. No. 20050037421.
Forms of "humanized" non-human (e.g., murine) antibodies include chimeric antibodies that contain the minimal sequence derived from a non-human Ig. For the most part, humanized antibodies are human Igs immunoglobulins (antibody receptor) in which one or more of the recipient's CDRs are replaced by CDRs from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and binding function. In some cases, one or more FR amino acid residues from human Ig are replaced by the corresponding non-human amino acid residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications can be made to refine the performance of the antibody, if necessary. A humanized antibody can comprise substantially all of at least one and, in some cases, two, variable domains, where all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are from one human immunoglobulin sequence. The humanized antibody, optionally, can also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., Nature 321: 522-525 (1986);
Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).
A humanized antibody also includes antibodies in which part or all of the light and heavy chain CDRs are derived from a monoclonal non-human antibody, substantially all of the remaining portions of the variable regions are derived from the human variable region (both heavy and light chain), and constant regions are derived from a human constant region. In a form of modality, the CDR1, CDR2 and CDR3 regions of the heavy and light chains are derived from a non-human antibody. In yet another embodiment, at least one CDR (for example, a CDR3) of the heavy and light chains is derived from a non-human antibody. Various combinations of CDR1, CDR2 and CDR3 can be derived from a non-human antibody and are contemplated here. In a non-limiting example, one or more of the CDR1, CDR2 and CDR3 regions of each of the heavy and light chains are derived from a murine chimeric monoclonal antibody clone TRC105. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that comprise the population are identical except for possible naturally occurring mutations that can be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations, which may include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be interpreted as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or they can be made by recombinant DNA methods (see, for example, US Pat. No. 4 , 816, 567). In certain embodiments, monoclonal antibodies can be isolated from antibody phage libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example.
Antibodies can be isolated and purified from the culture supernatant or ascites mentioned above by saturated ammonium sulfate precipitation, euglobulin precipitation method, capronic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52) , or affinity chromatography using an anti-Ig antibody column or a protein A, G or L column as described in more detail below.
Exemplary antibodies for use in the compositions and methods described herein are intact immunoglobulin molecules, such as, for example, a humanized antibody or those portions of a humanized Ig molecule that contain the antigen binding site (i.e., pacamundongope) or a single heavy chain and single light chain, including portions known in the art as Fab, Fab ', F (ab)', F (ab ') 2, Fd, scFv, a variable heavy domain, a variable light domain, a variable domain Variable NAR, bispecific scFv, bispecific Fab2, triespecific Fab3 and single-stranded polypeptides and others, also referred to as antigen-binding fragments. When constructing an immunoglobulin molecule or its fragments, variable regions or portions thereof, they can be fused with, linked to, or otherwise joined with one or more constant regions or portions thereof to produce any of the antibodies or their fragments here described. This can be achieved in a variety of ways known in the art, including, but not limited to, molecular cloning techniques or by direct synthesis of the nucleic acids encoding the molecules. Non-limiting examples of methods of building such molecules can also be found in the examples described herein.
In an exemplary form, the application contemplates a single polypeptide binding chain having a variable region heavy chain, and / or a variable region light chain, which binds to the endoglobin and optionally has an immunoglobulin Fc region. In an exemplary form, the application contemplates a single polypeptide link chain having a variable region heavy chain, and / or a variable region light chain, which binds to endoglobin and inhibits angiogenesis and has, optionally, an Fc region immunoglobulin. Such a molecule is a fragment of variable single chain optionally having the effector function or increased half-life through the presence of the immunoglobulin Fc region. Methods of preparing single-stranded polypeptides are known in the art (for example, U.S. Patent Application, No. 2005 / 0.238,646).
The terms "germline gene segments" or "germline sequences" refer to the germline genes (the haploid gametes and the diploid cells from which they are formed). Germinal DNA contains multiple segments of genes that encode a single heavy or light Ig chain. These segments of genes are carried out on germ cells, but cannot be transcribed and translated into heavy and light chains, until they are arranged in functional genes. During the differentiation of B cells in the bone marrow, these segments of genes are randomly mixed by a dynamic genetic system capable of generating more than 108 specificities. Most of these gene segments are published and collected by the germline database.
As used here, "immunoreactive" refers to binding agents, antibodies or fragments that are specific to a sequence of amino acid residues ("binding site" or "epitope"), still cross-reacting with other peptides / proteins, are not toxic to the levels at which they are formulated for administration for human use. The term "mandatory" refers to a direct association between two molecules, due, for example, to covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding, interactions under physiological conditions, including interactions such as salt and water bridges and bridges any other conventional means of connection. The term "preferably binds" means that the binding agent binds to the binding site with greater affinity than it binds unrelated amino acid sequences. Preferably, the affinity is at least once greater, at least twice greater, at least three times greater, at least four times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater at least 8 times greater, at least nine times greater, 10 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least at least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, or at least 1000 times greater than the binding agent's affinity for independent amino acid sequences. The terms "immunoreactive" and "preferentially bind" are used interchangeably here.
As used herein, the term "affinity" refers to the equilibrium constant of the reversible bond of two agents, and is expressed as KD. Affinity of a linker binding protein such as the affinity of an antibody for an epitope can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nm to about 1 picomolar (pM), or from about 100 nm to about 1 femtomolar (fM). As used herein, "avidity" refers to the resistance of a complex of two or more dissociating agents after dilution. Apparent affinities can be determined by methods such as an enzyme immunoadsorption assay (ELISA) or any other technique known to a person skilled in the art. Avidity can be determined by methods such as a Scatchard analysis or any other technique known to a person skilled in the art. "Epitope" refers to the part of an antigen or other macromolecule capable of forming a binding interaction with the binding receptacle of the variable region of an antibody. Such binding interactions can be manifested as intermolecular contact with one or more amino acid residues from one or more CDRs. Antigen binding may involve, for example, a CDR3 or a CDR3 pair or, in some cases, interactions of up to all six CDRs in the VH and VL chains. An epitope can be a linear polypeptide sequence (i.e., "continuous") or it can be composed of non-contiguous amino acid sequences (for example, "conformational" or "discontinuous"). An antibody is capable of recognizing one or more amino acid sequences and, therefore, the epitope can define more than one distinct amino acid sequence. Epitopes recognized by the antibodies can be determined by peptide mapping techniques and the sequence of analysis well known to one skilled in the art. Binding interactions are manifested as intermolecular contacts with one or more amino acid residues from a CDR. TRC105 is a chimeric antibody that has the same variable amino acid sequence as the murine antibody described as Y4-2F1 or SN6j in U.S. Patent Nos. 5,928,641; 6,200,566; 6,190,660; and 7,097,836. Epitopes recognized by Y4-2F1 and SN6j and, therefore, TRC105, had previously been identified. The term "specific" refers to a situation in which an antibody does not show any significant binding to other molecules than the antigen containing the epitope recognized by the antibody. The term is also applicable when, for example, an antigen binding domain is specific to a particular epitope that is carried by a number of antigens, in which case the antibody or antigen binding fragment thereof that carries the binding domain of the antigen will be able to bind to the various antigens that carry the epitope. The terms "preferentially bind" or "specifically bind" mean that antibodies or their fragments bind to an epitope with greater affinity than it binds to unrelated amino acid sequences and, if cross-reactive to other polypeptides that contain the epitope, they are not toxic at the levels at which they are formulated for administration for human use. In one aspect, the affinity is at least 1 times greater, at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater , at least 8 times greater, at least 9 times greater, 10 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least at least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, or at least 1000 times greater than the affinity of the antibody or its fragment for independent amino acid sequences. The terms "immunoreactive", "binds", "preferably binds" and "specifically binds" are used interchangeably here. The term "mandatory" refers to a direct association between two molecules, due, for example, to a covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding, interactions under physiological conditions, and includes interactions with saline and water bridges, as well as any other conventional means of bonding. The phrase "conservative amino acid substitution" refers to a grouping of amino acids based on certain common properties. A functional way to define the common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between the corresponding proteins of homologous organisms (Schulz, GE and Schirmer's RH, Principies of Protein Structure, Springer-Verlag) .According to such analyzes, groups of amino acids can be defined in which amino acids within an exchange group preferentially with each other, and therefore more closely resemble each other in their impact on the structure of total protein (Schulz, GE and RH Schirmer, Principles of Protein Structure, Springer-Verlag). Examples of amino acid groups defined in this way include: (i) a charged group, consisting of Glu and Asp, Lys, Arg and His, (ii) a positively charged group, consisting of Lys, Arg and His, (iii) a group negatively charged, consisting of Glu and Asp, (iv) an aromatic group, consisting of Phe, Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large aliphatic non-polar group , consisting of Val, Leu and Ile, (vii) a slightly polar group, consisting of Met and Cys, (viii) a group of small residues, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro, (ix) the group consisting of an aliphatic group Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group consisting of Ser and Thr.
In addition to the groups shown above, each amino acid residue can form its own group, and the group formed by an individual amino acid can be referred to simply by one and / or three letters of abbreviation for the amino acid commonly used in the art, as described above.
A "conserved residue" is an amino acid that is relatively constant across a range of similar proteins. Conserved residues will often vary just by being replaced with a similar amino acid, as described above for "conservative amino acid substitution". The letter "x" or "xaa" as used in amino acid sequences here is intended to indicate that any of the twenty amino acids that can be placed in this position unless specifically indicated otherwise. For the purpose of peptidomimetic design, an "x" or "xaa" in an amino acid sequence can be replaced by one of the mimetic amino acid present in the target sequence, or the amino acid can be replaced by a spacer in essentially any way that does not interfere with peptidomimetic activity. "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence that can be aligned, for comparison purposes. When an equivalent position compared to the sequences is occupied by the same base or amino acid, then the molecules are identical in that position, when the equivalent location occupied by the same or a similar amino acid residue (for example, similar in nature and / or electronics), then the molecules can be referred to as homologous (similar) in that position. Expression as a percentage of homology / identity or similarity refers to a function of the number of identical or similar amino acids at the positions shared by the compared sequences. A sequence that is "unrelated" or "non-homologous" shares less than 40% identity, although preferably less than 25% identity with a sequence of the present invention. When comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of additional residues also decreases the identity and homology / similarity. The term "homology" describes a mathematically based comparison of similarities that is used to identify genes or proteins with similar functions or motifs. The nucleic acid (nucleotide, oligonucleotide) and amino acid (protein) sequences of the present invention can be used as a "query string" to perform a search on public databases to, for example, identify other family members, related or homologous sequences. These searches can be performed using the programs NBLAST and XBlast (version 2.0) by Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. Nucleotide BLAST searches can be performed with the NBLAST program, with punctuation = 100 and word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST amino acid searches can be performed with the XBlast program with punctuation = 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain spaced alignments for comparison, spaced BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and BLAST spaced programs, the standard parameters of the respective programs (for example, XBlast, and BLAST) can be used (see www.ncbi.nlm.nih.gov).
As used herein, "identity" means the percentage of identical acid residues of nucleotides or amino acids at the corresponding positions in two or more sequences, when the sequences are aligned to maximize sequence matching, that is, taking into account the gaps and insertions. Identity can be easily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW , ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods for determining identity are designed to provide the greatest similarity between the tested sequences. In addition, methods for determining identity are encoded in publicly available computer programs. determine the identity between two sequences uences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al ., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available through NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215 : 403-410 (1990). Smith Waterman's well-known algorithm can also be used to determine identity. "Isolated" (used interchangeably with "substantially pure") when applied to polypeptides means a polypeptide or a portion thereof that , by virtue of its origin or manipulation: (i) it is present in a host cell as the expression product of a portion of an expression vector, or (ii) it is attached to a protein or chemical portion other than that to which it is attached in nature, or (iii) does not occur in nature, for example, a protein that is chemically manipulated by attaching, or aRNA by attaching at least one hydrophobic portion to the protein so that the protein is in a form not found in nature. , means additionally a protein that is: (i) chemically synthesized, or (ii) expressed in a host cell and purified away from the association and contaminating proteins. The term generally means a polypeptide that has been separated from other proteins and nucleic acids with which it occurs naturally. Preferably, the polypeptide is also separated from substances such as antibodies or gel (polyacrylamide) matrices that are used to purify.
As used herein, the terms "angiogenesis inhibitor," "angiogenesis inhibition" or "antiangiogenic" include vasculogenesis, and are meant to effect a decrease in the extent, amount, or rate of neovascularization. Making a decrease in the extent, amount, or rate of endothelial cell proliferation or tissue migration is a specific example of inhibition of angiogenesis. The term "angiogenesis inhibitory composition" refers to a composition that inhibits angiogenesis mediated by processes such as endothelial cell migration, proliferation, tube formation and subsequently leading to the inhibition of the generation of new blood vessels from existing ones, and, consequently, the affect of conditions dependent on angiogenesis.
As used herein, the term "angiogenesis-dependent condition" is intended to mean a condition in which the process of angiogenesis or vasculogenesis maintains or augments a pathological condition or beneficially influences normal physiological processes. Therefore, treatment of a condition of dependent angiogenesis in which angiogenesis sustains a pathological condition could result in mitigation of the disease, while treatment of a condition of dependent angiogenesis in which angiogenesis beneficially influences normal physiological processes could result in, for example, improvement of a normal process. Angiogenesis is the formation of new blood vessels from pre-existing capillaries or post-capillary venules. Vasculogenesis results in the formation of new blood vessels resulting from angioblasts that are precursors to endothelial cells. Both processes result in the formation of new blood vessels and are included in the meaning of the term angiogenesis-dependent conditions. The term "angiogenesis" as used herein is intended to include the formation of new vessels, such as what will result in vasculogenesis, as well as those resulting from the branching and appearance of existing vessels, capillaries and venules. Angiogenesis may also be inclusive of induction of ALK1 signaling and related Smad 1/5/8 phosphorylation and / or signaling. CD105 is also known to be involved in the ALK1 signaling pathway and is therefore also included within the meaning of angiogenesis. "Inducing a host immune response" means that a patient experiences relief or reduction of disease signs or symptoms, and specifically includes, without limitation, prolonged survival. In certain preferred embodiments of the methods according to the invention, a CD8 + IFN-γ producing T cell is activated to induce the immune response of a cytotoxic T lymphocyte (CTL) in the patient in which the antagonist was administered. In certain forms of modality of the methods according to the invention, CD4 + IFN-y producing T cell is activated to induce an auxiliary T cell immune response in the patient in whom the composition was administered. These activated CD4 + IFN-y producing cells (ie helper T cells) provide the necessary immune help (for example, by releasing cytokines) to induce and maintain not only CTL, but also a humoral immune response mediated by B cells. Thus, in certain forms of method modality according to the invention, a humoral response to the antigen is activated in the patient in whom the composition was administered. In one aspect, an adjuvant can be added to the composition to enhance an immune response. Adjuvants are well known in the art. Activation of a CD8 + and / or CD4 + T cell means that T cells have the ability to produce cytokines (for example, IFN-γ) to actually produce one or more cytokines (s), or to increase their production of one or more cytokine (s). "Inducing the CTL response" means causing cytotoxic T lymphocytes to potentially expose antigen-specific cytotoxicity. "Antigen-specific cytotoxicity" means cytotoxicity against a cell that has an antigen that is associated with the cancer-associated antigen that is greater than an antigen that is not associated with a cancer. "Cytotoxicity" refers to the ability of the cytotoxic T lymphocyte to kill a target cell. Antigen-specific cytotoxicity, can be at least about 3 times, at least about 10 times greater, at least about 100 times greater, or more than cytotoxicity against a cell that lacks the antigen not associated with cancer. Antibody-mediated dependent cell cytotoxicity (CCDA), also includes the activation of natural killer cells ("NK cells") that mediate cell death through antibody binding. The antibodies and antigen-binding fragments described herein can mediate CCDA through NK cells by means of endoglobin binding.
As used herein, the terms "proliferative disease" and "proliferative condition" mean any non-pathological or pathological condition characterized by abnormal or undesirable proliferation. The terms "proliferative cell disorder" and "proliferative cell condition" mean any physiological pathological or non-pathological condition characterized by aberrant or undesirable cell proliferation, as well as conditions including characteristics of undesirable or unwanted cell proliferation or cell survival (for example , due to deficient apoptosis), conditions characterized by deficient or aberrant or deficient apoptosis, as well as conditions characterized by aberrant or undesirable or unwanted cell survival. "Differential disorder" means any pathological or non-pathological condition characterized by abnormal or deficient physiological differentiation.
Proliferative or differentiating disorders amenable to treatment include non-pathological, benign and neoplastic diseases and physiological conditions, characterized by the number of abnormal or undesirable cells, cell growth or cell survival. Such disorders or conditions may therefore constitute a disease state and include all types of cancerous tumors or oncogenic processes, malignant transformed metastatic tissues or cells, tissues or organs, or may be non-pathological, that is, a deviation from normal , but that is not typically associated with the disease. A specific example of a pathological condition that cannot be treated according to the invention is a re-growth of wound repair tissue that results in scarring. Cells comprising the proliferative or differentiating disorder can be aggregated into a mass of cells or be dispersed. The "solid tumor" refers to neoplasms or metastases that usually aggregate and form a mass. Specific examples include visceral tumors such as gastric or colon cancer, hepatoma, venous carcinomas, lung and brain tumors / cancer. A "non-solid tumor" refers to neoplasms of the hematopoietic system, such as lymphomas, myelomas and leukemias, or neoplasms that are diffuse in nature, as they do not normally form a solid mass. Particular examples of leukemias include, for example, multiple, lymphoblastic and myeloblastic, acute and chronic myeloma.
These diseases include neoplasms or cancers, which can affect virtually any cell or tissue type, for example, carcinoma, sarcoma, melanoma, metastatic disorders or hematopoietic neoplastic disorders. A metastatic tumor can arise from a multitude of primary tumor types, including, but not limited to, breast, lung, thyroid, head and neck, brain, lymphoid, gastrointestinal (mouth, esophagus, stomach, intestine thin, colon, rectum), genital urinary tract (uterus, ovary, cervix, bladder, penis, testicles, prostate), kidney, pancreas, liver, bone, muscle, skin, etc.
Carcinomas refer to malignant neoplasms of epithelial or endocrine tissue, and include carcinomas of the respiratory system, carcinomas of the gastrointestinal system, carcinomas of the genitourinary system, testicular carcinomas, breast carcinomas, prostate carcinoma, carcinomas of the endocrine system and melanomas. Exemplary carcinomas include those that form from the cervix, lung, prostate, breast, head and neck, colon, liver and ovary. The term also includes carcinosarcomas, for example, which include malignant tumors composed of carcinomatous and sarcomatous tissues. Adenocarcinoma includes a carcinoma of a glandular tissue, or in which the tumor forms a gland like structure.
An eye tissue to be treated is, for example, a retinal tissue of a patient with diabetic retinopathy, macular degeneration or neovascular glaucoma, and the angiogenesis to be inhibited is the retinal tissue angiogenesis, where there is neovascularization of the retinal tissue.
A cancerous tissue to be treated is, for example, an endothelial tissue expressing an abnormal level of endoglobin. As used herein, the term "transformed cells" refers to cells that have been spontaneously converted to an uncontrolled growth state, i.e., that have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells can be characterized by terms such as neoplastic, anaplastic and / or hyperplastic, with respect to their loss of growth control. For the purposes of this invention, the terms "transformed phenotype of malignant mammalian cells" and "transformed phenotype" are intended to encompass, but are not limited to, any of the following phenotypic characteristics associated with the cellular transformation of mammalian cells: immortalization, moFRological or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immunocompetent or syngeneic animals. The term "tumor cell antigen" is defined herein as an antigen that is present in higher amounts of a tumor cell or in body fluids than unrelated tumor cells, normal cells, or in normal body fluid. The presence of antigen can be tested by any number of assays known to those skilled in the art and include, without limitation, negative and / or positive selection of antibodies, such as an ELISA assay, a radioimmunoassay, or by Western Blot.
The terms "apoptosis" or "programmed cell death" refer to the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Apoptosis is a modality of cell death that occurs under normal physiological conditions and the cell is an active participant in its own death ("cell suicide"). It is most often found during normal cell renewal and tissue homeostasis, in embryogenesis, in the induction and maintenance of immune tolerance, in the development of the nervous system and tissue atrophy dependent endocrine system. Apoptosis cells show moFRological and biochemical characteristics. These features include chromatin aggregation, nuclear and cytoplasmic condensation, the partition of cytoplasm and nucleus into attached membrane vesicles (apoptotic bodies), which contain ribosomes, mitochondria and moFRologically intact nuclear material. In vivo, these apoptotic bodies are quickly recognized and phagocyted by macrophages, dendritic cells or adjacent epithelial cells. Due to this effective mechanism for removing apoptotic cells in vivo, an inflammatory response is not induced. In vitro, the apoptotic bodies, as well as the remaining cell fragments, ultimately swell and ultimately lys. In this terminal stage of in vitro assays cell death was termed "secondary necrosis." Apoptosis can be measured by methods known to those skilled in the art such as DNA fragmentation, annexin V exposure, activation of caspases, release of cytochrome c etc. A cell that has been induced to die is referred to here as an "apoptotic cell". "Apoptosis-inducing agent" is defined herein to induce programmed apoptosis / cell death, and includes, for example, irradiation, chemotherapeutic agents or receptor binding agents, cells in which, for example, tumor cells are induced to undergo programmed cell death. Examples of apoptosis-inducing agents are described in more detail below. Apoptosis can be tested using an NIH Apextosis Apoptosis Assay standard: OVCAR-3 cells are cultured in 6-well plates (NUNC) and irradiated or treated with an antagonist (or in combination with another anti-cancer drug) 4-48 hours, washed and stained with annexin V-FITC (BD-Pharmingen) for 1 hour. The cells are analyzed by flow cytometry (Becton-RNAkinson, CellQuest), contrasted with propidium iodide and analyzed again on the flow cytometer.
B. METHODS FOR MAKING AND EXPRESSING HUMANIZED ANTIENDOGLIN ANTIBODIES
A chimeric monoclonal antibody that binds to endoglobin has been developed. This antibody was designated TR105 (also known as c-SN6j).
In one aspect, the antibodies and antigen-binding fragments thereof described herein were created by humanizing the VL and VH sequences of the chimeric monoclonal antibody TRC105 (SEQ ID NOS. 1 and 39, respectively).
Humanized immunoglobulins, including humanized antibodies, were constructed using genetic engineering. Most humanized immunoglobulins that have been previously described have a compound structure that is identical to that of a particular human immunoglobulin chain (ie, an acceptor or receptor), and three CDRs of a non-human immunoglobulin chain (ie, donor ). As described herein, humanization may also include criteria that a limited number of amino acids within a humanized immunoglobulin chain is identified and chosen to be the same as the amino acids at those positions in the donor, rather than in the acceptor, in order to increase or maintain the affinity of an antibody comprising the humanized immunoglobulin chain. The present invention is based, in part, on the model that two causes that contribute to the loss of affinity in previous means of producing humanized antibodies (using as examples mouse antibodies as the source of CDRs) are: (1) when CDRs of the mouse are combined with a human structure, the amino acids in the structures close to the CDRs become human instead of mouse. Without claiming to be limited by theory, these amino acids can be slightly distorting the CDRs (for example, they can create different electrostatic or hydrophobic forces than in the donor mouse antibody, and the distorted CDRs may not make contact with the antigen as effective as CDRs make in the donor antibody), (2) also, amino acids in the original mouse antibody that are close to, but not part of, the CDRs (that is, still part of the structure), can make contacts with the antigen that contribute to affinity. These amino acids are lost when the humanized antibody is because, in general, all amino acids are made of human frame. To circumvent these problems, and to produce humanized antibodies that have a very strong affinity for a desired antigen, humanized antibodies and antigen binding fragments can be constructed using one or more of the following principles.
A non-limiting principle is that, for example, as an acceptor, a structure is used from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or using a consensus structure of many human antibodies is used as an acceptor . For example, comparing the sequence of a variable (or light) mouse heavy chain region against variable human (or light) regions in a database (for example, the National BiomeRNAal Research Foundation Protein Identification Resource or the database protein sequence data from the National Center for Biotechnology Information -NCBI) shows that the extent of homology with different human regions can vary widely, for example, from about 40% to about 60%, about 70%, about 80% or superior. When choosing as the acceptor immunoglobulin one of the variable regions of the heavy chain that is more homologous to the variable region of the heavy chain of the donor immunoglobulin, less amino acids will be changed in the transition from the donor immunoglobulin to the humanized immunoglobulin. By choosing as the accepting human immunoglobulin one of the light chain variable regions that is more homologous to the donor immunoglobulin light chain variable region, less amino acids will be altered in the transition from donor immunoglobulin to humanized immunoglobulin. Generally, using such techniques, there is less chance of changing an amino acid close to one or more of the CDRs, which distorts their conformation. In addition, the general exact shape of a humanized antibody that comprises the humanized immunoglobulin chain may be more similar to the shape of the donor antibody, thus reducing the possibility of distorting the CDRs.
Light and heavy chains of the same human antibody can also be used as acceptor sequences, to improve the likelihood that humanized light and heavy chains make favorable contacts with each other. Alternatively, light and heavy chains of different sequences of human germline antibodies can also be used as acceptor sequences; when such combinations are used, one can easily determine whether the VH and VL bind an epitope of interest using conventional assays (for example, an ELISA). In one example, the human antibody will be chosen in which the sequences of the variable regions of the light and heavy chains, taken together, are in general more homologous to the donor light and the sequences of the variable region of the heavy chain. Sometimes more weight will be given to the heavy chain sequence. Regardless of how the acceptor immunoglobulin is chosen, the highest affinity can, in some cases, be achieved by selecting a small number of amino acids within the humanized immunoglobulin chain to be the same as the amino acids at positions in the donor before than in the acceptor. Affinity maturation methods are known in the art.
Humanized antibodies generally have at least three potential advantages over mouse or chimeric antibodies for use in human therapy. Because the effector portion of an antibody is human, it is believed to interact better with the other parts of the human immune system (for example, destroying target cells more efficiently by the complement of cytotoxicity dependent (CDC) or antibody dependent on cell cytotoxicity (CCDA)). In addition, the human immune system must not recognize the structure or constant region of the humanized antibody as foreign, and therefore the antibody response against that injected antibody must be less than against a totally foreign mouse antibody or a chimeric antibody. partially external. Finally, mouse antibodies are known to have a circulating human half-life that is much shorter than the half-life of human antibodies. Humanized antibodies can presumably have a half-life more similar to those that occur naturally in human antibodies, allowing smaller and less frequent doses to be administered. The humanization of antibodies and antigen-binding fragments thereof can be accomplished by a variety of methods known in the art and described herein. Likewise, the production of humanized antibodies can also be achieved by methods known in the art and described herein. Methods for modifying regions of the structure are known in the art and are contemplated herein. Selection of one or more structures relevant to amino acid positions for alteration depends on a variety of criteria. A criterion for selecting the relevant amino acid structures for exchange may be the relative differences in amino acid residue structures between the donor and acceptor molecules. Selection of the relevant positions and structures for exchange using this approach has the advantage of avoiding any subjective tendency in determining the residue or any bias in contribution to the CDR binding affinity for the residue.
Another criterion that can be used to determine the positions of relevant amino acids for exchange may be, for example, the selection of structural residues that are known to be important or contribute to the formation of CDR. For example, canonical structural residues are important for the formation of CDR and / or structure. Targeting a canonical structure residue as a relevant position for exchange can be used to identify an amino acid residue more compatible in context with the sequence associated with your CDR donor. The frequency of an amino acid residue at the position of a particular structure is another criterion that can be used to select positions of relevant amino acid structures to change. For example, comparing the selected structure with structural sequences from others in its subfamily can reveal residues that occur at lower frequencies in a particular position or positions. Positions harboring the less abundant residues are equally applicable for selection as a changing position of the structure of the acceptor's variable region.
The corresponding positions of amino acids for exchange can also be selected, for example, based on the proximity of a CDR. In certain contexts, RF residues may participate in the formation of the CDR and / or antigen binding. In addition, this criterion can also be used to give priority to the relevant positions selected by other criteria described here. Therefore, differentiating between proximal and distal residues to one or more CDRs represents a way to reduce the number of relevant positions to change.
Other criteria for selecting relevant positions of the amino acid structures to be exchanged include, for example, residues that are known or predicted to be in a three-dimensional space close to the CDR antigen inteFRace or intended to modulate CDR activity. Likewise, structural residues that are known for, or predicted for, the shape contacts between the interface of the variable region of heavy (VH) and light (VL) can be selected. Such structural positions can affect the conformation and / or affinity of a CDR, modulating the CDR receptacle binding, interaction with the antigen (epitope) or interaction with VH and VL. Therefore, the selection of these amino acid positions to build a diverse population for the screening of binding activity can be used to identify structural changes that replace residues that have detrimental effects on CDR conformation or compensate for the harmful effects of residues that occur anywhere else in the structure.
Other structural residues can be selected to include changes in amino acid positions that are inaccessible to the solvent. These residues are usually buried in the variable region and are therefore able to influence the conformation of the CDR or interactions with VH and VL. Solvent accessibility can be predicted, for example, from the relative hydrophobicity of the environment created by the polypeptide amino acid side chains and / or by known three-dimensional structural data. The following selection of relevant amino acid positions in the donor CDRs, as well as any relevant amino acid positions in the structure regions desired to be varied, the amino acid changes in some or all of the selected positions can be incorporated into nucleic acids that encode the structure of the variable and accepting region of donor CDRs. Altered CDR structures or sequences can be made and tested individually, or they can be sequentially or simultaneously combined and tested. The variability in any or all of the altered positions can vary from a few to a plurality of different amino acid residues, including all twenty naturally occurring amino acids or functional equivalents and their analogs. In some cases, non-naturally occurring amino acids can also be considered and are known in the art. The selection of the number and location of amino acid positions to vary is flexible and may depend on the intended use and the desired efficiency for identifying the altered variable region, with a desirable activity, such as, substantially the same or greater affinity compared to connection of the donor variable region. In this regard, the greater the number of changes that are incorporated into a population of the altered variable region, the more efficient it is to identify at least one species that exhibits a desirable activity, for example, substantially the same or higher binding affinity as the donor. Alternatively, when the user has empirical or real data to the effect that certain amino acid residues or positions disproportionately contribute to binding affinity, then it may be desirable to produce a limited population of altered variable regions that focus on changes within or around those identified residues or positions.
For example, if CDRs grafted with variable regions are desired, a highly diverse population of altered variable regions may include all positions of non-identical structural regions between the donor and acceptor structure and all unique changes in amino acid position in the CDR . Alternatively, a population of intermediate diversity may include subsets, for example, of only the positions of non-identical proximal structures to be incorporated together with all the unique changes in amino acid position in the CDR to, for example, increase the affinity of humanized antibodies or its antigen binding fragments. The diversity of the populations above can be further increased by, for example, additionally, including all of the pairs of changes in amino acid position in the CDR. In contrast, populations centered on predetermined residues or positions, which incorporate residues from variants with as little or less than a structure and / or an amino acid position in the CDR can similarly be constructed to detect and identify a variable region of the altered antibody. As with the populations above, the diversity of such concentrated populations can be further increased by further expanding the positions selected for the change to include other relevant positions in one or both of the structure and CDR regions. There are numerous other combinations ranging from few changes to many changes in one or both of the structure regions and the CDRs that can additionally be used, which will result in a population of altered variable regions that can be traced to identify at least one variable altered region grafted into the CDR and having the desired activity, for example, mandatory activity for endoglina. Those skilled in the art will know, or can determine, which selected positions of residues in the structures of CDRs or donors, or subsets, can be varied to produce a population for detection and identification of an altered antibody of the invention taking into account the teachings and guidelines here provided. Codons encoding amino acids are known in the art.
Another method of humanizing antibodies includes a method called superhumanization. Superhumanization involves the steps of obtaining a polypeptide sequence from a variable subject region encoded by a mature non-human antibody gene and identifying a first set of canonical CDR structure types for at least two CDRs within the non-human variable region antibody. Types of canonical CDR structures are the types of structure called Chothia (ISCED). Chothia and colleagues found that critical portions of the CDRs of many antibodies adopt conformations almost identical to the peptide skeleton, despite great diversity in terms of the amino acid sequence. Consequently, Chothia defined for each CDR in each chain one or a few "canonical structures." Each canonical structure mainly specifies a set of torsion angles of the peptide skeleton of a contiguous segment of amino acid residues that form a loop.
After identifying the type of canonical structure of the CDR, a library of polypeptide sequences for human antibody regions of variables for human antibodies is also obtained. This library contains sequences for humans from the variable regions of the germ line as encoded by nucleic acid segments of the germ line, and may include sequences of mature human antibodies. In any case, the method includes identifying canonical CDR structure types (i.e., a second set of canonical CDR structure types) for at least two CDRs for each sequence within the sequence library of the human variable region. From this library a subset of candidate sequences is selected, comparing the first set of canonical CDR structure types to the second set of canonical CDR structure types (for example, comparing the types of canonical CDR structures of mice with types of human canonical CDR structures, at corresponding locations within the variable region) and selecting human sequences, where the second set of canonical CDR structures is the same as the first set of canonical CDR structures types for CDR sequences , in corresponding locations within the non-human and human variable regions, respectively. The method uses these candidate sequences from the human variable region as a basis for the construction of a chimeric molecule that includes at least two of the CDR sequences from the non-human variable region (for example, from the mouse CDRs), combined with the framework regions. from the candidate sequence of the human variable region. The result of the construction is that the chimeric antibody contains each of the substituted non-human CDR sequences for each of the human CDR sequences at corresponding locations in the variable regions so that the chimeric antibody framework sequences differ from the candidate human framework sequences. The similarity with the subject CDRs of candidate human antibody sequences is assessed for each domain at two levels. First, identical three-dimensional conformations of the main structure of the CDR peptides are sought. Experimentally determined atomic coordinates of the subject CDRs are rarely available, hence three-dimensional similarity is approximated by determining Chothia's canonical structure types from the individual CDRs and excluding future consideration of candidates who have different canonical structures. Secondly, the residue for homology residue between individual CDRs and the remaining human CDR candidates is considered, and the candidate with the highest homology is chosen. The choice of the highest homology is based on several criteria used to classify candidate human variable regions that have the same canonical structure as the individual from non-human variable regions. The criterion for classifying the members of the selected set can be by amino acid sequence identity or amino acid homology or both. The identity of the amino acids is a simple position score by position matching of amino acid residues. The similarity by homology of amino acids is the position by similarity of position in the structure of the characteristic residue. Homology can be marked, for example, according to the tables and procedures described by Henikoff and Henikoff, (1992) substitution amino acid matrices from protein blocks, Proc. Natl. Acad. Sci. 89: 10915-10919, or by the BLOSUM series described by Henikoff and Henikoff, (1996). The steps are as follows: a) determining the polypeptide sequences of the variable and heavy chain variable domains of the subject antibody. These can be determined by any of several methods, such as DNA sequencing of the respective genes after conventional cDNA cloning; DNA sequencing of cloning products that have been amplified by polymerase chain reaction from inverted transcripts or DNA from the subject hybridoma line; or peptide sequencing of a purified antibody protein. b) Apply the Kabat numbering system (Kabat et al, Id .. 1991) with the heavy and light chain sequences of the subject non-human antibody. Determine the types of canonical structure for each of the CDRs of the subject non-human antibody. This determination is made by examining the peptide sequence according to the guidelines discussed in Chothia and Lesk (1987), Chothia et al (1992), Tomlinson et al (1995), Martin and Thornton (1996), and Al-Lazikani et al (1997).
The salient features of determining the canonical structure for each of the CDRs are as follows. For the heavy chain of CDR1, three types of canonical structures are known today. Assigning a new sequence is simple, because each type of canonical structure has a different number of residues. As described in Al-Lazikani et. al (1997), when the Kabat numbering is assigned to the sequence, the residue numbering from 31-35 will be as follows for the respective canonical structures.
Canonical structure of type 1: 31, 32, 33, 34, 35.
Canonical structure type 2: 31, 32, 33, 34, 35, 35a.
Canonical structure of type 3: 31, 32, 33, 34, 35, 35a, 35b.
For the heavy chain CDR2, four types of canonical structures are currently known. Several have unique residue numbers, and are easily distinguished from their unique Kabat numbers for positions 52-56, viz .: Canonical structure type 1: 52, 53, 54, 55, 56.
Canonical structure type 4: 52, 52a, 52b, 52c, 53, 54, 55, 56.
Canonical structure types 2 and 3 for heavy chain CDR2 have equal numbers of residues, so they must be distinguished by clues within their sequence, as discussed by Chothia et al (1992). The Kabat numbering of the segment containing these tracks is: 52, 52a, 53, 54, 55. The type of canonical structure 2 has Pro or Ser in position 52a and Gly or Ser in position 55, with no restrictions in other positions. Canonical structure type 3 has Gly, Ser, Asn, Asp or in position 54, with no restrictions in other positions. These criteria are sufficient to resolve the correct assignment in most cases. In addition, residue frame 71 is commonly Ala, Val, Leu, Ile, Thr or for the canonical structure type 2 and commonly Arg for the canonical structure type 3. The CDR3 heavy chain is the most diverse of all CDRs. It is generated by genetic processes, some of a random nature, unique to lymphocytes. Consequently, the canonical structures for CDR3 have been difficult to predict. In any case, segments of human germline V genes do not encode any part of CDR3; because the final V gene segments at position of Kabat 94, while positions 95-102 encode CDR3. For these reasons, the canonical structures of CDR3 are generally not considered when choosing candidate human sequences.
For CDR1 light chain, six types of canonical structures are currently known for CDR1 in kappa chains. Each type of canonical structure has a different number of residues, so the assignment of a type of canonical structure to a new sequence is apparent from the Kabat numbering of residue positions 27-31.
Canonical structure of type 1: 27, 29, 30, 31.
Canonical structure type 2: 27, 28, 29, 30, 31.
Canonical type of structure 3: 27, 27a, 27b, 27c, 27d, 27e, 27F, 28, 29, 30, 31.
Canonical structure type 4: 27, 27a, 27b, 27c, 27d, 27e, 28, 29, 30, 31.
Canonical type of structure 5, 27, 27a, 27b, 27c, 27d, 28, 29, 30, 31.
Canonical structure type 6, 27, 27a, 28, 29, 30, 31.
For the CDR2 light chain, only one type of canonical single structure is known as CDR2 in kappa chains, therefore, except for exceptional sequences of subject antibodies, the assignment is automatic. For light chain CDR3, up to six types of canonical structures have been described for CDR3 in kappa chains, but three of these are rare. The three most common can be distinguished by their length, reflected in the Kabat numbering of the residue positions 91-97: Canonical structure type 1: 91, 92, 93, 94, 95, 96, 97 (also with a mandatory Pro in the position 95 and Gln, Asn, or His in position 90).
Canonical structure type 3: 91, 92, 93, 94, 95, 97.
Canonical structure type 5: 91, 92, 93, 94, 95, 96, 96a, 97.
After identifying the types of canonical CDR structures of the non-human antibodies of the beginning, human genes of the same type of chain (heavy or light), which have the same combination of types of canonical structures as the subject antibody are identified to form a group candidate human sequences. Most of these gene fragments have been discovered and have already been attributed to a type of canonical structure (Chothia et al, 1992, Tomlinson et al, 1995).
For the heavy chain, the conformity of CDR1 and CDR2 for the types of canonical structure of the mouse is assessed, and genes that do not conform are excluded. For the light chain, the compliance of CDR1 and CDR2 of each human sequence for the types of canonical structures of the subject antibody is first assessed. The potential of 89-95 residues of a candidate Vk gene to form a CDR3 of the same canonical structure type as the subject antibody is evaluated by placing a fusion of the gene with a J region and applying criteria for determining the type of CDR structure canonical CDR3 with the fused sequence, and non-conforming sequences are excluded.
Alternatively, when a variable domain of the subject antibody is of a type of canonical structure not available in the human genome, human germline V genes that have three-dimensionally similar but not identical canonical structure types are considered for comparison. Such a condition often occurs with chain in murine kappa antibodies CDR1, including two of the examples described below. All 6 possible types of canonical structures have been observed in this murine antibody CDR, while the human genome encodes only canonical types 2, 3, 4 and 6. In these circumstances, a type of canonical CDR structure has length of amino acid residues within two lengths of the amino acid residues of the subject's non-human sequence can be selected for comparison. For example, where a canonical structure type 1 is found in the subject antibody, human Vk sequences with canonical structure type 2 are used for comparison. Whenever a type of canonical structure 5 is found in the murine antibody, human Vk sequences with any type of canonical structure 3 or 4 are used for comparison.
Sequences of mature and rearranged human antibodies can be considered for sequence comparison. Such consideration can be warranted under a variety of circumstances, including, but not limited to, cases where the mature human sequence (1) is too narrow for germline, (2) is not known to be immunogenic in humans; or (3) contains a type of canonical structures identical to that of the subject antibody, but not found in the human germline.
For each of the candidate V genes with which it satisfies the types of canonical structures, residues for the identification of the residues of the sequence and / or homology with the subject sequence are also evaluated to classify the candidate human sequences. For example, the evaluated residues are as follows: (1) Kappa (κ) CDR light chain amino acid residue positions are CDR1 (2632), CDR2 (50-52), CDR3 (91-96), and (2) heavy chain CDR positions of amino acid residues are CDR1 (31-35) and CDR2 (50-60). In addition, the heavy chain of CDR3 positions of amino acid residues 95-102 can also be considered. The waste-to-waste homology is first marked by the number of identical amino acid residues between the subject and the candidate human sequences. The human sequence used for the subsequent construction of a converted antibody is chosen from among the 25 percent of candidates with the highest score. When appropriate, such as when multiple candidate sequences have similar identity scores, the similarity between non-identical amino acid residues can additionally be considered necessary. Aliphatic to aliphatic, aromatic to aromatic, or polar to polar fittings between subject and subject residues are added to the scores. In another example, quantitative evaluation of sequence homology can be performed using substitution matrices with amino acids, such as the BLOSUM62 matrix of Henikoff and Henikoff.
A subject sequence for the C-terminal structure region and the CDR3 sequence can be selected from the set of human segments J known from the germline. The J polypeptide sequence is selected by assessing the residue-residue homology for each J segment for the sequence positions for which CDR3 and J overlap, using the specified scoring criteria for the evaluation of candidate V genes as mentioned above. The segment of the polypeptide sequence of gene J used for the later construction of a converted antibody is chosen from among the 25 percent of candidates with the highest score.
As an example, the variable chimeric chain contains at least two CDRs from a subject non-human sequence, and structure sequences from a candidate human sequence. In another example, the chimeric light chain contains three CDRs from a subject non-human sequence and structure sequences from a candidate human sequence. In other examples, a chimeric heavy chain contains at least two CDRs from a subject heavy chain, and the structure sequence of a candidate human heavy chain, or a chimeric heavy chain each contains a heavy chain of the subject CDRs and candidate structural sequences from a human heavy chain. In yet another example, a chimeric antibody heavy chain contains CDRs 1 and 2 from a subject non-human sequence and residues 50-60 of CDR3 and residues 61-65 of a heavy chain human candidate CDR, along with the structural sequences of the candidate human sequence. In another example, a chimeric heavy chain sequence contains each CDR from the subject's non-human sequence; structures of the 27-30 sequences form the subject sequence, and the framing sequences from the candidate sequences. In all cases, however, the chimeric antibody molecule contains no more than 10 amino acid residues in the sequence of structures that differ from those of the candidate human variable feed structure sequence.
When an increase in the affinity of a humanized antibody is desired, residues within the converted antibody's CDRs can be additionally substituted with other amino acids. Typically, no more than four amino acid residues in a CDR are altered, and more typically no more than two residues in the CDR will be altered, except for the heavy chain of CDR2, where as many as 10 residues can be altered. Changes in affinity can be measured by conventional methods, such as those described here (for example, Biacore).
The methods of superhumanized antibodies are described in more detail in U.S. Patent No. 6,881,557 which is incorporated herein by reference in its entirety.
Humanized antibodies or antigen binding fragments can be constructed and produced using conventional techniques known in the art. In addition, recombinantly prepared antibodies can often be produced in large quantities, particularly when using high level expression vectors.
Antibodies can be sequenced using conventional techniques known in the art. In one aspect, the amino acid sequences of one or more of the CDRs are inserted into a synthetic sequence of, for example, a human antibody structure (or antigen binding fragment thereof) to create a human antibody that can limit side reactions adverse effects of treating a human patient with a non-human antibody. The amino acid sequences of one or more of the CDRs can also be inserted into a synthetic sequence of, for example, a binding protein such as an AVIMER ™ to create a construct for administration to a human patient. Such techniques can be modified depending on the species of animal to be treated. For example, for veterinary uses, an antibody, antigen binding fragment or binding protein can be synthesized for administration to a non-human (for example, a primate, a cow, a horse, etc.).
In another aspect, using techniques recognized in the art, such as those provided and incorporated herein, nucleotides encoding amino acid sequences from one or more of the CDRs can be inserted, for example, by recombinant techniques into endonuclease restriction sites on an existing polynucleotide encoding an antibody, antigen binding fragment or binding protein.
For expression, an expression system is one that uses the GS system (Lonza), using a glutamine synthetase gene as the selectable marker. Briefly, a transfection is performed on CHO cells by electroporation (250V) using the GS system (Lonza), using the glutamine synthetase gene as the selectable marker. Wild-type CHO cells are cultured in DMEM (Sigma) containing 10% dialysed fetal calf serum (FCS) with 2mM glutamine. 6x107 CHO cells are transfected with 300pg of DNA linearized by electroporation. After electroporation, the cells are resuspended in DMEM with glutamine and plated in 36x96 well plates (50pl / well) and incubated at 37 ° C in 5% CO2. The next day, 150pL / well of selective medium (DMEM without glutamine) is added. After about 3 weeks, colonies are screened by ELISA (see below) using an irrelevant antibody as a negative control. All colonies producing> 20pg / ml are expanded into 24 wells and then into duplicate T25 flasks.
For high-level production, the most widely used mammalian expression system is one that uses the gene amplification procedure offered by deficient dihydrofolate reductase ("dhfr") Chinese hamster ovary cells. The system is well known to the person skilled in the art. The system is based on the "dhfr" dehydrofolate reductase gene, which encodes the DHFR enzyme, which catalyzes the conversion of dehydrofolate to tetrahydrofolate. In order to achieve high production, CHO dhfr cells are transfected with an expression vector containing a functional DHFR gene, along with a gene that encodes a desired protein. In this case, the desired protein is recombinant antibody heavy chain and / or light chain.
By increasing the amount of the competitive methotrexate DHFR inhibitor (MTX), recombinant cells develop resistance by amplifying the dhfr gene. In normal cases, the amplification unit used is much larger than the size of the dhfr gene, and as a result, the antibody heavy chain is co-amplified.
When large-scale production of the protein, such as the antibody chain, is desired, both the level of expression and the stability of the cells to be employed are taken into account. In long-term culture, recombinant CHO cell populations lose homogeneity with respect to their specific antibody productivity during amplification, although they are derived from a single parental clone. The present application provides an isolated polynucleotide (nucleic acid) that encodes an antibody or antigen binding fragment thereof as described herein, the vectors containing these polynucleotides, and host cells and expression systems for transcribing and translating such polynucleotides into polypeptides . The present application also provides constructs in the form of plasmids, vectors, transcripts or expression cassettes that comprise at least one polynucleotide as described above. The present application also provides a recombinant host cell that comprises one or more constructs as described above. A nucleic acid encoding any antibody or antigen binding fragments described herein, as provided in itself, is an aspect of the present application, as does a method of producing the antibody or antigen binding fragments described herein. , a method that includes expression based on the encoding of nucleic acid. Expression can be conveniently achieved by culturing under appropriate conditions of recombinant host cells containing the nucleic acid. After production by expression, an antibody or antigen binding fragment thereof can be isolated and / or purified using any suitable technique and then used as appropriate.
Specific antibodies or antigen-binding fragments thereof, and nucleic acid coding molecules and vectors described herein can be supplied isolated and / or purified, for example, from their natural environment, in substantially pure or homogeneous form. In the case of nucleic acid, free or substantially free of nucleic acid or genes of origin, other than the sequence encoding a polypeptide with the required function. The nucleic acid can comprise DNA or RNA and can be totally or partially synthetic. Purification methods are well known in the art.
Systems for cloning and expressing a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for the expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and NS0 mouse myeloma cells and many others. A common bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells, such as E. coli, is well established in the art. For a review, see, for example, Pluckthun, A. Bio / Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for the production of antibodies and antigen-binding fragments described herein, see for recent reviews, for example, Raff, ME (1993) Curr. Opinion Biotech. 4: 573-576; Trill J.J. et al. (1995) Curr. Opinion Biotech 6: 553-560, each of which is incorporated herein as a reference in its entirety.
Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, termination sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors can be plasmids, viruses, for example, phage, or phagemids, as appropriate. For more details see, for example, Molecular Cloning: a Labocamundongory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Labocamundongory Press. Many known techniques and protocols for nucleic acid manipulation, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in detail in Short Protocols in Molecular Biology , Second edition, Ausubel et al. eds., John Wiley & Sons, 1992. The methods disclosed by Sambrook et al. and Ausubel et al. they are incorporated herein by reference in their entirety and are well known in the art.
Thus, an additional aspect provides a host cell containing nucleic acid as described herein. An additional aspect further provides a method comprising introducing such a nucleic acid into a host cell. The introduction can employ any available technique. For eukaryotic cells, suitable techniques may include, for example, calcium phosphate transfection, DEAE Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, for example, vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include, for example, calcium chloride transformation, electroporation and transfection using bacteriophages. The introduction can be followed by causing or allowing expression from the nucleic acid, for example, by culturing host cells under conditions for expression of the gene.
In one embodiment, the nucleic acid is integrated into the genome (chromosome, for example) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome, according to standard techniques. Ig increases can be initiated as needed to maximize expression. The present application also provides a method which comprises using a construct as described above, in an expression system to express antibodies or antigen-binding fragments thereof, as reported above. The present application also relates to isolated nucleic acids, such as recombinant DNA molecules or cloned genes, or their degenerate variants, mutants, analogs, or fragments thereof, which encode an antibody or antigen binding fragment thereof as described herein. , which binds to endogline.
In one aspect, the present application provides for a nucleic acid encoding an antibody or antigen binding fragment thereof as described herein, which binds to endoglobin.
In a further embodiment, the complete DNA sequence of the recombinant DNA molecule or cloned gene of an antibody or antigen binding fragment thereof as described herein can be operably linked to an expression control sequence that can be introduced into an appropriate host. The application of the agreement extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the VH and / or VL, or portions thereof, of the antibody.
Another characteristic is the expression of the DNA sequences disclosed here. As is well known in the art, DNA sequences can be expressed by operatively linking them to an expression control sequence in a suitable expression vector and employing that expression vector to transform an appropriate unicellular host.
Such a device connecting a DNA sequence to an expression control sequence is clear, it includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading structure upstream of the sequence of DNA.
Polynucleotides and vectors can be provided in an isolated and / or purified form (for example, free or substantially free of polynucleotides of origin other than the polynucleotide that encodes a polypeptide with the required function). As used herein, "substantially pure", and "substantially free" refer to a solution or suspension containing less than, for example, about 20% or less foreign material, about 10% or less foreign material, about 5% or less of foreign material, about 4% or less of foreign material, about 3% or less of foreign material, about 2% or less of foreign material, or about 1% or less of foreign material .
A wide variety of expression vector / host combinations can be employed to express the DNA sequences of the present invention. Useful expression vectors may consist, for example, of chromosomal, non-chromosomal and synthetic DNA sequence segments. Suitable vectors include, but are not limited to, SV40 derivatives and known bacterial plasmids, for example, E. coli col El plasmids, Pcr1, pBR322, Pmb9 and their derivatives, plasmids such as RP4; Phage DNAs, for example, the numerous λ phage derivatives, for example, NM989, and other phage DNAs, for example, M13 and single-stranded phage DNA; yeast plasmids, such as plasmid 2u or its derivatives; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
Also provided here is a recombinant host cell that comprises one or more polynucleotide constructs. A polynucleotide encoding an antibody or antigen binding fragment thereof as described herein constitutes an aspect of the present application, as does a method of producing the antibody or antigen binding fragment thereof, which method comprises expression from polynucleotide. Expression can be achieved, for example, by culturing under appropriate conditions of recombinant host cells that contain the polynucleotide. An antibody or antigen binding fragment thereof can then be isolated and / or purified using any suitable technique, and used as appropriate.
Any of a wide variety of expression sequence control sequences that control the expression of a DNA sequence operably linked to it can be used in these vectors to express DNA sequences. Such useful expression control sequences include, for example, SV40 early or late promoters, CMV, vaccinia, adenovirus or polyoma, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the main operator and λ phage promoter regions, the fd coating protein control regions, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the acid phosphatase promoters (for example, PHO5), the promoter factors for mating of yeast, and other known sequences to control the expression of genes from prokaryotic or eukaryotic cells or their viruses, and various combinations of these.
Systems for cloning and expressing a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for the expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse myeloma cells and many others. A common bacterial host can be, for example, E. Coli. The expression of antibodies or antigen-binding fragments thereof in prokaryotic cells, such as E. coli, is well established in the art. For a review, see, for example, Pluckthun, A. Bio / Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art (Raff, ME (1993) Curr. Opinion Biotech 4: 573-576; Trill JJ et al (1995) Curr. Opinion Biotech 6: 553-560).
A wide variety of single-celled host cells are also useful for expressing DNA sequences. These hosts include well-known eukaryotic and prokaryotic hosts, such as E. coli strains, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO and YB / 20, NS0, SP2 / 0, RI cells .I, BW and LM, African green monkey kidney cells (eg, COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (eg, Sf9), and human cells and plant cells grown in tissues.
It will be understood that not all vectors, expression control sequences and hosts will work equally well to express DNA sequences. Not all hosts will function equally well with the same expression system. However, a person skilled in the art will be able to select the vectors, expression control sequences, and suitable hosts, without undue experimentation to perform the desired expression, without departing from the scope of the present application. For example, when selecting a vector, the host must be considered, since the vector must work on it. The number of copies of the vector, the ability to control that number of copies, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered. A person skilled in the art can select the appropriate vectors, expression control sequences, and hosts to perform the desired expression, without departing from the scope of the present application. For example, when selecting a vector, the host is considered because the vector works on it. The number of copies of the vector, the ability to control the number of copies, and the expression of any other proteins encoded by the vector, such as antibiotic markers, can also be considered. The present application also provides constructs in the form of plasmids, vectors, transcription or expression cassettes, as described elsewhere in the present application which comprises at least one polynucleotide as described above. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, termination sequences, polyadenylation sequences, enhancer sequences, selectable markers and other sequences as appropriate. Vectors can be viral plasmids, for example, phage, phagemid, etc., as appropriate. For more details see, for example, Molecular Cloning: the Labocamundongory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Labocamundongory Press. Many known techniques and protocols for nucleic acid manipulation, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in detail in Short Protocols, in Molecular Biology, Second edition, Ausubel et al. eds., John Wiley & Sons, 1992. The methods and disclosures by Sambrook et al. and Ausubel et al. are incorporated herein by reference.
In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, in particular with regard to potential secondary structures. Suitable single-cell hosts will be selected considering, for example, their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by DNA sequences to be expressed, and the ease of purifying the expression products.
An additional aspect provides a host cell containing one or more polynucleotides as disclosed herein. In addition, an additional aspect provides a method of introducing such one or more polynucleotides into a host cell, whatever the technique available. For eukaryotic cells, suitable techniques may include, for example, calcium phosphate transfection, DEAEDextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus (for example vaccinia) or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include, for example, calcium chloride transformation, electroporation, and transfection using bacteriophages. The introduction can be followed by causing or allowing expression from one or more polynucleotides, for example, by culturing host cells under conditions for the expression of one or more polypeptides of one or more polynucleotides. Inducible systems can be used and expression induced by the addition of an activator.
In one embodiment, polynucleotides can be integrated into the genome (for example, the chromosome) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome, according to standard techniques. In another embodiment, the nucleic acid is maintained in an episomal vector in the host cell.
The methods provided herein include the use of a construct as noted above, in an expression system to express a specific polypeptide.
Considering these and other factors, a person skilled in the art will be able to construct a variety of vector / expression control / host combinations that will express DNA sequences in fermentation or in large-scale animal culture.
A polynucleotide that encodes an antibody, or antigen binding fragment thereof, or a binding protein can be prepared recombinantly / synthetically in addition to, or instead of cloned. The polynucleotide can be designed with the appropriate codons for the antibody, or antigen binding fragment thereof, or a binding protein. In general, one will select the preferred codons for a desired host if the sequence is used for expression. The complete polynucleotide can be assembled from overlapping oligonucleotides prepared by standard methods and aggregated in a complete coding sequence. See, for example, Edge, Nature, 292: 756 (1981); Nambair et al, Science, 223: 1299 (1984); Jay et al, J. Biol. Chem., 259: 6311 (1984).
A general method for incorporating the specific site of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244: 182-188 (April 1989 ). This method can be used to create analogs with unnatural amino acids.
As mentioned above, a DNA sequence encoding an antibody or antigen binding fragment thereof can be prepared synthetically, rather than cloned. The DNA sequence can be designed with the appropriate codons for the antibody or antigen-binding fragment thereof from the amino acid sequence. In general, one will select preferred codons for the intended host, if the sequence is used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and aggregated into a complete coding sequence. See, for example, Edge, Nature, 292: 756 (1981); Nambair et al, Science, 223: 1299 (1984); Jay et al, J. Biol .. Chem., 259: 6311 (1984), each of which is incorporated herein by reference, in its entirety.
C. LMUNOGENICITY ANALYSIS IN SITU
If necessary, an antibody or antigen-binding fragment described herein can be evaluated for immunogenicity and, when necessary, be de-immunized (that is, the antibody is made less immune reactive by changing one or more epitopes of cell T). Analysis of immunogenicity and T cell epitopes present in the humanized anti-endoglobin antibodies or antigen binding fragments thereof as described herein can be performed using specific software and databases. Examples of software and databases include iTope ™ developed by Antitope of Cambridge, England. iTope ™ is an in silico technology for the analysis of peptide binding to human MHC class II alleles. ITope ™ software predicts binding peptides to human MHC class II alleles and thus provides an initial scan for the location of such "potential T cell epitopes." ITope ™ software predicts favorable interactions between the amino acid side chains of a peptide and specific binding sets within the binding streaks of 34 human MHC class II alleles. The location of the main binding residues is achieved by the in silico generation of overlapping peptides by a 9mer amino acid that measures the sequence of the variable region of the test antibody. Each 9mer peptide can be tested against each of the 34 MHC class II allotypes and labeled based on their potential "fits" and interactions with the MHC class II binding streak. Peptides that produce a high average binding score (> 0.55 in the iTope ™ scoring function) against> 50% of the MHC class II alleles are considered to be potential T cell epitopes. In such regions, the 9 core amino acid sequence for binding of the peptide in the streak of MHC class II is analyzed to determine the sets of MHC class II residues (P1, P4, P7, P6 and P9) and the possible contact residues of the T cell receptor (TCR) ( P-1, P2, P3, P5, P8).
After identifying any T cell epitopes, changes in amino acid residues, substitutions, additions and / or deletions can be introduced to remove the identified T cell epitope. Such changes can be made in order to preserve the antibody structure and function while still removing the identified epitope. Examples of modifications may include, but are not limited to, conservative amino acid changes. Techniques that explore the soluble complexes of recombinant MHC molecules in combination with synthetic peptides have come into use. These reagents and procedures can be used to identify the presence of T cell clones from samples of peripheral blood from human subjects or experimental animals that are capable of binding to particular MHC peptide complexes and are not adapted for scanning potential epitopes multiple for a wide variety of MHC allotypes.
Biological T cell activation assays remain the best practical option for providing a reading of the ability of a test peptide / protein sequence to evoke an immune response. Examples of this type of approach include the use of T cell proliferation assays for bacterial protein staphylo kinase, followed by epitope mapping using synthetic peptides to stimulate T cell lines. Likewise, T cell proliferation assays using synthetic peptides of the tetanus toxin protein resulted in the definition of regions of immunodominant epitopes of the toxin. In one embodiment, epitopes of T cells in a test protein can be determined using isolated subsets of cells from the human immune system, promoting their differentiation in vitro and culturing cells in the presence of synthetic peptides of interest and measuring any proliferation induced in T-cell culture. Other techniques can also be used. Such a technique involves the careful application of cell isolation and cell culture techniques with multiple cytokine supplements to obtain the desired immune cell subsets (dendritic cells, CD4 + and / or CD8 + T cells). In another embodiment, the presence of T cell epitopes in an antibody can be determined by adding the antibody to isolated subsets of human immune cells, and assessing their differentiation in vitro and measuring any proliferation induced in T cell cultures. silico to define MHC class II ligands for multiple proteins of therapeutic interest can also be used. However, for reasons such as the requirement for proteolytic processing and other physiological steps that lead to the presentation of immunogenic peptides in vivo, a subset of the entire peptide repertoire defined by computer-based regimens may have ultimate biological relevance. Thus, ex vivo human T cell activation assays can be used to identify regions within the protein sequence of a polypeptide that are capable of supporting T cell activation and are thus more biologically relevant to the problem of immunogenicity in this protein. As used herein, "T cell epitope" refers to an amino acid sequence that is capable of binding to MHC class II, capable of stimulating T cells and / or also of binding (without necessarily measurable activation) cells T in complex with MHC class II.
According to a method disclosed in this document, synthetic peptides or whole antibodies are tested for their ability to evoke a proliferative response in human T cells cultured in vitro. T cells are present within a peripheral blood mononuclear cell layer (PBMC) readily obtainable by well-known means from whole blood samples. In addition, the PBMC preparation contains physiological rays from T cells and cells presenting antigen and is therefore a good source of materials with which to conduct an immune replacement reaction in vitro. In the operation of such an assay, a stimulation index that approaches or exceeds 2.0 is a useful measure of induced proliferation. However, the stimulation index can be different depending on the antibody, or the antigen binding fragment thereof, and can be established with reference to a baseline for each antibody, or antigen binding fragment thereof, and the a corresponding peptide library. In an example of such a test, the stimulation index (SI) can conventionally be derived by dividing the proliferation score (eg, counts per minute of radioactivity using, for example, 3H-thymidine incorporation) measured for the test peptide by the score measured in cells not in contact with a test peptide. Peptides that do not evoke any response can generate an SI = 1.0, although SI values in the range 0.8-1.2 may also be normal. A number of technical procedures can be constructed for the operation of such tests, in order to ensure confidence in the recorded scores. Normally all determinations are made at least in triplicate and the average score must be computed. When an SI b 2.0 is computed, the individual results of the triplicate can be examined for evidence of the away data. Test peptides are placed in contact with cells in at least two different concentrations and the concentrations typically span a minimum difference in concentration of two times. Such a concentration range provides compensation for the kinetic dimension for the assay and can be useful where a determination of a single point of time, for example, on the 7th plus day, is being conducted. In some assays, multiple time course determinations can be conducted and these can also be done using peptide immunogen delivered at a minimum of two different concentrations. Likewise, the inclusion of control peptides for which there is an expectation that the majority of PBMC samples from donors will generate residue must be included in each assay plate. The influenza hemagglutinin peptide 307-309, sequence PKYVKQNTLKLA (SEQ ID NO: 104), and the peptide Chlamydia HSP 60 sequence KVVDQIKKISKPVQH (SEQ ID NO: 105) are examples of control peptides to be used in such an assay. Alternatively, or in addition, the assays may also use a potent complete protein antigen, such as keyhole limpet hemocyanin, for which all PBMC samples would be expected to exhibit an SI significantly greater than 2.0. Other control antigens for such use are well known in the art.
The methods described here can provide an epitope map of antibodies, or antigen binding fragments thereof, where the map is relevant to a wide spectrum of possible MHC allotypes. The map may be sufficiently representative to allow the design or selection of a modified protein for which the protein's ability to evoke a T cell immune response can be eliminated, or at least improved, for most patients for whom protein is likely to be administered. Improvement can refer to a reduction in an immune response (i.e., reduced immunogenicity) compared to an unmodified protein (for example, about 1.5 times less, about 2 times less, about 5 times less, about 10 times less, about 20 times less, about 50 times less, about 100 times less, about 200 times less, about 500 times less or more, or any range referred to here). Alternatively, antibodies, or antigen-binding fragments thereof, with reduced immunogenicity may refer to a percentage reduction in their ability to elicit an immune response compared to an unmodified protein (for example, about 1% less, about 2% less, about 3% less, about 4% less, about 5% less, about 10% less, about 20% less, about 50% less, about 100% less, and any range referred to here). Thus, in the practice of the screening process, T cells derived from PBMC from natural donors are collected from a set of donors of sufficient immunological diversity to provide a sample of at least more than 90% of the class II MHC repertoire ( HLA-DR) in the human population. Where a normal T cell response is to be detected for a given synthetic peptide (or antibody), the peptide (or antibody) is, in practice, brought into contact with PBMC preparations derived from multiple donors in isolation; donor numbers (or "donor pool size") is for practical purposes only and is not likely to be less than 20 unrelated individuals and all samples in the donor pool can be pre-selected according to their MHC haplotype class II.
As used herein, the term "normal donor" refers to a subject who has not previously been exposed to antibodies, or antigen binding fragments thereof, described herein, either environmentally, by vaccination, or by other means, such as such as blood transfusions.
When scanning for T cell epitopes, T cells can be supplied from a peripheral blood sample from a multiplicity of different healthy donors, but who have not been receiving the therapeutic protein. If necessary, the patient's blood samples can be tested for the presence of a particular polypeptide using conventional assays, such as an ELISA that uses antibodies to identify the presence or absence of one or more polypeptides. The assay is conducted using PBMC grown in vitro using conventional procedures known in the art and involves contacting PBMC with synthetic polypeptide species representative of the protein of interest (i.e., a library), or an entire protein, such as an antibody, and followed of an appropriate incubation period, measurement of induced T cell activation such as cell proliferation. The measurement can be made by any suitable means and can, for example, be conducted using incorporation of H3-thymidine in which the accumulation of H3 in the cellular material is readily measured using long-term laboratory instruments. The degree of cell proliferation for each sample combination of PBMC and synthetic peptide or whole protein can be examined against that observed in an untreated PBMC sample. Reference can also be made to the proliferative response observed after treatment with a peptide or peptides or whole proteins for which there is an expected proliferative effect. In this regard, it is advantageous to use a whole peptide or protein with known broad MHC restriction and especially MHC restricted peptide epitopes for DP or DQ isotypes, although the invention is not limited to the use of such restricted peptides or proteins. Such peptides have been described above, for example, with respect to influenza hemagglutinin and chlamydia HSP60.
In a non-limiting example, T cell epitopes are mapped and subsequently modified using the methods described here. To facilitate the assembly of an epitope map, a library of synthetic peptides is produced. Each of the peptides is 15 amino acid residues in length and each overlaps the next peptide in the series by 12 amino acid residues, that is, each successive peptide in the series incrementally assists 3 more amino acids for analysis. In this way, any given adjacent pair of peptides maps 18 amino acids of the contiguous sequence. A method for defining a T cell map using normal T cell assays is illustrated in the Examples below. Each of the peptides identified using the method for defining a T-cell map is suggested to be able to bind MHC class II and involve at least one TCR cognate with sufficient affinity to evoke a detectable proliferative explosion in the assay system.
In another non-limiting example, the potential of an antibody to be processed to generate T cell epitopes that bind MHC class II and involve at least one TCR cognate with sufficient affinity to evoke a detectable proliferative explosion in the assay system is assessed.
The molecules described herein can be prepared in any of several ways, including the use of recombinant methods. The protein sequences and the information provided here can be used to deduce a polynucleotide (DNA) encoding an amino acid sequence. This can be achieved, for example, using computer software tools, such as DNAstar software [DNAstar, Inc., Madison, Wisconsin, USA] or similar. Any polynucleotide encoding the polypeptides or significant homologues, variants, truncations, elongations, or additional modifications thereof, are contemplated herein.
This document provides methods for mapping (identifying) T cell epitopes and modifying the epitopes in such a way that the modified sequence reduces (partially or completely) the induction of a response assisting T. Modification includes substitutions, deletions, or insertion of amino acids made in codons of a polynucleotide encoding polypeptides modified to affect similar changes. Codons encoding amino acid residues are well known in the art. Recombinant DNA methods can be used to achieve targeted mutagenesis of the target sequences and many of these techniques are available in this application, and are known in the art as described above. In general, the technique of site-specific mutagenesis is well known. Briefly, a bacteriophage vector, which produces a single chain model for oligonucleotide-directed PCR mutagenesis is employed. Phage vectors (e.g., M13) are commercially available and their use is generally well known in the art. Likewise, double-stranded plasmids are also routinely used in site-directed mutagenesis, which eliminates the step of transferring the polynucleotide of interest from a phage to a plasmid. Synthetic oligonucleotide primers that carry the desired mutated sequence can be used to direct the in vitro synthesis of modified DNA (desired mutation) from this model and heteroduplex DNA is used to transform E. coli competent for growth selection and clone identification desired. Alternatively, a pair of primers can be annealed to two separate strands of a double-stranded vector to simultaneously synthesize both corresponding complementary strands with the desired mutation (s) in a PCR reaction.
In one embodiment, the rapid change site-directed mutagenesis method using plasmid DNA models can be employed. PCR amplification of the plasmid model containing the insert insertion target gene is achieved using two synthetic oligonucleotide primers that contain the desired mutation. The oligonucleotide primers, each complementary to the opposite strands of the vector, are extended during temperature cycles by degree of DNA polymerase PfuTurbo mutagenesis. Upon incorporation of the oligonucleotide primers, a mutated plasmid containing stepped notches is generated. Amplified non-methylated products are treated with DPn I to digest methylated parental DNA template and select for newly synthesized DNA containing mutations. Since DNA isolated from most of the E. coli strain is poorly methylated, it is susceptible to digestion of DPn I, which is specific for methylated and hemimethylated DNA. The reaction products are transformed into highly efficient E. coli strains to obtain the plasmids that contain the desired modifications. Additional methods for introducing amino acid modifications into a polypeptide are well known in the art and can also be used herein.
Appropriate modifications to a protein may include substitution of particular residues or combinations of amino acid residues. For the elimination of T cell epitopes, amino acid substitutions are made at appropriate points or at amino acid residues within an amino acid sequence predicted to achieve the reduction or elimination of T cell epitope activity. In practice, a point appropriate amino acid residue, preferably amounts to a binding amino acid residue within one of the sets provided within the MHC class II binding strand. Such modifications can exchange the bond within the first slot assembly in the so-called "P1" or "anchor P1" position of the peptide. The quality of the binding interaction between the P1 anchor residue of the peptide and the first set of the MHC class II binding streak is recognized as being a major determinant of the overall binding affinity for the entire peptide. A suitable substitution at this position in the amino acid sequence will generally incorporate an amino acid residue less easily accommodated within the set (e.g., substitution for a more hydrophilic residue). Amino acid residues in the peptide at positions equivalent to binding within other regions of the package within the MHC binding slot are also considered and are within the scope of the present invention.
It is understood that unique amino acid modifications within a given potential T cell epitope represent a pathway by which one or more T cell epitopes can be eliminated. Combinations of modifications within a single epitope can be contemplated and may be appropriate when individually defined epitopes are in overlap with each other. In addition, modifications of amino acids (either alone within a given epitope or in combination within a single epitope) can be made in positions not equivalent to "pouch residues" relative to the MHC class II binding streak, but, at any point within the amino acid sequence. Modifications can be made with reference to a homologous structure, or structural method produced using in silico techniques known in the art and described herein can be based on known structural characteristics of the polypeptide. A change (modification) can be envisaged to restore the structure or biological activity of the variant molecule. Such compensation changes and changes may also include deletion or addition (insertion) of particular amino acid residues from a polypeptide. In addition, modifications can be made that alter the structure and / or reduce the biological activity of the molecule and also eliminate a T cell epitope, thus reducing the immunogenicity of the molecule. All types of modifications are contemplated here.
An additional means of removing epitopes from protein molecules is the combined use of a normal T cell activation assay method as described here in conjunction with an in silico tool developed according to the scheme described in WO 02 /069.232, which is also incorporated here in its entirety, for reference. The software simulates the antigen presentation process at the level of MHC class polypeptide binding interaction to provide a binding score for any given polypeptide sequence. This score is determined by many of the predominant MHC class II allotypes in the population. Since this scheme is capable of testing any polypeptide sequence, the consequences of the addition or deletion of amino acid substitutions with respect to the ability of a polypeptide to interact with a class II MHC binding streak can be predicted. Consequently, new sequence compositions can be designed, which contain reduced numbers of amino acids capable of interacting with a class II MHC and thus functioning as immunogenic T cell epitopes. When the biological assay using any of the given donor samples can assess binding to a maximum of four DR allotypes, the in silico process can test the same polypeptide sequence using over 40 allotypes simultaneously. In practice, this approach is able to drive the design of new sequence variants that are altered in their ability to interact with multiple MHC allotypes. As will be clear to a person skilled in the art, multiple alternative sets of substitutions can achieve the goal of removing unwanted epitopes. The resulting sequences would, however, be recognized as being closely homologous to the specific compositions described herein and, therefore, falling within the scope of the present application.
A combined approach of using an in silico tool for the identification of class II MHC ligands and design of sequence analogs that do not have class II MHC ligands, in concert with epitope mapping and retesting, optionally, using base assays biological activation of T cells is an additional method and embodiment of the present application. The general method according to this embodiment comprises the following steps: i) use of normal T cell and synthetic peptide activation assays collectively covering the protein sequence of interest to identify regions of epitope susceptible to T cell activation; ii) use of a computational scheme for simulating the binding of the peptide ligand with one or more MHC allotypes to analyze the regions of the epitope identified in step (i) and, thus, identify MHC class II ligands within the region of the epitope ; iii) use of a computational scheme for simulating the binding of the peptide ligand with one or more MHC allotypes to identify analogs of MHC ligand sequences encompassed in the region of the epitope (s) that no longer bind to MHC class II or bind with reduced affinity to a lower number of MHC allotypes and, optionally, iv) use of normal T cell and synthetic peptide activation assays covering all or together that encompasses the regions of the epitope identified within the protein of interest and testing the sequence analogs in the normal T cell activation assay in parallel with the wild type (parental) sequences.
In one embodiment, a method of producing a modified antibody, or antigen binding fragment thereof, exhibiting reduced immunogenicity compared to an unmodified antibody, or antigen binding fragment thereof, comprises identifying at least one epitope of T cell within the amino acid sequence of an antibody, or antigen binding fragment thereof, and modification of at least one amino acid residue within at least one identified T cell epitope.
In another embodiment, a modified antibody, or antigen binding fragment thereof, exhibiting reduced immunogenicity compared to an unmodified antibody, or antigen binding fragment thereof, is produced by a process of identifying at least one T cell epitope within the amino acid sequence of an antibody, or its antigen binding fragment, and modifying at least one amino acid residue within at least one identified T cell epitope.
In yet another embodiment, a method of selecting a modified antibody, or an antigen binding fragment thereof, which exhibits reduced immunogenicity compared to an unmodified antibody, or an antigen binding fragment thereof, comprises identifying at least one T cell epitope within the amino acid sequence of an antibody, or an antigen binding fragment thereof, modifying at least one amino acid residue within at least one identified T cell epitope, and selecting a modified antibody, or an antigen binding fragment thereof, which has reduced immunogenicity compared to an unchanged antibody, or an antigen binding fragment thereof.
T cell epitopes described herein can be further characterized depending on the regions of the epitope. Such regions include the nucleus of the epitope, the N-terminus and the C-terminus. As used herein, "doepitope nucleus" refers to the 9-mer nucleus of the T cell epitope amino acid sequences. The nucleus of the epitope may further include 0, 1, 2 or 3 amino acid residues adjacent to the 9-mer nucleus of the amino acid sequence at the N-terminus and / or the C-terminus. Thus, the nucleus of the epitope, in certain embodiments, can vary in length from about 9 amino acids to about 15 amino acids.
As used herein, "N-terminus" refers to amino acids adjacent to the N-terminus of the epitope nucleus and includes at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids adjacent to and to amount of the N-terminal of the nucleus of the epitope.
As used herein, "C-terminus" refers to the amino acids adjacent to the C-terminus of the epitope nucleus and includes at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids adjacent to the and downstream of the C-terminus of the epitope nucleus.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains one or more modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains two modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains three modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains four modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains five modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains six modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains seven modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains eight modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains nine modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains ten modifications.
In one embodiment, a modified antibody, or an antigen binding fragment thereof, contains up to twenty modifications. An antibody, or antigen binding fragment thereof, is provided herein, comprising a light chain variable region having an amino acid sequence shown as SEQ ID NO: 93 - (VK1AA) and a heavy chain variable region having a sequence amino acid shown as SEQ ID NO: 89 (VH1A2). An antibody, or antigen binding fragment thereof, is provided herein, comprising a variable region of heavy chain having an amino acid sequence shown as any of SEQ ID NOS: 88, 89, 90, 91 and 92. It is here An antibody, or antigen binding fragment thereof, is provided, comprising a light chain variable region having an amino acid sequence shown as any of SEQ ID NOS: 93, 94, 95, 96, 97, 100, 102, and 103. An antibody, or antigen-binding fragment thereof, which binds to endoglobin is provided herein, comprising a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 89 and a variable chain region light chain having an amino acid sequence presented as SEQ ID NO: 93, in which: (i) the variable region of the heavy chain further comprises one or more modifications selected from the group consisting of a substit use of glycine (G) by alanine (A) or serine (S) at position 49; a replacement of alanine (A) with isoleucine (I) at position 51; a replacement of lysine (K) with arginine (R) or asparagine (Q) at position 52b; a replacement of leucine (L) with valine (V) at position 78, using the Kabat numbering system; and (ii) the light chain variable region further comprises one or more modifications selected from the group consisting of a replacement of methionine (M) with leucine (L) in position 4; a substitution of alanine (A) for valine (V) in position 19; a replacement of threonine (T) by serine (S) at position 22; a substitution of alanine (a) for isoleucine (I) at position 48, and a replacement of threonine (T) with serine (S) at position 51, using the Kabat numbering system. An antibody, or antigen binding fragment thereof, is provided herein, comprising a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 88, 89, 90, 91 and 92, and a light chain variable region having an amino acid sequence established as SEQ ID NO: 93, 95, 96, 97, 100, 102, or 103.
In addition to the above-mentioned examples and embodiments, a modified antibody, or antigen-binding fragment thereof, with one or more amino acid modifications in one or more T cell epitopes is contemplated herein. In a non-limiting example, provided herein are antibodies, or antigen binding fragments thereof, having at least one modification in at least one T cell epitope. In another non-limiting example, provided here are antibodies, or antigen binding fragments. of them, having at least one amino acid modification in 1, 2, 3, 4, 5, 6 or 7 of the T cell epitopes described herein. Additional non-limiting examples include antibodies, or antigen-binding fragments thereof, with more than one amino acid modification in more than one T-cell epitope. Any combination of the amino acid modifications in any number of antibodies, or antibody-binding fragments antigen thereof, T cell epitopes described above are contemplated herein. T-CELL EPITHES AND ALLOTYPE FREQUENCY Individual epitopes found in antigens may preferably be presented by specific MHC class II allotypes and, similarly, other specific epitopes in the same antigen may not be shown at all in MHC class II molecules. These associations of particular epitopes with specific MCH class II molecules have been shown to be dependent on the individual MHC class II allotype. The association of a specific epitope with a specific allotype can also be considered when modifying antibodies, or antigen-binding fragments thereof, to remove T cell epitopes. Such considerations may allow for the highly specific modification of an antibody, or antigen binding fragment thereof, by specific allotypes (for example, for specific populations of individuals with certain MHC class II allotypes). The class II MHC allotype of a subject or subjects can be easily determined by genotyping methods known in the art, and the association of T cell epitopes with the given allotype, therefore, easily identified, for consideration in antibody modification, or antigen-binding fragments thereof, adapted for said allotype. The identification of associations between T cell epitopes and MHC class II allotypes are described in more detail in the examples below. Modified antibodies, or antigen binding fragments thereof, are contemplated herein, which have T cell epitope modifications adapted to the MHC class II associations identified for the given epitopes.
D. ANTI-ENDOGLINE ANTIBODIES
Simultaneous incorporation of all FR and / or CDRs encoding nucleic acids and all selected amino acid position changes can be achieved by a variety of methods known to those skilled in the art, including, for example, chemical synthesis and recombination. For example, simultaneous incorporation can be performed by, for example, chemically synthesizing the nucleotide sequence for the acceptor variable region, fused together with CDR donors that encode nucleic acids, and incorporating at the selected positions to house variable amino acid residues one plurality of codons of corresponding amino acids. Antibodies and binding fragments of the antigen thereof that bind to endoglobin are provided herein. Antibodies and antigen-binding fragments thereof are also provided that bind to the endoglobin and inhibit (partially or totally) or administer / treat (partially or totally) angiogenesis / neovascularization, dilation of small vessels and / or diseases associated with excessive angiogenesis. Likewise, inhibition of endoglobin function (for example, signaling, activation, binding, and the like) is also included within the scope of the meaning of inhibition or binding of endoglobin. In yet another embodiment, an antibody or antigen-binding fragment thereof inhibits angiogenesis by binding to endoglobin. The application also provides cell lines that can be used to produce antibodies, methods for producing cell lines, methods for expressing antibodies or antigen binding fragments therefrom and purifying them.
It can be recognized that antibodies and antigen-binding fragments thereof that specifically bind to the endoglobin generated using the methods described herein can be tested using the assays provided or known in the art for the ability to bind to the endoglobin using conventional methods , including, but not limited to, ELISA. The affinity of antibodies described herein can also be determined using conventional methods, including, but not limited to, or Biacore or surface plasma resonance.
The antibodies and antigen binding fragments described herein were constructed by humanizing the Vh and Vl sequences of the TRC105 antibody. To achieve this humanization, a three-dimensional model of the VH and VL chains of TRC105 was created and analyzed. The VH and VL sequences were then individually compared to a human germline sequence database, from which the human VH and VL sequences were chosen based on their homology with the TRC105 VH and VL sequences. The human VL sequence chosen for humanization was O2 / O12 (VK1-39) (SEQ ID NO. 2). O2 / O12 has a sequence identity with 65% TRC105 and the gene is highly expressed in the human germline repertoire. The human VH sequence chosen for humanization was VH3-15 (SEQ ID NO. 40). VH3-15 has sequence identity with 70% TRC105, and is expressed with reasonable frequency in the human germline repertoire. The positions of amino acids that were different between TRC105 and human sequences were examined in the 3D model of TRC105 to determine which substitutions would be considered for the modification. Criteria for selecting amino acids based on the analysis of the 3D model include, but are not limited to, for example, steric effects related to the amino acid, relative charge of the amino acid, and location of the amino acid within the heavy and / or light variable chain. The substitutions identified and proposed for the human structure regions are incorporated into the human structure O2 and VH3-15 regions, and the TRC105 CDRs are grafted into the corresponding human structure O2 and VH3-15 regions, resulting in a large number of antibodies humanized or binding fragments of the antigen thereof. In addition, the light chain FR-4 is derived from the human J germline Jk4 sequence. Similarly, the heavy chain FR-4 is derived from the JH4 human J germline sequence. Humanized antibodies and antigen-binding fragments thereof that bind to endoglobin are described herein. Also described herein are humanized antibodies and antigen-binding fragments thereof that bind to endoglobin and inhibit angiogenesis. Antibodies and antigen-binding fragments described herein were generated as described above.
Antibodies and antigen binding fragments thereof may have a heavy variable chain (Vh), a light variable chain (VL), both, or binding portions thereof. In one embodiment, the Vh chain has an amino acid sequence shown as either one of SEQ ID NOS: 41-43, or a linking portion thereof. These Vh chains can have sequences of the structural regions defined as any of SEQ ID NOS: 44-62. In another embodiment, the VL chain has an amino acid sequence shown as any of SEQ ID NOS: 3-5, or a linking portion thereof. Such VL chains can have sequences of the structural regions defined as any of SEQ ID NOS: 6-38. An antibody, or antigen binding fragment thereof, is provided herein, comprising a light chain variable region having an amino acid sequence shown as SEQ ID NO: 3 and a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 41. An antibody, or antigen binding fragment thereof, is provided herein, comprising a light chain variable region having an amino acid sequence shown as SEQ ID NO: 3 and a heavy chain variable region having a sequence of amino acids presented as SEQ ID NO: 41, where: the heavy chain variable region further comprises one or more modifications selected from the group consisting of a substitution of glycine (G) for alanine (A) at position 49; a replacement of asparagine (N) with serine (S) at position 76; a replacement of threonine (T) with arginine (R) at position 77; a replacement of leucine (L) with valine (V) at position 78; a replacement of asparagine (N) with isoleucine (I) at position 82a; a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89; a replacement of threonine (T) with arginine (R) or glycine (G) at position 94; a replacement of leucine (L) with threonine (T) at position 108; a substitution of valine (V) for leucine (L) in position 109, and a substitution of serine (S) for alanine (A) in position 113, using the Kabat numbering system, and the variable region of light chain also comprises one or more modifications selected from the group consisting of a substitution of aspartic acid (D) for glutamine (Q) in position 1; a substitution of glutamine (Q) for valine (V) in position 3; a substitution of methionine (M) for leucine (L) in position 4; a replacement of threonine (T) by serine (S) at position 5; a substitution of tyrosine (Y) for phenylalanine (F) at position 36; a replacement of leucine (L) with proline (P) at position 46; a replacement of leucine (L) with tryptophan (W) at position 47; a replacement of serine (S) by valine (V) or alanine (A) at position 60; a replacement of aspartic acid (D) with serine (S) at position 70; a substitution of phenylalanine (F) for tyrosine (Y) at position 71; a replacement of glutamine (G) with alanine (A) at position 100, and a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system. An antibody, or antigen binding fragment thereof, is provided herein which binds to the endoglobin comprising a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 41, 42, or 43, and a variable region of light chain having an amino acid sequence defined as SEQ ID NO: 3, 4 or 5. An antibody, or antigen binding fragment thereof, can comprise a variable region of heavy chain having an amino acid sequence shown as SEQ ID NO: 41 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 3. An antibody, or antigen binding fragment thereof, can comprise a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 41 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 4. An antibody, or antigen binding fragment it may comprise a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 41 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 5. An antibody, or fragment of antigen binding thereof, can comprise a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 42 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 3. An antibody, or antigen binding fragment thereof, can comprise a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 42 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 4. An antibody , or antigen binding fragment thereof, may comprise a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 42 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 5. An antibody, or antigen binding fragment thereof, can comprise a heavy chain variable region having a sequence of amino acids shown as SEQ ID NO: 43 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 3. An antibody, or antigen binding fragment thereof, may comprise a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 43 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 4. An antibody, or antigen binding fragment thereof, can comprise a variable chain region heavy having an amino acid sequence shown as SEQ ID NO: 43 and a light chain variable region having an amino acid sequence presented as SEQ ID NO: 5. Such antibodies can bind to endoglobin and inhibit angiogenesis.
In any of the embodiments of this type, a heavy chain variable region may further comprise one or more modifications selected from the group consisting of: a replacement of asparagine (N) by serine (S) at position 76; a replacement of threonine (T) with arginine (R) at position 77; a replacement of asparagine (N) with isoleucine (I) at position 82a; a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89; a replacement of threonine (T) with glycine (G) at position 94; a replacement of leucine (L) with threonine (T) at position 108; a substitution of valine (V) with leucine (L) at position 109, and a substitution of serine (S) with alanine (A) at position 113, and the light chain variable region may further comprise one or more modifications selected from the group consisting of: a replacement of aspartic acid (D) with glutamine (Q) in position 1; a substitution of glutamine (Q) for valine (V) in position 3; a replacement of threonine (T) by serine (S) at position 5; a substitution of tyrosine (Y) for phenylalanine (F) at position 36; a replacement of serine (S) by valine (V) or alanine (A) at position 60; a replacement of aspartic acid (D) with serine (S) at position 70; a substitution of glycine (G) for alanine (A) at position 100, and a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system. An antibody, or antigen binding fragment thereof, is provided herein which binds to the endoglobin, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises: (i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQ ID NO: 68; (ii) a heavy chain FR1 with the amino acid sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 44, except for one or more conservative substitutions; (iii) a heavy chain FR2 with the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence of SEQ ID NO: 45, except for a substitution of glycine (G) with alanine (A) at position 49, using the Kabat numbering system; and (iv) a heavy chain FR3 with the amino acid sequence of SEQ ID NO: 47 or the amino acid sequence of SEQ ID NO: 47, except for one or more substitutions selected from the group consisting of: (a) a replacement of an asparagine (N) with serine (S) at position 76; (b) a replacement of threonine (T) with arginine (R) at position 77; (c) a replacement of leucine (L) with valine (V) at position 78; (d) a replacement of asparagine (N) with isoleucine (I) at position 82a; (e) a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89, and (f) a replacement of threonine (T) with arginine (R) or glycine (G) at position 94, using the Kabat numbering system, and (v) a heavy chain FR4 with the amino acid sequence of SEQ ID NO: 56 or the amino acid sequence of SEQ ID NO: 56, except for one or more substitutions selected from the group consisting of: (a) a replacement of leucine (L) with threonine (T) at position 108; (b) a substitution of valine (V) with leucine (L) at position 109, and (c) a substitution of serine (S) with alanine (A) at position 113, using the Kabat numbering system; and said light chain variable region comprises: (i) a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65; (ii) a light chain FR1 having the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6, except for one or more substitutions selected from the group consisting of: (a) a substitution aspartic acid (D) by glutamine (Q) in position 1; (b) a substitution of glutamine (Q) for valine (V) in position 3; (c) a replacement of methionine (M) with leucine (L) at position 4; and (d) a replacement of threonine (T) by serine (S) in position 5; using the Kabat numbering system and (iii) a light chain FR2 having the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence of SEQ ID NO: 20, except for one or more substitutions selected from the group consisting of: (a) the replacement of a tyrosine (Y) with phenylalanine (F) at position 36; (b) a substitution of leucine (L) with proline (P) at position 46, and (c) a substitution of leucine (L) with tryptophan (W) at position 47, using the Kabat numbering system and (iv) a light chain FR3 having the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence of SEQ ID NO: 28, except for one or more substitutions selected from the group consisting of: (a) a serine substitution ( S) by valine (V) or alanine (A) at position 60; (b) a substitution of aspartic acid (D) for serine (S) at position 70, and (c) a substitution of phenylalanine (F) for tyrosine (Y) at position 71, using the Kabat numbering system and (v ) a light chain FR4 having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35, except for one or more substitutions selected from the group consisting of: (a) a glycine substitution (G) with alanine (A) at position 100, and (b) a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system.
An antibody, or antigen binding fragment thereof, provided herein can comprise a heavy chain CDR1 variable region having an amino acid sequence as shown in SEQ ID NO: 66, a heavy chain CDR2 variable region having an amino acid sequence as defined in SEQ ID NO: 67, a heavy chain CDR3 variable region having an amino acid sequence as shown in SEQ ID NO: 68, a light chain CDR1 variable region having an amino acid sequence as shown in SEQ ID NO: 63, a light chain CDR2 variable region having an amino acid sequence as shown in SEQ ID NO: 64, and a light chain CDR3 variable region having an amino acid sequence as shown in SEQ ID NO: 65.
In one embodiment, the antibody, or antigen binding fragment thereof, binds to the endoglobin and comprises a heavy chain FR1 variable region having an amino acid sequence as shown in SEQ ID NO: 44; a heavy chain FR2 variable region having an amino acid sequence as described in SEQ ID NO: 45; a heavy chain FR3 variable region having an amino acid sequence as shown in SEQ ID NO: 47; a heavy chain FR4 variable region having an amino acid sequence as shown in SEQ ID NO: 56.
In another embodiment, the antibody, or antigen binding fragment thereof, binds to the endoglobin and comprises a heavy chain variable FR1 region having an amino acid sequence as shown in SEQ ID NO: 44; a heavy chain FR2 variable region having an amino acid sequence as described in SEQ ID NO: 46; a heavy chain FR3 variable region having an amino acid sequence as shown in SEQ ID NO: 48; a heavy chain FR4 variable region having an amino acid sequence as shown in SEQ ID NO: 56.
In another embodiment, the antibody, or antigen binding fragment thereof, comprises a light chain FR1 variable region having an amino acid sequence as shown in SEQ ID NO: 6; a light chain FR2 variable region having an amino acid sequence as defined in SEQ ID NO: 20; a light chain FR3 variable region having an amino acid sequence as shown in SEQ ID NO: 28, and a light chain FR4 variable region having an amino acid sequence as shown in SEQ ID NO: 35.
In another embodiment, the antibody, or antigen binding fragment thereof, binds to the endoglobin and comprises a light chain variable FR1 region having an amino acid sequence as shown in SEQ ID NO: 6; a light chain FR2 variable region having an amino acid sequence as described in SEQ ID NO: 21; a light chain FR3 variable region having an amino acid sequence as shown in SEQ ID NO: 29, and a light chain FR4 variable region having an amino acid sequence as shown in SEQ ID NO: 35.
In another embodiment, the antibody, or its antigen binding fraction, binds to the endoglobin and comprises a light chain variable FR1 region having an amino acid sequence as shown in SEQ ID NO: 7; a light chain FR2 variable region having an amino acid sequence as described in SEQ ID NO: 21; a light chain FR3 variable region having an amino acid sequence as shown in SEQ ID NO: 29, and a light chain FR4 variable region having an amino acid sequence as shown in SEQ ID NO: 35. An antibody is provided herein, or antigen binding fragment thereof, comprising a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 42 and a light chain variable region having an amino acid sequence shown as SEQ ID NO: 4. It is provided herein an antibody, or antigen binding fragment thereof, that binds to the endoglobin, comprising a light chain variable region having an amino acid sequence shown as SEQ ID NO: 4 and a heavy chain variable region having an displayed amino acid sequence as SEQ ID NO: 42, where: said heavy chain variable region further comprises one or more modifications selected a from the group consisting of a substitution of glycine (G) for alanine (A) in position 49; a replacement of asparagine (N) with serine (S) at position 76; a replacement of threonine (T) with arginine (R) at position 77; a replacement of leucine (L) with valine (V) at position 78; a replacement of asparagine (N) with isoleucine (I) at position 82a; a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89; a replacement of arginine (R) with threonine (T) or glycine (G) at position 94; a replacement of leucine (L) with threonine (T) at position 108; a substitution of valine (V) with leucine (L) at position 109, and a substitution of serine (S) with alanine (A) at position 113, using the Kabat numbering system; and the light chain variable region further comprises one or more modifications selected from the group consisting of a substitution of aspartic acid (D) with glutamine (Q) in position 1; a replacement of glutamine (Q) with valine (V) in position 3, a replacement of methionine (M) with leucine (L) in position 4; a replacement of threonine (T) by serine (S) at position 5; a substitution of tyrosine (Y) for phenylalanine (F) at position 36; a substitution of proline (P) for leucine (L) at position 46; a substitution of tryptophan (W) for leucine (L) at position 47; a replacement of serine (S) by valine (V) or alanine (A) at position 60; a replacement of aspartic acid (D) with serine (S) at position 70; a substitution of tyrosine (Y) for phenylalanine (F) at position 71; a replacement of glutamine (G) with alanine (A) at position 100; and a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system. An antibody, or antigen binding fragment thereof, is provided herein which binds to the endoglobin, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises: (i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQ ID NO: 68; (ii) a heavy chain FR1 with the amino acid sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 44, except for one or more conservative substitutions; (iii) a heavy chain FR2 with the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence of SEQ ID NO: 45, except for a substitution of glycine (G) with alanine (A) at position 49, using the Kabat numbering system; and (iv) a heavy chain FR3 with the amino acid sequence of SEQ ID NO: 47 or the amino acid sequence of SEQ ID NO: 47, except for one or more substitutions selected from the group consisting of: (a) a replacement of asparagine (N) by serine (S) at position 76; (b) a replacement of threonine (T) with arginine (R) at position 77; (c) a replacement of leucine (L) with valine (V) at position 78; (d) a replacement of asparagine (N) with isoleucine (I) at position 82a; (e) a replacement of valine (V) with isoleucine (I) or leucine (L) at position 89, and (f) a replacement of arginine (R) with threonine (T) or glycine (G) at position 94, using the Kabat numbering system and (v) a heavy chain FR4 with the amino acid sequence of SEQ ID NO: 56 or the amino acid sequence of SEQ ID NO: 56, except for one or more substitutions selected from the group consisting of of: (a) a substitution of leucine (L) with threonine (T) at position 108; (b) a substitution of valine (V) with leucine (L) at position 109, and (c) a substitution of serine (S) with alanine (A) at position 113, using the Kabat numbering system; and said light chain variable region comprises: (i) a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65; (ii) a light chain FR1 having the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6, except for one or more substitutions selected from the group consisting of: (a) a substitution aspartic acid (D) by glutamine (Q) in position 1; (b) a substitution of glutamine (Q) for valine (V) in position 3; (c) a replacement of methionine (M) with leucine (L) at position 4; and (d) a replacement of threonine (T) by serine (S) in position 5; using the Kabat numbering system and (iii) a light chain FR2 having the amino acid sequence of SEQ ID NO: 21 or the amino acid sequence of SEQ ID NO: 20, except for one or more substitutions selected from the group consisting of: (a) a replacement of tyrosine (Y) with phenylalanine (F) at position 36; (b) a substitution of proline (P) with leucine (L) at position 46, and (c) a substitution of tryptophan (W) with leucine (L) at position 47, using the Kabat numbering system and (iv) a light chain FR3 having the amino acid sequence of SEQ ID NO: 29 or the amino acid sequence of SEQ ID NO: 28, except for one or more substitutions selected from the group consisting of: (a) a serine substitution ( S) by valine (V) or alanine (A) at position 60; (b) a substitution of aspartic acid (D) with serine (S) at position 70, and (c) a substitution of tyrosine (Y) with phenylalanine (F) at position 71, using the Kabat numbering system and (v ) a light chain FR4 having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35, except for one or more substitutions selected from the group consisting of: (a) a glycine substitution (G) with alanine (A) at position 100, and (b) a replacement of isoleucine (I) with leucine (L) at position 106, using the Kabat numbering system.
A substantial portion of a variable domain will include three CDR regions, together with their intervening structural regions. The portion may also include at least about 50% of one or both of the first and fourth structural regions, the 50% being the C-terminal 50% of the first structural region and the N-terminal 50% of the fourth structural region. Additional residues at the N-terminus or C-terminus of the substantial part of the variable domain can be those that are not normally associated with naturally occurring variable domain regions. For example, the construction of humanized endoglobin antibodies and antigen binding fragments thereof produced by recombinant DNA techniques can result in the introduction of N-or C-terminal residues encoded by binding editors introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linker editors to join variable domains to additional protein sequences including immunoglobulin heavy chains, other variable domains (for example, in the production of diabody) or protein labels as discussed in more detail below.
Humanized endoglobin CDR3 regions that have amino acid sequences substantially as defined as in the CDR3 regions of the antibodies described herein will be carried out in a structure that allows the binding of the CDR3 regions to the endoglobin. The structure for transporting the CDR3s can be a light or heavy chain sequence of the antibody or a substantial portion of them where the CDR3 regions are located at sites corresponding to the CDR3 region of naturally occurring antibody VH and VL variable domains encoded by genes of immunoglobulin rearranged.
In a non-limiting example, antibodies or antigen binding fragments thereof are provided herein, containing a variable heavy chain having a CDR3, which has an amino acid sequence shown as SEQ ID NO: 68 and / or a variable light chain having a CDR3 , which has an amino acid sequence shown as SEQ ID NO: 65. In one embodiment, the heavy chain variable has an amino acid sequence shown as SEQ ID NO: 40 except for the replacement of CDR3 by the defined amino CDR3 sequence as SEQ ID NO: 68. In another embodiment, the variable light chain has an amino acid sequence shown as SEQ ID NO: 2, with the exception of a replacement of CDR3 by the sequence of CDR3 amino acid defined as SEQ ID NO: 65. In addition In addition, CDR3s containing variable regions / chains may comprise one or more FR sequences of amino acids defined as, for example, described above (or FRs such as containing one or more m additional modifications), where antibodies or antigen-binding fragments of the same have 3 CDRs and 4 FRs in each of the VH and VL regions, have specific binding activity for endoglobin and are capable of inhibiting angiogenesis. In addition, several J segments of antibodies can also be substituted within these variable regions for further variation within the chains of the variable region.
In one respect, heavy and light variable chains described herein can also be created by further replacing FR4 sequences. In one embodiment, FR4 heavy chain sequences can be replaced by one of the following: In one embodiment, FR4 light chain sequences can be replaced by one of the following: In addition, humanized versions of anti-endoglobin antibodies are contained in this document. alternatively called "superhumanized" anti-endoglobin antibodies or antigen-binding fragments thereof. Such superhumanized antibodies, or antigen binding fragments thereof, may comprise a light chain variable region having an amino acid sequence shown as SEQ ID NOS: 71 or 72 and a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 75.
In another aspect, the present application provides a humanized antibody capable of competing with a humanized anti-endoglobin antibody or antigen binding fragment thereof described herein under conditions in which at least 5% of an antibody having the antibody's VH and VL sequences is blocked from binding to endoglobin by competition with such an antibody in an ELISA assay. Neutralizing antibodies or antigen-binding fragments thereof are provided herein that bind to endoglobin and modulate endoglobin activity. The neutralizing antibody can, for example, inhibit angiogenesis by binding to endoglobin. The percentage (%) of inhibition of angiogenesis by a humanized anti-endoglobin antibody or antigen binding fragment thereof at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least twenty times, at least thirty times, at least forty times, at least fifty times, at least sixty times, or greater than those Negative controls are indicative that an antibody or antigen binding fragment thereof inhibits angiogenesis. Percentage (%) of inhibition of angiogenesis by a humanized anti-endoglobin antibody or antigen binding fragment of it less than 2 times greater than the negative controls is indicative that an antibody or antigen binding fragment of the same does not inhibit angiogenesis.
The binding of an antibody or antigen binding fragment thereof to the endoglobin may partially (for example, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95%, 98%, 99%) or completely inhibit angiogenesis. The inhibiting or neutralizing activity of an antibody or antigen binding fragment thereof can be determined using an in vitro and / or in vivo assay using assays known in the art, such as those described herein or otherwise known in the art.
In one aspect, the antigen binding fragment of any of the humanized antibodies described above, is a Fab, Fab ', Fd, F (ab') 2, Fv, scFv, single polypeptide binding chain (for example, a scFv with Fc portion) or any other functional fragment thereof, as described herein.
Antibodies or antigen-binding fragments thereof described herein are useful in detection or diagnostic applications, as described in more detail below. Antibodies or antigen-binding fragments thereof described herein are useful for binding to endoglobin, which, in turn, can inhibit angiogenesis as described herein.
Antibodies, or antigen binding fragments thereof, as described herein, can be further modified to alter the specific properties of the antibody, while maintaining the desired functionality, if necessary. For example, in one embodiment, the compound can be modified to alter a compound's pharmacokinetic property, such as in vivo stability, solubility, bioavailability, or half-life. Antibodies, or antigen-binding fragments thereof, may further comprise a therapeutic portion, a detectable portion, or both, for use in diagnostic and / or therapeutic applications.
Antibodies or antigen-binding fragments thereof can also be used as immunoconjugates. As used herein, for the purposes of the specification and claims, immunoconjugates refer to conjugates consisting of the humanized anti-endoglobin antibodies or fragments thereof in accordance with the present invention and with at least one therapeutic indication. Therapeutic indications include antitumor agents and angiogenesis inhibitors. Such antitumor agents are known in the art and include, but are not limited to, toxins, enzymes, drugs, cytokines, radionuclides, photodynamic agents, and angiogenesis inhibitors. Toxins include, but are not limited to, ricin A chain, Pseudomonas mutant exotoxins, diphtheria toxoid, streptonigrin, boamycin, saporin, gelonin, and antiviral protein from phytolaca. Drugs include methotrexate, daunorubicin, and calicheamicins. Radionuclides include radiometals. Cytokines include, but are not limited to, transformed growth factor (TGF) -p, interleukins, interferons, and tumor necrosis factors. Photodynamic agents include, but are not limited to, poFRirins and their derivatives. Additional therapeutic indications are known in the art and are also contemplated herein. Methods for complexing anti-endoglobin mAbs or their fragment with at least one anti-tumor agent are well known to those of skill in the art (i.e., conjugated antibodies as reviewed by Ghetie et al., 1994, Pharmacol. Ther. 63: 209 -34 ). Such methods can use one of several available heterobifunctional reagents used for coupling or linking molecules. Additional radionuclides are further described here, along with additional methods for binding molecules, such as therapeutic and diagnostic indications.
Antibodies or antigen-binding fragments thereof can be modified using techniques known in the art for various purposes, such as, for example, by adding polyethylene glycol (PEG). Modification with PEG (PEGylation) can lead to one or more improved circulation times, improved solubility, improved resistance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and greater ease of formulation (for a review see , Francis et al., International Journal of Hematology 68: 1-18, 1998).
In the case of an antigen binding fragment that does not contain an Fc portion, an Fc portion can be added (e.g., recombinantly) to the fragment, for example, to increase the half-life of the circulating antigen binding fragment in the blood when administered to a patient. The choice of an appropriate Fc region and methods of incorporating such fragments are known in the art. Incorporating an IgG Fc region into a polypeptide of interest, in order to increase its circulatory half-life, but in order not to lose its biological activity, can be performed using conventional techniques known in the art, such as, for example , described in U.S. Patent No. 6,096,871, which is incorporated herein by reference in its entirety. Fc portions of antibodies can be further modified to increase the half-life of the antigen-binding fragment circulating in the blood when administered to a patient. Modifications can be determined using conventional means in the art, such as, for example, that described in U.S. Patent No. 7,217,798, which is incorporated herein by reference in its entirety.
Other methods for improving the half-life of proteins based on circulating binding antibodies are also known, such as, for example, that described in U.S. Patent Nos. 7,091,321 and 6,737,056, each of which is incorporated herein. by reference. In addition, antibodies or antigen-binding fragments can be produced or expressed so that they do not contain fucose in their complex linked N-glycoside sugar chains. Removal of fucose from complex n-glycoside-linked sugar chains is known to increase the effector functions of antibodies or antigen-binding fragments thereof, including, but not limited to, antibody-dependent cell cytotoxicity (CCDA) and cytotoxicity complement-dependent (CDC). Likewise, antibodies or antigen-binding fragments thereof that can bind to the endoglobin can be attached at its C-terminus to all or part of an immunoglobulin heavy chain derived from any antibody isotype, eg, IgG, IgA , IgE, IgD and IgM and any subclasses of the isotype, namely IgG1, IgG2a, IgG2b, IgG3 and IgG4.
In addition, antibodies or antigen-binding fragments thereof can also be modified so that they are able to cross the blood-brain barrier. Such modification of the antibodies or antigen-binding fragments thereof described herein allows the treatment of diseases of the brain, such as glioblastoma multiforme (GBM). Examples of modifications to allow proteins such as antibodies or antigen-binding fragments thereof to cross the blood-brain barrier are described in U.S. Patent Application Publication 2007 / 0.082,380 which is incorporated herein by reference in its entirety. Glycosylation of immunoglobulins has been highlighted for exhibiting significant effects on their effector functions, structural stability, and secretion rate of antibody-producing cells (Leatherbarrow et al., Mol. Immunol. 22: 407 (1985)). The carbohydrate groups responsible for these properties are generally linked to the constant (C) regions of the antibodies. For example, glycosylation of IgG in asparagine 297 in the CH2 domain is necessary for the total IgG capacity to activate the classic complement-dependent cytolysis pathway (Tao and Morrison, J. Immunol. 143: 2595 (1989)). The glycosylation of IgM in asparagine 402 in the CH3 domain is necessary for the correct assembly and cytolytic activity of the antibody (Muraoka and Shulman, J. Immunol. 142: 695 (1989)). The removal of glycosylation sites, such as positions 162 and 419 in the CH1 and CH3 domains of an IgA antibody, led to intracellular degradation and at least 90% inhibition of secretion (Taylor and WLLA, Mol. Cell. Biol. 8: 4197 (1988)). In addition, antibodies or antigen-binding fragments can be produced or expressed so that they do not contain fucose in their complex n-glycoside-linked sugar chains. Removal of fucose from complex n-glycoside-linked sugar chains is known to increase the effector functions of antibodies or antigen-binding fragments thereof, including, but not limited to, antibody-dependent cellular cytotoxicity (CCDA) and complement-dependent cytotoxicity (CDC). These "defucosylated" antibodies or antigen binding fragments thereof can be produced through a variety of systems using molecular cloning techniques known in the art, including, but not limited to, transgenic animals, transgenic plants, or cell lines that have been genetically modified so that they no longer contain the enzymes and biochemical pathways necessary for the inclusion of a fucose in the sugar chains linked to the complex n-glycoside (also known as fucosyltransferase knock-out animals, plants, or cells). Non-limiting examples of cells that can be modified to be knock-out fucosyltransferase include CHO cells, SP2 / 0 cells and NS0 cells, and YB2 / 0 cells. Glycosylation of immunoglobulins in the variable region (V) has also been observed. Sox and Hood reported that about 20% of human antibodies are glycosylated in region V (Proc. Natl. Acad. USA 66: 975 (1970)). V-domain glycosylation is believed to arise from random occurrences of the Asn-Xaa-Ser / Thr N-linked glycosylation signal in the V region sequence and has not been recognized in the art as playing a role in immunoglobulin function. Glycosylation in a variable domain structure residue can exchange the antibody-binding interaction with the antigen. The present invention includes criteria that a limited number of amino acids in the structure or CDRs of a humanized immunoglobulin chain are chosen to be mutated (for example, by substitution, deletion or addition of residues) in order to increase the affinity of an antibody .
Affinity for the binding of a predetermined polypeptide antigen can generally be modulated by introducing one or more mutations within the V region, typically in areas adjacent to one or more CDRs and / or in one or more regions of structure. Typically, such mutations involve the introduction of conservative amino acid substitutions to destroy or create sequences at the glycosylation site, but do not substantially affect the structural properties of hydropathic polypeptides. Typically, mutations that introduce a proline residue are prevented. Glycosylation of antibodies or antigen-binding fragments thereof is further described in U.S. Patent No. 6,350,861, which is incorporated herein by reference with respect to glycosylation.
Antibodies or antigen-binding fragments can be formulated in the short term or extended (in the long term).
Antibodies or antigen-binding fragments thereof that bind to endoglobin may also be used for the purification of endoglobin and / or to detect levels of endoglobin in a sample or patient to detect or diagnose a disease or disorder associated with endoglobin, as described in more detail below.
Humanized antibodies or antigen binding fragments thereof, and binding proteins that bind to the endoglobin generated using such methods can be tested for one or more of their binding affinity, avidity and neutralizing capacity. Useful humanized antibodies, or antigen binding fragments thereof, and binding proteins can be used for administration to a patient to prevent, inhibit, control or treat a disease or condition / disorder associated with angiogenesis. Methods of identifying humanized antibodies or antigen-binding fragments thereof that bind to endoglobin are provided herein. Antibodies or binding fragments of the antigen can be evaluated for one or more binding affinity, association rates, dissociation rates and avidity. In one aspect, antibodies can be assessed for their ability to neutralize the activity of endoglobin or a polypeptide in which the endoglobin binding sequence is present. Measurement of binding affinity, association rates, dissociation rates and avidity can be performed using techniques recognized in the art, including surface plasma resonance assays, but not limited to, an ELISA assay, Scatchard analysis, BIACORE analysis, etc. , as well as other tests commonly used and known to those skilled in the art. The measurement of antibody binding to endoglobin and / or the ability of antibodies or antigen binding fragments thereof, for example, to inhibit angiogenesis, can be determined using, for example, an ELISA assay, a competitive assay for binding, an ELISPOT assay, or any other useful assay known in the art. These tests are commonly used and well known to those skilled in the art.
In a non-limiting embodiment, an ELISA assay can be used to measure the binding capacity of specific antibodies, or antigen-binding fragments thereof, which bind to endoglobin.
Assays, such as ELISA, can also be used to identify antibodies or antigen-binding fragments that exhibit increased specificity for endoglobin compared to other antibodies or antigen-binding fragments therein. Assays, such as ELISA, can also be used to identify antibodies or antigen-binding fragments that bind to epitopes on one or more polypeptides and through one or more endoglobin species. The specificity assay can be conducted by running parallel ELISAs in which a test antibody, or antigen binding fragments, are concurrently screened in separate test chambers for the ability to bind one or more epitopes on different species polypeptide containing endoglobin epitopes to identify antibodies or antigen-binding fragments that bind to endoglobin. Another technique for measuring binding affinity, apparently familiar to those skilled in the art, is a surface plasma resonance technique (analyzed in a 2000 BIACORE system) (Liljeblad, et al., Glyco. J. 2000, 17: 323- 329). Standard and traditional measures of binding assays are described by Heeley, RP, Endocr. Res. 2002, 28: 217-229.
Humanized antibodies to endogline can also be analyzed for their ability to treat various diseases and conditions associated with angiogenesis, for example, various forms of eye diseases characterized by angiogenesis / neovascularization (eg, macular degeneration, diabetic retinopathy), diabetic nephropathy , chronic inflammatory diseases (eg, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancer (primary tumors and metastases). Any suitable assay known to a person skilled in the art can be used to monitor such effects. Several of these techniques are described here. In one example, antibodies or antigen-binding fragments thereof described herein are analyzed for their ability to bind to endogline. In another example, the affinity constants for the antibodies or antigen binding fragments thereof described herein are determined by surface plasma resonance (SPR). In yet another example, antibodies or antigen-binding fragments described herein are analyzed for their effect on inhibition of angiogenesis. II. Compositions Each of the compounds described herein can be used as a composition when combined with an acceptable carrier or excipient. Such compositions are useful for in vitro or in vivo analysis or for administration to a subject in vivo or ex vivo for treating the subject with the described compounds.
Thus, pharmaceutical compositions may include in addition to the active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.
Pharmaceutical formulations comprising a protein of interest, for example, an antibody or antigen binding fragments thereof, identified by the methods described herein that can be prepared for storage by mixing the protein having the desired degree of purity with optional vehicles, excipients or physiologically acceptable stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable vehicles, excipients, or stabilizers are those that are non-toxic to the receptors at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbon carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, Zn-protein complexes), and / or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).
Acceptable vehicles are physiologically acceptable for the patient to be treated and retain the therapeutic properties of the compounds with / in which it is administered. Acceptable vehicles and their formulations are generally described in, for example, Remington's Pharmaceutical Sciences (18th edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990). An exemplary carrier is physiological saline. The term "pharmaceutically acceptable carrier", as used herein, means a pharmaceutically acceptable carrier material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulation material, involved in loading or transporting the compounds in question from the site of administration of an organ, or body portion, to another organ, or body portion, or in an in vitro test system. Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to a subject to whom it is administered. Nor is an acceptable vehicle to alter the specific activity of the compounds in question.
In one aspect, pharmaceutically acceptable or physiologically acceptable compositions including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonics and absorption that promote or delay agents compatible with pharmaceutical administration are provided herein. Pharmaceutical compositions or pharmaceutical formulations, therefore, refer to a composition suitable for pharmaceutical use in a subject. The pharmaceutical compositions and formulations include an amount of a compound described herein and a pharmaceutically or physiologically acceptable carrier.
The compositions can be formulated to be compatible with a particular route of administration (i.e., systemic or local). Thus, the compositions include carriers, diluents, or excipients suitable for administration by various routes.
In another embodiment, the compositions may further comprise, if necessary, an acceptable additive, in order to improve the stability of the compounds in the composition and / or to control the rate of release of the composition. Acceptable additives do not alter the specific activity of the compounds in question. Examples of acceptable additives include, but are not limited to, a sugar such as mannitol, glucose, sorbitol, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Acceptable additives can be combined with acceptable vehicles and / or excipients, such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase the stability of the peptide and decrease the gelation of the solution. The suFRactant can be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition in storage. The pharmaceutical composition can be administered, for example, by injection, including, but not limited to, subcutaneous, intravitreal, intradermal, intravenous, intraarterial, intraperitoneal or intramuscular. Excipients and carriers for use in formulating compositions for each type of injection are contemplated here. The following descriptions are, for example, only and are not intended to limit the scope of the compositions. Compositions for injection include aqueous solutions (when soluble in water) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable or dispersion solutions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, liquid glycerol, propylene glycol, and polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using suFRactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, ascorbic acid, phenol, and thimerosal. Isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride can be included in the composition. The resulting solutions can be packaged for use or lyophilized; the lyophilized preparation can then be combined with a sterile solution before administration. For intravenous injection or injection at the site of the affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is free of pyrogens and has adequate pH, isotonicity and stability. Technicians in the relevant art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, ringer injection, ringer lactate injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as needed. Sterile injectable solutions can be prepared by incorporating an active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active ingredient in a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which produces a powder of the active ingredient plus any desired additional ingredient from a solution previously sterilized by filtration.
The compositions can be administered conventionally intravitreally, subcutaneously, or via intravitreal implants.
The compositions can conventionally be administered intravenously, such as by injection of a unit dose, for example. For injection, an active ingredient can be in the form of a parenterally acceptable aqueous solution that is substantially free of pyrogens and has adequate pH, isotonicity and stability. Suitable solutions can be prepared using, for example, isotonic vehicles such as sodium chloride injection, ringer injection, ringer lactate injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as needed. In addition, the compositions can be administered via aerosolization (Lahn et al, anti-T cell antibody receptor aerosol are effective against airway inflammation and hyperresponsiveness, Int Arch LLAegery Immuno, 134: 49-55 (2004 )).
In one embodiment, the composition is lyophilized, for example, to increase shelf life in storage. When the compositions are considered for use in medicaments or any of the methods provided herein, it is contemplated that the composition may be substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human patient . Test compositions for pyrogens and compositions substantially free of pyrogens are well understood by a person skilled in the art and can be made using commercially available kits.
Acceptable vehicles may contain a compound that stabilizes, increases or delays absorption or clearance. Such compounds include, for example, carbon carbohydrates, such as sucrose, glucose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides, or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monoesterates and gelatin. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. To protect from digestion, the compound can be complexed with a composition to be resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in a suitably resistant carrier such as a liposome. Digestion protection compounds are known in the art (see, eg, Fix (1996) Pharm Res 13: 1760 1764; Samanen (1996) J. Pharm Pharmacol 48: 119 135; and U.S. Patent No. 5,391,377, describes lipid compositions for oral administration of therapeutic agents). The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not normally produce an inconvenient or similar allergic reaction, such as gastric disorders, dizziness and the like, when administered to a human. The term "unit dose", when used in reference to a therapeutic composition, refers to physically discrete units suitable as a unit dose for humans, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in association with the required diluent, ie vehicle or carrier.
The compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the ability of the subject's immune system to use the active ingredient, and the degree of binding capacity desired. Precise amounts of active ingredient needed to be administered will depend on the practitioner's discretion and are unique to each individual. Suitable regimens for initial administration and booster doses are also variable, but are typified by an initial administration followed by repeated doses at one or more hourly intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusions sufficient to maintain blood concentrations are contemplated.
One embodiment contemplates the use of the compositions described herein to produce a medicament for the treatment of a condition, disease or disorder described herein. Medicines can be formulated based on the physical characteristics of the patient / subject in need of treatment, and can be formulated in single or multiple formulations based on the stage of the condition, disease or disorder. Medicines can be packaged in an appropriate package, with appropriate labels for distribution to hospitals and clinics where the label is for indicating the treatment of a subject with a disease described here. Medicines can be packaged as single or multiple units. Instructions for dosing and administering the compositions can be included with the packages as described below. The invention is further directed to drugs of a humanized anti-endoglobin antibody or antigen binding fragments thereof as described above and a pharmaceutically acceptable carrier. Compositions of humanized antibodies or antigen-binding fragments thereof that bind to endoglobin are provided herein and include those such as those described herein. Humanized antibodies or antigen-binding fragments thereof that bind to endoglobin as described herein can be used for the treatment of various forms of eye diseases characterized by angiogenesis / neovascularization (eg macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (eg, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancer (primary tumors and metastases).
A composition (an antibody or antigen binding fragment thereof described herein) can be administered alone or in combination with a second composition, simultaneously or sequentially depending on the condition to be treated. In one embodiment, a second therapeutic treatment is an inhibitor of angiogenesis (as described herein). When two or more compositions are administered, the compositions can be administered in combination (either sequentially or simultaneously). A composition can be administered in a single dose or in multiple doses.
In an embodiment of the present invention, the compositions are formulated free of pyrogens such that they are acceptable for administration to human patients. Test compositions for pyrogens and preparation of pyrogen-free pharmaceutical compositions are well understood by one skilled in the art.
An embodiment of the present invention contemplates the use of any of the compositions of the present invention to produce a medicament for the treatment of a disorder of the present invention. Medicines can be formulated based on the physical characteristics of the patient / subject in need of treatment, and can be formulated in single or multiple formulations based on the disorder. Medicines of the present invention can be packaged in a suitable pharmaceutical package with labels suitable for distribution to hospitals and clinics where the label is for indicating the treatment of a disorder as described herein in a subject. Medicines can be packaged as single or multiple units. Instructions for dosing and administering the pharmaceutical compositions of the present invention can be included with the pharmaceutical product packages. III. Methods of Use Here is provided a method of eliciting a response from a patient (human or non-human) by administering to the patient a composition of an antibody or antigen binding fragment thereof which preferably binds to endogline. The binding site to which the antibody binds can be a continuous or discontinuous conformation epitope.
An effective response of the present invention is obtained when the patient experiences partial or total relief or a reduction in signs or symptoms of disease, and specifically includes, without limitation or prolongation, survival and / or visual acuity. The expected progression of survival can be measured in months or years, depending on prognostic factors, including the number of relapses, stage of the disease, and other factors. Extending survival includes, without limitation, about at least 1 month, about at least 2 months, about at least 3 months, about at least 4 months, about at least 6 months, about at least one year, about at least 2 years, about at least 3 years, etc. Global survival can also be measured in months or years. Alternatively, an effective response may be that a patient's symptoms remain static. Other indications for handling indications are described in more detail below.
Antibody compositions or antigen-binding fragments thereof described herein can be used as non-therapeutic agents (for example, as affinity purifying agents). Generally, in one embodiment, such that a protein of interest is immobilized on a solid step by a resin such as Sephadex resin or filter paper, using conventional methods known in the art. The immobilized protein is contacted with a sample containing the target of interest (or its fragment) to be purified and, subsequently, the support is washed with a suitable solvent that will remove substantially all material in the sample, except the target protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, which will release the target protein. In addition to purification, the compositions can be used for the diagnosis, detection and therapy of diseases and disorders associated with endoglobin and angiogenesis. The term "contacting" as used herein refers to the addition of a solution or composition of a compound with a liquid medium that bathes an organism's polypeptides, cells, tissue or organ. Alternatively, "counting" refers to mixing together a solution or composition of a compound, with a liquid such as blood, serum, plasma or an organism derivative. For in vitro applications, a composition can also comprise another component, such as dimethyl sulfoxide (DMSO). DMSO facilitates the absorption of the compounds or the solubility of the compounds. The solution comprising the test compound can be added to the medium that bathes the cells, tissues or organs, or mixed with another liquid, such as blood, using a diffusion device, such as a pipette-based device or a syringe. For in vivo applications, contacting can occur, for example, by administering a composition to a patient by any suitable means; compositions with pharmaceutically acceptable excipients and carriers have been described in more detail above.
A "patient" (for example, a mammal such as a human or a non-human animal, such as a primate, cow, horse, pig, sheep, etc.) according to an embodiment of the present application, is a mammal that has one or more clinical manifestations and / or symptoms of a disease or disorder described herein. In certain situations, the patient may be asymptomatic and still have the clinical manifestations of the disease or disorder. An antibody or antigen binding fragment thereof can be conjugated to a therapeutic moiety or be a fusion protein containing a therapeutic moiety. An antibody or antigen binding fragment thereof can be conjugated to a detectable moiety or be a fusion protein containing a detectable moiety. In one embodiment, the antibody or its antigen-binding fragment can be conjugated to either a therapeutic portion or a detectable portion. An antibody or antigen binding fragment thereof can be conjugated to, or made recombinantly, with an affinity tag (e.g., a purification tag). Affinity tags, such as, for example, His6 tags (SEQ ID NO: 85) are conventional in the art.
Antibodies or antigen binding fragments thereof provided herein are such that they can be conjugated or linked to a therapeutic moiety and / or an image or a detectable moiety and / or an affinity marker. Methods for conjugating or attaching polypeptides are well known in the art. Associations (bonds) between the compounds and labels include any means known in the art, including, but not limited to, covalent and non-covalent interactions, chemical conjugation, as well as recombinant techniques. A. Endoglobin binding and endoglobin angiogenesis (CD105) is expressed on the cell surface as a 180 kDa homodimeric transmembrane protein. The external domain links TGF- βΐ and -3 isoforms with high affinity (50 nM), and the transmembrane and intracellular domains of CD105 share a sequence similarity with 71% betaglycam. The human CD105 gene is located on chromosome 9q34, identified using fluorescence in situ hybridization, and the coding region contains 14 exons, and two different CD105 isoforms (L and S) capable of binding to TGF-β were characterized. L-CD105 consists of 633 amino acid residues with 47 amino acid residues in the cytoplasmic tail as opposed to S-CD105, which consists of 600 amino acid residues with a cytoplasmic tail of 14 amino acids. However, L-CD105 is the predominant form. CD105 is constitutively phosphorylated in endothelial cells, mainly on serine and threonine, and this phosphorylation is due to the constitutively active TGF-β RII within the cell. Binding of TGF-β to CD105 results in poor regulation of phosphorylation, similar to the effects observed with protein kinase C inhibitors. The human CD105 amino acid sequence contains arginine-glycine-aspartic tripeptide (RGD), located in an exposed region of the domain extracellular. The RGD peptide is a key recognition structure found in ECM proteins, such as fibronectin, vitronectin, von Willebrand factor (vWF), type I collagen, and fibrinogen and is recognized by integrins on the cell surface. Integrin adhesion has been implicated in hemostasis, thrombosis, angiogenesis and inflammation, processes in which the endothelium plays a critical role. (Duff et al., ESTÁGIOB J., 17: 984-992 (2003)). CD105 is a member of the TGF-β receptor family that is expressed by proliferative endothelial cells. Normal levels of CD105 are necessary for the proliferation of endothelial cells. CD105 expression is increased by cellular hypoxia through the production of the inducible factor 1-a (HIF-1-a) hypoxia and protects hypoxic cells from apoptosis. Several functions of CD105 are associated with TGF-β signaling. TGF-β signals through heterodimeric receptors consist of serine kinases, receptor I (RI), and receptor II (RII). The binding of TGF-β to the external domains of the receptor unmasks the activity of the cytoplasmic kinase RII that phosphorylates TGF-β RI, which can then interact with downstream signalers, such as Smad proteins. CD105 is part of the TGF-β receptor complex, but it can exist independently on the cell surface. In many cells in vitro, CD105 suppresses TGF-β signaling. CD105 also binds to other growth factors such as activin A and bone moFRogenetic protein (BMP) -10, -9, -7 and -2. The binding of TGF-β or other growth factors to CD105 requires the presence of at least the RII receptor, and cannot bind ligands alone. The association of CD105 with the receptors does not alter its affinity with the ligand. After association, the cytoplasmic domain of CD105 is phosphorylated by TGF-β RI and TGF-β RII, then TGF-β RI, but not TGF-β RII, dissociates kinase from the receptor complex.
Expression of CD105 inhibits phosphorylation levels of TGF-β RII, but increases that of TGF-β RI, resulting in increased phosphorylation of Smad 2, but not of Smad 3. Since Smad 2 can interact with a variety of transcription factors , co-activators, and eliminators, phosphorylated Smad 2 can act as an integrator of multiple signals to modulate gene transcription. Thus, CD105 modulates TGF-β functions through interaction with TGF-β RI and TGF-β RII and modifies protein phosphorylation downstream from Smad. CD105 acts to modulate the signaling of multiple kinase receptor complexes of the TGF-β family, including TGF-β receptors (TGF ^ R), activin receptor-type kinases (ALK) and activin receptors. In the absence of CD105, activation of TGF-β receptors results in the phosphorylation of SMAD proteins (SMAD 2 and 3) that inhibit the growth of endothelial cells. However, activation of CD105 by TGF-β modulates the phosphorylation of the SMAD protein (including the phosphorylation of SMAD 1, 5 and 8). The end result is the release of the inhibitory growth effects of TGF-β receptor activation in endothelial cells (see Figure 3). Not surprisingly, the prevention of CD105 activation by anti-CD105 antibody or antisense oligonucleotide acts synergistically with TGF-β to suppress the growth of endothelial cells. The CD105 promoter is 2.6kb in length, but does not contain TATA or CAAT transcription initiation sets. However, it has two GC-rich regions, reasons for consensus for SP1, ETS, GATA, AP-2, NGF-β, and Mad, as well as TGF-β response elements. However, CD105 has a relatively restricted cell distribution. The baseline level of transcription appears to require an ets site at the -68 position and the SP1 sites, but the relative expression restriction, for example, for endothelial cells, appears to involve multiple regulatory regions, in particular, one at -1294 to -932 and another very close to the place where the transcription started. CD105 is over-regulated by TGF-β, and this has been shown to require a Sp1 site at -37 to -29, also involving one or more sites juxtaposed upstream from SBE binding Smads 3 and / or 4 (which are activated by TGF-β signaling). Hypoxia is a common feature of ischemic tissues and tumors, and is a potent stimulant for CD105 gene expression in vascular endothelial cells (ECS). This effect is enhanced in combination with TGF-β 1. The CD105 regulated above can play a role of self-protection in ECs under hypoxic stress.
EC vessels are the main source of CD105. Other types of vascular cells, including smooth muscle cells, fibroblasts, macrophages, leukemic cells of pre-B and myelomonocytic origin and erythroid precursors express CD105 to a lesser extent. CD105 is involved in angiogenesis. Antisense experiments have shown that suppression of CD105 expression in HUVEC results in marked inhibition of angiogenesis in vitro in combination with ΊΌΕ-β1, indicating that CD105 is a proangiogenic component in endothelial cells. Further evidence of the importance of CD105's role in angiogenesis comes from CD105 knockout mice. Mice that do not have CD105 have several vascular and cardiac defects, leading to death in an embryonic stage. Severe vascular damage seen in null mice on CD105 indicates that CD105 is necessary for the formation of mature blood vessels in the extra-embryonic vasculature, further confirming the direct role of endoglobin in angiogenesis. Endoglobin, also known as, inter alia, CD105 or edg-1, is a homodimeric type I membrane glycoprotein that is expressed in high levels in proliferative vascular endothelial cells. Thus, endoglobin is primarily a proliferation-associated marker for endothelial cells subjected to angiogenic activity. However, there may be limited expression of endoglobin by the vascular endothelium of normal tissues. Human endogline is known to bind specifically to growth transforming factor-β (TGF-β), and the deduced amino acid sequence of endoglobin has strong homology to β-glycan, a type of TGF-β receptor. Endoglobin (EDG) has been the target of methods based on antibodies to reduce tumor vasculature, since EDG is an antigen associated with proliferation in endothelial cells and leukemia. Its expression is over-regulated in association with the vascular endothelial tumor, and EDG is essential for angiogenesis. Angiogenesis includes the formation of new capillary blood vessels that lead to neovascularization, as well as the maintenance of the existing vasculature. It is a complex process that includes a series of sequential steps, including endothelial cell-mediated degradation of the basal vascular membrane and interstitial matrices, endothelial cell migration, endothelial cell proliferation, and capillary formation by endothelial cells. Humanized antibodies that bind to endogline are provided here. Endoglina can be found in cells that comprise and support existing vasculature, as well as cells that are promoting the growth of, and become part of, new vascularization. Antibodies can bind to endogline and thus inhibit angiogenesis, inhibit the existing vasculature or the maintenance of the existing vasculature, and / or inhibit the dilation of small vessels. In addition to their use for the purification of endoglobin, these antibodies are useful for detection, purification and diagnostic purposes, as well as therapeutic purposes. The antibodies provided herein can be used for formulating medicaments for the treatment of a variety of conditions and diseases, methods for treating such conditions and diseases, and methods of detection or diagnosis.
Murine monoclonal antibodies (mAbs) have been raised against endoglobin, which modulate endoglobin activity and thus inhibit angiogenesis and / or inhibit vasodilation of small blood vessels. These murine antibodies are described in US patents 5,928,641, 6,200,566, 6,190,660, and 7,097,836, each of which is incorporated herein in its entirety. In addition, ex vivo and in vivo efficiency of a number of these antibodies has been demonstrated; monoclonal antibodies that bind to endogline are of interest as endoglobin modulating compounds. The therapeutic use of murine antibodies is not feasible, however, as the administration of murine antibodies has a number of limitations, including immunogenicity in, for example, in the form of human anti-mouse antibodies (HAMA). The term "angiogenesis" is used here to include all aspects of blood vessel maintenance and development. Thus, angiogenesis includes the formation of new capillary blood vessels that lead to neovascularization, as well as the maintenance and control of the existing vasculature and small blood vessels. Angiogenesis is a complex process that includes a series of sequential steps, including endothelial cell-mediated degradation of the basal vascular membrane and interstitial matrices, endothelial cell migration, endothelial cell proliferation, and capillary formation by endothelial cells. Angiogenesis includes the growth and / or development of new blood vessels (also referred to as neovascularization), dilation of small vessels, prolonged or excessive vascular growth, and maintenance of the existing vasculature. Endoglina is known to be involved in the regulation of angiogenesis and is believed to be involved in several biochemical pathways related to the induction of angiogenesis. (Duff et al, FASEB J., 17: 984-992 (2003); Bernabeu et al, J. Cell Biochem, 102 (6): 1375-1388 (2007)).
As used herein, the terms "inhibitory angiogenesis," "inhibition of angiogenesis" or "anti-angiogenic" include inhibition of vasculogenesis, and are intended to mean that there is a decrease in the degree, amount, or rate of neovascularization. Making a decrease in the degree, amount, or rate of endothelial cell proliferation or tissue migration is a specific example of inhibition of angiogenesis. The term "angiogenesis inhibitory composition" refers to a composition that inhibits angiogenesis by mediating processes such as endothelial cell migration, proliferation, tube formation and subsequently leading to the inhibition of the generation of new blood vessels from existing ones, and consequently affects conditions dependent on angiogenesis. The term "disease associated with angiogenesis" is used here, for the purpose of specification and claims, to mean certain pathological processes in humans, where angiogenesis is abnormally prolonged. This further includes conditions of angiogenesis and diseases, such as related diseases and conditions, caused by or associated with angiogenesis. Non-limiting examples of such diseases include various forms of eye diseases characterized by angiogenesis / neovascularization (eg, macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (eg, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancers and metastases. Antibodies or antigen-binding fragments thereof described herein can be used to treat a disease associated with angiogenesis by endoglobin binding and inhibition of angiogenesis. The term "anti-angiogenic therapy" is used here, for the purposes of the specification and claims, to mean therapy directed at cells and / or vasculature that express endoglobin (expressed at higher levels in vasculature proliferation, compared to quiescent vasculature ); this further includes therapy that is directed against angiogenesis (that is, the formation of new capillary blood vessels that lead to neovascularization), therapy that is directed against existing vasculature and / or excessive vascularization or blood vessel growth therapy, directed at dilation of small vessels, and therapy directed at a disease or condition (for example, vascular segmentation therapy). Examples of diseases or conditions contemplated within the scope of the invention include, but are not limited to, various forms of eye diseases characterized by angiogenesis / neovascularization (for example, macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (for example, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancer, solid tumors and metastases. The term "ocular disease characterized by neovascularization" is used herein, for the purposes of the specification and claims, to mean any eye disease caused by, or resulting in, increased angiogenesis within any portion of the eye, including the retina, cornea, pupil, iris, vitreous humor and watery humor. Such diseases include, for example, those related to age, macular degeneration, diabetic and non-diabetic retinopathy, retinopathy, choroidal neovascularization (NVC) and sub-retinal neovascularization (SRN or SRNV) and neoplasms of the eye. B. Diagnostic Applications Humanized anti-endoglobin antibodies and fragments thereof can be used for in vivo and in vitro detection, diagnosis and / or control effects. Endogline is believed to be involved in several diseases and disorders, as described below. Treatment of diseases related to endogline and dependent conditions, in part, through its diagnosis, and antibodies or antigen-binding fragments described herein are useful for the diagnosis of excess endoglobin or for the diagnosis of diseases and associated conditions with the activity of the endoglina. Provided herein is a method of detecting levels of endoglobin in a sample or subject comprising (i) making contact between an antibody or antigen binding fragment thereof described herein with the sample or subject, and (ii) detecting a complex of the antibody or antigen-binding fragment thereof and endoglobin. An angiogenesis imaging or diagnostic method or an angiogenic-dependent disease or disorder is provided herein, comprising contacting a composition of an antibody or antigen binding fragment thereof as described herein with a sample. The sample can be, for example, blood, serum, plasma, platelets, biopsy fluid, spinal fluid, meninges and urine. Imaging or diagnostic method can occur in an in vitro assay. Alternatively, when contact is by administering the composition to a patient, angiogenesis or angiogenic-dependent disease or disorder is photographed or diagnosed in vivo.
In one embodiment, the antibody or antigen binding fragment thereof further comprises a detectable portion. Detection can occur in vitro, in vivo or ex vivo. In vitro assays for the detection and / or determination (qualification, quantification, etc.) of endoglobin with antibodies or antigen-binding fragments thereof include, but are not limited to, for example, ELISAs, RIAs and western blots. In vitro detection, diagnosis or monitoring of endoglobin can occur by taking a sample (eg a blood sample) from a patient and testing the sample in, for example, a standard ELISA assay. For example, a 96-well microtiter plate can be coated with an antibody or antigen binding fragments thereof described here, washed and coated with PBS-Tween / BSA to inhibit non-specific binding. The blood sample can be serially diluted and placed in duplicate wells compared to a diluted endoglobin standard curve. After incubation and washing of the wells, a biotin-labeled anti-endoglobin antibody can be added, followed by the addition of streptavidin-alkaline phosphatase. The wells can be washed and a substrate (horseradish peroxidase) added to develop the plate. The card can be read using a conventional card reader and software.
When detection occurs in vivo, contact occurs through administration of the antibody or antigen binding fragment thereof using any conventional means such as those described herein. In such methods, the detection of endoglobin in a sample or an object can be used to diagnose a disease or disorder associated with, or correlated with, the activity of endoglobin such as the diseases and disorders described herein.
In the diagnosis, detection or monitoring of in vivo endogline, an antibody or antigen binding fragment thereof that binds to the endogline is administered to a patient, which antibody or antigen binding fragment thereof is attached to a detectable portion. The detectable portion can be visualized using techniques recognized in the art such as, but not limited to, magnetic resonance imaging (MRI), fluorescence, radio imaging, light sources provided by endoscopes, laparoscopes, or intravascular catheter (ie, through detection of agents photoactives), positron emission tomography (TEP), whole body nuclear magnetic resonance (NMR), computed tomography (CT), infrared region (IR), X-rays, ultrasound, etc., as described, for example , in US patent No. 6,096,289, US patent No. 7,115,716, US patent No. 7,112,412, US application No. 20030003048 and American application No. 20060147379, each of which is incorporated herein in its entirety by reference. . Indications for detecting compounds using such methods are also known in the art and described in such patents and applications and are incorporated herein by reference. Visualization of the detectable portion may allow the detection, diagnosis and / or monitoring of a condition or disease associated with endoglin and / or angiogenesis.
Additional diagnostic assays using antibodies specific for the desired target protein, i.e., endoglobin, are known in the art and are also contemplated herein. Non-limiting conditions, diseases and disorders to be considered for these methods include, but are not limited to, those associated with angiogenesis, such as, for example, various forms of eye diseases characterized by angiogenesis / neovascularization (for example, macular degeneration , diabetic retinopathy, diabetic nephropathy, chronic inflammatory diseases (eg, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancer, primary tumors and metastases). In the detection, diagnosis or monitoring of such diseases, the patient is subjected to the administration of a composition of an antibody or antigen binding fragment thereof described herein, which antibody or antigen binding fragment thereof is conjugated to a detectable portion . The portion can be viewed using methods recognized in the art such as those described above. Visualization of the detectable portion can allow the detection, diagnosis and / or monitoring of such conditions and diseases.
For in vitro detection methods, samples to be obtained from a patient include, but are not limited to, blood, tissue samples and biopsy fluids.
Thus, the present invention provides humanized antibodies or antigen-binding fragments thereof against endoglobin that are useful for the detection or diagnosis of levels of endoglobin associated with a disease or disorder, potentially indicating the need for therapeutic treatment. In certain embodiments, the antibodies comprise a humanized anti-endoglobin antibody described herein. In other embodiments, the antibody further comprises a second agent. Such an agent can be a molecule or portion, such as, for example, a reporter molecule or a detectable marker. Detectable markers for detection methods are known in the art and are described in more detail below. Reporter molecules are any portion that can be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to polypeptides include enzymes, radioactive markers, haptens, fluorescent, phosphorescent markers, chemiluminescent molecules, chromophores, luminescent molecules, photo-affinity, molecules, colored particles or ligands, such as biotin. Detection markers include compounds and / or elements that can be detected, due to their specific functional properties, and / or chemical characteristics, the use of which allows the polypeptide to which they are attached can be detected, and / or even quantified, if desired. Many detection agents are known in the art, such as the method for attaching them to polypeptides (see, for example, U.S. Patent Nos. 5,021,236; 4,938,948; and 4,472,509, each of which is incorporated herein by reference).
Methods of joining polypeptides, such as antibodies with detectable moieties, are known in the art and include, for example, recombinant DNA technology to form fusion and conjugation proteins (for example, chemical conjugation). Methods for the preparation of fusion proteins by chemical conjugation or recombinant engineering are well known in the art. Methods that bind the components covalently and non-covalently are also known in the art. See, for example, Williams (1995) Biochemistry 34: 1787 1797; Dobeli (1998) Protein Expr. Purif. 12: 404-414; and Kroll (1993) cellular DNA. Biol. 12: 441-453.
In some cases, it may be necessary to introduce an unstructured polypeptide binding region between a marker or medium and one or more portions of the antibodies, antigen binding fragments or binding proteins, described herein. The binder can facilitate greater flexibility, and / or reduce the steric impediment between any two fragments. The binder can also facilitate the occurrence of the appropriate rate in each fragment. The linker can be of natural origin, such as a sequence determined between two domains of a protein. A linker sequence is the linker found between the domains of a C-terminal and N-terminal subunit of the RNA polymerase. Other examples of naturally occurring ligands include ligands found in proteins 1CI and LexA.
In a linker, an amino acid sequence can be varied according to the function of its characteristics, determined empirically or as shown by modeling. Considerations when choosing a ligand include its flexibility, its load, and the presence of some amino acids in the ligand in naturally occurring subunits. The ligand can also be designed so that the nucleic acid residues come into contact with deoxyribose (DNA), thereby influencing the binding affinity or specificity, or to interact with other proteins. In some cases, such as when it is necessary to understand a greater distance between the subunits, or when the domains must be carried out in a specific configuration, the ligand may, optionally, contain an additional folded domain. In some of the configurations, the design of a linker may involve an array of domains that requires the linker to cover a relatively short distance, for example, less than about 10 Angstroms (Ã). However, in certain configurations, the binders span a distance of up to about 50 Angstroms.
In a ligand, the sequence of amino acids can be varied according to its characteristics, determined empirically or as shown by modeling. Considerations when choosing a ligand include its flexibility, its load, and the presence of some amino acids in the ligand in naturally occurring subunits. The ligand can also be designed so that contact residues in the binding DNA influence binding affinity or specificity, or to interact with other proteins. In some cases, when it is necessary to understand a greater distance between the subunits, or when the domains must be carried out in a specific configuration, the ligand may optionally contain an additional folded domain. Methods for coupling polypeptides (free or cell-bound) to spheres are known in the art. Methods for selecting coupled polypeptides or cells that have a polypeptide are also known in the art. Briefly, polystyrene paramagnetic microparticles are commercially available (Spherotech, Inc., Libertyville, IL; Invitrogen, Carlsbad, CA) in which the peptide pair had the microparticle surfaces modified with functional groups or coated with various antibodies or ligands, such as , for example, avidin, streptavidin or biotin. The paramagnetic property of microparticles allows them to be separated from the solution using a magnet. The microparticles can be easily resuspended when the magnet is removed. Polypeptides can be attached to polystyrene paramagnetic microparticles coated with a polyurethane layer in a tube. Hydroxy groups on the microparticle surface are activated by reaction with p-toluenesulfonyl chloride (Nilsson K and Mosbach K. "p-toluenesulfonyl chloride as an agarose activating agent for the preparation of immobilized affinity binders and proteins." Eur J. Biochem. 1980: 112: 397-402). Alternatively, paramagnetic polystyrene microparticles containing carboxylic acid on the surface can be activated with a carboDICmide, followed by coupling to a polypeptide, resulting in a stable amide bond between a primary amino group on the polypeptide and the carboxylic acid groups on the surface of the microparticles ( Nakajima N and Ikade Y, Mechanism of formation of amide by carboDICmide by bioconjugation in aqueous medium, Chemical Bioconjugation. 1995, 6 (1): 123-130; Gilles MA, Hudson AQ and Borders CL Jr, Stability of water soluble carboDICmide, in an aqueous solution, Anal Biquimica. 1990 Feb 1; 184 (2): 244-248; Sehgal D and Vijay IK, a method for measuring the high amidation efficiency of water-soluble carboDICmide, Anal Biochem. 1994 Apr; 218 ( 1): 87-91; Szajani B et al, Effects of carboDICmide structure on enzyme immobilization, Appl Biochemistry Biotechnology. 1991 Aug; 30 (2): 225-231). Another option is for the pair of biotinylated polypeptides to the paramagnetic polystyrene microparticles whose surfaces have been covalently linked with a streptavidin monolayer. (Argarana CE, Kuntz ID, Birken S, Axel R, Cantor CR molecular cloning and the nucleotide sequence of the streptavidin gene Nucleic Acids Res. 1986; 14 (4): 1871-82; Pahler A, Hendrickson WA, Gawinowicz Kolks MA , Aragana CE, Cantor CR Characterization, and crystallization of the streptavidin nucleus J Biol Chem 1987: 262 (29): 13933-13937).
Polypeptides can be conjugated to a wide variety of fluorescent dyes, suppressors and haptens, such as fluorescein, R-phycoerythrin, and biotin. Conjugation can occur during the synthesis of the polypeptide or after its synthesis and purification. Biotin is a small vitamin (244 kilodaltons) that binds with high affinity to the proteins avidin and streptavidin and can be conjugated to most peptides, without altering their biological activities. Biotin-labeled polypeptides are easily purified from unlabeled polypeptides using immobilized streptavidin and affinity avidin gels, and probe-conjugated streptavidin or avidin can be used to detect biotinylated polypeptides in, for example, ELISA, dot blot or Western applications blot. N-hydroxy succinimide esters of biotin are the most widely used type of biotinylation agent. Biotin-activated N-hydroxysuccinimide react efficiently with primary amino groups in physiological buffers to form stable amide bonds. Polypeptides have N-terminal primary amines and may also have several primary amines in the side chain of lysine residues that are available as targets for labeling with biotin-activated N-hydroxysuccinimide reagents. Several biotin esters other than N-hydroxysuccinimide are available, with varying properties and length of the spacer arm (Pierce, Rockford, IL). Sulfo-N-hydroxysuccinimide reagents are soluble in water, allowing reactions to be carried out in the absence of organic solvents. The molar ratio of biotin to polypeptide can be estimated using a 2- (4'-Hydroxyazobenzene-2-carboxylic acid) assay using techniques recognized in the art (Green, NM, (1975) "Advances in avidin protein. Chemistry" Academic publisher, New York 29, 85-133 ;. Green, NM, (1971) "). The use of bifunctional biotinyl compounds to determine the arrangement of avidin subunits" Biochem J. 125, 781-791 ;. Green, NM, (1965) "spectrophotometric assay one for avidin and biotin based on dye binding by avidin." J. Biochem 94: 23c-24c). The formation of the biotin-avidin bond is very fast and stable in organic solvents, extreme pH and denaturing reagents. To quantify biotinylation, a solution containing the biotinylated polypeptide is added to a mixture of 2- (4'Hydroxyazobenzene-2-carboxylic acid) and avidin. Because biotin has a greater affinity for avidin, it displaces 2- (4'-Hydroxyazobenzene-2-carboxylic acid) and the absorbance at 500 nanometers decreases proportionately. The amount of biotin in a solution can be quantified in a single cuvette by measuring the absorbance of 2- (4'-Hydroxyazobenzene-2-carboxylic acid) -avidine solution before and after adding the biotin-containing peptide. The change in absorbance is related to the amount of biotin in the sample by the extinction coefficient of the 2- (4'-Hydroxyazobenzene-2-carboxylic acid) -avidine complex.
Alternatively, an antibody, an antigen binding fragment or a binding protein can be conjugated to a fluorescent moiety through polypeptide conjugation (eg, R-phycoerythrin, fluorescein isothiocyanate (FITC), etc.) and can be performed using recognized techniques described in, for example, Glazer, AN and Stryer L. (1984). Trends Biochem. Sci. 9: 423-7; Kronick, MN and Grossman, PD (1983) Clin. Chem. 29: 1582-6; Lanier, LL and Loken, MR (1984) J. Immunol, 132: 151-156 ;. Parks, DR et al. (1984) Cytometry 5: 159-68; Hardy, RR et al. (1983) Nature 306: 270-2; Hardy RR et al. (1984) J. Exp. Med. 159: 1169-88; Kronick, MN (1986) J. Immuno. Meth. 92: 1-13; Der-Balian G, Kameda, N and Rowley, G. Anal (1988). Biochem. 173: 59-63.
In a non-limiting embodiment, an antibody to the antigen-binding fragment can be associated (conjugated with) to a detection marker, such as a radionuclide, iron compound, a dye, an imaging agent, or a fluorescent agent for immunodetection of endoglobin that can be used to visualize binding of antibodies to endoglobin in vitro and / or in vivo.
Non-limiting examples of radioactive markers include, for example, 32P, 33P, 43K, 52Fe, 57Co, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 77Br, 81Rb / 81MKr, 87MSr, 90Y, 97Ru , 99Tc, 100Pd, 101Rh, 103Pb, 105Rh, 109Pd, 111Ag, 111In, 113In, 119Sb, 121Sn, 123I, 125I, 127Cs, 128Ba, 129Cs, 131I, 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169Eu, 177Lu, 186Re , 188Re, 189Re, 191Os, 193Pt, 194Ir, 197Hg, 199Au, 203Pb, 211At, 212Pb, 212Bi and 213Bi. Radioactive markers can be attached to compounds using the conventional known chemistry of antibody images. The radiolabeled compounds are useful in in vitro diagnostic techniques, in vivo radioimaging techniques and in radioimmunotherapy. For example, in the case of in vivo imaging, antibodies and antigen binding fragments can be conjugated to an imaging agent instead of a radioisotope (s), including, but not limited to, an enhanced magnetic resonance imaging agent, where, for example, an antibody molecule is charged with a large number of paramagnetic ions, through chelating groups. Examples of chelating groups include EDTA, poFRirins, crown ethers and polyamines and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanum, holmium and ferbium. Such detectable portions also include: metals; metal chelators; lanthanides; lanthanide chelators; radiometals; radiometal chelators; nucleus emitting positron; microbubbles (for ultrasound); liposomes; microencapsulated molecules in liposomes or nanospheres; monocrystalline iron oxide nanocomposites, contrast agents for magnetic resonance; light absorbing, reflecting and / or dispersing agents; colloidal particles; fluorophores, such as near-infrared fluorophores. In many embodiments, the secondary functionality of the medium will be relatively large, for example, at least 25 amu in size, and in many cases, it may be at least 50, 100 or 250 amu in size. In certain embodiments, the secondary functionality is a chelate portion of a metal chelator, for example, an ion chelator or paramagnetic radiometal. In a certain way, it is a chelator of a radionuclide useful for radiotherapy or imaging procedures.
Antagonists of the invention can also be tested for their ability to modulate angiogenesis in a tissue. Any suitable test known to a specialist can be used to monitor such effects. Several of these techniques are described here.
A measure of angiogenesis is the rabbit eye model in vivo, and is referred to as the rabbit eye test. The rabbit eye test has been described in detail by others, and has still been used to measure both angiogenesis and neovascularization in the presence of angiogenic inhibitors, such as thalidomide. See D'Amato et al. (1994) Proc. Natl.
Acad. Sci. 91: 4082-4085. The rabbit eye test is a well-recognized test model for in vivo angiogenesis, as the neovascularization process, exemplified by growing rabbit blood vessels from the corneal rim to the cornea, is easily visualized through the naturally transparent cornea. eye. In addition, both the extent and amount of stimulus or inhibition of neovascularization or regression of neovascularization can be easily monitored over time.
Finally, the rabbit is exposed to any test reagent, and therefore the health of the rabbit is an indication of the toxicity of the test reagent.
Another test measures angiogenesis in the chimeric mouse: human mouse model and is referred to as the chimeric mouse test. The test has been described in detail by others, and it has also been described here to measure angiogenesis, neovascularization and tumor tissue regression. See Yan, et al. (1993) J. Clin. Invest. 91: 98699 6. The chimeric mouse test is a useful test model for in vivo angiogenesis, because transplanted skin grafts are histologically similar to normal human skin and all tissue neovascularization is taking place in human blood vessels and is growing at from the human skin grafted into the tumor tissue, on the surface of the grafted human skin. The origin of neovascularization in the human graft can be demonstrated by immunohistochemical staining of neovascularization with specific markers of human endothelial cells. The chimeric mouse test shows regression of neovascularization based on both the amount and extent of regression of new vessel growth. In addition, it is easy to monitor the effects on the growth of any transplanted tissue on top of the grafted skin, such as tumor tissue. Finally, the test is useful because there is an internal control for toxicity in the test system. The chimeric mouse is exposed to any test reagent and, therefore, the health of the mouse is an indication of toxicity. Other animal models described herein and known in the art can also be used in the methods described herein. C. Treatment with Humanized Endoglobin Antibodies Methods are provided here for preventing or treating one or more diseases or disorders associated with angiogenesis / neovascularization, excessive vascularization, or dilation of small vessels involving administration of a composition comprising a humanized antibody or binding fragments. of antigens described herein that bind to endoglobin associated with the disease or disorder and prevents angiogenesis, thereby preventing, treating, ameliorating, or decreasing the disease or its severity. Provided herein are methods of preventing or treating one or more diseases or disorders associated with angiogenesis / neovascularization comprising administering a composition involving a humanized antibody or antigen-binding fragment described herein that binds to endoglobin associated with the disease or disorder , decreases angiogenesis or prevents excessive angiogenesis.
As used herein, "prevention" refers to prophylaxis, preventing the onset of symptoms, preventing the progression of a disease or disorder associated with angiogenesis or correlated with endoglobin activity. "Inhibition", "Treatment" and "treatment" are used interchangeably and refer, for example, to stasis of symptoms, prolongation of survival, partial or complete improvement of symptoms, and total or partial eradication of a condition, disease or disorder associated with angiogenesis or correlated with endoglobin activity.
The compositions can be administered to a patient in an effective therapeutic amount, which are effective in producing any desired therapeutic effect from an inhibitor's disease or disorder as described herein, which can be associated with endogline, with a benefit / reasonable risk, applicable to any medical treatment. For the administration of the present compositions to human patients, the compositions can be formulated by a methodology known to a person skilled in the art. A therapeutically effective amount is an amount that at least partially achieves the desired therapeutic or prophylactic effect on an organ or tissue. The amount of a humanized anti-endoglobin antibody or antigen binding fragment thereof necessary to lead to the prevention and / or therapeutic treatment of a disease or disorder is not fixed in itself. The amount of humanized anti-endoglobin antibody or antigen binding fragment administered can vary with the type of disease, extent of the disease, and the size of the mammal suffering from the disease or disorder. In a certain embodiment, two or more humanized anti-endoglobin antibodies described herein are administered in combination to a patient. The combination includes concomitant or subsequent administration of the antibodies. The term "administering" is defined herein as a means of providing the composition to the patient in a manner that results in the composition being within the patient's body. Such administration can be by any route, including, without limitation, locally, regionally or systemically, subcutaneously, intravitreal, intradermal, intravenous, intraarterial, intraperitoneal, or intramuscular (for example, injection). "Concurrent administration" means administration within a relatively short period of time; such a period of time, can be less than 2 weeks, less than 7 days, less than 1 day and could even be administered simultaneously.
The actual dosage levels of the active substances in the compositions can vary in order to obtain the amount of the active ingredient that is effective in achieving the desired therapeutic response for a specific patient, of the composition, and mode of administration, without being toxic to the patient. The dosage level selected will depend on a variety of factors including the activity of the particular compound used, the route of administration, the time of administration, the rate of excretion of the particular compound to be employed, the duration of treatment, other drugs, the compounds and / or materials used in combination with the particular composition employed, age, sex, weight, condition, general health status, previous medical history of the patient to be treated and similar factors well known in meRNAin. The antibodies and binding fragments of the antigen described herein can be administered to a patient in dosage amounts and over several different periods of time. Non-limiting doses include about 0.01mg / kg, about 0.05mg / kg, about 0.1mg / kg, about 0.5mg / kg, about 1mg / kg, about 5mg / kg, about about 10mg / kg, about 20mg / kg, about 30mg / kg, about 40mg / kg, about 50mg / kg, about 60mg / kg, about 70mg / kg, about 80mg / kg, about 90mg / kg, about 100mg / kg, about 125mg / kg, about 150mg / kg, about 175mg / kg, about 200mg / kg, or any integer between the two. In addition, the dose (s) of antibody or antigen binding fragment can be administered twice weekly, weekly, every two weeks, every three weeks, every 4 weeks, every 6 weeks, every 8 weeks , every 12 weeks, or any combination of weeks contained therein. Dosing cycles are also contemplated, such as, for example, administration of antibodies or antigen binding fragment thereof once or twice a week for 4 weeks, followed by two weeks without therapy. Additional administration cycles, including, for example, different combinations of weekly doses and cycles described herein are also contemplated in the invention. "Contacting" is defined herein as a means of carrying a composition as provided herein in physical proximity to a cell, organ, tissue or fluid as described herein. Contacting includes systemic or local administration of any of the compositions provided herein and includes, without limitation, in vitro, in vivo and / or ex vivo procedures and methods. "Combination" and "contacting" are used interchangeably here and are intended to be defined in the same way.
A response is achieved when the patient experiences partial or total relief, or a reduction in signs or symptoms of illness and, specifically, includes, without limitation, prolonged survival. The predicted survival time can be measured in months or years, depending on prognostic factors, including the number of relapses, stage of the disease, and other factors. Extending survival includes, without limitation, times of at least 1 month, about at least 2 months, about at least 3 months, about at least 4 months, about at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years, or more. Global survival can also be measured in months or years. The patient's symptoms may remain static or may subside.
A doctor or veterinarian with knowledge of the art can readily determine and prescribe the effective amount (ED50) of the required composition. For example, the doctor or veterinarian could start with doses of the compounds used in the composition at levels lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a dose can remain constant.
The compositions can be administered to a patient by any convenient route, as described above. Regardless of the route of administration selected, the compounds of the present invention that can be used in an appropriate hydrated form, and / or the compositions, are formulated in acceptable dosage forms as described below or by other conventional methods known to those skilled in the art .
The antibodies can be combined with a therapeutic moiety or a detectable moiety (image) using methods known in the art such as, for example, chemical conjugation, covalent or non-covalent bonds or recombinant techniques to create conjugates or fusion proteins as described in more detail below. Alternatively, antibodies and / or other agents can be combined in separate compositions for simultaneous or sequential administration. The toxicity and therapeutic efficacy of the compound can be determined by standard pharmaceutical procedures in cell cultures or in experiments using animals, for example, for the determination of LD50 (the lethal dose for 50% of the population) and ED50 (the therapeutically effective dose 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. While compounds that exhibit toxic side effects can be used, care must be taken when creating a delivery system that targets such compounds to the affected tissue site in order to minimize potential damage to healthy cells and thereby reduce side effects.
The data obtained from cell culture assays and animal experiments can be used in formulating a dosage range for use in humans. The dosage of these compounds is preferably within a range of circulating concentrations that include ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animals to achieve a circulating plasma concentration that includes the IC 50 (i.e., the concentration of the test compound that achieves semi-maximum inhibition) as determined in cell culture. Plasma levels can be measured, for example, by high performance liquid chromatography. Such information can be used to more accurately determine useful doses in humans. The pro-angiogenic role of endoglobin has been established in several models, including the culture of endothelial cells and knock-out mouse models. The endothelium and associated cells are well known for expressing endoglobin (CD105), as well as the role of endoglobin in angiogenesis in general, as well as cardiac development has also been confirmed in numerous studies, culture models and in animal models. (Duff et al, ESTÁGIOB J., 17: 984-992 (2003); Bernabeu et al, J. Cell Biochem, 102 (6): 1375-1388 (2007); U.S. Patent No. 7,097,836).
Thus, methods that inhibit angiogenesis in a diseased tissue improve the symptoms of the disease and, depending on the disease, can contribute to its cure. In one embodiment, the invention contemplates the inhibition of angiogenesis in a tissue. The extent of angiogenesis in a tissue, and therefore the extent of inhibition carried out by the present methods, can be assessed by a variety of methods, as described herein. The unique specificity of antibodies that recognize (for example, bind) an epitope in the endoglobin, inhibits angiogenesis and provides diagnostic uses and therapeutic uses for diseases characterized by angiogenesis (neovascularization), dilation of small vessels, and / or excessive vascularization such as described here. Humanized anti-endoglobin antibodies and fragments thereof can be administered to an individual such as a mammal (for example, a human), who suffers from a medical disorder, for example, various forms of eye diseases characterized by angiogenesis / neovascularization (for example, macular degeneration, diabetic retinopathy), diabetic nephropathy, chronic inflammatory diseases (eg, DIC), rheumatoid arthritis, osteoarthritis, and various forms of cancer (primary tumors and metastases). This document contains a method for the treatment of an individual who has an eye disease characterized by angiogenesis by the administration of a humanized antibody or its fragment that binds to endoglobin and inhibits angiogenesis. Also provided here is a method for treating an individual who has a chronic inflammatory disease by administering a humanized antibody or its fragment described here, which binds to endogline and inhibits angiogenesis. Examples of such chronic inflammatory diseases include, but are not limited to, Crohn's disease and ulcerative colitis. In addition, a method is presently provided for the treatment of an individual who has diabetic nephropathy by administering a humanized antibody or fragment thereof described herein. Here is provided a method for treating a subject who has rheumatoid arthritis or osteoarthritis by administering a humanized antibody or fragment thereof described herein.
It can be understood that anti-endoglobin antibodies can be effective for the treatment of angiogenesis, this is contemplated in this document in which an individual can also be treated with one or more additional angiogenesis inhibitors. The term "angiogenesis inhibitor" is used here, for the purpose of specification and supports, to mean a compound or molecule including, but not limited to, peptides, proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, recombinant vectors, and drugs whose function is to inhibit angiogenesis. Angiogenesis inhibitors are known in the art and all types are contemplated here. Non-limiting examples of compounds and molecules include natural and synthetic biomolecules such as paclitaxel, O- (chloroacetyl-carbomyl) fumagilol ("TNP-470" or "AGM 1470"), thrombospondin-1, thrombospondin-2, angiostatin, human chondrocytes of angiogenesis inhibitor ("hCHIAMP"), the cartilage derived from the angiogenic inhibitor, platelet factor-4, gro-beta, the human inducible protein Interferon 10 ("IP10"), interleukin 12, Ro 318.220, tricyclodecan-9-il xanthate ("D609"), irsogladine, 8,9-dihydroxy-7-methyl-benzo [b] quinolizine bromide ("GPA 1734"), medroxyprogesterone, a combination of heparin and cortisone, glucosidase inhibitors, genistein, thalidomide, diaminoanthraquinone, herbimycin, ursolic acid and oleanolic acid. Non-limiting examples of antibodies specifically include molecules such as VEGF, VEGF receptor, or epitopes other than endogline. In addition, small molecular VEGF receptor inhibitors are known and contemplated herein. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (Lucentis), aflibercept (VEGF-Trap), sunitinib (Sutent), sorafenib (Nexavar), axitinib, pegaptanib and pazopanib.
The antibodies and antigen binding fragments described herein can be administered in combination with VEGF receptor inhibitors for the combination therapy of any conditions related to angiogenesis or diseases described herein. In a certain non-limiting embodiment, the VEGF receptor inhibitor is bevacizumab. The exemplary dosage for bevacizumab is about 7.5, about 10 or about 15 mg / kg, administered every 2 or 3 weeks.
In a certain non-limiting embodiment, the VEGF receptor inhibitor is ranibizumab. Eye dosages for ranibizumab include about 0.5 mg, administered intravitreally monthly. In a certain non-limiting modality, the VEGF receptor inhibitor is aflibercept (VEGF-Trap). Dosages for VEGF-Trap include about 0.5 to about 10 mg / kg administered every 2 or 3 weeks. Exemplary eye dosages for VEGF-Trap include about 0.5 to about 2.0 mg administered directly into the eye monthly or quarterly.
In another non-limiting embodiment, the VEGF receptor inhibitor is sunitinib. Exemplary regimens for sunitinib include about 50 mg administered over 4 weeks, followed by 2 weeks of no drugs. Therapeutic regimens can be repeated on a cyclic or acyclic basis.
In another non-limiting embodiment, the VEGF receptor inhibitor is sorafenib. Exemplary dosages for sorafenib include about 400 mg administered daily.
In another non-limiting modality of the VEGF receptor inhibitor is axitinib. Exemplary dosages for axitinib include about 3, 5 or 10 mg administered twice daily.
In another form of non-limiting mobility of the VEGF receptor inhibitor is pegaptanib. Exemplary dosages for pegaptanib include about 0.3 to 3mg administered directly to the eye every 6 weeks.
In yet another non-limiting modality, the VEGF receptor inhibitor is pazopanib. Exemplary dosages for pazopanib include about 200 to 1000 mg administered daily. Multiple combinations of these VEGF receptor inhibitors can be administered with antibodies and antigen binding fragments, described herein. In one embodiment, the combinations may result in the use of lower doses for the antibodies or antigen binding fragments described herein, VEGF receptor inhibitors, or both. Such change in dosage may result from synergistic effects with the combination of the antibodies and antigen binding fragments described herein, with VEGF receptor inhibitors.
EYE CONDITIONS INVOLVING ANGIOGENESIS
CONDITIONS OF MACULAR EYE DEGENERATION AND RETINOPATHY
DIABETIC
In one aspect, the present invention provides a method for treating diabetic retinopathy, macular degeneration, choroidal neovascularization or neovascular glaucoma in a patient, by administering a therapeutically effective amount of any of the compositions provided herein. Endoglobin is a receptor associated with angiogenesis and the extracellular matrix. Macular degeneration (DM) is the loss of photoreceptors in the portion of the central retina, called the macula, responsible for high vision acuity. Macular degeneration is associated with abnormal deposition of extracellular matrix components and other residues on the membrane between the retinal pigment epithelium and the vascular choroid. This residual material is called druse. The drusen is observed with an ophthalmic examination of the fundus. Normal eyes may have druse-free macules, although druse may be abundant on the periphery of the retina. The presence of soft druses in the macula, in the absence of any loss of macular vision, is considered an early stage of AMD. Macular degeneration is characterized by choroidal neovascularization (NVC), the development of abnormal blood vessels under the pigmented layer of the retinal epithelium (RPE). These vessels break the Bruch's membrane, interrupting the pigmented epithelium of the retina, hemorrhage and eventually cause macular scars - which results in profound loss of central vision (disciform scar). Choroidal neovascularization (NVC) usually occurs in macular degeneration, in addition to other eye disorders and is associated with the proliferation of choroidal endothelial cells, the overproduction of extracellular matrix and the formation of a sub-retinal fibrovascular membrane. The proliferation of cells in the retinal pigmented epithelium and the production of angiogenic factors appears to effect choroidal neovascularization. Diabetic retinopathy (RD) is an eye disease characterized by excessive angiogenesis that develops in patients with diabetes due to thickening of the basal capillary membranes and lack of contact between pericytes and the capillary endothelial cells. The loss of pericytes increases the leakage of capillaries and leads to the rupture of the blood-retina barrier. Diabetic retinopathy is the result of microvascular changes in the retina. The hyperglycemia induced by the death of pericytes and the thickening of the basement membrane leads to incompetence of the vascular walls. These damages alter the formation of the blood-retina barrier and also make the blood vessels in the retina become more permeable. Small blood vessels, such as those in the eye, are especially vulnerable to control of low blood sugar (blood glucose). An excessive accumulation of glucose and / or fructose damage to the tiny blood vessels in the retina. Macular edema can also develop when damaged blood vessels leak fluids and lipids into the macula. These fluids cause the macula to swell, which blurs the vision. This damage also results in a lack of oxygen in the retina. As the disease progresses, the lack of oxygen in the retina stimulates angiogenesis throughout the retina and in the free space, a vitreous humor similar to the gel that fills the inside of the eye. Without proper treatment, these new blood vessels can bleed, cloud vision and destroy the retina. Fibrovascular proliferation can also cause detachment of traction retina. The new blood vessels can also grow to the angle of the anterior chamber of the eye and cause neovascular glaucoma.
Proliferative vitreoretinopathy is associated with cell proliferation of cell and fibrotic membranes within the vitreous membranes and on the surfaces of the retina. The proliferation of pigment cells in the retinal epithelium and migration is common with this eye disorder. The membranes associated with proliferative vitreoretinopathy contain components of the extracellular matrix, such as types of collagen I, II and IV and fibronectin and progressively become fibrotic. Age-related macular degeneration (AMD) and diabetic retinopathy are the two main causes of blindness in developed countries. The recent approval of the LUCENTIS®, AVASTIN®, and MACUGEN® macromolecules has improved the treatment options available to patients with AMD. LUCENTIS® is a Fab and AVASTIN® is a monoclonal antibody. They bind to vascular endothelial growth factor (VEGF) and have shown the most impressive results to date when treating AMD, however, only a minority of treated patients experience a significant improvement in visual acuity. Anti-angiogenic therapy focused on a target other than VEGF may overcome some of the limitations associated with agents that target the VEGF pathway.
Humanized antibodies and antigen-binding fragments that bind to endoglobin can be used to treat or prevent macular degeneration, NVC, diabetic retinopathy, or proliferative vitreoretinopathy. Here are described methods of treating or preventing macular degeneration, CNV, diabetic retinopathy, or proliferative vitreoretinopathy by administering antibodies and antigen binding fragments. Humanized antibodies and antigen-binding fragments that bind to endoglobin can also narrow blood vessels, inhibit proliferation of endothelial cells associated with eye disease, evident bleeding symptoms, treat blurred vision, provide stasis of vision loss, and / or prevent loss of blood vessels. Humanized antibodies and antigen binding fragments can also be used in medications for the treatment of macular degeneration, NVC, diabetic retinopathy or proliferative vitreoretinopathy.
In addition, the humanized antibodies and antigen binding fragments described herein can also be used in combination with known therapies and / or compounds for the treatment of macular degeneration, CNV, diabetic retinopathy or proliferative vitreoretinopathy. Examples of such compounds include, but are not limited to, bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept retinopathy (VEGF-Trap), sunitinib (SUTENT®), sorafenib compounds for the treatment of macular degeneration, CNV, diabetic or vitreoretinal proliferation. Examples of such compounds include, but are not limited to, bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib, pazopanib or MACUGENIB ®. In addition to the modes of administration described herein, humanized anti-endoglobin antibodies and antigen binding fragments can be administered intravitreally. Non-limiting examples of modes of administration include intravitreal injection and the use of intravitreal implants.
Patients can be assessed for improvement and responsiveness to treatment. Treatment includes, but is not limited to, decreased macular edema, decreased NVC areas and increased visual acuity. Symptom measurements are as known in the art and are further described in the examples below.
CHRONIC INFLAMMATORY DISEASES
Any of a variety of tissues or organs composed of organized tissues can withstand angiogenesis in disease conditions, including, skin, muscle intestinal tissue, connective tissue, joints, bones and the tissue into which blood vessels can invade and provide angiogenic stimuli . Thus, in a certain modality, a tissue to be treated is an inflamed tissue and the angiogenesis to be inhibited is the angiogenesis of the inflamed tissue, where there is neovascularization of the inflamed tissue.
INFLAMMATORY ENTEROPATHIES Angiogenesis plays an important role in inflammatory bowel disease (IHD). DIC is a general term for intestinal joint and intestinal diseases or conditions including Crohn's disease and ulcerative colitis. Crohn's disease is typically characterized by inflammation of the small and large intestine, while ulcerative colitis is usually located in the colon. Abnormal or pathological angiogenesis is central to both Crohn's disease and ulcerative colitis. Both diseases involve increased density and microvascular dysfunction and this angiogenesis is temporally related to the tissue of the pathology and the inflammatory cycles found in both diseases. Endoglobin is known to be expressed in these tissues and to play a role in the dysregulation of angiogenesis during IHD. (Chidlow et al., Am. J. Physiol. Gastrointest. Physiol Liver., 293: 5-18 (2007)).
The humanized antibodies and antigen binding fragments that bind to endoglobin and are described herein can be used to treat IHD. In addition, humanized antibodies and antigen-binding fragments that bind to endoglobin can be used to treat Crohn's disease and ulcerative colitis. Humanized anti-endoglobin antibodies and antigen-binding fragments can also be used in combination with surgery and / or known therapies for IHD, Crohn's disease or ulcerative colitis. Examples of such known therapies include, but are not limited to, aminosalicylates (eg, Mesalamine), corticosteroids (eg, budesonide, prednisone, etc.), antibiotics (eg, metronidizole, etc.), immunosuppressive drugs (eg, azathioprine , 6-mercaptopurine, methotrexate, tacrolimus and cyclosporine, etc.), and biological drugs such as proteins and antibodies (eg, infliximab, etc.) The treatment of DIC can be assessed by the decreased vascularization of inflamed tissue. Treatment can also be assessed by stasis, resolution and / or healing of ulcerative lesions that characterize IHD.
DIABETIC NEPHROPATHY AND KIDNEY TRANSPLANT ISCHEMIA Diabetic nephropathy is one of the main causes of morbidity and mortality in both types of diabetics: 1 and 2. It is the main cause of end-stage renal disease worldwide. Diabetic nephropathy is characterized by microvascular glomerular injury due to increased synthesis of pro-angiogenic factors. These pro-angiogenic factors cause the proliferation of endothelial cells and increase angiogenesis, and endoglobin is known to be regulated in chronic kidney disease. This angiogenesis results in the destruction of the glomeruli and, finally, renal failure. (Zent et al, Seminars in Nephrology, 27 (2): 161-171 (2007); Roy-Chaudhury et al, Exp Nephrol, 5: 55-60 (1997)).
Similar effects are seen in kidney transplantation resulting in ischemia and failure of the transplanted organ. The positive regulation induced by endoglobin results in angiogenesis and inflammation in the kidney. On the other hand, studies with endoglobin in mice show significantly reduced kidney damage after transplantation / ischemia and increased organ survival. (Docherty et al., Nephol. Dial. Transplant., 21: 2106-2119 (2006)).
The humanized antibodies and antigen binding fragments that bind to endogline and are described herein can be used to treat or prevent diabetic nephropathy, kidney failure after transplantation, and / or ischemic kidney injury after transplantation.
Described below are the methods of treatment or prevention of diabetic nephropathy, kidney failure after transplantation, and / or ischemic kidney injury after transplantation through the administration of antibodies and antigen binding fragments, described here. Humanized antibodies and antigen binding fragments, described herein, can also be used in medications for the treatment of diabetic nephropathy, kidney failure after transplantation, and / or ischemic kidney injury after transplantation. In addition, the humanized antibodies and antigen binding fragments described herein can also be used in combination with known therapies and / or compounds for the treatment of diabetic nephropathy, kidney failure after transplantation, and / or ischemic kidney injury after transplantation.
Patients can be assessed for treatment effectiveness, for example, improvement of kidney function.
Rheumatoid arthritis and osteoarthritis Rheumatoid arthritis is characterized by excessive angiogenesis, and is well understood in this regard. The inflammation and destruction found in synovial fluids are directly related to angiogenesis and the increase in the surrounding synovial tissues found. Numerous pro-angiogenic factors are present in the affected tissues of patients with rheumatoid arthritis. (Koch and Distler, Arthritis & Ther Res, 9 (suppl. 2): S3, 1-9 (2007). Osteoarthritis is a group of chronic disabling conditions that affects synovial joints. Angiogenesis and inflammation are integral processes in pathophysiology that contribute to joint damage through a variety of mechanisms, including, but not limited to, stimulation of MMP production and endochondral ossification. In addition, angiogenesis in osteoarthritis induces complementary innervation, which develops in a circuit feedback where each one continues to stimulate the other (Bonnet & Walsh, Rheumatology, 44: 7-16 (2005)).
The humanized antibodies and antigen binding fragments that bind to endoglobin and are described herein can be used to treat or prevent rheumatoid arthritis and osteoarthritis. Described below are methods of treating or preventing rheumatoid arthritis and osteoarthritis by administering the antibodies and antigen binding fragments described herein. Humanized antibodies and antigen binding fragments, described herein, can also be used in medications for the treatment of rheumatoid arthritis and osteoarthritis.
Two composite measures of RA improvement well accepted in studies are: the Paulus Criteria and the American College of Rheumatology Criteria (ACR). The Paulus criteria is defined as improvement in 4 of the following: tender and number of swollen joints, morning stiffness, patient's assessment of disease activity, medical assessment of disease activity and erythrocyte sedimentation rate (SE) rate . The level of improvement is defined as a percentage improvement in each of these variables, that is, a Paulus 20 indicates a response rating with a 20% improvement in 4 of the 6 parameters. Rheumatoid arthritis can also be assessed through the American College of Rheumatology Scoring (ACR). Briefly, ACR classification criteria for determining clinical remission in rheumatoid arthritis is assessed by the presence of 5 or more of the following factors present for at least two consecutive months: a. morning stiffness <15 minutes; B. Without fatigue; ç. No joint pain; d. No joint sensitivity or pain on movement; and. Non-soft tissue in the joints or tendon sheaths and f. SE (Westergren method) <30mm / hour for females or 20mm / hour for males.
Exclusions may occur and include: the clinical manifestations of active vasculitis, pleuritis pericarditis, or myositis, and recent unexplained weight loss or fever attributable to rheumatoid arthritis will prohibit the designation of complete clinical remission (Pinals RS, et.al .: Rheumatpide 24: 1308,1981). In addition, ACR classification criteria for functional status in Rheumatoid Arthritis includes classification based on the skills of patients: Class I: Completely capable of performing usual activities of daily living (self-care, professional and free activities);
Class II: Able to perform usual self-care and professional activities, but limited to free activities;
Class III: Able to perform habitual self-care activities, but limited in vocational activities and free activities and Class IV: limited capacity to perform usual self-care, professional, and free activities. Osteoarthritis can also be assessed using an ACR score. Clinical criteria for ACR classifications for hip osteoarthritis is assessed using the patient's history, physical examination and long-term labocamundral findings: the patient is assessed for hip pain and one of the following options: (1) the inner hip pathway, less 15 degrees and SE less than or equal to 45 degrees mm / hour or hip flexion less than or equal to 115 degrees if SE is not available; or (2) via the inner hip, less than 15 degrees, pain associated with the inner hip, morning hip stiffness for less than or equal to 60 minutes, and the patient is over 50 years of age.
Using history, physical examination, laborological and radiographic findings, traditional format is hip pain and two of the following indications: IF less than 20 mm / hour, femoral and / or acetabular radiographic osteophytes, or narrowing of the radiographic joint space (upper, axial, and / or medial). A classification tree is as follows: hip pain, in association with (1) radiographic femoral and / or acetabular osteophytes or (2) SE less than or equal to 20 mm / hour and narrowing of the common axial radiographic space (Altman, R, et al: Rheumatoid Arthritis 34: 505, 1991).
CLINICAL ACR CLASSIFICATION CRITERIA FOR KNEE OSTEOARTHRITIS
Clinical ACR classification criteria for knee osteoarthritis is assessed through history and physical examination, using the following criteria: knee pain in connection with three (3) of the following: (1) a patient over 50 years of age; (2) less than 30 minutes of morning stiffness; (3) Crackling in active movement; (4) bone tenderness; (5) bone widening, and (6) the non-palpable heat of the synovial membrane.
Using the patient's history, physical and radiographic examination, knee pain can be assessed in relation to one of the following patient characteristics: (1) the patient is over 50 years of age; (2) less than 30 minutes of morning stiffness; and (3) Crackling in active movement and osteophytes. Using history, physical and laboratory examination: knee pain can be assessed in connection with five (5) of the following characteristics: (1) a patient over 50 years of age; (2) less than 30 minutes of morning stiffness; (3) Crackling in active movement; (4) bone tenderness; (5) bone enlargement; (6) Non-palpable heating of the synovial membrane; (7) FHS is less than 40 mm / hour; (8) Rheumatoid factor (RF) of less than 1:40, and (9) Signs of osteoarthritis in synovial fluid (LS).
See, for example, Altman, Ret al :. Rheumatoid Arthritis 29: 1039, 1986.
Clinical ACR classification criteria for osteoarthritis of the hand can be assessed by the following: distress, pain or stiffness in the hand in connection with three (3) of the following: (1) the enlargement of the hard tissue of two or more of the following joints (second and third distal interphalangeal), second and third proximal interphalangeal, and the first carpal joints of both hands, (2) the enlargement of the hard tissue of two or more distal interphalangeal joints; (3) less than three MCP swollen joints and (4) deformity of at least one of the joints listed above in (1).
CD105 CANCER is associated with tumor angiogenesis and is strongly upregulated in the endothelium of the various tumor tissues compared to normal tissues. CD105 is over-regulated in a wide range of tumor endothelium, including, for example, the colon, breast, brain, lung, prostate, endometrium, kidney, liver, stomach, head and neck and cervical cancer. In addition, it is known that there is no stronger expression of CD105 in the corresponding endothelial tumor in normal tissues. (Duff et al, ESTÁGIOB J., 17: 984-992 (2003); Bernabeu et al, J. Cell Biochem, 102 (6): 1375-1388 (2007); U.S. Patent No. 7,097,836). Thus, inhibition of angiogenesis with humanized anti-endoglobin antibodies represents the treatment option for cancerous tumors. The humanized antibodies and antigen-binding fragments that bind to endoglobin and are described herein can be used to treat cancerous tumors. Humanized antibodies and antigen binding fragments, described herein, can also be used in medications for the treatment of cancerous tumors. The term "tumor" is used here to refer to a cancerous tissue expressing endoglobin (compared to expression by normal tissue of the same type). Tumors can include solid and semi-solid tumors. Non-limiting examples of tumors include human leukemias, including the non-T cell type of acute lymphoblastic leukemia (ALL), myelomonocytic leukemia; and solid human and semi-solid tumors, with their surrounding vasculature expressing moderate to high levels of endoglobin (compared to expression by normal tissue of the same type), including angiosarcoma, breast carcinoma, stomach cancer, colon carcinoma , Hodgkin's lymphoma, lymphomas, glioblastoma multiforme (GBM), lung carcinoma, melanoma, myeloma, lymphoma, osteosarcoma, ovarian carcinoma, parotid tumor, pharyngeal carcinoma, prostate carcinoma, hepatocellular carcinoma, renal carcinoma, renal carcinoma, carcinoma .
A cancerous tissue to be treated consists, for example, of an endothelial tissue expressing an abnormal level of endoglobin.
In the absence of neovascularization of the tumor tissue, the tumor tissue does not obtain the necessary nutrients, retarding growth, ceasing further growth, regressing and, finally, becoming necrotic resulting in tumor death. The present invention provides a method of inhibiting tumor neovascularization by inhibiting angiogenesis according to the present methods. Likewise, the invention provides a method of inhibiting tumor growth by practicing methods of inhibiting angiogenesis.
The methods are also particularly effective against the formation of metastases because their formation requires the vascularization of a primary tumor so that metastatic cancer cells can leave the primary tumor and attach to a secondary site requiring neovascularization to support the growth of metastases. It should be noted that a "cancer / metastasis patient" can express a mutant protein (tumor-associated antigen) or a mutant gene and is not yet symptomatic of the disease. In a non-limiting example, where cancer is colon cancer (which is associated with the mutant K-ras protein), a patient with a mutant K-ras protein in some colon cells is a patient according to the invention. , although the patient may not yet be symptomatic for colon cancer. "Signs or symptoms of disease" are clinically recognitions, manifestations or indications of the disease.
By "treatment" it is understood that the symptoms of a cancer patient are partially or totally relieved, or remain under static treatment according to the invention. A patient who has been treated may experience partial or total decrease in symptoms and / or tumor burden. This is intended to encompass therapy, prophylaxis and healing. In a non-limiting example, a patient suffering from highly metastatic cancer (eg, breast cancer) is treated in which additional metastasis either does not occur, or is few in number, compared to a patient who has not received treatment. In another non-limiting example, a patient is treated in which the patient's solid cancer has either reduced in size or has not increased in size, compared to a patient who does not receive treatment. In yet another non-limiting example, the number of cancer cells in a treated patient does not increase or decrease compared to the number of cancer cells in a patient who does not receive treatment. The improvement can also be defined, for example, as the decrease in cell proliferation, the decrease in the number of cells, the increase in apoptosis, and / or greater survival of the patient to be treated.
As used herein, cancer treatment includes stasis, the total or partial elimination of cancerous growth or tumor. Treatment or partial elimination includes, for example, a reduction in the growth rate or tumor size and / or volume such as about 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, or any speed reduction between them. Likewise, treatment or partial elimination may include a percentage reduction in tumor growth or size and / or volume of about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30% , 40%, 50%, 60%, 70%, 80%, 90%, 95% or any percentage reduction in between.
A tumor or cancer to be treated by the methods described here includes, but is not limited to, lung cancer, malignant gynecological disease, melanoma, breast cancer, brain cancer (eg, glioblastoma multiforme, "GBM "), pancreatic cancer, ovarian cancer, uterine cancer, colorectal cancer, prostate cancer, kidney cancer, liver cancer (hepatocellular carcinoma), uterine cancer, neck cancer, kidney cancer (renal cell carcinoma), sarcoma, myeloma, and lymphoma. In one embodiment, a tumor to be treated is a solid or semi-solid tumor. In another embodiment, a tumor to be treated is a primary tumor. In another embodiment, a tumor to be treated is a metastatic tumor. In another modality, a tumor or cancer to be treated is of epithelial origin. In another modality, the cancer to be treated consists of myeloma. In another modality, the cancer to be treated is ovarian cancer. In another embodiment, the cancer to be treated consists of kidney / kidney cancer. In yet another modality, the cancer to be treated consists of hepatocellular / liver cancer.
LUNG CANCER
In one aspect, a method for treating lung cancer is provided in this document. The most common type of lung cancer is non-small cell lung cancer (NSCLC), which represents about 80 to 85% of lung cancers and is divided into squamous cell carcinomas, adenocarcinomas and large undifferentiated cell carcinomas. Small cell lung cancer accounts for 15 to 20% of lung cancers.
Lung cancer staging is an assessment of the degree of spread of cancer from its original source. It is an important factor that affects the potential prognosis of lung cancer treatment. Carcinoma in the non-small cells of the lung is seen from AI ("one A", better prognosis) to IV ("four"; worse prognosis). Small cell carcinoma of the lung is classified as the limited stage if it is confined to one half of the breast and within the scope of a single radiotherapy field, otherwise it is an extensive stage. Non-small cells of the lung can be seen with USE (endoscopic ultrasound) or computed tomography or magnetic resonance imaging or surgery to classify the extent of the disease according to the TNM system. These individuals suffer from stopping as part of the process of considering prognosis and treatment. AJCC recommends TNM staging followed by additional grouping. The primary tumor (T): TX: The primary tumor cannot be evaluated, or if there are malignant cells in the sputum or bronchoalveolar lavage, but not seen in the image or bronchoscopy; Tis: carcinoma in situ. T0: There is no evidence of a primary tumor. T1: Tumor smaller than 3 cm in its largest dimension, surrounded by lung or visceral pleura and without bronchoscopic invasion in the main bronchus. T2: A tumor with any one of: more than 3 cm in its largest dimension; extending to the main bronchus (but more than 2 cm from the carina) and obstructive pneumonitis (but not involving the entire lung). T3: A tumor with any of: invasion of the chest wall, diaphragm, mediastinal pleura or parietal pericardium; extending to the main bronchus, within 2 cm of the carina, but not involving the carina and obstructive pneumonitis of the entire lung. T4: A tumor with any of: invasion of the mediastinum, heart, great vessels, trachea, esophagus, vertebrae, or carina; separate tumor nodules in the same lobe and malignant pleural effusion. Lymph nodes (N): NX: Lymph nodes cannot be evaluated; N0: No lymph nodes involved; N1: metastases to ipsilateral peribronchial or ipsilateral hilar lymph nodes; N2: Metastases to ipsilateral or subcarinal mediastinal lymph nodes, and N3: metastasis to any of: ipsilateral supraclavicular lymph nodes; scalps ipsilateral lymph nodes, and contralateral lymph nodes. Distant metastases (M): MX: distant metastasis cannot be assessed; M0: Absence of distant metastasis, and M1: distant metastasis is present.
UTERUS CANCER / GYNECOLOGICAL MALIGNICITY Uterine cancer can refer to any of several different types of cancer that occur in the uterus, especially: uterine sarcomas (for example, myometrial sarcomas, or muscle layer of the uterus, are most commonly leiomyosarcomas); endometrium; and cervical cancer.
In another aspect, a method for treating endometrial cancer is provided here. Endometrial cancer is cancer that begins in the endometrium, the inner lining of the uterus. Some examples of cancer of the uterus and endometrium include, but are not limited to, adenocarcinomas, adenoacanthomas, adenosquamous carcinomas, serous papillary adenocarcinomas, clear cell adenocarcinomas, uterine sarcomas, stromal sarcomas, mixed malignant mesodermal tumors and leiomyosarcomas.
In another aspect, the method treats cervical cancer, preferably an adenocarcinoma in the epithelial cervix. There are two main types of this cancer: squamous cell carcinoma and adenocarcinomas. The first constitutes about 80 to 90% of all cervical cancers and develops where the ectocervix (portion closest to the vagina) and the endocervix (portion closest to the uterus) come together. The latter develops in the mucus-producing cells of the endocervical glands. Some types of cervical cancer have characteristics of both and are called adenosquamous carcinomas or mixed carcinomas.
OVARY CANCER
In another aspect, this document provides a method of treating ovarian cancer, including epithelial ovarian tumors. Ovarian cancer is classified according to the histology of the tumor, obtained in a pathology report. The surface of the epithelial-stromal tumor, also known as ovarian epithelial carcinoma, is the most common type of ovarian cancer. It includes serous tumor, endometrioid tumor and mucinous cystadenocarcinoma. Tumor of the stromal cord, including production of estrogen by the tumor of granular cells and virilizing tumor of Sertoli-Leydig cells or arrhenoblastoma, is responsible for 8% of ovarian cancers. Germ cell tumor reports in approximately 30% of ovarian tumors, but only 5% of ovarian cancers due to the majority of germ cell tumors are teratomas and most teratomas are benign tumors. Germ cell tumors tend to occur in young women and girls. The prognosis depends on the specific histology of the germ cell tumor, but in general it is favorable. Mixed tumors contain elements from more than one of the histological classes of the tumor above. Ovarian cancer can also be a secondary cancer, resulting from the metastasis of a primary cancer in other parts of the body. Common primary cancers are: breast cancer and gastrointestinal cancer (in which case ovarian cancer is Krukenberg cancer). The tumor of the epithelial-stromal surface may originate in the peritoneum (the lining of the abdominal cavity), in this case ovarian cancer is secondary to primary peritoneal cancer, but the treatment is basically the same as for the surface of the primary epithelial tumor- stromal surrounding the peritoneum.
Staging of ovarian cancer is through the FIGO staging system and uses information obtained after surgery, which may include total abdominal hysterectomy, removal of both ovaries and fallopian tubes, omentum and pelvic (peritoneal) washes for cytology. The AJCC stage is the same as the FIGO stage.
Stage I refers to ovarian cancer limited to one or both ovaries: IA- involves an ovary; intact capsule, without tumor on the ovarian surface, without malignant cells in ascites or peritoneal lavages; IB- involve both ovaries; intact capsule; no tumor on the ovarian surface; negative washes; and IC- tumor limited to the ovaries with any of the following: ruptured capsule, tumor on the surface of the ovary, positive washes.
Stage II refers to the pelvic extension or implants: IIA- extension or implants in the wall of the uterus or fallopian tube; negative washes; IIB- extension or implants for other pelvic structures; negative washes; IIC-pelvic extension or implants with positive peritoneal lavage.
Stage III refers to microscopic peritoneal implants outside the pelvis, or limited to the pelvis with extension to the small intestine or omentum: IIIA - microscopic peritoneal metastases outside the pelvis; IIIB - macroscopic peritoneal metastases in addition to the pelvis less than 2 cm in size, and IIIC - peritoneal metastases outside the pelvis of 2 cm or lymph node metastases.
Stage IV refers to remote metastases to the liver or outside the peritoneal cavity.
Metastases to para-aortic lymph nodes are considered regional lymph nodes (Stage IIIC).
In some embodiments, the methods described here treat ovarian cancer selected from the following: an adenocarcinoma of the ovary or an adenocarcinoma that has migrated from the ovary into the abdominal cavity.
MELANOMA
A melanoma is a malignant tumor of melanocytes that are found predominantly in the skin as well as in the intestine and eyes (uveal melanoma). It is one of the rarest types of skin cancer, but causes most deaths from related skin cancer. Malignant melanoma is a serious type of skin cancer caused by the uncontrolled growth of pigment cells, called melanocytes. Melanomas also include, but are not limited to, a choroidal melanoma, malignant melanomas, melanomas and intraocular melanomas.
Melanoma can be divided into the following types: malignant lentigo, malignant melanoma lentigo, superficially spreading melanoma, acrolentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma and uveal melanoma. Melanoma stages are as follows: Stage 0 - melanoma in situ (Clark I level).
Stage I / II - invasive melanoma: T1a: primary less than 1.00 mm, without ulceration, Clark II-III level; T1b: primary less than 1.00 mm, with ulceration or Clark IV-V level, and T2a: primary from 1.00 to 2.00 mm, without ulceration.
Stage II - High Risk Melanoma: T2b: primary from 1.00 to 2.00 mm, with ulceration; T3a: primer from 2.00 to 4.00 mm, without ulceration; T3b: primary from 2.00 to 4.00mm, with ulceration; T4a: primary of 4.00 mm or more without ulceration; T4b: primary of 4.00 mm or more with ulceration.
Stage III - Regional metastasis: N1: single positive lymph node; N2: 2 to 3 positive lymph nodes or regional / transit skin metastases and N3: 4 positive lymph nodes or regional lymph node and skin / transit metastases.
Stage IV - Distant metastasis: M1a: distant skin metastasis, normal LDH; M1a: Lung metastasis, normal LDH and M1c: metastases at other distances or metastases at any distance with high LDH.
In one embodiment, the methods described here deal with a melanoma.
COLON CANCER AND COLORECTAL CANCER Colorectal cancer (colon cancer or also called cancer of the large intestine) includes cancerous tumors in the colon, rectum (anus) and appendix. With 655,000 deaths worldwide each year, it is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. Many colorectal cancers have arisen from adenomatous polyps in the colon. These mushroom growths are generally benign, but some can turn into cancer over time.
In another embodiment, the Dukes classification can be used to classify colorectal cancer based on stages A-D. Stage A refers to colorectal cancer that is limited to the mucosa (that is, it has not invaded through the intestinal wall). Stage B1 refers to the extension of the musculature itself, but not penetrating through it (that is, the lymph nodes were not invaded), while in stage B2, the cancer penetrating into the musculature itself, but not penetrating through it (that is, the lymph nodes have not been invaded). Stage C1 refers to cancer that extends to the musculature itself, but does not penetrate through it (that is, the lymph nodes are involved), stage C2 refers to cancer that extends to the musculature itself and penetrates through it (or that is, lymph nodes are involved). Stage D refers to remote metastatic spread. The TNM system can also be used for the stage of colorectal cancer according to conventional means known in the art.
BREAST CANCER
There are several types of breast cancer that can be treated by the methods described here. Lobular carcinoma in situ and dutal carcinoma in situ are breast cancers that developed in the lobes and ducts, respectively, but have not spread to the adipose tissue around the breast or other areas of the body. Lobular and ductal carcinoma are cancers that have developed in the lobes and ducts, respectively, and have spread to any adipose tissue in the breast and / or other parts of the body. In one aspect, a method of treating breast cancer is contained in this document, such as a ductal carcinoma in the tissue duct in a mammary gland, a breast cancer that is Her2- and / or ER- and / or PR-. Other breast cancers that would benefit from treatment using the methods are medullary carcinomas, colloid carcinomas, tubular carcinomas and inflammatory breast cancer.
In one embodiment, breast cancer is tested according to the TNM system. The prognosis is closely linked to the test results, the internship is also used to allocate patients for treatments in both clinical studies and clinical practice.
Briefly, the test information is as follows: TX: primary tumor cannot be evaluated. T0: There is no evidence of tumor. Tis: carcinoma in situ, without invasion; T1: Tumor is 2 cm or less; T2: Tumor is more than 2 cm, but not more than 5 cm; T3: Tumor is more than 5 cm; T4: Tumor of any increasing size in the chest wall or skin, or inflammatory breast cancer. NX: Regional lymph nodes cannot be assessed N0: cancer has not spread to regional lymph nodes. N1: cancer has spread to 1 to 3 of the jaw or an internal mammary lymph node N2: cancer has spread from 4 to 9 maxillary lymph nodes or multiple internal mammary lymph nodes N3: one of the following situations: cancer has spread to 10 or more lymph nodes of the jaw, or cancer has spread to the lymph nodes under the clavicle (clavicle), or the cancer has spread to the lymph nodes above the clavicle, or cancer surrounds the lymph nodes and enlarged the internal mammary lymph nodes, or cancer involves 4 or more lymph nodes of the jaw, and minute amounts of cancer are found in the internal breast lymph nodes in the sentinel lymph node biopsy. MX: presence of remote spread (metastasis) cannot be assessed. M0: without remote propagation. M1: spreading to distant organs (not including the supraclavicular lymph node) occurred.
CANCER CANCER
Another aspect that is contained here is the method of treating pancreatic cancer selected from the following: an epithelial carcinoma in the pancreatic tissue duct and an adenocarcinoma in a pancreatic duct. The most common type of pancreatic cancer is an adenocarcinoma, which occurs in the lining of the pancreatic canal.
In one embodiment, the methods described here treat pancreatic cancer.
PROSTATE CANCER
In another aspect, a method for treating prostate cancer selected from the following is provided here: an adenocarcinoma or an adenocarcinoma that has migrated to the bone. Prostate cancer develops in the prostate organ of men, which surrounds the first part of the urethra. The prostate has several types of cells, but 99% of tumors are adenocarcinomas that develop in the gland cells responsible for generating seminal fluid.
There are two schemes commonly used in the stage of prostate cancer. The most common is the TNM system, which assesses the size of the tumor, the extent of the lymph nodes involved, and any metastasis (remote spread). As with many other cancers, these are often grouped into four stages (I-IV).
Another scheme, used less frequently, is the Whitmore-Jewett stage.
Briefly, in stage I of the disease, cancer is found incidentally in a small part of the sample when prostate tissue has been removed for other reasons, such as benign prostatic hypertrophy, and the cells resemble normal cells and the gland appears normal to touch examination. In stage II, more of the prostate is involved and a lump can be felt inside the gland. In stage III, the tumor extends through the prostatic capsule and the nodule can be felt on the surface of the gland. In stage IV of the disease, the tumor has invaded neighboring structures, or has spread to lymph nodes or other organs. The classification is based on cell content and tissue architecture from biopsies (Gleason), which provides an estimate of the potential final destructive prognosis of the disease.
In one embodiment, the methods described here treat prostate cancer.
HEAD AND NECK CANCER Head and neck cancers (for example, oral, larynx, nasopharynx, esophagus, etc.), refer to a group of biologically similar cancers from the upper aerodigestive tract, including the lip, the oral cavity (mouth ), the nasal cavity, the paranasal sinuses, the pharynx and the larynx. Most head and neck cancers are squamous cell carcinomas, originating from the mucosa (epithelium) of these regions. Head and neck cancer often spreads to lymph nodes in the neck, this is often the first (and sometimes only) manifestation of the disease at the time of diagnosis. Head and neck cancer is strongly associated with certain environmental and life-threatening factors, including smoking, alcohol consumption and certain strains of the sexually transmitted human papilloma virus. Treatment of head and neck cancer patients is still a formidable task. Cancers, such as hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, can be treated using the compounds described herein.
In one embodiment, the methods described here treat head or neck cancer.
KIDNEY CANCER
In another aspect, a method for treating kidney cancer is provided here. Kidney cancer (also called renal cell cancer, renal cell carcinoma, renal adenocarcinoma, hypernefroma) is a disease in which malignant cells are found in the lining of the tubules in the kidney. Renal cell carcinoma is the most common form of kidney cancer resulting from the renal proximal tubule. This is the most common type of kidney cancer in adults, accounting for approximately 80% of cases.
In one embodiment, the methods described herein treat kidney cancer.
LIVER CANCER
In another aspect, this document provides a method for the treatment of primary liver cancer (cancer that begins in the liver). Primary liver cancer can occur in both adults and children. Liver cancer is characterized by the presence of malignant liver tumors - tumors or growths on or in the liver. They can be discovered in medical imaging (even for a reason other than cancer itself), or they can be present in patients with an abdominal mass, abdominal pain, jaundice, or some other liver dysfunction. There are several types of liver cancer.
Hemangiomas: These are the most common types of benign liver tumors. They start in the blood vessels. Most of these tumors are asymptomatic, they do not need treatment. Some may bleed and need to be removed if they are mild to severe.
Hepatic adenomas: These benign epithelial tumors of the liver develop in the liver. They are, in most cases, located in the right hepatic lobe and are often seen as solitary. The size of the adenomas is in the range of 1 to 30 cm. The symptoms associated with adenomas are all associated with major injuries that can cause severe abdominal pain.
Focal nodular hyperplasia: Focal nodular hyperplasia (UFH) is the second most common tumor of the liver. This tumor is the result of the reaction of hepatocytes of congenital arteriovenous malformations. This process is one in which all normal components of the liver are present, but the pattern by which they are presented is abnormal. Even though these conditions still exist, the liver appears to function in the normal range.
Hepatocellular carcinoma: Hepatocellular cancer (HCC) is the most common liver cancer. It is associated with alcohol abuse and hepatitis B and is particularly dominant in Asia. Most HCCs are detected at the time that healing through surgical resection is not possible; Systemic treatment of non-operable HCC is associated with survival of less than one year.
In one embodiment, the methods described herein treat liver cancer.
LYMPHOMA Lymphoma is a type of cancer originating from the lymphocytes of the immune system. They often originate in the lymph nodes, which appear as an enlargement of the nodule (a tumor). Lymphomas are closely related to lymphoid leukemias, which also originate in lymphocytes, but typically involve only the circulating blood in the bone marrow (where blood cells are generated by a process called hematopoiesis) and do not usually form tumors. There are many types of lymphomas, and lymphomas, in turn, are a part of the broad group of diseases called hematological neoplasms. Some forms of lymphoma are indolent (for example, small lymphocytic lymphoma), compatible with a long life, even without treatment, while other forms are aggressive (for example, Burkitt's lymphoma), causing rapid deterioration and death. The WHO classification, published in 2001 and updated in 2008; http://en.wikipedia.org/wiki/Lymphoma-cite_note-isbn92-832-2411-6-2#cite_note-isbn92-832-2411-6-2 is the latest classification of lymphoma and is based on the bases established under the "European American magazine lymphoma classification" (REAL). This lymphoma of the cell type system group (that is, the normal cell type that most resembles the tumor) and defined phenotypes, molecular characteristics or genetic cyto. There are three major groups: B cells, T cells, and natural killer tumor cells. Other less common groups are also recognized. Hodgkin's lymphoma, although considered separately within the WHO classification (and precedent), is now recognized as a tumor, although clearly abnormal, lymphocytes of the mature B-cell lineage.
In one embodiment, the methods described here treat lymphoma.
SARCOMA
A sarcoma is a cancer of the connective tissue (bone, cartilage, fat), resulting in proliferation of the mesoderm.
This is in contrast to carcinomas, which are of epithelial origin (breast, colon, pancreas, and others). However, due to the evolution of the original tissue, the term "sarcoma" is sometimes applied to tumors now known from epithelial tissue. The term soft tissue sarcoma is used to describe soft tissue tumors, which includes elements that are in connective tissue, but not derived from it (such as muscles and blood vessels).
Sarcomas are given a number of different names, based on the type of tissue from which they develop. For example, osteosarcoma arises from bone, chondrosarcoma arises from cartilage, and leiomyosarcoma arises from smooth muscle. Sarcomas are severe in all age groups, but are very rare, representing only 1% of cancer cases. TEGI is the most common form of sarcoma, with about 3000 to 3500 cases per year in the United States. This must be compared with breast cancer, with about 200,000 cases per year in North America.
Approximately 50% of bone sarcomas and 20% of soft tissue sarcomas are diagnosed in people under the age of 35. Some sarcomas, such as leiomyosarcoma, chondrosarcoma, gastrointestinal stromal tumor (TEGI), are more common in adults than in children. Most of the series of bone sarcomas, including Ewing's sarcoma, osteosarcoma, which are much more common in children and young adults.
In one embodiment, the methods described here treat sarcoma.
CARCINOMA
A carcinoma is any malignant cancer that arises from epithelial cells. Carcinomas invade surrounding tissues and organs and can cause metastasis, or spread to lymph nodes and other locations.
Carcinoma, like all neoplasms, is classified by its histopathological appearance. Adenocarcinoma and squamous cell carcinoma are two common descriptive terms for tumors that reflect the fact that these cells may have appearance of glandular or squamous cells, respectively. Severely anaplastic tumors can be so undifferentiated that they do not have a distinct histological appearance (undifferentiated carcinoma).
Sometimes a tumor is referred to by the main presumed organ (for example, prostate carcinoma) or the supposed cell of origin (hepatocellular carcinoma, renal cell carcinoma).
Adenocarcinoma is a malignant tumor originating in the epithelial cells of glandular tissue and in the formation of glandular structures. This is common in the lung (forming 30 to 40% of all lung carcinomas). It can be found peripherally, arising from goblet cells or type II pneumocytes. Squamous cell carcinoma results from squamous metaplasia. This represents about 20 to 30 percent of lung tumors and is usually of hilar origin.
Small cell carcinoma is almost certainly due to smoking. These metastasize early, and can secrete ADH (decreased sodium concentration in the patient).
Large non-differentiated cell carcinomas represent 10 to 15 percent of lung neoplasms. These are aggressive and difficult to recognize, due to their undifferentiated nature. These are most commonly in the center of the lung.
Nasosinusal undifferentiated carcinoma.
In one embodiment, the methods described herein for treating a carcinoma.
MYELOMA Multiple myeloma (also known as MM, myeloma, myeloma cell plasma, or as Kahler's disease after Otto Kahler) is the cancer of plasma cells. These immune cells are formed in the bone marrow, are numerous in lymphatic vessels and produce antibodies. Myeloma is considered incurable, but remissions can be induced with steroids, chemotherapy, thalidomide and stem cell transplants. Myeloma is part of the broad group of diseases called malignant hematological diseases.
Multiple myeloma develops in the post-germinal B lymphocyte centers. A chromosomal translocation between the immunoglobulin heavy chain gene (on chromosome XIV, locus 14q32) and an oncogene (often 11q13, 4p16.3, 6p21, 16q23 20q11) is often seen in patients with multiple myeloma. This mutation results in deregulation of the oncogene that is thought to be an important event in the pathogenesis of myeloma onset. The result is the proliferation of a plasma cell clone and genomic instability that leads to additional mutations and translocations. Abnormality on chromosome 14 is seen in about 50% of all myeloma cases. Suppression (of parts) of the thirteenth chromosome is also seen in about 50% of cases.
Production of cytokines (especially IL-6) by the plasma cell causes much of its localized damage, such as osteoporosis, and creates a microenvironment in which malignant cells flourish. Angiogenesis (the attraction of new blood vessels) is increased.
In one embodiment, the methods described here treat myeloma.
STOMACH CANCER Stomach or gastric cancer can develop anywhere in the stomach and can spread along the stomach to other organs, particularly the esophagus, lung and liver. Stomach cancer causes about 800,000 deaths worldwide each year. Metastasis occurs in 80 to 90% of individuals with stomach cancer, with a six-month survival rate at a rate of 65% of those who were diagnosed in the early stages and less than 15% of those who were diagnosed in the late stages. Stomach cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. As the symptoms occur over time, the cancer usually has metastasized to other parts of the body, one of the main reasons for its poor prognosis.
In one embodiment, the methods described here treat stomach cancer.
THYROID CANCER
Thyroid neoplasms or thyroid cancer usually refer to any of the four types of malignant tumors of the thyroid gland: papillary, follicular, medullary or anaplastic. Papillary and follicular tumors are the most common. They grow slowly and can reappear, but are not usually fatal in patients under 45 years of age. Spinal tumors have a good prognosis, are restricted to the thyroid gland and a worse prognosis if metastasis occurs. Anaplastic tumors are fast-growing and respond poorly to treatment. Thyroid cancer is usually found in an euthyroid patient, but symptoms of hyperthyroidism or hypothyroidism can be associated with a large or well-defined metastatic tumor. The nodules deserve particular attention when they are found in people under 20 years of age. The presentation of benign nodules at this age is less likely, and thus the potential for malignancy is much greater. Thyroid cancers can be classified according to their pathological characteristics. The following variants can be distinguished (distribution by several subtypes may vary regionally): papillary thyroid carcinoma (up to 75%), follicular carcinoma of the thyroid (up to 15%), medullary thyroid carcinoma (up to 8%) and anaplastic cancer of the thyroid (less than 5%). The follicular and papillary types can well be classified as "differentiated thyroid cancer". These types have a more favorable prognosis than spinal and non-differentiated types. Thyroid adenoma is a benign tumor of the thyroid gland.
In one embodiment, the methods described here treat thyroid cancer.
BLADDER CANCER Bladder cancer refers to any of several types of malignant tumors of the urinary bladder. It is a disease in which abnormal cells multiply without control in the bladder. The bladder is a hollow muscular organ that stores urine and is located in the pelvis. The most common type of bladder cancer begins in cells of the inner lining of the bladder and is called transitional cell carcinoma (cell carcinoma, sometimes urothelial). 90% of bladder cancers are transitional cell carcinomas. The other 10% are squamous cell carcinomas, adenocarcinoma, sarcoma, small cell carcinoma and secondary deposits of cancers in other parts of the body.
The following stages are used to classify the location, size and spread of cancer, according to the TNM (tumor, lymph node and metastasis) staging system: Stage 0: Cancer cells are found only in the inner lining of the bladder. Stage I: Cancer cells proliferate beyond the inner lining of the urinary bladder, but not to the urinary bladder muscles. Stage II: Cancer cells proliferate for the muscles of the bladder wall, but not for the adipose tissue that surrounds the urinary bladder. Stage III: Cancer cells proliferate to the adipose tissue around the urinary bladder and to the prostate gland, vagina or uterus, but not to the lymph nodes or other organs. Stage IV: cancer cells have proliferated to the lymph nodes, pelvic or abdominal wall and / or other organs. Recurrence: Cancer has a recurrence in the urinary bladder or other nearby organ after being treated. Bladder CBT is performed according to the 1997 TNM system: Non-invasive papillary tumor Ta; Invasive T1, but not as far as the bladder muscle layer; T2 invasive within the muscle layer; T3 invasive in addition to the muscle in the fat and outside the bladder, and T4 invasive in neighboring structures, such as the prostate, uterus or pelvic wall.
In one embodiment, the methods described here treat bladder cancer.
According to the invention, humanized endoglobin antibodies or fragments thereof can be administered alone or in combination with active or inactive agents. When combinations are used, the invention contemplates the simultaneous or sequential administration of humanized endoglobin antibodies or antigen binding fragments and active or inactive agents.
The compounds of the present invention may, as necessary, be administered in combination with one or more therapeutic treatments, including, but not limited to, adriamycin, cyclophosphamide, paclitaxel, pemetrexed, temozolomide, oxaliplatin, bevacizumab, Erbitux, Vectibix, sorafenib, sunitinib , gefitinib, erlotinib, 5-fluorouracil (5-FU) irinotecan, topotecan, leucovorin, VELCADE®, lenalidomide, thalidomide, xeloda, taxotere and many other conventional therapies described here. It would be understandable that the listing of therapeutic regimens listed below represents conventional therapies, but the present invention encompasses other known therapeutic regimens that are not specifically disclosed herein.
As used herein, "radiation" refers to, for example, microwaves, ultraviolet (UV), infrared (IR), or alpha, beta or gamma radiation. The radiation can be "focused" or implanted at the local level using conventional techniques to target the radiation to the site of one or more tumors without irradiation from the entire body.
In one embodiment, cancer is ovarian cancer and one or more therapeutic treatments is surgery, chemotherapy (eg, doxorubicin, doxil, gemcitabine, Rubitecan, and platinum based on chemotherapeutics such as cisplatin, carboplatin and oxaliplatin), melphalan , paclitaxel, topoisomerase I inhibitors such as topotecan and irinotecan, taxane-based therapy, hormones, radiation therapy, whole body hypothermia, isoflavone derivatives, such as Phenoxodial, cytotoxic macrolides such as, epothilones, angiogenesis inhibitors such as bevacizumab, signal transduction inhibitors, such as gene therapy, trastuzumab, RNAi therapy, immunotherapy, monoclonal antibodies, phosphatidylinositol-like kinase inhibitors, such as rapamycin, or any combination thereof. In one embodiment, the combination is a humanized anti-endoglobin antibody or antigen-binding fragment thereof and doxyl. In another embodiment, the combination is a humanized anti-endoglobin antibody or antigen-binding fragment of the same and topotecan. In yet another embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and an inhibitor of the VEGF receptor. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib and pazopanib. The combination of antibody therapy and antigen binding fragments described herein with ovarian cancer therapies can also be provided for lower doses of either therapy, or both, due to a synergistic effect of co-administration of therapies.
In one embodiment, the cancer is renal / kidney cancer and one or more therapeutic treatments is surgery, chemotherapy, sunitinib, sorafenib, pazopanib, AVASTIN®, alpha-interferon, or IL-2. In one embodiment, the combination is a humanized anti-endoglobin antibody or antigen-binding fragment thereof and sorafenib. In a certain embodiment, the combination is a humanized antiendoglina antibody or antigen-binding fragment thereof and sunitinib. In one embodiment, the combination is a humanized anti-endoglobin antibody or antigen-binding fragment of the same and AVASTIN®. In yet another embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and an inhibitor of the VEGF receptor. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib and pazopanib. Combination therapy of antibodies and antigen-binding fragments described herein with kidney cancer therapies may also provide for lower doses of either therapy, or both, due to a synergistic effect of co-administration of therapies.
In one embodiment, cancer is myeloma and one or more therapeutic treatments is surgery, radiation therapy, VELCADE®, lenalidomide, or thalidomide. In one embodiment the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and VELCADE®. Doses for any of these therapies are known in the art and can be adjusted with the combination therapy accordingly.
In one embodiment, cancer is prostate cancer and one or more therapeutic treatments is surgery, radiation therapy (for example, external beam or brachytherapy), hormonal deprivation (androgen suppression), heat shock protein inhibitors 90 (HSP90) , chemotherapy (eg docetaxel, platinum-based chemotherapy such as cisplatin, carboplatin, satraplatin, and oxaliplatin, taxane, estramustine), prednisone or prednisolone, cholesterol-lowering drugs such as statins, luteinizing hormone (LHRH) agonists, RNAi therapy, whole tumor cells genetically modified to secrete macrophages and granulocytes - colony stimulating factor (GM-CSF) (also known as GVAX), or any combination of these. In yet another embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and an inhibitor of the VEGF receptor. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib and pazopanib.
In one embodiment, cancer is lung cancer and one or more therapeutic treatments is surgery, radiotherapy (radiotherapy, for example, thoracic, radiation therapy with charged particles, uracil-tegafur and platinum-based chemotherapy (for example , cisplatin, oxaliplatin, carboplatin, etc.) and vinorelbine, erlotinib (TARCEVA®), Gefitinib (IRESSA®), antibody receptor epidermal anti-growth factor (eg Cetuximab), anti-vascular factor for endothelial growth of antibodies (eg, bevacizumab), small molecule tyrosine kinase inhibitors, direct protein inhibitors involved in the proliferation of lung cancer cells, Aurora kinase inhibitors, thermotherapy-induced laser, RNAi therapy, whole tumor cells genetically modified for secrete macrophages and granulocytes - colony stimulating factor (GM-CSF) (also known as GVAX), or any combination of Additional therapeutic treatments include Taxol and pemetrexed. In one embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and erlotinib. In one embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and gefitinib. In a certain embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and pemetrexed. In yet another embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and an inhibitor of the VEGF receptor. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib and pazopanib. Doses for any of these therapies are known in the art and can be adjusted with the combination therapy accordingly.
In one embodiment, cancer is breast cancer and one or more therapeutic treatments is surgery, monoclonal antibodies (eg Her-2 antibodies, Herceptin), adjuvant chemotherapy as a single chemotherapy agent or combination chemotherapy ( for example, anthracycline and taxane based on polychemotherapies, taxol, or specific target trastuzumab with or without manipulation of the endocrine system, with or without PMRT, vinorelbine), adriamycin, cyclophosphamide, xeloda, taxotere, selective estrogen modulating receptors, such as tamoxifen and raloxifene, allosteric modulators of estrogen receptors such as Trilostane, radiation (for example, interstitial brachytherapy, Mammosite apparatus, three-dimensionally shaped external radiation and intraoperative radiation therapy), aromatase inhibitors that supply the total synthesis of the body (for example, anastrozole, exemestane and letrozole), RNAi therapy, intravenous analogs of rapamycin which is immun ossupressor and anti-proliferative, such as Temsirolimus (CCI779), or any combination thereof. A review of methods for performing three-dimensional models in vitro for breast cancer culture tissues are described by Kim et al, Research on Treatment of Breast Cancer 85 (3) :. 281-91 (2004). Other in vivo and in vitro models for cancer testing are known and can be used to test anti-endoglobin antibodies described herein. In a certain embodiment, the combination is a humanized anti-endoglobin antibody or antigen-binding fragment thereof, taxol, and AVASTIN®. In a certain embodiment, the combination is a humanized anti-endoglobin antibody or antigen-binding fragment thereof and adriamycin. In a certain embodiment, the combination is a humanized anti-endoglobin antibody or antigen-binding fragment of the same and xeloda. In a certain embodiment the combination is a humanized anti-endoglobin antibody or antigen-binding fragment thereof and taxotere. In yet another embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and an inhibitor of the VEGF receptor. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib and pazopanib. Doses for any of these therapies are known in the art and can be adjusted with the combination therapy accordingly.
In a certain embodiment, cancer is colon cancer and one or more therapeutic treatments is surgery, radiation therapy and chemotherapy (for example, 5-fluorouracil levamisole, leucovorin or semustine (methyl CCNU)), N- [2- ( dimethylamine) ethyl] acridine-4-carboxamide and other carboxamide anti-cancer drugs; non-topoisomerase II inhibitors, irinotecan, liposomal topotecan, taxane class anti-cancer agents (eg paclitaxel or docetaxel), a compound of the xanthenone acetic acid class (eg PMAA 5,6-dimethylantenone-4-acetic) , laminarin, selective sites analogous to cyclic AMP (for example, 8-chloroadenosine 3 ', 5'-cyclic phosphate), Cox-2 pyranoindole inhibitors, Cox-2 carbazole inhibitors, Cox-2 tetrahydrocarbazole inhibitors, inhibitors Cox-2 indene inhibitors, localized NSAID inhibitors (eg, anthranilic acids, aspirin (5-acetylsalicylic acid), azodisal sodium, heterocarboxylic acids, carprofen, chlorambucil, diclofenac, fenbufen, fenclofenac, fenprofen, flufenamic acid, flurbiprofen, fluurbiprofen, fluurbiprofen , furosemide, sodium thiomalate gold, ibuprofen, indomethacin, indoprofen, ketoprofen, lonazolac, Loxoprofen, mQLAophenamic acid, mefanamic acid, melphalan, naproxen, penicillamine, phenylac acids ethics, proprionic acids, salicylic acids, salazosulfapyridine, sulindac, tolmetin, propazone NSAID and pyrazolone butazone, meloxicam, oxicams, piroxicam, Feldene, piroxicam beta cyclodextran, tenoxicam, etodolac, and oxaprozine), a HER-2 inhibitor RNAi, GM-CSF, monoclonal antibodies (for example, anti-Her-2 / neu antibodies, anti-CEA antibodies, A33 (HB 8779) and 100210 (HB 11764) and 100-310 (HB 11028)), erbitux, vectibix, hormone therapy, pyrimidineamines, camptothecin derivatives (eg, CPT-11), folinic acid (AF), gemcitabine, Ara-C, platinum-based chemotherapeutics such as cisplatin, carboplatin and oxaliplatin, specific cGMP phosphodiesterase inhibitor, or any combination of these. In a certain embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and a combination of 5-FU, leucovorin and oxaliplatin (FOLFOX). In one embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and a combination of 5-FU, irinotecan and leucovorin (IFL). In one embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and erbitux. In one embodiment the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and a vectibix. In yet another embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and an inhibitor of the VEGF receptor. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib and pazopanib. Doses for any of these therapies are known in the art and can be adjusted with the combination therapy accordingly.
In a certain modality, cancer is pancreatic cancer and one or more therapeutic treatments is surgery, radiation therapy (RT), fluorouracil (5-FU) and RT, systemic therapy, stent, gemcitabine (GEMZAR®), gemcitabine and RT cetuximab, erlotinib (TARCEVA®), chemoradiotherapy, bevacizumab (AVASTIN®), or any combination of these. In yet another embodiment, the combination is a humanized anti-endoglobin antibody or antigen binding fragment thereof and an inhibitor of the VEGF receptor. Non-limiting examples of VEGF receptor inhibitors include bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), aflibercept (VEGF-Trap), sunitinib (SUTENT®), sorafenib (NEXAVAR®), axitinib, pegaptanib and pazopanib.
Patients can be assessed for symptoms at one or more times that include, before, during, and after treatment regimens. Treatment can lead to improvement in the individual's condition and can be assessed by determining whether one or more of the following factors occurred: decreased tumor size, decreased cell proliferation, decreased cell number, decreased neovascularization, increased apoptosis, or decreased survival of at least a part of the cells that constitute the cell proliferation disorder. One or more of these occurrences can, in some cases, result in the partial or total elimination of the cancer and prolong the patient's survival.
Alternatively, for terminal cancers, treatment may result in stasis of the disease, improved quality of life and / or prolonged survival.
EVALUATION OF BIOMARKERS
Certain genes can be expressed in increased or decreased levels of cancers. Changes in the levels of expression of cancer genes can be indicative of resistance or sensitivity to cancer therapy or treatment. A diagnostic method is currently provided to detect the expression of the chosen gene from a panel of genes whose expression has been correlated with the sensitivity or resistance to an angiogenesis inhibitor, in which the gene is at least: VEGF, VEGF receptor , HIF-Ια, placental or endogline growth factor receptor (CD105). The method may further include the step of comparing the level of expression of at least one gene detected in the patient sample to an expression level of at least one gene that has been correlated with sensitivity or resistance to the angiogenesis inhibitor. In a non-limiting modality, the angiogenesis inhibitor is a humanized anti-endoglobin antibody. In another embodiment, the angiogenesis inhibitor is a VEGF receptor inhibitor or a VEGF inhibitor.
As used herein, the term "expression", when used in connection with the detection of expression of a gene, can refer to the detection of transcription of the gene and / or to the detection of translation of the gene. To detect the expression of a gene it refers to the act of actively determining whether a gene is expressed or not. This may include determining whether gene expression is up-regulated compared to a control, suppressed compared to a control, or unchanged from a control. Therefore, the expression detection stage does not require that the expression of the gene is actually up-regulated or repressed, but instead it may also include the detection that the expression of the gene has not been altered (that is, the non-detection of expression or no change in gene expression).
Biomarkers to be evaluated in relation to the present invention include a VEGF receptor, placental growth factor, HIF-Ια and endoglina (CD105).
For the evaluation of the expression of biomarkers, patient samples containing endothelial tissue, tumor cells, or proteins or nucleic acids produced by tumor cells, can be used in methods described herein and still known in the art. Briefly, the level of expression of the marker can be assessed by evaluating the quantity (for example, absolute quantity or concentration) of the marker in a sample, for example, biopsy of the tumor obtained from a patient, or other patient sample material containing tumor derivative (for example, blood, urine, serum or other body fluids or excretions as described above). The cell sample can, of course, be subjected to a variety of well-known preparative post-collection and storage techniques (eg, nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation , centrifugation, etc.) before assessing the amount of marker present in the sample. Likewise, tumor biopsies can also be subjected to preparative post-collection and storage techniques, for example, fixation.
Protein expression of biomarkers can be detected by having at least a portion that is displayed on the surface of cells that express it. A marker protein, or a portion thereof, can be determined, which is exposed on the cell surface. For example, immunological methods can be used to detect such proteins in whole cells, or well-known methods of computer analysis based on the sequence can be used to predict the presence of at least one extracellular domain (i.e., including both secreted proteins and proteins that have at least cell surface domain). The expression of a marker protein having at least a portion that is displayed on the surface of an expressed cell can be detected without necessarily lysing the tumor cell (for example, using a classified antibody that specifically binds to a cell surface domain of the protein).
Expressions of biomarkers can be evaluated by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or protein. Non-limiting examples of such methods include, for example, immunological methods for detecting secreted, cell surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, acid hybridization methods nucleic acid reverse transcriptase methods and nucleic acid amplification methods or any other method known in the art.
A mixture of transcribed polynucleotides obtained from the sample can be contacted with a substrate having fixed the same complementary or homologous polynucleotide with at least one portion (for example, at least 7, 10, 15, 20, 25, 30, 40, 50 , 100, 500, or more nucleotide residues) of biomarkers of a nucleic acid. If polynucleotides complementary to, or homologous to, are differentially detected on the substrate (for example, detectable using different chromophores or fluorophores, or attached to different selected positions), then the expression levels of a plurality of biomarkers can be evaluated simultaneously using a single substrate (for example, a "gene chip" microarray of polynucleotides fixed at selected positions). When a method for evaluating the expression of biomarkers is used that involves hybridizing nucleic acid to another, hybridization can be carried out under stringent hybridization conditions.
When a plurality of biomarkers of the invention are used in the methods of the invention, the level of expression of each sample of the patient's biomarker can be compared with the normal level of expression of each of the plurality of biomarkers of non-cancerous samples of the same type, both in a single reaction mixture (that is, using reagents, such as different fluorescent probes, for each biomarker) or in individual reaction mixtures corresponding to one or more of the biomarkers. The level of expression of a normal (for example, non-cancerous) biomarker in human tissue can be assessed in several ways. This normal level of expression can be assessed by assessing the level of expression of the biomarker in a portion of cells that appears to be non-cancerous, and then comparing the normal level of expression with the level of expression in a portion of the tumor cells. As additional information, it becomes available as a result of the routine performance of the methods described here, the average population of values for the normal expression of the biomarkers can be used. Alternatively, the normal level of expression of a biomarker can be determined by evaluating the expression of the biomarker in a patient sample obtained from a non-cancer-afflicted patient, from a patient sample obtained from a patient before the onset of suspected cancer in the patient, from archived and similar patient samples.
An exemplary method for detecting the presence or absence of a protein or nucleic acid biomarker in a biological sample involving obtaining a biological sample from a test subject and finding the biological sample with a compound or agent capable of detecting the polypeptide or nucleic acid (for example, mRNA, genomic DNA, or cDNA). Detection methods can therefore be used to detect mRNA, proteins, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. In vitro techniques for mRNA detection include, for example, polymerase reverse chain reaction transcriptase (RT-PCR; for example, the experimental modality set out in Mullis, 1987, U.S. Pat No. 4,683,202.), Hybridization from the north and in situ hybridization. In vitro techniques for detecting a protein biomarker include, but are not limited to, immunosorbent enzyme (ELISA), western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detecting genomic DNA include, for example, southern hybridizations. In vivo techniques for mRNA detection include, for example, polymerase chain reaction (PCR), quantitative PCR, northern hybridizations and in situ hybridizations. In addition, techniques for the in vivo detection of a protein biomarker include the introduction of a characterized antibody directed against the protein or its fragment in an individual. For example, the antibody can be labeled with a radioactive marker, whose presence and location in an individual can be detected by standard imaging techniques. The general principle of such diagnostic and prognostic tests involves the preparation of a sample or reaction mixture that may contain a biomarker and probe, under suitable conditions and for a time sufficient to allow the biomarker and probe to interact and bond, forming thus a complex that can be removed and / or detected in the reaction mixture. These tests can be performed in a variety of ways, using a variety of methods. It is also possible to directly detect the biomarker / probe forming a complex without additional manipulation or labeling of any of the immunosorbent components (biomarker or probe), for example, using the fluorescence energy transfer technique (ie, FET, see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; and Stavrianopoulos, et al, U.S. Patent No. 4,868,103).
In another embodiment, the determination of a probe's ability to recognize a biomarker can be performed without the marking of any of the test components (probe or biomarker), using a technology such as real-time Biomolecular Interaction (BIA) analyzes, see , eg, Sjolander, S. and Urbaniczky, C., 1991, Anal. Chem. 63: 2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5: 699-705). As used herein, "BIA" or "surface plasma resonance" refers to a technology for studying biospecific interactions in real time, without marking any of the interactants (for example, BIAcore). Changes in mass at the bonding surface (indicative of a bonding event) result in changes in the refractive index of light near the surface (the optical phenomenon of resonance surface plasma (PSR)), resulting in a detectable signal that can be used as a real-time indication of biological reactions between molecules.
As an alternative to making determinations based on the biomarker's absolute expression level, the determinations can be based on the biomarker's normalized expression level. The expression levels are normalized, correcting the absolute expression level of a biomarker by comparing its expression to the expression of a gene that is not a biomarker, for example, a cleaning gene that is constitutively expressed. Genes suitable for normalization include maintenance genes, such as the actin gene, or specific epithelial cell genes. This normalization allows the comparison of the expression level of a sample, for example, a patient sample to another sample, for example, a non-tumor sample, or between samples from different sources.
Alternatively, the expression level can be provided as the relative expression level. To determine the relative level of expression of a biomarker (for example, VEGF receptor, placental growth factor, HIF-Ια and immunosorbent endoglobin (CD105)), the level of expression of the marker is determined to be 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more normal cell samples versus isolated cancer cells prior to determining the expression level, for the sample in question. The average level of expression of each of the genes tested in the largest number of samples is determined and this is used as a reference level of expression for the biomarker. The expression level of the biomarker determined for the test sample (absolute expression level) is then divided by the average expression value obtained for the biomarker. This provides the relative level of expression.
In another embodiment of the invention, a protein biomarker is detected. One type of agent for detecting the protein biomarker of the invention is an antibody capable of binding to such a protein or fragment thereof, such as, for example, a detectably labeled antibody. Antibodies can be polyclonal or monoclonal. An intact antibody, or antigen binding fragment thereof (e.g., Fab, F (ab ') 2, Fv, scFv, the single polypeptide binding chain) can be used. The term "labeled", with respect to the probe or antibody, is intended to encompass direct labeling of the probe or by coupling the antibody (i.e., physically binding) a detectable substance with the probe or antibody, as well as labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody, using a fluorescence-labeled secondary antibody and labeled end of a biotin DNA probe such that it can be detected with fluorescence-labeled streptavidin. A variety of formats can be used to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked enzyme immunoassay (ELISA). One skilled in the art can readily adapt known methods of protein / antibody detection for use in determining whether tumor cells express a biomarker of the present invention. A combination of two or more of the assays for detecting biomarkers (non-limiting examples include those described above) can also be used to evaluate one or more markers.
This document also provides a method of selecting a cancer patient for treatment with an angiogenesis inhibitor. The method comprises providing a sample of the patient's cancer cells, detecting the expression of one or more genes whose expression has been correlated with the sensitivity or resistance to an angiogenesis inhibitor, comparing the level of expression of the gene or genes detected in the sample of the patient with a level of gene expression or genes that have been correlated with sensitivity or resistance to the angiogenesis inhibitor. Non-limiting examples of genes that have been correlated with sensitivity or resistance to angiogenesis inhibitor include VEGF, VEGF receptor, HIF-Ια, placental growth factor receptor, and endoglobin (CD105). In an additional embodiment, a patient is selected as being expected to benefit from administration of the angiogenesis inhibitor if the expression of the gene or genes is similar to the expression of the gene or genes that have been correlated with the sensitivity to inhibition of angiogenesis. In a non-limiting modality, the angiogenesis inhibitor for which the individual or the individual's cancer cells are tested for sensitivity or resistance is an endoglobin (CD105) inhibitor (for example, humanized anti-endoglobin antibodies). In another embodiment, the angiogenesis inhibitor for which the individual or the individual's cancer cells are tested for sensitivity or resistance is an inhibitor of the VEGF receptor or a VEGF inhibitor.
IV. FUNCTIONAL TESTS
Antibodies and antigen-binding fragments thereof can be tested for a variety of functions, using a variety of in vitro assays and in in vivo methods, including, but not limited to, those known in the art and described herein. METHODS FOR ASSESSING THE SIGNALING AND FUNCTION OF CD105 CD105 (endogline) is a member of the TGF-β receptor family that is expressed by the proliferation of endothelial cells, and normal levels of CD105 are necessary for the proliferation of endothelial cells. CD105 expression is increased by cellular hypoxia through the production of factor-1-α induced hypoxia (HIF-1-α) and protects hypoxic cells from apoptosis. CD105 acts to modulate the signaling of multiple receptor complexes of the TGF-β superfamily kinase, including TGF-β receptors (TGF-βΕ), similar activin receptors such as kinases (ALK) and activin receptors. In the absence of CD105, the activation of TGF-β receptors results in the phosphorylation of SMAD proteins that inhibit the growth of endothelial cells. However, activation of CD105 by TGF-β modulates the phosphorylation of the SMAD protein. The end result is the release of the inhibitory growth effects of TGF-β receptor activation in endothelial cells. Prevention of CD105 activation by anti-CD105 antibody act synergistically with TGF-β to suppress the growth of endothelial cells. TGF-β can stimulate two distinct types of I / SMAD receptor signaling pathways with opposite effects on endothelial cells. The TGF-β / ALK5 (A) signaling pathway leads to inhibition of cell proliferation and migration, whereas the TGF-β / ALKl · (B) pathway induces proliferation of endothelial cells and migration. CD105, an accessory TGF-β receptor, highly expressed during angiogenesis, is essential for ALK1 signaling. In the absence of CD105, TGF ^ / ALK5 signaling is predominant and maintains quiescent endothelium. High expression of CD105 stimulates the ALK1 pathway and indirectly inhibits ALK5 signaling, thus promoting the activation state of angiogenesis.
In a non-limiting modality, the antibodies and antigen-binding fragments contained here block angiogenesis by blocking the TGF-β / ALKl signaling pathway. In another embodiment, antibodies and antigen binding fragments provided here block angiogenesis, preventing Smad1 / 5/8 phosphorylation and / or signaling. CD105 participates in the promotion of angiogenesis through TGF-e / ALKl signaling, which in turn involves decreasing and / or blocking the phosphorylation of Smad2 / 3 proteins. In yet another embodiment, the antibodies and antigen binding fragments provided herein block angiogenesis, increasing Smad2 / 3 phosphorylation and / or signaling. Methods and techniques for analyzing the blocking or inhibitory effect of antibodies and antigen-binding fragments provided herein on the TGF-e / ALK1 signaling pathway and / or Smad1 / 5 phosphorylation include, but are not limited to, known molecular techniques . For example, western blotting with antibodies specific to any of the proteins in the TGF-e / ALK5 or TGF-β / ALK1 pathways can be used to determine the inhibition and / or stimulatory effect of the antibodies and antigen binding fragments disclosed herein. in the TGF-e / ALK5 or TGE-e / ALK1 pathways. Likewise, mRNA detection or mRNA regulation for proteins involved in the TGF-e / ALK5 or TGF-e / ALKl pathway can be used to analyze the inhibitory and / or stimulatory effect of antibodies and antigen binding fragments here disclosed. Additional methods for cell signaling for the TGF-e / ALK5 or TGF-p / ALK1 pathways are known in the art and are contemplated herein.
In a non-limiting modality, antibodies can be evaluated for inhibition of angiogenesis and proliferation of endothelial cells. The binding of anti-endoglobin antibodies to HUVECs does not prevent the subsequent binding of TGF-β to HUVECs. Thus, the direct suppression of endothelial cell growth by anti-endoglobin antibodies represents one of the fundamental mechanisms by which the angiogenic and tumor-suppressive effects are observed in vivo. In another modality, antibodies can be evaluated for angiogenesis blockade, preventing phosphorylation of Smad1 / 5/8 and / or signaling. CD105 participates in the promotion of angiogenesis through TGF-e / ALK1 signaling, which in turn involves decreasing and / or blocking the phosphorylation of Smad2 / 3 proteins. In yet another modality, antibodies can be evaluated for blocking angiogenesis, increasing phosphorylation of Smad2 / 3 and / or signaling. Methods and techniques for analyzing the blocking or inhibitory effect of antibodies provided herein on the TGF-e / ALK1 signaling pathway and / or the phosphorylation of Smad1 / 5 include, but are not limited to, known molecular techniques. For example, western blotting with antibodies specific to any of the proteins in the TGF ^ / ALK5 or TGF ^ / ALK1 pathways can be used to determine the inhibitory and / or stimulatory effect of the anti-endoglobin antibodies disclosed here in the TGF-β pathways. / AL ^ or TGF ^ / ALK1. Likewise, the detection of mRNA or mRNA regulation in proteins involved in the TGF- and / ALK5 or TGF-p / ALKl pathway can be used to analyze the inhibitory and / or stimulatory effect of the antibodies described here. Additional signaling methods for cell assay in the TGF-p / ALK5 or TGF-p / ALK1 pathways are known in the art and are contemplated herein. The activity of the anti-endoglobin antibodies described herein can be assessed using tests recognized in the art by, for example, ELISAs binding tests, competitive ELISAs, surface plasma resonance and effect on HUVEC cells as described in more detail below.
METHODS OF ASSESSING CELL ADHESION Cell adhesion can be measured by methods known to those skilled in the art. Tests have been described previously, for example, by Lebrin, et al., J. Clin. Invest 1997, 99: 1390-1398. Briefly, cells can adhere to the substrate (i.e., an ECM component) in coated wells. Unfixed cells are washed away, and non-specific binding sites are blocked by incubation with BSA. Bound cells are stained with violet crystal, and cell adhesion is quantified by measuring the optical density of violet crystal eluted from cells bound at a wavelength of 600 nm.
METHODS OF ASSESSING CELL MIGRATION
Assays for cell migration have been described in the literature, for example, by Brooks, et al., J. Clin. Invest 1997, 99: 1390-1398 and measurement methods for cell migration are known to those skilled in the art. In a method for measuring cell migration described herein, the membranes of the Transwell migration chambers are coated with the substrate, the washed transwells, and the non-specific binding sites blocked with BSA. Tumor cells from subconfluent cultures are harvested, washed and resuspended in migration buffer in the presence or absence of test antibodies. After the tumor cells can migrate to the underside of the Transwell-coated membranes, the remaining cells on the top of the membrane are removed and the cells that migrate to the underside are stained with violet crystal. Cell migration is then quantified by direct cell count per microscopic field.
SCID / NUDE CAMUNDONGO
One method of measuring tumor growth uses mouse SCID, as follows: subconfluent human M21 melanoma cells are harvested, washed and resuspended in sterile PBS (20x106 per mL). The suspension with 100gL of human melanoma M21 cells (2x106) is injected subcutaneously into the SCID mouse. Three days after the injection of tumor cells, mice are either treated or treated intravenously or intraperitoneally (for example, 100 gg / mouse) with one or more controls or test compositions. Mice are treated daily for 24 days. The size of the tumor is measured with calipers and the volume is estimated using the formula V = (LxW2) / 2, where V is equal to volume, L is equal to length, and W is equal to width.
One method for measuring tumor growth uses the nude mouse, as follows: MDA-MB-435 tumor cells (0.4x106 cells / mouse) in 50gL of PBS are orthotypically implanted in the breast fat of nude female mice (5 to 6 weeks old). When the tumors reached an average volume of about 50 to 80 mm3, mice are randomized (at least 10 / group) and the treated intravenously or intraperitoneally with one or more antibodies in 1pg (0.05 mg / kg) per dose , 10pg (0.5 mg / kg), 100pg (5 mg / kg) or 200pg (10mg / kg), or 100pg of control antibody in ΙΟΟμΙ of PBS, or ΙΟΟμΙ of PBS being started twice a week; in some studies, an untreated group can also be assessed. The size of the tumor is measured with calipers and the volume is estimated using the formula V = (LxW2) / 2, where V is equal to volume, L is equal to length, and W is equal to width.
BALB / SINGENIC MODELS WITH MICE
Alternatively, BALB / single mouse models can also be used to assess tumor growth or even inhibition by the antibodies described here as exemplified by, for example, Tsujie et al., Int. J. Oncology: 29 1087-1094 (2006 ).
CHEMICAL MICE
Another test measures angiogenesis in a chimeric mouse: a human mouse model and is referred to as a chimeric mouse test. The test has been described in detail by others, and even more has been described here to measure angiogenesis, neovascularization and tumor tissue regression. See Yan, et al. (1993) J. Clin. Invest. 91: 986-996. The chimeric mouse test is a useful test model for in vivo angiogenesis, because the transplanted skin grafts histologically resemble normal human skin and the neovascularization of all tissue occurring in effective human blood vessels and are growing from the skin grafted into human tumor tissue, on the surface of the grafted human skin. The origin of neovascularization in the human graft can be demonstrated by immunohistochemical staining of the neovasculature with specific markers of human endothelial cells. The chimeric mouse test demonstrates regression of neovascularization based on both the amount and extent of regression of new vessel growth. In addition, it is easy to monitor the effects on the growth of any transplanted tissue on top of the grafted skin, such as tumor tissue. Finally, the test is useful because there is an internal control for toxicity in the test system. The chimeric mouse is exposed to any test reagent and therefore the health of the mouse is an indication of toxicity. Other animal models described herein and known in the art can also be used in the methods described herein.
BUNNY EYE TEST
Another measure of angiogenesis is an in vivo rabbit eye model and is referred to as the rabbit eye test. The rabbit eye test has been described in detail by others, and has been used to measure both angiogenesis and neovascularization in the presence of angiogenic inhibitors as exemplified by D'Amato et al. (1994) Proc. Natl. Acad. Sci. USA, 91 (9): 4082-4085. The rabbit eye test is a recognized model for analyzing angiogenesis in vivo because the neovascularization process, exemplified by rabbit blood vessels growing from the corneal border to the cornea, is easily visualized through the eye's naturally transparent cornea. In addition, both the extent and amount of stimulation or inhibition of neovascularization or regression of neovascularization can be easily monitored over time.
Finally, the rabbit is exposed to any test reagent, and therefore the health of the rabbit is an indication of the toxicity of the test reagent.
Briefly, the chicken chorioallantoic membrane (MCF) are tests performed and the effects on the development of the vasculature are registered after 48 hours of implantation of a 0.5% carboxymethylcellulose pellet containing one or more controls or test compounds. Corneal neovascularization is induced by an implanted poly (hydroxyethyl methacrylate) pellet (Hydron; Interferon Sciences, New Brunswick, NJ) containing 650 ng of the potent angiogenic basic fibroblast protein (bFGF) growth factor linked to sucralfate (sulfate sucrose aluminum; Bukh Meditec, Copenhagen). The addition of sucralfate to the pellet protects the bFGF from degradation and predicts its slow release, thus producing aggressive and consistent angiogenesis that is more evident than that induced by bFGF / Hydron alone. Release of bFGF from granules containing sucralfate / Hydron can be detected in vitro, for up to 4 days after pellets are formed compared to just one day of pellets with Hydron alone. The pellets are produced by mixing 110gL of saline solution containing 12gg of recombinant bFGF (Takeda, Osaka) with 40mg of sucralfate; this suspension is added to 80μL of 12% (weight / vol) Hydron in ethanol. Aliquots (10μΕ) of this mixture are then pipetted onto Teflon pins and allowed to dry, producing approximately 17 pellets.
A pellet is implanted in the corneal micro-bags of each anesthetized eye of a New Zealand white female rabbit, from 2 mm from the limbus, followed by a single topical application of erythromycin ointment on the corneal surface. Histological examination on consecutive days demonstrates the progressive growth of blood vessels within the cornea to the pellet with only rare inflammatory cells observed. This angiogenic response is not altered by severe immune suppression with total body irradiation, and sucralfate pellets alone do not induce angiogenesis. New vessels are primarily induced by bFGF, rather than by inflammation. The animals are fed daily from 2 days after implantation by gastric lavage with one or more compounds suspended in 0.5% carboxymethylcellulose or in a vehicle alone. Immunodepressed animals receive total body radiation of 6 Gy for 6 minutes, just before the implantation of the pellets. This radiation dose results in marked leukocytopenia with a> 80% reduction in leukocyte count on day 2 and a> 90% reduction on day 3, the results are consistent with previous reports.
The animals are examined with a slit lamp every other day in a masked manner by the same corneal specialist (M.S.L.). The area of neovascularization of the cornea is determined by measuring with a reticle the length of the vessel (L) of the limbus and the number of clock hours (C) of the limbus involved. A formula is used to determine the area of a circular band segment: C / 12x3,1416 [r2- (R-L) 2], where r = 6mm, the measured radius of the rabbit cornea. The contiguous uniform band of neovascularization adjacent to the pellet is measured, thus, the total inhibition of neovascularization can be assessed.
ANGIOGENESIS BUFFER TESTS ON MATRIGEL IN
MOUSE
To confirm the effects of a composition on angiogenesis, a buffer angiogenesis assay on matrigel in mice can be used. Various growth factors (for example, IGF-1, bFGF or VEGF) (250 ng) and heparin (0.0025 units per mL) are mixed with Matrigel's reduced growth factor as previously described (Montesano, et al, J Cell Biol 1983, 97: 1648-1652; Stefansson, et al, J. Biol. Chem. 2000, 276: 8135-8141). The compositions described herein or control antibodies can be included in Matrigel preparations using one or more dosage groups of animals. In control experiments, Matrigel is prepared in the absence of growth factors. In mice, 0.5 ml of the Matrigel preparation is injected subcutaneously and allowed to incubate for one week. After the incubation period, the mice are sacrificed and the Matrigel tampons surgically polymerized removed. Angiogenesis in Matrigel buffers is quantified by two established methods, including immunohistochemical analysis and hemoglobin content (Furstenberger, et al., Lancet. 2002, 3: 298-302; Volpert, et al, Cancer Cell 2002, 2 (6): 473-83, .. E Su, et al, Cancer Res. 2003, 63: 3585-3592). For immunohistochemistry analysis, Matrigel buffers that are incorporated in OCT, are quickly frozen and prepared in 4 gm sections. Frozen sections are fixed in methanol / acetone (1: 1). Frozen sections are stained with polyclonal antibody directed to CD31. Angiogenesis is quantified by counting microvascular density in 20 microscopic high-power fields (200x).
Hemoglobin content can be quantified as previously described (Schnaper, et al., J. Cell Physiol. 1993, 256: 235-246; Montesano, et al, J. Cell Biol .. 1983, 97: 1648-1652; Stefansson , et al., J. Biol. Chem. 2000, 276: 8135-8141; and Gigli, et al, J. Immunol. 1986, 100: 1154-1164). Matrigel implants are quickly frozen on dry ice and freeze dried overnight. The dry implants are resuspended in 0.4mL of 1.0% saponin (Calbiochem) for one hour, and interrupted by vigorous pipetting. The preparations are centrifuged at 14,000Xg for 15 minutes to remove any particles. The concentration of hemoglobin in the supernatant is then directly determined by measuring the absorbance at 405nm and compared to a standard concentration of purified hemoglobin.
TUMOR GROWTH TEST METHODS
Tumor growth can be assayed by methods known to those skilled in the art, for example, the SCID mouse model, the nude mouse model and BALB / c mice with syngeneic tumors. SCID mouse models for tumor growth are performed as follows: subconfluent human M21 melanoma cells (or any type of tumor cell desired) are harvested, washed and resuspended in sterile PBS (20x106 per mL). A 100pL suspension of human M21 melanoma cells (2 x 106) is injected subcutaneously into the SCID mice. Three days after the injection of tumor cells, the mice are intraperitoneally untreated or treated with an antagonist in the desired dose ranges. Mice are treated daily for 24 days. The size of the tumor is measured with calibrators and the estimated volume using the formula V = (LxW2) / 2, where V is equal to volume, L is equal to length, and W is equal to width.
Alternatively, nude mouse models, SCID mouse models and / or syngenic BALB / c mouse models can also be used to assess tumor growth and inhibition by humanized anti-endoglobin antibodies or antigen binding fragments here described. (Tsujie et al, Int. J. Oncology, 29: 1087-1094 (2006)).
TEST METHODS OF CELL PROLIFERATION Cell proliferation can be assayed by methods known to those skilled in the art. As described herein, subconfluent human endothelial cells (CEHSs) can be resuspended in proliferation buffer containing little (5.0%) serum, in the presence or absence of CM (25pL) ECV or ECVL cells and endothelial cells allowed to proliferate during 24 hours. Proliferation can be quantified by measuring mitochondrial dehydrogenase activity using a commercially available WST-1 assay kit (Chemicon). In addition, as described herein, proliferation can be quantified by measuring 3H uptake using standard methods. (She et al, Int. J. Cancer, 108: 251-257 (2004)).
Other methods of assessing cell proliferation are known in the art and are contemplated in this document. Other non-limiting examples are described in more detail in the examples.
METHODS OF INDUCING CDC, CCDA AND OPSONIZATION Several therapies have been targeted to increase the body's natural immune response to transformed cells. Classical effector methods include complement-dependent cytolysis (“CDC”), antibody-dependent cell cytotoxicity (“CCDA”) and phagocytosis (separation by reticuloendothelial system after the target cell is coated with immunoglobulins). It is known that in the presence of antibodies, certain effector cells, such as lymphoid cells, have receptors coupled to the surfaces of the Fc regions of the antibodies, mediating an antibody-dependent cellular cytotoxicity reaction (“CCDA”) against target cells. Through CCDA, these effector cells exert cytolytic activity against such target cells.
Two types of CCDA reactions have been demonstrated in vitro. In classic CCDA reactions, effector cells attach to antibody-coated target cells and subsequently cause cytolysis of the target cells (AH Greenberg et al., "Characteristics Of The Effector Cells Mediating Cytotoxicity Against Antibody-Coated Target Cells." I. , Immunology, 21, p. 719 (1975)). This fixation between effector and target cells results from the interaction of the antibody's Fc region lining the target cell and the effector cell's Fc receptor. A disadvantage of this type of CCDA reaction is that it can be hampered by the circulation of antigen-antibody complexes, often associated with various diseases, which compete with the target cell's bound antibody for effector cell Fc receptors (ICM MacLennan, “Competition For Receptors For Immunoglobulin On Cytotoxic Lymphocytes, ”Clin. Exp. Immunol., 10, p. 275 (1972)). Due to this disadvantage of the classic CCDA, a second type of CCDA reaction - antibody-directed CCDA- has been proposed. In CCDA - antibody-directed, the specific target antibody is first fixed to the effector cell and the resulting complex is then "directed", through the antibody, to its specific antigen on the surface of the target cell. Advantageously, antibody-directed CCDA cannot be affected by the presence of antigen-antibody complexes that circulate in the host system. The interaction between antibodies and effector cells through the binding of the Fc / Fc receptor region is usually weak. And in some cases, antibodies do not remain associated with effector cells for a period of time sufficient to allow lysis of target cells. In view of this potential problem, antibodies have been linked to effector cells using pretreatment with polyethylene glycol and a mixture of phthalate oils (JF Jones and DM Segal, "Antibody-Dependent Cell Mediated Cytolysis (CCDA) With Antibody-Coated Effectors: New Methods For Enhancing Antibody Binding And Cytolysis, "J. Immunol., 125, pp 926-33 (1980)). The applicability of this method for in vivo treatments, however, can be diminished by the toxic effects that any residue of polyethylene glycol and phthalate oil on the antibody effector cell complex may have on the body.
Alternatively, a method has been proposed to increase antibody-directed CCDA through adjuvant chemotherapy with cytotoxic drugs (IR Mackay et al., “Effect On Natural Killer And Antibody-Dependent Cellular Cytotoxicity Of Adjuvant Cytotoxic Chemotherapy Including Melphalan In Breast Cancer,” Cancer Immunol, Immunother., 16, pp 98-100 (1983)). Testing assays for CCDA are well known in the art, such as, for example, U.S. Patent No. 5,756,097.
Accordingly, the present invention provides antibodies (e.g., humanized anti-endoglobin antibodies) that can bind cells having a role in neovascularization or angiogenesis that can increase cell phagocytosis and death and thus increase protection in vivo. Other antibodies and functional fragments thereof are also provided that immunoreact, specifically they bind to, or preferentially bind to an epitope or binding site to which such antibodies can bind and which have the same effect.
The antibodies of the invention can also be opsonic, or have opsonic activity, for cells that have a role in neovascularization or angiogenesis (for example, endothelial cells). As those skilled in the art recognize, “opsonic activity” refers to the ability of an opsonin (in general, or an antibody or serum factor C3b) to bind to an antigen or cell receptor to promote the attachment of the antigen or receptor to cell in a phagocyte and thus increase phagocytosis. Certain cells become extremely attractive to phagocytes, such as neutrophils and macrophages, when coated with an opsonic antibody and their rate of separation from the blood stream is markedly improved. Opsonic activity can be measured in any conventional manner, as described, for example, in U.S. Patent No. 6,610,293.
In a non-limiting modality, a patient with a neovascular disorder or a disorder of dependent angiogenesis releases antigens / peptides (eg, endoglobin) from angiogenesis. These antigens / peptides can be "tumor-associated antigens". Such patients can be systemically administered with an antibody to the antigen / peptide (e.g., endoglobin) and can initiate any path described herein to induce CDC, CCDA, opsonization or any other form of cell-mediated extermination.
V. PACKAGING AND KITS
Still in additional embodiments, the present patent application concerns kits for use with the compounds described above. Humanized antibodies or antigen binding fragments that bind endoglobin can be provided in a kit. The kits thus comprise, in a suitable container, a composition comprising an antibody or antigen-binding fragment thereof that binds endoglobin. The kit may comprise an antibody or antigen binding fragment thereof that binds to endogline in a suitable container.
The kit container means generally include at least one vial, test tube, flask, flask, syringe and / or other container means, into which the polypeptide can be placed, and / or preferably suitably aliquoted. The kits may include a medium containing at least one fusion protein, detectable moiety, reporter molecule and / or any other confined reagent container for commercial sale. Such containers may include injection and / or blow-molded plastic containers in which the desired vials are retained. Kits can also include printed material for using the materials in the kit.
Packages and kits can additionally include a buffering agent, a preservative and / or a stabilizing agent in a pharmaceutical formulation. Each component of the kit can be placed in an individual container and all the various containers can be contained in a single package. Kits of the invention can be designed for cold storage or storage at room temperature.
In addition, the preparations may contain stabilizers to increase the shelf life of the kits and include, for example, bovine serum albumin (ASB). When the compositions are lyophilized, the kit may contain other preparations of solutions to reconstitute the lyophilized preparations. Acceptable reconstitution solutions are well known in the art and include, for example, pharmaceutically acceptable phosphate buffered saline (FTS).
In addition, the packages or kits provided herein may further include any of the other portions provided herein such as, for example, one or more reporter molecules and / or one or more detectable portions or agents.
Packages and kits can also include one or more components for an assay, such as, for example, an ELISA assay. The samples to be tested in this application include, for example, blood, plasma, sections of tissue and secretions, urine, lymph and products thereof. Packages and kits may also include one or more components for collecting a sample (for example, a syringe, a cup, a swab, etc.).
Packages and kits may also include a label specifying, for example, a description of the product, method of administration and / or indication of treatment. Packages provided herein may include any of the compositions, as described herein. The package may also include a label for the treatment of eye diseases characterized by angiogenesis / neovascularization (eg, macular degeneration, NVC, diabetic retinopathy), diabetic nephropathy, a chronic inflammatory disease (eg DIC), rheumatoid arthritis, osteoarthritis , forms of cancer and their metastases. The term "packaging material" refers to a physical structure housing of the kit components. The packaging material can keep the components sterile and can be made of a material commonly used for these purposes (for example, paper, wrinkled fibers, glass, plastic, blades, blisters, etc.). The label or packaging insertion can include instructions appropriate writings. Kits, therefore, may additionally include labels or instructions for the use of kit components, in any method of the invention. A kit can include a compound in a package, or dispenser along with instructions for administering the compound in a method described herein. The invention also provides kits that use the diagnostic and testing methods described herein. In some embodiments, a kit according to the invention comprises reagents for the detection of a gene or genes whose levels of expression have been correlated with the sensitivity or resistance to an angiogenesis inhibitor in a sample of cancer cells from a patient. In some embodiments, the gene or genes are selected from VEGF, VEGF receptor, HIF-Ια, placental growth factor receptor and CD105. In some embodiments, the kit comprises VEGF. In some embodiments, the kit comprises the receiving VEGF. In some embodiments, the kit comprises HIF-1a. In some embodiments, the kit comprises the placental growth factor receptor. In some embodiments, the kit comprises CD105. In some embodiments, the kit comprises at least two of: VEGF, VEGF receptor, HIF-1a, placental growth factor receptor and CD105. In some embodiments, the kit comprises at least two genes that have been correlated with sensitivity to an angiogenesis inhibitor. In some embodiments, the kit comprises at least two genes that have been correlated with resistance to an angiogenesis inhibitor. In some embodiments, the kit comprises at least one gene that has been correlated with sensitivity to an angiogenesis inhibitor and one gene that has been correlated with resistance to an angiogenesis inhibitor.
In yet other embodiments, a kit according to the invention comprises reagents for the detection of VEGF, VEGF receptor, HIF-Ια, placental growth factor receptor and CD105 expression levels in a sample of tumor cells from a patient to to be treated; and a dose or doses of an inhibitor, including, but not limited to, humanized anti-endoglobin antibodies or antigen binding fragments described herein, in a variety of dosage forms, such as capsules, pills, gel capsules, powders for suspension, etc. It is further contemplated within the scope of the invention that the kit comprising reagents for the detection of VEGF, VEGF receptor, HIF-Ια, placental growth factor receptor, and CD105 expression levels in a sample of tumor cells from a patient to being treated will further comprise any of the aforementioned modalities of kits for the co-administration of at least one additional angiogenesis inhibitor.
Instructions may include instructions for practicing any of the methods described here, including methods of treatment. Instructions may additionally include indications of a satisfactory clinical outcome or any adverse symptoms that may occur, or additional information needed by regulatory bodies, such as the FDA (Food and Drug Administration) for use in a human individual.
The instructions can be in "printed matter", for example, on paper or cardboard, inside or attached to the kit, or on a label attached to the kit or packaging material, or attached to a bottle or tube containing a kit component. Instructions can also be included on a computer-readable medium, such as a disk (floppy or hard drive), optical CD, such as CD or DVD-ROM / RAM, magnetic tape, electrical storage media such as RAM and ROM , IC terminal and hybrids thereof, such as magnetic / optical storage media.
The embodiments of the compounds and methods of the present invention are intended to be illustrative and not limiting. Modifications and variations can be made by people skilled in the art in the light of the above teachings, specifically those that can refer to changes in antibody fragments or antigen binding that bind to the endoglobin around the described modifications, while maintaining functionality native to the binding of endogline. Therefore, it should be understood that changes can be made to the embodiments in the particular modalities that have elapsed that are within the scope of what is described.
EXAMPLES The application can be better understood with reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are presented in order to more fully illustrate the modalities of the invention and should in no way be understood, however, as limiting the scope of the report. While certain embodiments of the present invention have been shown and described here, it will be obvious that such embodiments are provided by way of example only. Numerous variations, changes and substitutions can occur for those skilled in the art without leaving the invention; it should be understood that several alternatives to the modalities described here can be used in the practice of the methods described here. EXAMPLE 1 Generation and binding of humanized Anti-CD105 antibodies and humanized / de-immunized antibodies Construction, expression and purification of antibodies The entire humanized and humanized / de-immunized gene from the VK and VH regions were synthesized using a series of overlapping oligonucleotides that were annealed, linked and amplified by PCR to give full length of the synthetic V regions. The assembled variants were then cloned directly into the Antitope Ltd.'s pANT expression vector system for IgG1 heavy chains and kappa light chains.
All humanized heavy and light chain combinations (ie, a total of 4 pairs) and humanized / de-immunized heavy and light chain combinations (ie, a total of 24 pairings) were stably transfected into NS0 cells via electroporation and selected using 200 nM methotrexate (Cat Sigma. No. M8407). For each methotrexate-resistant colony, IgG expression levels were tested using an IgG1 ELISA. The best expression lines were selected and frozen under liquid nitrogen. Successful transfection and selection of clones have been achieved for all variants and expression levels of humanized and humanized / de-immunized antibodies in static saturated cultures are shown in Table 1.
Twenty-four IgG1 variants were therefore purified from NS0 cell culture supernatants on a protein A sepharose column (GE Cat Healthcare. No. 110.034-93) and quantified by OD280nm using an extinction coefficient, EC (0.1 %) = 1.62, based on the predicted amino acid sequence. Approximately 500gg of each variant antibody was purified and lead variants were analyzed by SDS-PAGE reduction. Briefly, the reduction of SDS-PAGE Coomassie blue stain in variant antibody gel. 1gg of each sample was loaded onto a NuPag 4-12% Bis-Tris gel (Cat Invitrogen. No. NP0322BOX) and run at 200V for 30 minutes. The marker was Bio-Rad Precision Plus (Cat. No. 161-073). The bands corresponding to the predicted sizes of the heavy and light chains were observed without evidence of any contamination, on any track (data not shown).
ELISA Methodology
An ELISA was used for humanized and humanized / de-immunized anti-endoglobin antibody binding assay. Briefly, an ELISA test was performed according to the following steps: 1. Coating a Nunc Maxisorp plate with MAB9811-01 (polyclonal anti-endoglobin antibody) in 1500ng / ml PBS in 100 gL / well. Cover the plate with a sealer and incubate overnight (16-24 hours) at 4 ° C. 2. Wash plate 2X with - 200gL of PBS (without Tween). 3. Add 200 μΐ / well of BSA blocking solution (1% BSA) and incubate for 60 minutes at room temperature. 4. Wash the plate 3x with PBS containing Tween (PBS-T), using the Biotek plate washer. 5. Add 100 μΐ / well of CD105 (Cat R and D Systems 1097-PT) at 100 ng / ml in PBS-T with 0.1% BSA and incubate for 60 minutes at room temperature. 6. Wash the plate 3x with PBS-T using the Biotek plate washer. 7. In the test wells: add 100 μΐ / well of anti-endoglobin antibodies in 20, 10, 4, 2, 1, 0.5 and 0.2ng / ml (diluted in PBS-T with 0.1% BSA ) and incubate for 60 minutes at room temperature. In negative control wells: add 100 μΐ / well of paired isotopes of the corresponding antibody. 8. Wash the plate 3x with PBS-T using the Biotek plate washer. 9. Add 100 μΐ / well of Goat anti-human IgG conjugated with HRP (Jackson Immunoresearch), diluted 1: 10,000 in PBS-T with 0.1% BSA to all wells; incubate 30 to 60 minutes at room temperature. 10. Wash the 5X plate with PBS-T using the Biotek plate washer. 11. Add 100 μΐ / well of TMB substrate solution and incubate uncovered in the dark for 15 minutes. 12. Stop the reaction by adding 100 μL / well of TMB stopped solution.
Samples are performed in triplicate and the optical density is read to construct a standard curve and determine the binding constant. Statistical analysis is performed using Student's t-test or other standard test.
ELISA COMPETITION
The antibodies were tested in a competition ELISA for the binding of CD105 against chimeric anti-CD105 biotinylated. Briefly, the chimeric anti-CD105 was biotinylated using a micro-biotinylation kit (Sigma, Catalog No. BTAG-1KT), following the manufacturer's instructions. 96-well Nunc Immuno Maxisorp flat-bottom microtiter plates were coated with mouse anti-human CD105 (Southern Biotechnologies Catalog, No. 9811-01) at 1.5 pg / mL in phosphate saline buffer (PBS) overnight at 4 ° C. The next day, 100ng / ml of human CD105 (Systems R and D, Catalog No. 1097-EN) in PBS / 2% BSA was added to the pre-coated plate and incubated at room temperature for 1 hour. Varying concentrations of both chimeric, humanized or humanized / de-immunized antibodies (4 pg / mL to 0.0018 pg / mL in three dilutions) were mixed with a fixed concentration of biotinylated chimeric anti-CD105 antibody (6.25 ng / ml) and added to the plate. Binding of the biotinylated chimeric antibody was detected using streptavidin-HRP (Sigma, catalog No. S5512) and the TMB substrate (Sigma, catalog No. T0440). OD450nm values were measured on an MRX Dynex TCII plate reader. The results of the competition analysis are illustrated in Figures 7 and 8. The curves were adjusted through the straight line part of each of the absorbance graphs against the sample concentration log and the line equations were used to calculate the concentrations of humanized or humanized / de-immunized antibodies needed to inhibit the binding of the biotinylated chimeric antibody to CD105 by 50% (IC50). To allow comparisons with and between experiments, the IC50 values of humanized or humanized / de-immunized variants were normalized against the reference antibody that was included in each plate to give a value for the speed difference. IC50 values are relative to the chimeric anti-CD105 and are representative of three experiments. Summary ELISA data are shown in Table 1 and include the levels of antibody expression (pg / ml) as measured in static saturated cultures.
Table 1. Characteristics of humanized and humanized / de-immunized antibody variants. IC50 values are relative to the chimeric anti-endogline antibody and are representative of three experiments. The levels of antibody expression (pg / ml) were assayed in static saturated cultures. The level of de-immunization is represented by an arbitrary scale based on the location in the epitopes of the mutations. EXAMPLE 2 BIAcore analysis (Plasma Surface Resonance -RPS) of humanized and humanized / de-immunized anti-endogline antibody binding Antibody affinity can be assessed using, for example, BIAcore analysis using standard protocols. In short, protein A is chemically coupled to a CM5 BIAcore chip, with the amount of immobilized protein A corresponding to ~ 2000 RU. Subsequent steps are performed in a 10mM HEPES, buffer, 150 mM NaCl, 3 mM EDTA, 0.05% Tween, pH = 7.4, at 25 ° C using a data collection rate of 10 Hz. The anti-endoglobin antibody (10 nM) is captured at a flow rate of 10 uL / min by the immobilized protein A on the BIAcore chip: typically, capture times of 20, 40 and 80 seconds allow for density capture of antibody corresponding to 130 RU, 330RU and 570 RU, respectively. Initiation cycles are performed using execution buffer at a flow rate of 40 uL / min, a contact time of 90 seconds and a dissociation time of 90 seconds. Sample cycles are performed using recombinant endoglobin in concentrations ranging from 0 to 40 nM. Endogline is passed over the BIAcore chip containing antibody captured at a flow rate of 40 uL / min with a contact time of 525 seconds and dissociation time of 2500 seconds. Eight sample cycles are typically performed at each antibody capture regeneration density of the chip using 10 mM glycine with pH = 1.7. Data analysis is performed using BIAcore T100 v1.1 signal evaluation software generated using BIAcore chips with which different densities of captured antibodies are compared and data generated in the absence of recombinant endoglobin are used to adjust the blank intra-signal test. For data adjustment, Rmax is allowed to fluctuate to account for the variation in the capture levels of each antibody in each cycle. The data for each capture density is adjusted simultaneously during the analysis of each antibody. BIAcore data are shown in Table 2 for chimeric, humanized and humanized / de-immunized anti-endoglobin antibodies, including ka (1 / Ms), kd (1 / s), KD (M) and Chi2 (RU2).
Table 2. BIAcore binding data for chimeric anti-endoglobin antibody, humanized anti-endoglobin antibody VK1VH and humanized / de-immunized anti-endoglobin antibodies VK1AAVH1R, VK1AAVH1Q and VK1AAVKVH1A2. EXAMPLE 3 Avidity of antibodies and the number of epitopes available in cells expressing endoglobin Avidity of antibodies and the number of epitopes available in cells expressing endoglobin can be evaluated using Scatchard plot analyzes using standard protocols.
In summary, Scatchard plot analyzes of direct binding of humanized radiolabeled anti-endogline antibodies to leukemia cells that express KM-3 endogline and subconfluent HUVECs proliferation are performed. The purified anti-endoglobin antibodies are individually radiolabeled with 125I using GEN-Iodine and according to standard methods known to those skilled in the art. The radiolabeled humanized anti-endogline antibodies are assayed for the average number of iodine atoms per IgG molecule. Titration experiments are performed using a fixed amount (0.1 pg) of each 125I-labeled mAb and 2 times in series of increments of cells expressing HUVEC endogline to determine antigen binding activity. Scatchard plot analysis of the link data is performed according to known methods. An equilibrium constant and a maximum mean number of bound mAb / cell are estimated by this analysis. EXAMPLE 4 Western Blot Assay for Anti-Endoglobin Antibody Activity The ability of humanized anti-endoglobin antibodies to modify intracellular signaling in proliferation of CD105-expressing endothelial cells can be assayed using Western Blots to detect phosphorylation of proteins involved in the pathway CD105 signaling.
Western blot analyzes are performed to identify phosphorylated Smad1 / 5/8 or Smad2 / 3 according to known Western Blotting techniques in untransfected endothelial cells. Primary antibodies against phosphorylated Smad1, Smad2, Smad5, ID1 (Santa Cruz) and endogline are used to detect molecules in the samples. Detection is performed by increased chemiluminescence (QLA). EXAMPLE 5 HUVEC growth inhibition and 3H-thymidine incorporation assay A number of assays are available to assess inhibition of cell growth.
In one example, the HUVEC E6 / E7 P3-17 cell line was grown in EBM2 media with supplements (Lonza-Clonetics), containing 5% fetal calf serum. The cells were cultured in 75-cm2 flasks (Falcon, Becton-RNAkinson, Franklin Lakes, NJ) in a CO2 incubator at 37 ° C under subconfluency conditions. Cells are separated by incubation with Hanks balanced saline with 15 mM EDTA in 25 mM HEPES buffer, ph = 7.3 at 37 ° C for 15 min. After washing twice with cold PBS, the cells are resuspended in an endothelial cell growth medium at a concentration of 25,000 cells / ml. In additional experiments, human umbilical vein endothelial cells (HUVECs) are suspended and cultured in an endothelial cell growth medium free of FBS and bovine brain extracts. A 200μ1 aliquot of cell suspension containing 2,500 cells is seeded on each 96-well culture plate. The cells are cultured at 37 ° C in a CO2 incubator overnight before being added in triplicate of 100 µg / ml of humanized anti-endoglobin antibody VK1AAVH1A2 or control IgG or PBS. The culture plates are kept in the incubator for 72 hours, during which fresh media and humanized anti-endoglobin antibody, control IgG or PBS are replaced every 24 hours. 3H-thymidine (1 μθί) is added to each well and the plates are incubated for 20 hours. The cells are washed with PBS, followed by treatment with 100 μΐ / well of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA) at 37 ° C for 15 min. Cells are harvested on fiberglass filters (Wallac printed FiltermatA) using Harvester 96 (TOMTEC, HDMRIen, CT) and 3H-radioactive is determined in a Trilux 1540 MicroBeta liquid scintillation and luminescence counter (Wallac, Turku, Finland). In a second example, the cells used were a primary culture of HUVEC cells, HUVEC 2517C, humanized antibody TRC105 inhibited the growth of the HUVEC cell line E6 / E7 and the primary HUVEC culture, derived from a single donor HUVEC 2517C, compared to the control antibody and PBS (Table 3). E6 / E7 cells Table 3. Inhibition of human endothelial cell growth by humanized / de-immunized anti-endogline antibody VK1AAVH1A2. EXAMPLE 6 Assay for Inhibition of Cell Migration by Anti-Endoglobin Antibodies Migration (chemokinesis) as a measure of cell proliferation and activation is measured using a Boyden chamber.
Briefly, cell migration is assessed as follows: a Costar Nucleopore filter (8mm pore) is coated with fibronectin overnight at 4 ° C. The chamber is washed with phosphate buffered saline (PBS) and the lower chamber was filled with DMEM with or without serum and with or without TGF-ββ. The cells are trypsinized and suspended at a final concentration of 50,000 cells / ml in DMEM with anti-endoglobin antibody. A 150 µl aliquot of the cell suspension is added to the upper chamber and incubated at 37 ° C. After 16h, the cells are washed and the upper surface is cleaned to remove non-migrating cells. The membranes are fixed in methanol, washed with water, dyed and the number of cells on the bottom surface are counted. EXAMPLE 7 CCDA Assay for Humanized / Deimmunized Anti-Endoglobin Antibodies The anti-endoglobin antibodies described herein can be evaluated for their ability to bind to activated IL-2 killer (NK) natural cells and to induce antibody-dependent cytotoxicity cells (CCDA) of HUVECs using, for example, the following protocols.
Isolation NK and Generation of IL-2 NK cells activated PBMC are isolated and allowed to stand for 24 hours at 4 ° C in RPMI with 10% FBS. PBMC are then placed in RPMI medium with 2% FBS (total volume = 50 ml), and 10 ml of the cell suspension is plated in a Petri dish. PBMC are incubated for 2 hours at 37 ° C and non-adherent cells are collected. NK cells are cultured at 8x106 / ml with 1000U / ml IL-2 for 48 hours, followed by normal culture for 5 to 8 days before use in a CCDA assay.
Cytotoxicity and CCDA assays NK cells are scraped from the culture and collected in a 50 ml conical tube. The cells are washed once with complete RPMI medium and centrifuged at 1200 rpm for 10 minutes. The NK cells are then resuspended in 5mL of complete RPMI medium and counted. Before performing the test, the NK cell count is normalized to an effector: target 10: 1 ratio. Standardized NK cells are bathed and 10μ1 of antiendoglina antibody is added to designated wells and incubated for 30 minutes at 37 ° C.
Control samples include populations of untreated cells or treated antibody control. All samples and controls are tested in quintuplicate. Target cells of interest are collected (HUVEC cells), washed, centrifuged at 1200 rpm for 10 minutes and resuspended in 5 ml of complete RPMI medium. The target cells are washed again and resuspended in free RPMI serum to a final concentration of 1x10 6 cells / ml. The target cells are then labeled with a final concentration of 5μg / ml of calcein AM for 1 hour at 37 ° C, followed by two washes with the complete RPMI media. The target cells are then resuspended and added to the NK cell wells. The target cell / NK cell combination is incubated at 37 ° C for 4 hours. After incubation, the plates are centrifuged at 1200 rpm for 5 minutes and the cells are washed and resuspended in DPBS. Fluorescence is read using 450/530 nm excitation / emission and the emission is a measure of antibody-mediated cell death. The average fluorescence intensity and standard deviation are calculated and used to calculate% of CCDA according to the following formula:% CCDA = 100% * [(f sample fmedia) (control physotype fTriton)], where: famoustra = mean of fluorescence in wells containing anti-endoglobin antibody fmedia = mean fluorescence in wells containing medium without the control physotype antibody = mean fluorescence in wells containing control IgG isotype fTriton = mean fluorescence in wells containing Triton detergent (to lyse target cells) Antibody Humanized / de-immunized VK1AAVH1A2 demonstrated dose-dependent CCDA of HUVECs that was significantly greater than the control antibody isotype (Table 4A and 4B). Observe the experiments summarized in Table 4A and 4B performed with NK cells from different donors.
Table 4A. CCDA of humanized / deimmunized antiendoglina antibody VK1AAVH1A2 versus isotype compatible with control IgG in HUVEC cell.
Table 4B. CCDA of humanized / deimmunized antiendoglina antibody VK1AAVH1A2 versus isotype compatible with control IgG in HUVEC cell. EXAMPLE 8 Effect of humanized / de-immunized entiendoglina antibodies on a murine model Choroidal neovascularization The effect of humanized / de-immunized antiendoglina antibodies can be evaluated on a murine model of choroidal neovascularization.
Briefly, C57BL / 6 mice aged 4 to 5 weeks are anesthetized with ketamine hydrochloride (100mg / kg) and pupils dilated with 1% tropicamide (Alcon Labocamundongories, Inc Fort Worth, TX). Three 532nm diode laser photocoagulation burns (75 pm- site size, 01 seconds long, 120 mW) are distributed to each retina using a photocoagulator slit lamp distribution system (OcuLight; IRIDEX, Mountain View, CA ) and a hand slide as a contact lens. Burns are performed at positions 9, 12 and 3 o'clock on the posterior pole of the retina. The production of a bubble, at the time of the laser, which indicates the rupture of the Bruch's membrane, which is an important factor in obtaining choroidal neovascularization (CNV); thus, only burns in which a blister is produced are included in the study.
Four independent experiments are carried out to investigate the effect of intraocular injections on day 0 after the rupture of Bruch's membrane. Group 1 mice receive an intraocular injection of about 0.5 to about 5 pg of an anti-endoglobin antibody or antigen binding fragment in 1 pL of PBS in one eye and 1 pL of PBS in the other eye . Group 2 mice are given an intraocular injection of about 1.5 to about 10 pg of anti-endoglobin antibody or antigen binding fragment in 1 pL of PBS in one eye and 1 pL of PBS in the other eye . Group 3 mice are given an intraocular injection of about 5 to about 25 pg of anti-endoglobin antibody or antigen binding fragment in one eye and 1 pL of PBS in the other eye. Group 4 receives PBS in both eyes.
After 14 days, the mice are anesthetized and sprayed with fluorescein-labeled dextran (2 x 106 average molecular weight, Sigma-Aldrich) and flat choroidal assemblies are prepared. Briefly, the eyes are removed, fixed for 1 hour in 10% formalin phosphate buffer, and the cornea and lens are removed. The entire retina is carefully dissected from the eyepiece, radial cuts are made from the edge of the equator eyepiece in all four quadrants, and the retina is prepared planarly in aqueous preparation medium (Aquamount; BDH, Poole, UK). Flat preparations are examined by fluorescence microscopy (Axioskop; Carl Zeiss Meditec, Thornwood, NY), and the images are digitized with a color video camera with three charge-coupled devices (CCD) (1K-TU40A, Toshiba, Tokyo, Japan) . Image capture analysis software is used to measure the area of each CNV lesion. Statistical comparisons are made using ANOVA with Dunnett's correction for multiple comparisons. EXAMPLE 9 Anti-angiogenic therapy of human breast cancer tumors preformed on human skin grafted onto SCID mice The effect of the humanized / de-immunized anti-endoglobin antibodies described here can be appreciated in relation to their anti-angiogenic effect on cancer tumors preformed human breast cells grown on human skin and grafted into SCID mice.
Briefly, MCF-7 cells (8x106 cells in 0.1 ml of PBS) are transplanted intradermally in full thickness of human skin grafted into SCID mice, when the grafts showed no signs of inflammation, contraction or rejection. The mice are left untreated, until distinct palpable tumors (3 to 6 mm in diameter, in most cases) appear. Mice with distinct tumors are divided into groups for therapeutic studies. Monoclonal humanized / de-immunized anti-endoglobin antibody (mAb) and a corresponding control IgG isotype are diluted with sterile PBS containing mouse serum albumin (0.05% final concentration). For antibody therapy, 1 to 20 mg / kg of anti-endoglobin antibody or control IgG is administered intravenously (VI) through the tail vein of mice. Administration is given every two to three days.
During treatment, mice are monitored daily for tumor size and morbidity. The mice are weighed twice a week using an electronic scale (OhausTM Model GT210). Tumor size is measured three times a week using an electronic caliper (PRO-MAX 6-inch caliber; Fowler Co., Newton, MA) connected to a computer using OptoDemo ™ software (Fowler Co.). The measurements of the tumor diameters are converted into tumor volumes using the following formula: V = length x width x height x pi / 6. Statistical analysis of data for the comparison of different groups of mice is performed using Student's t test. EXAMPLE 10 Mouse model of ovarian cancer To determine the ability of the humanized / de-immunized anti-endoglobin antibody, or antigen binding fragments thereof, to treat ovarian cancer, an ovarian cancer cell line can be used in SCID mice or rats.
In summary, ovarian cancer cells are implanted in immunodeficient or nude mice to generate ovarian tumors. Groups of established tumor-bearing mice are treated by VI administration of increasing doses (from 1.8 mg / kg body weight) of humanized / de-immunized anti-endoglobin antibody or control IgG. Treatment is carried out 2 or 3 times a week. A VEGF inhibitor and / or anti-cancer agent can be used in some or all groups. Mice are monitored and tumor growth is measured 2 or 3 times a week. EXAMPLE 11 Colorectal Cancer Mouse Model To determine the ability of the humanized / de-immunized anti-endoglobin antibody, or antigen-binding fragments thereof, to treat colorectal cancer, a colorectal cancer cell line can be used in SCID mice, nude or immunocompetent.
In summary, colorectal cancer cells are implanted in immunodeficient, naked or immunocompetent mice to generate colorectal tumors. Groups of established tumor-bearing mice are treated by VI administration of increasing doses (from 1.8 mg body weight / kg) of humanized / de-immunized anti-endoglobin antibody or control IgG. Treatment is carried out 2 or 3 times a week. A VEGF inhibitor and / or anti-cancer agent can be used in some or all groups. Mice are monitored and tumor growth is measured 2 or 3 times a week. Tumors can be visualized by standard image imaging, including PET and ultrasound. Treated tumors can be explored to assess intracellular signaling or vascular pathways by immunohistochemistry. EXAMPLE 12 Kidney Cancer Mouse Model To determine the humanized / de-immunized anti-endoglobin antibody capacity or antigen binding fragments thereof for the treatment of kidney cancer, a kidney cancer cell line is used in SCID mice or naked.
Briefly, kidney cancer cells are implanted in SCID or nude mice to generate kidney tumors. Groups of established tumor-bearing mice are treated by VI administration of increasing doses (from 1.8 pg / g body weight) of humanized / de-immunized anti-endoglobin antibody or control IgG. The treatment is carried out with an interval of 3 days for the first three injections and an interval of 7 days for the fourth injection. A VEGF inhibitor and / or other anti-cancer agents can be used in some or all groups. Mice are monitored and tumor growth is measured by sacrificing animals on a weekly basis. EXAMPLE 13 Myeloma mouse model To determine the humanized / de-immunized anti-endoglobin antibody capacity or antigen binding fragments thereof for the treatment of myeloma, a myeloma cell line is used in SCID or nude mice.
Briefly, myeloma cancer cells are implanted in SCID or nude mice to generate myeloma tumors. Groups of established tumor-bearing mice are treated by VI administration of increasing doses (from 1.8 mg / kg body weight) to humanized / de-immunized anti-endoglobin antibody or control IgG. Treatment is carried out 2 or 3 times a week. A VEGF inhibitor and / or anti-cancer agent can be used in some or all groups. Mice are monitored and tumor growth is measured 2 or 3 times a week. EXAMPLE 14 Sarcoma mouse model To determine the humanized / de-immunized anti-endoglobin antibody capacity or antigen binding fragments thereof for the treatment of sarcoma, a sarcoma cell line is used in SCID or nude mice.
Briefly, Sarcoma cancer cells are implanted in SCID or nude mice to generate sarcoma tumors. Groups of established tumor-bearing mice are treated by VI administration of increasing doses (from 1.8 mg / kg body weight) of humanized / de-immunized anti-endoglobin antibody or control IgG. Treatment is carried out 2 or 3 times a week. A VEGF inhibitor and / or anti-cancer agent can be used in some or all groups. Mice are monitored and tumor growth is measured 2 or 3 times a week. EXAMPLE 15 Mouse model of breast cancer To determine humanized / de-immunized anti-endoglobin antibody capacity or antigen binding fragments thereof for the treatment of breast cancer, a breast cancer cell line is used in SCID mice or naked.
In summary, breast cancer cells are implanted in immunodeficient or nude mice to generate breast tumors. Groups of established tumor-bearing mice are treated by VI administration of increasing doses (from 1.8 mg / kg body weight) humanized / de-immunized anti-endoglobin antibody. Control animals are administered with a control IgG. Treatment is carried out 2 or 3 times a week. A VEGF inhibitor and / or anti-cancer agent can be used in some or all groups. Mice are monitored and tumor growth is measured 2 to 3 times a week. EXAMPLE 16 Combination Therapy Clinical Trial for Colorectal Cancer This example describes a randomized, blinded, placebo-controlled, multicenter, phase 2 study designed to provide a preliminary assessment of the safety and efficacy of a humanized / de-immunized anti-endoglobin antibody in patients with colorectal cancer. Approximately about 100 to about 800 patients are enrolled, with about 50 to about 400 patients being assigned to a treatment group and about 50 to about 400 patients being assigned to a placebo group. Screening will consist of administration of repeated intravenous doses of humanized / de-immunized anti-endogline antibody at about 0.1 to about 20 mg / kg or placebo each, two or three weeks for 6 to 10 cycles. A VEGF inhibitor and / or anti-cancer agent can be used in all groups. The study period is estimated to be about 6 months to about 5 years, with continued therapy for those who responded to treatment as indicated at the end of the initial study. Additional outcome measures are as follows: Primary outcome: overall response rate. One of the objectives of the study is to demonstrate a 35% increase in progression-free survival with humanized / de-immunized anti-endoglobin antibody.
Secondary outcome measures that can be assessed include the overall response rate, duration of response, overall survival, serious and non-serious adverse events. For example, a treatment may prevent the disease from progressing (ie, stasis), or may result in an improvement. As an alternative or, in addition, other objectives can be measured with respect to one or more of the following: decreased tumor burden, decreased vascularization, reduced side effects, decreased adverse reactions and / or increased patient compliance. EXAMPLE 17 Myeloma Combination Therapy Clinical Trial This example describes a randomized, blinded, placebo-controlled, multicenter, phase 2 study designed to provide a preliminary assessment of the safety and efficacy of combining the humanized / de-immunized anti-endoglobin antibody with bortezomib in patients with myeloma.
Approximately about 100 to about 800 patients are enrolled, with about 50 to about 400 patients being assigned to a treatment group and about 50 to about 400 patients assigned to a placebo group. Screening will consist of administration of repeated intravenous doses of humanized / de-immunized anti-endogline antibody at about 1 to about 20 mg / kg or placebo each, two or three weeks combined with bortezomib at 1.3 mg / kg weekly. The study period is estimated to be about 6 months to about 5 years, with continued therapy for those who responded to treatment as indicated at the end of the initial study. Additional outcome measures are as follows: Primary outcome: overall response rate. One of the objectives of the study is to demonstrate an increase in the overall response rate of about 40% with bortezomib plus placebo plus by about 60% (or more) with bortezomib plus humanized / de-immunized anti-endogline antibodies.
Secondary outcome measures that can be assessed include response time, free survival, overall survival, serious and non-serious adverse events. For example, a treatment may prevent the disease from progressing (ie, stasis), or may result in an improvement. As an alternative or, in addition, other objectives can be measured with respect to one or more of the following: decreased tumor burden, decreased vascularization, reduced side effects, decreased adverse reactions and / or increased patient compliance. EXAMPLE 18 Combination Therapy Clinical Trial for Kidney Cancer This example describes a randomized, blind, placebo-controlled, multicenter, phase 2 study designed to provide a preliminary assessment of the safety and efficacy of the humanized anti-endoglobin antibody combination / de-immunized with sunitinib (Sutent®) in patients with kidney cancer cells (kidney cancer). Approximately about 100 to about 800 patients are enrolled, with about 50 to about 400 patients being assigned to a treatment group and about 50 to about 400 patients being assigned to a placebo group. Screening will consist of intravenous administration of repeated doses of humanized / de-immunized anti-endoglobin at about 0.1 to about 20 mg / kg or placebo each, two or three weeks combined with sunitinib at about 5 to about 50 mg administered daily for 4 weeks, with 2 weeks off before repeating the 4-week dosing cycle. The study period is estimated to be about 6 months to about 5 years, with therapy continuing for those who responded to treatment as indicated at the end of the initial study. Additional outcome measures are as follows: Primary outcome: progression-free survival. One of the objectives of the study is to demonstrate an increase in progression-free survival from about 9 to 13 months on sunitinib plus placebo to about 14 to 18 months (or more) on sunitinib plus humanized / de-immunized anti-endogline antibody.
Secondary outcome measures that can be assessed include response rate, duration of response, time to tumor progression, overall survival, serious and non-serious adverse events. For example, a treatment may prevent the disease from progressing (ie, stasis), or may result in an improvement. As an alternative or, in addition, other objectives can be measured with respect to one or more of the following: decreased tumor burden, decreased vascularization, reduced side effects, decreased adverse reactions and / or increased patient compliance. EXAMPLE 19 Combination Therapy Clinical Trial for Hepatocellular Cancer This example describes a randomized, blind, placebo-controlled, multicenter, phase 2 study designed to provide a preliminary assessment of the safety and efficacy of the humanized / de-immunized anti-endoglobin antibody combination. with sorafenib (NEXAVAR®) in patients with hepatocellular carcinoma (liver cancer). Approximately about 100 to about 800 patients are enrolled, with about 50 to about 400 patients being assigned to a treatment group and about 50 to about 400 patients being assigned to a placebo group. The trait will consist of the intravenous administration of repeated doses of humanized / de-immunized anti-endoglobin antibody at about 0.1 to about 20 mg / kg or placebo every two or three weeks, combined with sorafenib at about 400 mg per day for 3 to 6 cycles or until progression. The study term is estimated to be about 6 months to about 5 years, with continued therapy for those who responded to treatment as indicated at the end of the initial study.
Additional outcome measures are as follows: Main outcome measures: free of survival progression. One of the objectives of the study is to demonstrate an increase in progression-free survival from about 3 to 9 months on sorafenib plus placebo to about 6 to 12 months (or more) on sorafenib plus humanized / de-immunized anti-endoglobin antibody.
Secondary outcome measures that can be assessed include duration of response, time to tumor progression, overall survival, serious and non-serious adverse events. For example, a treatment may prevent the disease from progressing (ie, stasis), or may result in an improvement. As an alternative or, in addition, other objectives can be measured with respect to one or more of the following: decreased tumor burden, decreased vascularization, reduced side effects, decreased adverse reactions and / or increased patient compliance. EXAMPLE 20 Combination Therapy Clinical Trial for Kidney Cancer This example describes a randomized, blinded, placebo-controlled, multicenter, phase 2 study designed to provide a preliminary assessment of the safety and efficacy of the humanized / de-immunized anti-endoglobin antibody combination with bevacizumab (AVASTIN®) in patients with kidney cancer cells (kidney cancer). Approximately about 100 to about 800 patients are enrolled, with about 50 to about 400 patients being assigned to a treatment group and about 50 to about 400 patients being assigned to a placebo group. The screening will consist of the intravenous administration of repeated doses of humanized / de-immunized anti-endogline antibody at about 0.1 to about 20 mg / kg or placebo each, two or three weeks combined with bevacizumab at about 7.5, about 10, or about 15 mg / kg given intravenously every two weeks. The study period is estimated to be about 6 months to about 5 years, with continued therapy for those who responded to treatment as indicated at the end of the initial study. Additional outcome measures are as follows: Primary outcome: progression-free survival. One of the objectives of the study is to demonstrate an increase in progression-free survival of about 8 to 12 months in the bevacizumab plus placebo group in about 13 to 18 months (or more) in the bevacizumab plus humanized / de-immunized antibody group.
Secondary outcome measures that can be assessed include the overall response rate, duration of response, time to tumor progression, overall survival, serious and non-serious adverse events. For example, a treatment may prevent the disease from progressing (ie, stasis), or may result in an improvement. As an alternative or, in addition, other objectives can be measured with respect to one or more of the following: decreased tumor load, decreased vascularization, reduced side effects, decreased adverse reactions and / or increased patient compliance. EXAMPLE 21 Combination Therapy Clinical Trial for Ovarian Cancer This example describes a randomized, blinded, placebo-controlled, multicenter, phase 2 study designed to provide a preliminary assessment of the safety and efficacy of the DOXIL humanized / deimmunized antiendogline antibody combination ® in patients with ovarian cancer. Approximately about 100 to about 800 patients are enrolled, with about 50 to about 400 patients being assigned to a treatment group and about 50 to about 400 patients being assigned to a placebo group. Screening will consist of administration of repeated intravenous doses of humanized / deimmunized antiendoglina antibody at about 0.1 to about 20 mg / kg or placebo each, two or four weeks combined with DOXIL at about 5 to about 50 mg / m2, administered once every 4 weeks. The study term is estimated to be about 6 months to 5 years, with therapy continuing for those who responded to treatment as indicated at the end of the initial study. Other outcome measures are as follows: Primary outcome: progression-free survival. One of the objectives of the study is to demonstrate an increase in progression-free survival from about 3 to 6 months in DOXIL® plus placebo group to about 4 to 12 months (or more) in DOXIL® plus humanized / de-immunized anti-globin antibody.
Secondary outcome measures that can be assessed include duration of response, time to tumor progression, overall survival, serious and non-serious adverse events. For example, a treatment may prevent the disease from progressing (ie, stasis), or may result in an improvement. As an alternative or, in addition, other objectives can be measured with respect to one or more of the following: decreased tumor weight, decreased vascularization, reduced side effects, decreased adverse reactions and / or increased patient compliance. EXAMPLE 22 Platinum-Based Combination Therapy Trial for Ovarian Cancer This example describes a randomized, blind, placebo-controlled, multicenter, phase 2 study designed to provide a preliminary assessment of the safety and efficacy of the humanized / de-immunized anti-globin antibody combination with platinum-based chemotherapy in patients with ovarian cancer. Approximately about 100 to about 800 patients are enrolled, with about 50 to about 400 patients being assigned to a treatment group and about 50 to about 400 patients being assigned to a placebo group. The experiment will consist of administering repeated intravenous doses of humanized / deimmunized antiendoglina antibody at about 0.1 to about 20 mg / kg or placebo each, two or three weeks in combination with a platinum-based chemotherapy regimen ( for example, carboplatin, and paclitaxel) by intravenous infusion with repeat courses throughout the study. The study period is estimated to be about 6 months to about 5 years, with treatment continuing for those who responded to treatment as indicated at the end of the initial study. Additional outcome measures are as follows: Primary outcome: progression-free survival. One of the objectives of the study is to demonstrate an increase in progression-free survival from about 12 to 18 months, in topotecan plus placebo to about 12 to 24 months (or more) in platinum-based chemotherapy plus humanized / de-immunized anti-globin antibody.
Secondary outcome measures that can be assessed include duration of response, time to tumor progression, overall survival, serious and non-serious adverse events. For example, a treatment may prevent the disease from progressing (ie, stasis), or may result in an improvement. As an alternative or, in addition, other objectives can be measured with respect to one or more of the following: decreased tumor burden, decreased vascularization, reduced side effects, decreased adverse reactions and / or increased patient compliance. EXAMPLE 23 The use of antiendoglina antibody for the treatment of diabetic retinopathy Study Design To assess the biological activity of multiple intriendrinous injections of antiendoglina antibody in patients with nuclei clinically involving significant diabetic macular edema (EMD) and to report the adverse events associated with a single, open, marked, scaled-dose nucleus to begin the pilot study. Patients with DME involving the center of the macula and best corrected visual acuity (AVCC) in the study eye, between 20/40 and 20/400 are enrolled.
Treatment Study Eligible patients are randomly assigned in a 1: 1 ratio to receive three intravitreal injections of humanized / de-immunized anti-globin antibodies (about 0.25 to 2.5 mg per injection) administered monthly and observations are continued until 24 months . Primary end points are: ocular frequency and severity and systemic adverse events. Secondary outcomes are: 1) best corrected visual assessment, assessed with the Initial Diabetic Retinopathy Treatment Study (ETRDI) graph, using standardized refraction and testing protocol at an initial test distance of 2 meters and 2) measurement retinal thickness by optical coherence tomography. The evaluating physician is not aware of the patient's treatment assignment, the doctor administering the injection is aware of the patient's treatment assignment in relation to the humanized / de-immunized anti-globin antibody or placebo treatment, but is unaware of the dose of the humanized anti-globin antibody / de-immunized. Other people at each study site (except to help those with injections), patients and people at the central reading center are not aware of the patient's treatment assignment.
The efficacy and safety analyzes The efficacy analyzes are carried out based on the intention to treat among all patients using a method of last observation extended for missing data. For all pairs of comparisons, the statistical model is adjusted to score visual acuity (less than 55 letters versus 55 letters).
Comparisons between groups of dichotomous end points are performed using the Cochran chi-square test. Change of the visual acuity reference is analyzed using models of analysis of variance. At the end points for the characteristics of the lesion, covariance analysis adjustment models for the reference value are used. The Hochberg-Bonferroni multiple comparison procedure is used to adjust the comparisons of the two treatment pairs to the primary end point. Safety analyzes include all treated patients.
Conclusion Humanized / de-immunized antiendoglina antibodies will be a well-tolerated therapy for patients with DME. This pilot study demonstrates that humanized / deimmunized antiendoglina antibody therapy has the potential to maintain or improve visual acuity and reduce retinal thickness in patients with clinically significant DME involved nucleus. EXAMPLE 24 Clinical Trial of Antiendoglina Antibodies and Age-Related Macular Degeneration Study Design In several locations in the United States, patients are included in a 2-year, prospective, randomized, double-blind, placebo-controlled study of the safety and efficacy of repeated intravitreal injection of humanized / deimmunized antiendoglina antibodies in patients with choroidal neovascularization associated with age-related macular degeneration. The primary efficacy analysis is performed in 12 months. The end point of primary efficacy is the proportion of patients who had lost less than 15 letters (approximately 3 lines) of reference visual acuity, as assessed with the Initial Diabetic Retinopathy Treatment Study (ETRDI) chart, using standardized refraction and test protocol at a test distance from 2m. The eligibility of injuries is confirmed by an independent central reading center, using criteria of standardized and trained evaluators, who are unaware of the patients' treatment assignments. Patients provide written informed consent prior to determining their full eligibility. Screening can take 28 days.
To be included in the study, patients must be at least 50 years old; have a better corrected visual acuity from 20/40 to 20/320 (Snellen equivalent determined using an ETDRS graph); having primary or recurrent choroidal neovascularization associated with age-related macular degeneration, involving the foveal nucleus; having a type of lesion that had been assessed using fluorescein angiography and background photography as minimally classical or hidden without classical choroidal neovascularization; have a lesion of maximum size of 12 areas of optical disc (1 area of optical disc equal to 2.54 mm2 based on 1 diameter of optical disc of 1.8 mm), with neovascularization composition of 50% or more of all the injury; and who has presumed recent disease progression, as evidenced by observable blood, recent loss of vision, or a recent increase in the largest linear diameter of an injury of 10% or more. There are no exclusion criteria for preexisting cardiovascular, cerebrovascular or peripheral vascular conditions.
First Study Fifty to 500 patients (50 to 500 eyes) with AMD will participate in the study at several locations. Eligible patients are randomly assigned in a 1: 1: 1 ratio to receive humanized / de-immunized anti-globin antibodies at a dose of about 0.25 mg to 2.5 mg or a monthly injection simulation (within 23 to 37 days) for 2 years (24 injections) in one eye. The evaluating physician is unaware of the patient's treatment assignment, the doctor administering the injection is aware of the patient's treatment assignment for humanized / de-immunized anti-globin antibody or placebo treatment, but is not aware of the dose of humanized / de-immunized anti-globin antibody . Other staff at each study site (except to help those with injections), patients and staff at the central reading center are not aware of the patient's treatment assignment. Intervention therapy (eg photodynamic verteporfin therapy) is allowed if choroidal neovascularization in the study eye becomes predominantly classic.
As the first treatment step, patients will receive a complete eye exam to establish an eye health reference. The eye examination includes indirect ophthalmoscopy, slit lamp biomicroscopy, examination of the peripheral retina, measurements of intraocular pressure, visual acuity symptomatology (unaided and with the best corrected), color retinography, fluorescein angiography, optical coherence tomography, electroretinography and A-scan measurements.
After the preliminary examination, an intravitreal injection as described above is given to the affected eye of a patient with AMD. If both eyes are affected, they can be treated separately. The eye to be treated is injected with an ophthalmic solution.
After treatment, patients' eyes will be examined on days one (1), two (2), seven (7), fifteen (15), thirty (30) and sixty (60) and every month thereafter for 2 years. Because of the possibility of recurrence, patients must return for periodic checkups on a monthly basis. On each day of analysis, the patient is monitored for vitreous liquefaction. In addition, patients are monitored for posterior vitreous detachments using indirect ophthalmoscopy with scleral depression. Finally, the extent of AMD presented by the patient is continuously monitored through periodic retinal examinations, optical coherence tomography and fluorescein angiography to monitor the presence of subretinal fluid, blood, exudates, RPE detachment, retinal cystic changes, or the presence of a greyish green subretinal neovascular membrane. Additional treatments may be necessary if signs of recurrence of neovascularization are observed. Additional treatments can be given on a weekly or monthly basis. In a preferred embodiment, an initial treatment is followed by subsequent treatments between 1 to 6 months apart.
Efficacy analyzes are performed based on the intention to treat among all patients using a last observation method extended for missing data. For all pairs of comparisons, the statistical model is adjusted for scoring for visual acuity (less than 55 letters versus b 55 letters) and choroidal neovascularization subtype (minimally classic versus hidden with no classic disease). Between groups of dichotomous endpoint comparisons are performed using the Cochran chi-square test. Changes from the visual acuity reference are analyzed using models of analysis of variance.
For the end points for the characteristics of the lesion, covariance analysis models of adjustment for the reference value are used. The Hochberg-Bonferroni multiple comparison procedure is used to adjust for comparisons of the two treatment pairs for the primary end point. Safety analyzes include all treated patients.
Second study Patients with age-related macular degeneration are treated according to the methods of the first study (see above) with an intravitreal injection of (1) individualized humanized / de-immunized anti-globin antibody, (2) individualized ranibizumab, (3) humanized / anti-globin antibody de-immunized in combination with ranibizumab in the same or different compositions or (4) control antibody to reduce or prevent the development of neovascularization, macular disease and damage to the retina.
Conclusion Humanized / de-immunized antiendoglina antibodies will be a well-tolerated therapy for patients with AMD. This clinical trial demonstrates that humanized / deimmunized antiendoglina antibody therapy has the potential to maintain or improve visual acuity and reduce choroidal neovascularization in patients with AMD. In addition, humanized / de-immunized antiendoglina antibody will demonstrate superior activity compared to ranibizumab and with the combination of humanized / de-immunized antiiendogline antibody therapy and ranibizumab and will demonstrate increased activity against an individual antibody. EXAMPLE 25 Systemic toxicology in cynomolgus monkeys Cynomolgus monkeys are used in a study for systemic toxicology of humanized / de-immunized anti-globin antibodies.
Briefly, monkeys are medicated weekly for three weeks with 10.0 mg / kg, 30.0 mg / kg or 100.0 mg / kg of humanized / de-immunized anti-globin antibody. Animals are medicated with placebo in the same schedule with an appropriate solution, in the absence of antibody. Doses are administered intravenously in bolus for 30 to 60 minutes and at least six animals are medicated at each dose level.
Toxicology is assessed using one or more of the following indications: body weight measurements, basic clinical physiological measurements, serial serum chemistry, hematological assessments and histopathological assessments. EXAMPLE 26 Systemic toxicology in combination with bevacizumab in Cynomolgus monkeys Cynomolgus monkeys are used in a study for the systemic toxicity of humanized / de-immunized anti-globin antibodies in combination with ranibizumab (LUCENTIS®).
Briefly, monkeys are medicated weekly for three weeks with 10.0 mg / kg, 30.0 mg / kg or 100.0 mg / kg of the humanized / de-immunized antiendoglina antibody in combination with about 10 mg / kg for 100 mg / kg of bevacizumab. Other animals received humanized / de-immunized or bevacizumab antibodies individually. Animals are medicated with placebo in the same scheme, with an appropriate solution, in the absence of antibody. Doses are administered intravenously in bolus for more than 30 to 60 minutes and at least six animals are medicated at each dose level. Toxicology is assessed using one or more of the following indications: body weight measurements, basic clinical physiological measurements, serial serum chemistry, hematological and histopathological assessments. EXAMPLE 27 Regional Toxicology Study in Cynomolgus Monkeys Cynomolgus Monkeys are used in a study for the regional toxicology of humanized / deimmunized antiendoglina antibodies.
Briefly, monkeys are medicated by weekly intravitreal injection for six weeks with 0.25, 1.25 and 2.5 mg of humanized / de-immunized antiendoglina antibody. Animals are medicated with placebo in the same schedule with an appropriate solution, in the absence of antibody. Doses are administered as intravitreal injections and at least six animals are medicated at each dose level. Toxicology is assessed using one or more of the following indications: body weight measurements, basic clinical physiological measurements, serial serum chemistry, hematological and histopathological assessments.
Regional combination toxicology study Cynomolgus monkeys are used in a study for the toxicology of humanized / deimmunized antiendoglina antibodies in combination with ranibizumab (LUCENTIS®) when administered by intravitreal injection.
Briefly, monkeys are medicated by intravitreal weekly injection for six weeks with 0.25, 1.25 and 2.5 mg of humanized / de-immunized anti-globin antibodies and 0.5 mg of ranibizumab (LUCENTIS®).
Other animals receive an antibody syndividually, with the same dose and schedule. Placebo in animals is dosed on the same schedule with an appropriate solution in the absence of antibody. Doses are administered as intravitreal injections and at least six animals are dosed for each dose. Toxicology is assessed using one or more of the following indications: body weight measurements, basic clinical physiological measurements, serial serum chemistry, hematological assessments and histopathological assessments. EXAMPLE 28 Germination assays Angiogenesis can be tested in a three-dimensional in vitro model of germination. HUVECs are isolated from umbilical cords and grown in M199 supplemented with 10% fetal bovine serum (SBF) (GIBCO, Carlsbad, CA) and endothelial cell growth supplement (SCCE) (BD Biosciences, Bedford, MA) a 3 ° C and 5% CO2, part 2 to 4 of HUVEC are used for all experiments (part 0 being the primary culture). Lung fibroblasts (FP) are routinely grown in DMEM (GIBCO, Carlsbad, CA) supplemented with 10% FBS at 37 ° C and 5% CO2 and used between P10 and P15. Other fibroblast lines, obtainable from ATCC, can also be used.
Preparing the HUVEC cells and fibroblasts are expanded in 10% M199 FBS / Pen-Strep (1: 100) 1 to 2 days before application. For HUVEC, the medium is switched to EGM-2 (Clonetics, Walkersville, MD) the day before application. For fibroblasts, the medium is changed to EGM-2 the day before incorporation. Counting requires approximately 400 HUVEC per granule. Fibroblasts are used in 20,000 cells per well for a 24-well plate. Ninety-six well plates can also be used with quantities sized accordingly.
Preparation of the Cytodex 3 granule Cytodex 3 microcarrier granules, for example, can be used in the assay (Amersham Pharmacia Biotech, Piscataway, NJ).
Dry granules (0.5 g) are hydrated and swollen in 50 ml of PBS (pH = 7.4) for at least 3 hours at room temperature (RT) in a 50 ml tube and placed on a rocker.
The granules are allowed to settle (about 15 min). The supernatant is discarded and the granules are washed for a few minutes in fresh PBS (50 ml). The washed PBS is discarded and replaced with fresh PBS: The granule suspension is placed in a siliconized glass bottle (from windshield wiper, for example, or Sigrnacote). The granules are sterilized by autoclaving for 15 min at 115 ° C and then stored at 4 ° C.
Reagents Fibrinogen solution A fibrinogen solution is made by dissolving 2 mg / ml of fibrinogen in DPBS in a water bath at 37 ° C. The solution is then mixed by inverting the tube instead of vortexing. The percentage of clotting protein can be determined and adjusted accordingly. The solution is then passed through a 0.22pm filter to sterilize.
Aprotinin Freeze-dried aprotinin can be reconstituted at 4U / ml in DI water and sterilized by filtration. Aliquots of 1ml each are made and stored at -20 ° C.
Thrombin Thrombin is reconstituted in sterile water at 50 U / ml. 0.5 ml aliquots are made and stored at -20 ° C.
Coating the granules with HUVEC (day 1) HUVEC cells are trypsinized. Granules are allowed to settle (do not centrifuge), the supernatant is aspirated, and the granules are briefly washed in 1 ml in hot EGM-2 medium. Granules (2500) are mixed with 1x106 HUVEC in 1.5 ml of hot EGM-2 medium in a FACS tube and placed vertically in the incubator. (This will be enough for about 10 cavities; scale if necessary). The mixture is incubated for 4 hours at 37 ° C, inverting and mixing the tube every 20 min. (granules should look like mini-golf balls after granulation that indicates sufficient coating for germination).
After 4 hours, the coated granules are transferred to a T25 tissue culture flask (Falcon, Bedford, MA) and incubated overnight in 5 ml of EGM-2 medium at 37 ° C and 5% CO2.
Incorporation of fibrin gel-coated granules (day 0) A 2.0 mg / ml fibrinogen solution is prepared as described above and 0.15 Units / ml aprotinin is added to the fibrinogen solution.
Coated granules are transferred to a 15 ml conical tube and the granules are allowed to settle.
Granules are resuspended in 1 ml of EGM-2 medium and transferred to a 1.5 ml centrifuge tube. The granules are washed three times with 1 ml of EGM-2 medium, mixing by pipetting up and down slowly with a P1000 pipette. The granules are counted on a slide and resuspended in a fibrinogen solution at a concentration of 500 granules / ml.
Thrombin (0.625 Units / ml) is added to each well of a 24-well plate. Fibrinogen / granule suspension (0.5 ml) to each well by changing the pipette tip to each well. Thrombin and fibrinogen / granules are mixed by gently pipetting up and down about four to five times; avoiding the creation of bubbles in the fibrin gel. The control sample is treated in the absence of antibodies or one or more control antibodies. The test samples are treated with anti-globin antibodies alone, anti-VEGF antibodies alone, or a combination thereof. Multiple concentrations of agents can be tested. The fibrinogen / granule solution is allowed to clot for 5 minutes at room temperature and then at 37 ° C and 5% CO2 for 15 min. It is important that the plaque is not disturbed during the 5 min before coagulation to minimize fibrin shear, which can result in reduced germination. EGM-2 (1ml) is added dropwise to each well. Lung fibroblasts are seeded at the top of the clot, at a concentration of 20,000 cells / well. The culture medium is replaced with fresh EGM-2 medium every other day until the desired growth is achieved.
When the fibrin gel is formed, small bubbles may be present in the gel, they will disappear in 3 to 4 days. Germination should be apparent between days 2 and 4. The formation of the lumen starts around 4 days to 5 and shoots continue to elongate. Newly formed tubes start to branch around day 4 to 6. On days 6 to 7, microvessel-like structures begin to anastomose with adjacent tubes, increasing the number of granules per cavity resulting in anastomosis previously. The germination distance is measured by conventional techniques. EXAMPLE 29 Immunocytochemistry of Angiogenic In Vitro Shoots For staining of endothelial cell nuclei (EC), fibrin gels are washed twice with 1 X PBS and then fixed overnight in 2% paraformaldehyde. After two washes with an additional 1 X PBS, the gels are then stained with 4 ', 6-diamidine-2-phenylindole (DAPI) (Sigma, St. Louis, MO).
By immunostaining, lung fibroblasts (FP) are first removed by briefly treating the gels with 10X trypsin. Digestion is stopped with serum as soon as all fibroblasts are removed. The gels are then washed extensively with HBSS, 1X (Cellgro, Herndon, VA). The cultures are then fixed for 10 minutes in 10% formalin and permeabilized with 0.5% Triton X-100 for 5 minutes. Non-specific binding is blocked with a solution of 5% BSA in PBS for 2 hours.
Primary antibodies are used at a 1/100 dilution in blocking buffer and incubated overnight at 4 ° C. After extensive washing, the bound antibody is detected by specific species Alexa Fluor 488-conjugate or Alexa Fluor 568-secondary antibodies conjugated to a 1/1000 dilution (Molecular Probes, Carlsbad, CA). Specific isotypes of non-binding antibodies are used as controls. If high bottom occurs, the concentration of the primary or secondary antibody can be reduced and, if necessary, incubation and / or wash times can be increased. F-actin is stained with TRITC-phalloidin (Sigma, St. Louis, MO) at a concentration of 0.2 μΜ.
Phase contrast and fluorescent images are captured on an Olympus IX70 microscope coupled with a digital camera. Stacks of Z series fluorescent images are captured in one of two photon microscopes from Carl Zeiss Microlmaging LSM Meta 510 and compiled in three-dimensional renderings with Metamorph software (Universal Imaging Corporation, Downingtown PA). Thus, the expression of several markers can be easily detected.
Stacks of fluorescent optical images along the z-axis of the cultures can be captured to create 3D representations of the vessels. The nuclei are marked by DAPI (green), and the vessel walls are stained with vimentin (orange). Widely, hollow lumens are clearly visible, surrounded by a single layer of endothelial cells. These images confirm that the lumens present in the in vitro assay are intercellular and non-intracellular cracks as seen frequently in Matrigel assays. In addition, it can be confirmed that HUVECs are polarized, in which they have an apical membrane, facing the lumen, and a basal membrane, different from an IV-rich collagen-based membrane and fibrin gel. EXAMPLE 30 Suppression of choroidal neovascularization in Cynomolgus monkeys The effect of the compositions described herein on laser-induced choroidal neovascularization is evaluated in adult cynomolgus monkeys.
In this experiment, (1) humanized / de-immunized antiendoglina antibodies alone, (2) anti-VEGF antibody alone, (3) humanized / de-immunized antiiendogline antibodies in combination with anti-VEGF antibody in the same composition or different compositions or (4) control antibody being administered by intravenous or intravitreal injection. Each animal receives nine or ten laser burns on each retina, and the development of active choroidal neovascular lesions is assessed by fluorescent angiography, once before the start of treatment and 15, 20 and 29 days after laser treatment. The compositions are administered intravenously once a week, starting one week before the laser injury. Intravitreal injections are made once every two weeks starting one week before the laser, or once, two weeks after the laser, at which point the active NVC lesions have already formed. Control animals receive weekly intravenous or fortnightly intravitreal injections of placebo, starting one week before the laser.
NVC lesions are visualized by fluorescent angiography and classified according to standard procedures. EXAMPLE 31 Injury-induced Corneal Neovascularization Inhibition Corneal neovascularization is induced in male C57BL / 6 mice by intrastromal placement of 3 nylon sutures, or by chemical injury (NaOH) and mechanical debridement of the corneal epithelium. Multiple experiments are conducted in which (1) humanized / de-immunized antiendogline antibody alone, (2) anti-VEGF antibody alone, (3) humanized / de-immunized antiiendogline antibody in combination with anti-VEGF antibody alone in the same composition or different compositions or (4 ) control antibody is administered intraperitoneally once or at multiple time points immediately before or after the injury.
The growth of corneal neovessels is assessed by a slit lamp microscopy and histological evaluation. The vasculature is marked with a specific fluorescein conjugated endothelial cell lectin, and corneal neovascularization is evaluated in flat assemblies, as well as in cross sections using PECAM immunohistochemistry. The presence of corneal edema is assessed using a slit lamp microscopy, and the corneal thickness is measured in cross sections; increases in corneal thickness reflect the amount of edema. The numbers of polymofornuclear leukocytes (PMN) and macrophages are determined by staining the cross sections with HEMA-3 or F4 / 80 mouse anti-mouse monoclonal antibody, respectively. EXAMPLE 32 Identification of T cell epitopes in humanized anti-endoglobin antibody Sequences of humanized variable regions were tested by iTopeTM analysis. Sequences from the humanized variable region were divided into overlapping 9 to 15mer peptides. The variable region sequences were analyzed for high promiscuous binding affinity for human class II MHC (potential T cell epitopes) using iTopeTM, an in silico analytical tool that determines the peptide affinity for class II MHC by computer analysis. Sequences with the lowest frequencies of potential T cell epitopes from iTopeTM analysis are identified as drivers for generating a humanized antibody. Humanized variable region sequences can be redesigned by including mutations to remove potential T cell epitopes. Mutations are designed using iTopeTM to reduce or eliminate Class II MHC binding. Alternatively, human sequence line germ can be substituted at potential T cell epitope sites or alternative sequences can be substituted.
Figures 19-23 show the predicted binding of 9mer peptides for the humanized antiendoglina antibody containing the HuVK_v0 light chain and the HuVH_v0 heavy chain, seen in Figure 4. EXAMPLE 33 Humanized / de-immunized antibodies design Anti-CD105 This example describes the design of antibodies Humanized / de-immunized monoclonal therapies targeting human CD105 that exhibit reduced immunogenicity. The promiscuous high affinity of MHC class II linking sequences identified using iTopeTM (see Example 31) was further analyzed by iTopeTM in order to identify the amino acid substitutions in pocket positions in the MHC class II key that reduce or eliminate peptide binding to MHC class II. Since all CDR sequences overlapped, changes in the CDR site (potential contact antigen residues) and the physical and chemical characteristics of the original and substituted amino acids were considered. TCR contact residues and residues outside the main binding groove involved in the peptide / MHC stabilization of class II - TCR interactions were also considered for substitution.
In VHV1, a 9mer peptide being completely within CDR2 and starting at residue 51 was identified as a high promiscuous MHC class II affinity binding the peptide. The most successful method for eliminating MHC class II binding is to reach the first 9-mer amino acid (the pocket 1 or p1 position) where removal of the hydrophobic side chain or replacement with a hydrophilic side chain eliminates the class II MHC connection. However, this type of amino acid radical substitution may not always be successful in maintaining antibody affinity, therefore secondary pocket positions (p4, P7, p6 or p9), alone or in combination, have also been evaluated. ITope ™ analysis revealed that the segmentation of the p4 position of this peptide, changing from K52b to Q or R is predicted to significantly reduce MHC class II binding, while the substitution of 151 pl with A is predicted to remove binding entirely (Table 5).
Table 5 Table 5: Analysis of the effect of substitutions on the VHV1 immunogenic region using iTope ™. The nucleus of the 9mer peptide is underlined in the "sequence" column and the substitutions are highlighted in bold. Flanking waste is not underlined. The prediction of binding of each 9mer peptide nucleus to each class II MHC allele is indicated by "0" if the binding result was 0.55 to 0.6 and "X" if the binding result was> 0 , 6. The numbers of MHC class II alleles predicted to bind are shown in the "total" and "high affinity" columns.
Table 5 discloses SEQ ID NOS 106 to 109, respectively, in order of appearance.
Crystalline structures of antibody / antigen complexes suggest that I51 may infrequently be in contact with the antigen, however, a radical change from I to A (a substitution to interrupt an anchor position p1) at this position could affect the overall conformation of the CDR. Therefore, relatively conservative changes in K52b (anchor position p4) have also been included since this residue is exposed to a solvent, but cannot come into contact with antigen. Finally, additional mutations outside the CDR were also designed (G49 for A or S) to assess the destabilizing effect on peptide / MHC class II / TCR interactions. Table 6 lists humanized and humanized / de-immunized VH variant regions that were built; the SEQ ID NOS for the corresponding nucleotide sequences and amino acid sequences are indicated next to the constructs.
Table 6 Two high-affinity promiscuous MHC class II binding peptides were identified in VKV2 and VKV1. The first, with an anchor p1 in V19, partially overlaps CDR1 and the second, with an anchor p1 in I48, overlaps CDR2. Both anchors p1 are outside the CDRs and have been targeted by mutation to A, which can completely remove the MHC class II binding (Table 7). However, both of these residues may be involved in maintaining the conformations of CDRs 1 and 2 and, therefore, additional mutations have been designated that significantly reduced class II MHC binding (Table 7). In both cases, p4 residues were targeted by T to S. mutation. T22S is also found outside the CDR and is less likely to affect CDR conformation compared to V19A. T51 is inside CDR2; however, evidence of crystalline structures of antibodies complexed with antigen suggests that this residue rarely finds the antigen. Table 6 lists the humanized and humanized / de-immunized VK regions that were built.
Table 7: Analysis of the effect of substitutions in the immunogenic regions of VKV2 or VKV1 using iTope ™. The 9mer peptide nucleus is underlined in the "sequence" column and the substitutions are highlighted in bold. Flanking waste is not underlined. Predicted binding of each 9mer peptide nucleus to each class II MHC allele is indicated by "0" if the binding result was 0.55 to 0.6 and "X" if the binding result was> 0, 6. The numbers of MHC class II alleles predicted for binding are shown in the "total" and "high affinity" columns. Table 7 discloses SEQ ID NOS 110 to 115, respectively, in order of appearance. EXAMPLE 34 This example describes a method of screening antiendogline antibodies for T cell epitopes. The interaction between MHC, polypeptide and the T cell receptor (TCR) provides the structural basis for the specific T cell recognition antigen. T cell proliferation tests the binding of processed polypeptides from antibodies with MHC and the recognition of MHC / polypeptide complexes by TCR. In vitro T cell proliferation assays of the present example, involve stimulation of peripheral blood mononuclear cells (PBMC), containing antigen-presenting cells (APCs) and T cells. Stimulation is performed in vitro, using intact anti-globin antibodies. Stimulated T cell proliferation is measured using 3H-thymidine (3H-Thy) and the presence of incorporated 3H-Thy is assessed by the scintillation count of fixed washed cells.
All genes from the humanized and humanized / de-immunized VH and VK regions were synthesized using a series of overlapping oligonucleotides that were annealed, linked and amplified by PCR to give full-length synthetic V regions. The assembled variants were then cloned directly into the expression system vector antitope Ltd.'s pANT for heavy IgG1 and kappa light chains.
Purification of Antibodies Antiendoglina antibodies were purified from mammalian culture supernatants by protein A chromatography. Buffer exchange and protein concentration was done using PBS pH = 7.4. Antiendoglina antibody was further purified by size exclusion chromatography using a Sephacryl S200 column (GE Healthcare, Amersham, UK). The main peak is collected, sterilized by filtration and shown to have endotoxin levels <5 EU / mg using an Endosafe-PTS (Charles River, Margate, UK). The purified antibodies are stored at 4 ° C. Final concentrations were determined by UV absorption using calculated molar extinction coefficients, where A280 1.0 = 1.62 mg / mL. Each antibody was then diluted to 100 µg / ml in AIMV culture medium.
PBMC donor preparation and selection Peripheral blood mononuclear cells (PBMC) are isolated from a buffer donor community (from blood drawn within 24 hours), which are obtained from the UK National Blood Transfusion Service (Addenbrooke's Hospital, Cambridge, UK) according to the approval given by the Addenbrooke Local Research Ethics Hospital. PBMCs are isolated from Lymphoprep density centrifugation buffer (Axis-Shield, Dundee, Scotland) and CD8 + T cells are depleted using CD8 + RossetteSepTM (StemCell Technologies, Inc.). Donors are characterized by identifying HLA-DR haplotypes using a type of Biotest HLA SSP-PCR kit (Biotest, Landsteinerstrape, Denmark). T cell responses to a control antigen, “Keyhole Limpet Haemocyanin” (KLH) (Pierce, Rockford, IL, USA) are determined for positive control. PBMC were then frozen and stored in liquid nitrogen until needed. When necessary for use, the cells are quickly thawed in a 37 ° C water bath before transferring to 10 ml of pre-heated AIM V medium.
A group of 20 donors is selected to better represent the number and frequency of HLA-DR allotypes expressed in the world population. Analysis of allotypes expressed in the group versus those expressed in the world population revealed that coverage> 80% is achieved and that all major HLA-DR alleles (individual allotypes with a frequency> 5%, expressed in the world population) are well represented. A summary of donor haplotypes is provided in Figure 23, and a comparison of the frequency of donor allotypes used in the study versus those present in the world population is made. PBMCs from each donor are thawed, counted and viability assessed. The cells were revived and resuspended in AIMV culture medium to 4 to 6x106 PBMC / mL. For each donor, the culture volume was established in which a total of 1mL of cell proliferation lineage was added to a 24-well plate. A total of 1mL of each test dilute sample was added to the PBMC to give a final concentration of 50 µg / ml per antibody sample. For each donor, a positive control (cells incubated with 100 qg / mL of KLH) and a negative control (cells incubated with culture medium only) were also included. For the first 4 donors, an additional control was included to test the modulation of T cell responses by the test samples, where the test sample and KLH were added to the PBMC. Comparison of these samples with KLH alone can be used to assess the effects of test samples on proliferation. The cultures were incubated for a total of 8 days at 37 degrees Celsius, with 5% carbon dioxide. On days 5, 6, 7 and 8, the cells in each well are gently resuspended and three 100 µl aliquots are transferred to individual wells of a 96-well round bottom plate. Cultures are pulsed with 1pCi 3 [H] -Thy (Perkin Elmer, Waltham, MA) in 100uL of AIMV culture medium and incubated for an additional 18 hours before harvesting for filter element using a TomTec Mach III cell harvester. Counts per minute (cpm) for each well are determined by MeltilexTM scintillation counting (Perkin Elmer®, Waltham, Massachusetts, USA) on a Beta microplate counter (Perkin Elmer®, Waltham, Massachusetts, USA) in paralux, counting mode low bottom.
The results are expressed as stimulation indices, where the stimulation index (IE) is derived by dividing the proliferation score (counts per minute, for example, radioactivity), measured in the test antiendoglina antibody by the score measured in cells that are not are in contact with a test antiendoglina antibody. The entire basal cpm for the control wells is above the minimum dosing limit of 150 cpm.
For proliferation assays, an empirical threshold of a stimulation index (IE) equal to or greater than 2 (IE> 2) was previously established through which samples induce proliferative responses that above this threshold are considered positive (where included, boundary of IEsk1.90 are featured). Development of the extensive test and previous studies have shown that this is the minimum noise limit signal, allowing maximum sensitivity without detecting large numbers of false positive responses. Positive responses are defined by the following statistics and empirical limits: 1. Significance (p <0.05) of the response by comparing test wells cpm against the control wells medium using two unpaired samples from Student's t-test. 2. Stimulation index greater than 2 (IE> 2), where IE = half of test wells (cpm) / half of control wells (cpm).
In addition, the intra-assay variation is assessed by calculating the coefficient of variance and standard deviation (SD) of the raw data from identical cultures.
Results for the EpiScreen time course proliferation assay with the antiendoglina antibody are shown in Figure 24 and summarized as a table (Table 8). The chimeric antibody stimulated responses in 4 of 20 donors (20% of the study group) and, although two of the donor responses were borderline (1.92 and 1.95 for donors 11 and 17, respectively), they were significantly different from the historical (p <0.05). The humanized antibody VK1VH1 stimulated responses in 2 of 20 donors (10% of the group study), including a borderline response (1.91 for donor 20) that was significantly different from the historical (p <0.05). It is noted that donors 11 and 20 responded to both of these antibodies suggesting that there could be a shared T cell epitope. In contrast, none of the donors in the study group responded positively to the deimmunized antiendoglina antibody VK1AA VH1A2. The results with the KLH control antigen showed that there was a good correlation between the positive and negative results, indicating a high level of reproducibility in the assay.
Table 8. Stimulation T cells, as a measure of immunogenicity, culture induced with antiendoglina and KLH antibodies, where "P *" indicates a stimulation boundary above normal and "P" indicates a stimulation index greater than 2. EXAMPLE 35 T cell epitope mapping EpiScreen ™ EpiScreen ™ is an ex vivo technology for measuring T cell epitopes, on every antibody or for mapping the location of the T cell epitope sequence as described in more detail below.
EpiScreen donor selection Peripheral blood mononuclear cells (PBMC) are isolated from a healthy buffer donor community (from blood drawn within 24 hours), which are obtained from the UK National Blood Transfusion Service (Hospital Addenbrooke, Cambridge, UK) in accordance with the approval granted by the Local Hospital for Ethics in Addenbrooke Research. PBMCs are isolated from Lymphoprep density centrifugation buffer (Axis-Shield, Dundee, Scotland) and CD8 + T cells are depleted using CD8 + RossetteSepTM (StemCell Technologies, Inc.). Donors are characterized by identifying HLA-DR haplotypes using a type of Biotest HLA SSP-PCR kit (Biotest, Landsteinerstrape, Denmark). T cell responses to a control antigen, Keyhole Limpet Haemocyanin (KLH) (Pierce, Rockford, IL, USA) are determined for positive control. The group of 54 donors is selected to better represent the number and frequency of HLA-DR allotypes expressed in the world population. Analysis of allotypes expressed in the group against those expressed in the world population revealed that coverage> 80% is achieved and that all major HLA-DR alleles (individual allotypes with a frequency> 5%, expressed in the entire world population) are well represented . A summary of donor haplotypes is provided, and a comparison with the frequencies of donor allotypes used in the study versus those present in the world population is made.
Details of donors and haplotypes. Donor responses (IE) to KLH are tested in two independent experiments. Test 1 is run on newly isolated PBMCs and an antibody is the re-test in the current study. Answers that do not produce the same result (that is, positive or negative) in both tests are highlighted. Donors with very low baseline cpm (<150cpm) are excluded from the analysis.
EpiScreen analysis: proliferation assays EpiScreen ™ is used to test the overlapping peptides derived from the chimeric, humanized and humanized / de-immunized antibody sequence. Overlapping peptides are designed. A series of 128 x 15 mere peptides overlaid by 12 amino acids are synthesized together with 1 x 14 mere and 1 x 11 mere used to stimulate peripheral blood mononuclear cells (PBMC) derived from a group of 51 healthy donors, using mapping EpiScreen ™ of T cell epitopes. Individual peptides are tested in replicated cultures and responses are evaluated using T cell proliferation assays to identify the precise location of epitopes. PBMC of each donor is thawed, counted and evaluated for viability. The cells are revived in AIM V culture medium at room temperature (Invitrogen, Carlsbad, California) before adjusting the cell density to 2.5 x 106 CMSP / ml (cell proliferation line). The peptides are dissolved in DMSO (Sigma-Aldrich , St. Louis, MO, USA) for a final concentration of 10 mM. Peptide culture stocks are then prepared by dilution in AIM V culture medium to a final concentration of 5 μΜ. For each peptide and each donor, six times duplicate cultures are established by adding 100 μΐ of the peptide culture line to 100 μΐ of cell proliferation line in a 96-well flat bottom plate. Positive and negative control cultures are also established in hexaplicate. A total of 9 x 96 well plates are used for each donor, and each plate is sufficient to test 15 peptides with a negative control (individual carrier) six times duplicated. On the end plate, a positive control is added.
Cultures are incubated for a total of 6 days before 0.5 MCi 3 [H] -Thymidine (Perkin Elmer®, Waltham, Massachusetts, USA) is added to each well. Cultures are incubated for more than 18 hours before harvesting for filter element using a TomTec Mach III cell harvest. Counts per minute (cpm) for each well are determined by Meltilex ™ (Perkin Elmer □, Waltham, Massachusetts, USA) in a Beta Microplate Counter (Perkin Elmer ™, Waltham, Massachusetts, USA) in paralux, background counting mode low.
For proliferation assays, an empirical threshold of a stimulation index (IE) equal to or greater than 2 (IEÚ2), has previously been established through which samples induce proliferative responses that above this threshold are considered positive (where included, borders with IEsbl, 90 are highlighted). The development of the extensive test and previous studies have shown that this is the minimum noise limit signal, allowing maximum sensitivity without detecting large numbers of false positive responses. Positive responses are defined by the following statistics and empirical limits: 1. Significance (p <0.05) of the response by comparing cpm of test wells against the control well medium using two unpaired samples from the Student t test. 2. Stimulation index greater than 2 (IE> 2), where IE = half of test wells (cpm) / half of control wells (cpm).
In addition, the intra-assay variation is assessed by calculating the coefficient of variance and standard deviation (SD) of the raw data from identical cultures.
Proliferation assays are created on cultures duplicated six times (“Unadjusted data”). To ensure that the intra-assay variability is low, the data is also analyzed after removing the maximum and minimum cpm values (“adjusted data”) and the donor response IE is compared using both joint data. Details of donor IEs from both adjusted and unadjusted data sets are prepared. T cell epitopes are identified by calculating the average frequency of responses to all peptides in the study +2 x SD (history of response rate). Any peptide (s) that induce proliferation above this threshold is considered to contain a T cell epitope.
Peptide Analysis in Silico iTope ™ The peptide sequences that are positive in the proliferation assay are analyzed using ITopeTM antitope predictor software. This software predicts favorable interactions between amino acid side chains and specific peptide binding pockets with the Class II MCH binding groove. The location of the main binding residues is determined by generating 10-mer peptides that are overlapped by an amino acid that spans a long peptide sequence. Each 10-mer is tested against the Antitope database of MHC class II allotypes and based on their fit and interactions with MHC class II molecules. Peptides that produced a high binding score against a large number of alleles are considered to contain the 9mer nucleus.
Identification of T cell epitopes
All peptides identified using the EpiScreenTM analysis described above are successfully synthesized to be tested against 51 healthy donors (54 donors are originally selected; donors can be excluded from the analysis due to low baseline cpm, that is, below the cutoff value 150cpm). Positive responses are defined by donors who produced a significant (p <0.05) response with an IE> 2 to any given peptide. Frontier responses (a significant response (p <0.05) with an IE> 1.90) are also included. The outputs of the unadjusted and adjusted data analysis are compared to ensure that the intra-assay variability is low and that positive responses are not the result of false proliferation in individual wells. The results of each analysis showed little difference between the methods, consequently, the T cell epitope map is compiled using the adjusted data analysis. Donor stimulation indices from both unadjusted and adjusted analyzes are prepared. T cell epitopes are identified by calculating the average frequency of responses to all peptides under study plus twice the standard deviation (called the “historic response rate”). This is estimated to be 5.6% and is the equivalent to induce a positive response in three or more donors. Peptides that induce proliferative responses above this threshold are considered to contain a T cell epitope.
Immunogenicity testing of lead variants using EpiScreen ™ Lead variants are purified and compared to the wild-type polypeptide using EpiScreen T cell time course proliferation assays. A large number of healthy donors representing the world population according to the expression of HLA allotypes are selected from a donor library, as described above. Donors are stimulated with each protein in separate culture volumes containing 2 to 4x106 CD8 + T cells from depleted PBMC. Replicated samples (from T blasts) are removed from the culture volumes on days 5 to 8, proliferation and, together with IL-2 secretion (ELISPOT) is evaluated. For further validation of the evaluation between wild type and variants, the study group is complemented with response donors from the study of EpiScreenTM T cell mapping of epitopes (sufficient number of CD8 + T cells remaining from depleted PBMC provided). In order to confirm the loss of immunogenicity in lead variants, an analysis of immunogenicity with T cell assays by EpiScreenTM stroke time is performed as follows: (i) healthy donor buffer, (with> 80% allotypic coverage DRB1 for the world population) are used to isolate PBMCs that contain physiological levels of APC and CD4 + T cells; (ii) each donor is tested for positive control antigens, including keyhole limpet haemocyanin (a potent neoantigen); (iii) CD8 + T cells are depleted to exclude the detection of class I MHC response from restricted T cells; (iv) lead variants and wild-type polypeptides are compared against each other to assess relative ability to activate CD4 + T cells from T cells; (v) data are analyzed using test parameters previously validated with positive responses of IE> 2 supported by additional information, including statistics and frequency analysis; (vi) EpiScreenTM T cell stroke time data provides information on the magnitude and kinetics of T cell responses, for individual molecules; (vii) any remaining PBMC from donors that produce positive responses is archived and available for use in repeat assay studies, and (VIII) an assessment of the association between donor allotype and wild type polypeptide responses and any responses to variant connections.
Aspects of the present invention can be incorporated into other forms or carried out in other forms without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, and all changes that come within the meaning and range of equivalence are intended to be covered here. EXAMPLE 36 Cross-reactivity of Antiendoglina antibodies Antiendoglina antibody has been shown to be cross-reactive with human and mouse endothelial cells (Matsuno et al, 1999). Humanized / deimmunized antiendoglina antibodies are tested for their ability to bind to the human and murine endothelium by the radioiumunoassay (ERI) according to the method of Haruta et al, 1986. Briefly, purified antiendoglina antibodies are individually radiolabeled with 125I using iodine- gen and according to standard methods known to those skilled in the art. The radiolabeled anti-humanized / de-immunized antibodies are assayed for the average number of iodine atoms per IgG molecule. The counts per minute are compared by testing for anti-globin antibodies or IgG isotype control found in human and murine endothelial cell cultures. The binding of murine and human subconfluent endothelial cells can also be demonstrated using FITC-labeled antiendoglina antibodies and analyzed by Becton RNAkinson FACScan for comparison of mean fluorescence intensity according to the method of Matsuno et al, 1999. Endothelial binding murine can also be demonstrated by the image of the biodistribution of the radiolabeled antiendoglina antibody in mice with syngeneic tumors.
Briefly, immunocompetent mice are implanted with syngeneic 4T1 breast carcinomas. Tumors are allowed to grow to a palpable size and animals are treated with antibody chelated to a radioisotope, such as 64Cu. The distribution of labeled antiendoglina antibody in BALB / c tumor granules in mice by autoradiography or PET scanning is compared with the similar distribution of isotype-labeled controlled antibody. Tumor uptake of labeled antibody is reported in relation to the absorption of solid organs and blood.

权利要求:
Claims (18)
[1]
1. Antibody, or antigen-binding fragment thereof, that binds to endoglobin, characterized by the fact that it comprises: a heavy chain variable region having an amino acid sequence presented as SEQ ID NO: 89, and a variable region of light chain having an amino acid sequence shown as SEQ ID NO: 93, 94, 95 or 96; a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 90, and a light chain variable region having a sequence and amino acids shown as SEQ ID NO: 93 or 94; or a heavy chain variable region having an amino acid sequence shown as SEQ ID NO: 91, and a light chain variable region having a sequence and amino acids shown as SEQ ID NO: 93 or 94.
[2]
2. Antibody, or antigen binding fragment thereof, according to claim 1, characterized by the fact that it comprises a variable region of heavy chain having an amino acid sequence presented as SEQ ID NO: 89, and a variable region of light chain having an amino acid sequence shown as SEQ ID NO: 93.
[3]
3. Antibody, or antigen binding fragment thereof, according to claim 1, characterized by the fact that it comprises a heavy chain variable region having an amino acid sequence presented as SEQ ID NO: 89, and a variable region of light chain having an amino acid sequence shown as SEQ ID NO: 94, 95 or 96.
[4]
Antibody, or antigen binding fragment thereof, according to any one of claims 1 to 3, characterized in that the antigen binding fragment is a Fab fragment, a Fab 'fragment, an F fragment (ab ') 2, an Fv fragment, an scFv fragment, or single-stranded binding polypeptide.
[5]
5. Use of an antibody or antigen binding fragment thereof, as defined in any one of claims 1 to 4, characterized in that it is in the preparation of a medicament for the treatment of a disease related to angiogenesis in an individual.
[6]
6. Use according to claim 5, characterized in that the antibody, or antigen binding fragment thereof, is marked with a therapeutic label, a detectable label, or both.
[7]
7. Use, according to claim 5, characterized by the fact that the disease related to angiogenesis is an eye disease characterized by angiogenesis / neovascularization.
[8]
8. Use, according to claim 7, characterized by the fact that the eye disease is macular degeneration.
[9]
9. Use, according to claim 8, characterized by the fact that macular degeneration comprises age-related macular degeneration.
[10]
10. Use, according to claim 7, characterized by the fact that the eye disease is diabetic retinopathy.
[11]
11. Use, according to claim 5, characterized by the fact that the disease related to angiogenesis is a cancer or a metastasis of it.
[12]
12. Use according to claim 11, characterized by the fact that the cancer or its metastasis is a solid tumor.
[13]
13. Use, according to claim 11, characterized by the fact that the cancer or its metastasis is epithelial based.
[14]
14. Use, according to claim 11, characterized by the fact that the cancer is selected from lung cancer, melanoma, breast cancer, pancreatic cancer, ovarian cancer, uterine cancer, colorectal cancer, prostate cancer, kidney cancer, head cancer, liver cancer, neck cancer, sarcoma, myeloma, brain cancer, and lymphoma.
[15]
15. Use according to claim 14, characterized by the fact that brain cancer is a glioblastoma multiforme.
[16]
16. Use according to any one of claims 5 to 15, characterized in that it is for use in combination with one or more angiogenesis inhibitors.
[17]
17. Pharmaceutical composition characterized by the fact that it comprises the antibody or antigen binding fragment as defined in any one of claims 1 to 4, and an acceptable carrier or excipient.
[18]
18. Pharmaceutical composition according to claim 17, characterized in that the pharmaceutical composition further comprises one or more angiogenesis inhibitors.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-01-22| B65X| Notification of requirement for priority examination of patent application|

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2019-02-12| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

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2019-04-24| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-02-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-03-10| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/09/2010, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-08-10| B21F| Lapse acc. art. 78, item iv - on non-payment of the annual fees in time|Free format text: REFERENTE A 11A ANUIDADE. |

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2021-11-30| B24J| Lapse because of non-payment of annual fees (definitively: art 78 iv lpi, resolution 113/2013 art. 12)|Free format text: EM VIRTUDE DA EXTINCAO PUBLICADA NA RPI 2640 DE 10-08-2021 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDA A EXTINCAO DA PATENTE E SEUS CERTIFICADOS, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |

优先权:

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申请号 | 申请日 | 专利标题

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US24729009P| true| 2009-09-30|2009-09-30|

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US61/247,290|2009-09-30|

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US12/751,907|2010-03-31|

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US12/751,907|US8221753B2|2009-09-30|2010-03-31|Endoglin antibodies|

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PCT/US2010/050759|WO2011041441A1|2009-09-30|2010-09-29|Endoglin antibodies|

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