human or humanized monoclonal antibody, bispecific molecule, expression vector, transformed cell, co

专利摘要:
HUMAN OR HUMANIZED ISOLATED MONOCLONAL ANTIBODY THAT CONNECTS TO HUMAN CD27, BIESPECIFIC MOLECULES, ISOLATED ANTIBODY, EXPRESSION VECTOR, TRANSFORMED CELL, COMPOSITION, AND, METHODS FOR INDUCING OR INTENSIFYING AN EMOTION IN AN EMPTY RESPONSE CELLS THAT EXPRESS CD27, WHICH INHIBITS THE BINDING OF CD70 TO CD27 IN CELLS IN A SUBJECT HAVING A DISORDER, NEGATIVE REGULATION OF A T-CELL RESPONSE IN AN INDIVIDUAL HAVING A DISTRIBUTION, TO DETECT THE PRESENCE OF HEALTH IN A HEALTHY DEATH OF HEALTH IN HEALTH OF HEALTH. , TO INTENSIFY AN IMMUNE RESPONSE AGAINST AN ANTIGEN ON A SUBJECT IN NEED OF THE SAME. Isolated monoclonal antibodies that bind to human CD27 and related antibody-based compositions and molecules are disclosed. Therapeutic and diagnostic methods for using antibodies are also disclosed.
公开号:BR112012026227A2
申请号:R112012026227-0
申请日:2011-04-13
公开日:2020-08-04
发明作者:Tibor Keler；Henry C. Marsh；Lizhen He；Laura A. Vitale；Lawrence J. Thomas
申请人:Celldex Therapeutics, Inc.；
IPC主号:

专利说明:

“HUMAN OR HUMANIZED MONOCLONAL ANTIBODY, BIESPECIFIC MOLECULE, EXPRESSION VECTOR, TRANSFORMED CELL, COMPOSITION, AND, USES OF AN ANTIBODY.” Background of the invention 5 Interactions between T cells and antigen presenting cells involve a variety of accessory molecules that facilitate the generation of an immune response. One such molecule is CD27, which binds CD70 and belongs to the tumor necrosis factor receptor (TNF-R) superfamily (Ranheim EA, et al., Blood. June 15, 1995; 85 (12): 3556-65) . CD27 normally exists as a glycosylated type I transmembrane protein, often in the form of homodimers with a disulfide bridge, linking the two monomers. The disulfide bridge is from the extracellular domain near the membrane (Camerini et al., J Immunol. 147: 3165-69 (1991). CD27 can also be expressed in a soluble form (see, for example, van Oers MH, et al. , Blood. 1993 Dec.1; 82 (11): 3430-6 and Loenen WA, et al., Eur. J.
Immunol. 22: 447, 1992). Cross-linking of CD27 antigen to T cells provides a co-stimulation signal that, in conjunction with T-cell receptor cross-linking, can induce cell proliferation and immune activation.
CD27 is expressed in mature thymocytes, most CD4 + and CD8 + peripheral blood T cells, natural killer cells and B cells (Kobata T, et al., Proc.
Natl.
Acad.
Scie.
U S A. 1995 Nov 21; 92 (24): 11249-553). CD27 is also highly expressed in non-Hodgkin B-cell lymphomas and chronic B-cell lymphocytic leukemias (Ranheim EA, et al., Blood. 1995 Jun 15; 85 (12): 3556-65). In addition, increased levels of soluble CD27 protein have been identified in serum or disease activity sites in parasitic infections, cytomegalovirus (CMV) infection, sarcoidosis, multiple sclerosis and chronic B-cell lymphocytic leukemia (Loenen WA, et al., Eur. J.
Immunol. 22: 447, 1992). Monoclonal agonist antibodies against CD27 have recently been shown to promote T cell responses and show promise as anti-cancer therapy (see, for example, Sakanishi T, et al., Biochem Biophys.
Res.
Commun. 2010 Feb 18 and WO 2008/051424). However, while the results obtained so far have established CD27 as a useful target for immunotherapy, it is unknown that particular characteristics of anti-CD27 monoclonal antibodies are especially advantageous for therapeutic purposes.
As such, there is a need in the art for more insight into the specific functional properties that generate therapeutically effective anti-CD27 antibodies, as well as better therapeutic antibodies against CD27 that are more effective in treating or preventing disease.
Summary of the Invention The present invention provides, among others, isolated anti-CD27 antibodies, containing particular functional properties that may be related to the desirable and advantageous therapeutic effects.
Specifically, anti-CD27 monoclonal antibodies capable of up-regulating T cell mediated by immune responses (for example, as evidenced by inducing or improving antigen-specific T cell responses), which are particularly well suited for combination with therapies vaccine, were generated and characterized by means of the present invention.
In one embodiment, agonist anti-CD27 antibodies can improve the immune response against cancers or infectious diseases by combination with active vaccination, or by improving the endogenous immune response.
Said antibodies can also directly or indirectly induce the expression of cytokines.
In addition, anti-CD27 antibodies that down-regulate immune responses mediated by T cells, which are particularly suitable for the treatment of immune disorders, such as graft rejection, allergy and autoimmune diseases, have been generated and characterized.
In addition, anti-CD27 antibodies that inhibit the growth of cells that express CD27 by direct cell death mechanisms (for example, antibody dependent cell mediated cytotoxicity (ADCC) and / or complement dependent cell cytotoxicity (CDCC)), which are particularly effective in treating a variety of diseases that involve cell proliferation (eg, cancers), have been generated and characterized.
In one embodiment, the anti-CD27 antibodies of the present invention exhibit one or more of the following properties: (a) it blocks the binding of sCD70 to CD27 by at least about 70% at an antibody concentration of 10 µg / ml; (b) binds human CD27 with a Kd equilibrium dissociation constant of 10-9 M or less, or alternatively, a Ka equilibrium association constant of 10 + 9 M-1 or more; (c) induces specific complement-mediated cytotoxicity (CDC) of cells expressing CD27 of at least 10% at an antibody concentration of 3 µg / ml and approximately 6% serum rabbit complement; (d) induces antibody-dependent cell-mediated cytotoxicity (ADCC) specific lysis of cells expressing CD27 of at least 10% at an antibody concentration of 3 µg / ml and effector: target cells ratio of 75: 1; (e) improves mean survival by at least 20% in 5 severe combined immunodeficiency (SCID) in mice after inoculation of tumor cell in vivo (5 x 105 Raji cells or 1 x 106 Daudi cells) when administered at 0.3mg (ip ) at least twice a week for 3 weeks compared to mice to which the antibody is not administered; (f) induces or enhances antigen-specific immune responses in combination with a vaccine or endogenous antigen; (g) induces or enhances antigen-specific TH1 immune responses in combination with a vaccine or endogenous antigen; (h) induces or increases proliferation or activation of antigen-specific T cells in combination with a vaccine or endogenous antigen; (i) reduces or inhibits T cell proliferation or activation; (j) induces or increases T cell activity when combined with simultaneous, separate or sequential TCR activation; (k) blocks the binding of sCD70 to CD27 by at least about 70% at an antibody concentration of 10 µg / ml and reduces or inhibits T cell activation when it is unable to bind to, or containing reduced binding to Fc receptors; (l) results in less than 50% depletion of CD3 + T cells (in addition to NK cells) in monkeys when administered at 3 mg / kg (i.v.) for the period of 29 days immediately after administration; or (m) results in less than 50% depletion of memory B cells in monkeys when administered at 3 mg / kg (i.v.) for the period of 29 days immediately after administration.
In a particular embodiment, the antibodies of the invention exhibit combinations of these functional properties.
Thus, in one aspect, the invention provides anti-CD27 antibodies that induce and / or reinforce an immune response (for example, a T cell-mediated immune response). In another embodiment, the antibodies inhibit the binding of CD70 to CD27 in cells.
Specific antibodies containing these combinations of properties include mAb 1F5 comprising heavy and / or light variable region sequences comprising SEQ ID NOs: 37 and / or 43, respectively). Alternatively, the antibodies do not inhibit the binding of CD70 to CD27 in cells.
Specific antibodies containing these combinations of properties include mAb 3H8 comprising heavy and / or light variable region sequences comprising SEQ ID NOs: 7 and / or 13, respectively, or 7 and / or 19, respectively). Said anti-CD27 antibodies can also be linked to a second molecule (for example, as a bispecific molecule), containing a binding specificity that is different from antibodies, such as a T cell receptor (for example, CD3, CD25, CD137, CD154_), or an Fc receptor (for example, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB1 (CD32), FcγRIIB2 (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcεRI, FcεRII (CD23), F23 CD89) Fcα / μR and FcRn), or an NK receptor (for example CD56), or a B cell receptor (for example CD19, CD20). Antibodies intended to be used to induce or improve immune responses according to the present invention can have a functional Fc domain, allowing binding to Fc receptors and can include a mutant Fc domain, with increased levels of binding to Fc receptors.
In another aspect, the invention provides anti-CD27 antibodies that down-regulate T cell-mediated immune responses by inhibiting the binding of CD27 to CD70 in cells that express these proteins.
In a particular embodiment, the antibodies inhibit the binding of soluble CD70 (sCD70) to cells that express at least about 70% CD27. Specific antibodies within this class include, for example, mAb comprising sequences of variable regions of heavy and / or light chain 5 comprising SEQ ID NOs: 37 and / or 43 (mAb 1F5), SEQ ID NOs: 49 and / or 55 (mAb 1H8), SEQ ID NOs: 103 and / or 109 (mAb 3 H 12). In yet another aspect, the invention provides anti-CD27 antibodies that induce or improve the effector cell function (for example, the cell killing via or ADCC and / or CDC). In one embodiment, the antibody induces at least about 30% specific lysis of CD27 expressing cells through ADCC at an antibody concentration of 10µg / ml and / or induces at least about 30% CDC of cells expressing CD27 at a concentration of 10 µg / ml.
Specific antibodies within this class having an ADCC effector function include, for example, (for example, mAb) comprising sequences of heavy and / or light chain variable regions comprising SEQ ID NOs: 61 and / or 67 (mAb 1G5), SEQ ID NOs : 85 and / or 91, 85 and / or 97 (mAb 3A10), SEQ ID NOs: 37 and / or 43 (mAb 1F5), SEQ ID NOs: 7 and / or 13, 7 and / or 19 (mAb 3H8) , SEQ ID NOs: 49 and / or 55 (mAb 1H8), or SEQ ID NOs: 103 and / or 109 (mAb 3 H 12). In another embodiment, antibodies also inhibit the binding of CD70 to CD27 in cells.
Specific antibodies containing these combinations of functions include, for example, (for example, mAb comprising heavy and / or light chain variable region sequences comprising SEQ ID NOs: 37 and / or 43 (mAb 1F5), SEQ ID NOs: 49 and / or 55 (mAb 1H8), SEQ ID NOs: 103 and / or 109 (mAb 3H12) Alternatively, the antibody induces ADCC and / or CDC as described above, but does not inhibit the binding of CD70 to CD27 in cells.
Specific antibodies containing these characteristics include, for example, mAb comprising heavy and / or light chain variable region sequences comprising SEQ ID NOs: 61 and / or 67 (mAb 1G5), SEQ ID NOs: 85 and / or 91, 85 and / or 97
(mAb 3A10), SEQ ID NOs: 7 and / or 13, 7 and / or 19 (mAb 3H8). Anti-CD27 antibodies capable of inducing or improving effector cell function (for example, ADCC and / or CDC) can also be constructed to include an Fc region that adequately contributes binding specificity to a specific Fc receptor (for example , FcγRI (CD64), FcγRIIA (CD32), FcγRIIB1 (CD32), FcγRIIB2 (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcεRI, FcεRII (CD23), FcαRI (CD89), Fcα / μ. In another embodiment, a method is provided to improve an immune response against an antigen in an individual in need of administration of the individual: i) a human or humanized anti-CD27 antibody and ii) an antigen, in which the anti-CD27 antibody is administered separately from and before the antigen is administered.
Typically in said method the anti-CD27 antibody can be administered between at least 2 and 96 hours before the antigen.
For example, in said method, the anti-CD27 antibody can be administered at least 2 hours before the antigen, for example, at least 12 hours before the antigen, properly at least 24 hours before the antigen, at least 48 hours before the antigen or at least 72 hours before the antigen in which the TLR agonist is a TLR3 agonist. In another embodiment, a method is provided to improve an immune response against an antigen in an individual in need of it by simultaneously, separately or sequentially administering to the individual: i) an anti-CD27 antibody; ii) a TLR agonist; and iii) optionally, the antigen.
In a preferred embodiment of said method the agonist is a TLR agonist and a TLR3 agonist, for example, among others, Poly IC: LC.
CD27-expressing cells include any and all CD27-expressing cells, including but not limited to B cells, NK cells and T cells.
In a particular embodiment, cells that express CD27 include cancer cell lines, such as Jurkat cells, Raji cells, Ramos cells and Daudi cells.
In another embodiment, the cells that express CD27 are tumor cells or cancer cells.
In another embodiment, cells that express CD27 include B cells, NK cells and T cells including T cells that are found in infiltrating tumors, also called tumor infiltrating lymphocytes.
The specific antibodies of the present invention comprise the variable regions of heavy and light chains that use certain human germ lines, that is, they are encoded by the germ line genes, but include genetic rearrangements and mutations, for example, the somatic mutations that occur during maturation of antibodies.
In one embodiment, the variable region of the antibody heavy chain of the present invention is derived from a human germline gene 3-7 or 3-33. In another embodiment, the variable region of the antibody light chain is derived from a human germline gene 3-20, 3-11, 24, 1D-16, 1-13. In a particular embodiment, the antibody heavy chain variable region is derived from a VH 3-7 or VH 3-33 germline gene and the antibody light chain variable region is derived from a VK 3 human germline gene -20, VK 3-11, VK 1D-16, or VK 1-13. A VH 3-33 germline sequence is provided (Gene Bank Accession No. AAP44382) as follows: 1 vqlvesgggv vqpgrslrls caasgftfst ygmhwvrqap gkglewvaii wfdgsntyya 61 dsvrgrftis rdssrktyy NO (3s) germline VH 3-7 is provided (Gene Bank Accession No. AAP44389) as follows: 1 vqlvesgggl vqpggslrls caasgftfsn symtwvrqap gkglewvani kpdgsdknyi
61 nsvrgrftis rdnaekssyl qmnslraedt aiyycvt (SEQ ID NO: 4) In another embodiment, the sequence of the variable region of the heavy chain CDR3 is selected from the group consisting of SEQ ID NOs: 10, 28, 40, 52, 64, 76, 88, 106, and conservative sequence modifications thereof (for example, conservative amino acid substitutions). The 5 antibodies can also include a CDR3 light chain variable region sequence selected from the group consisting of SEQ ID NOs: 16, 22, 34, 46, 58, 70, 82, 94, 100, 112, and the modifications conservative sequences.
In another embodiment, the heavy chain sequences CDR2 and CDR1 are selected from SEQ ID NOs: 9, 27, 39, 51, 63, 75, 87, 105, and SEQ ID NOs: 8, 26, 38, 50, 62, 74, 86, 104, respectively, and conservative sequence modifications thereof.
The light chain sequences CDR2 and CDR1 are selected from SEQ ID NOs: 15, 21, 33, 45, 57, 69, 81, 93, 99, 111, and SEQ ID NOs: 14, 20, 32, 44, 56, 68, 80, 92, 98, 110, respectively, and conservative sequence modifications thereof.
In yet another embodiment, the invention provides an isolated antibody that binds CD27 and includes CDR1, CDR2 and CDR3 heavy chain variable region sequences from the group consisting of: (i) a CDR1 heavy chain variable region comprising SEQ ID NO: 38; a heavy chain variable region CDR2 comprising SEQ ID NO: 39; a heavy chain variable region CDR3 comprising SEQ ID NO: 40; a light chain variable region CDR1 comprising SEQ ID NO: 44; a light chain variable region CDR2 comprising SEQ ID NO: 45;
a light chain variable region CDR3 comprising SEQ ID NO: 46; or conservative sequence modifications thereof; (ii) a heavy chain variable region CDR15 comprising SEQ ID NO: 50; a heavy chain variable region CDR2 comprising SEQ ID NO: 51; a heavy chain variable region CDR3 comprising SEQ ID NO: 52; a light chain variable region CDR1 comprising SEQ ID NO: 56; a light chain variable region CDR2 comprising SEQ ID NO: 57; a light chain variable region CDR3 comprising SEQ ID NO: 58; or conservative sequence modifications thereof; (iii) a heavy chain variable region CDR1 comprising SEQ ID NO: 104; a heavy chain variable region CDR2 comprising SEQ ID NO: 105; a heavy chain variable region CDR3 comprising SEQ ID NO: 106; a light chain variable region CDR1 comprising SEQ ID NO: 110; a light chain variable region CDR2 comprising SEQ ID NO: 111; a light chain variable region CDR3 comprising SEQ ID NO: 112; or conservative sequence modifications thereof;
(iv) a heavy chain variable region CDR1 comprising SEQ ID NO: 86; a heavy chain variable region CDR2 comprising SEQ ID NO: 87; 5 is a heavy chain variable region CDR3 comprising SEQ ID NO: 88; a light chain variable region CDR1 comprising SEQ ID NO: 92 or 98; a light chain variable region CDR2 comprising SEQ ID NO: 93 or 99; a CDR3 light chain variable region comprising SEQ ID NO: 94 or 100, or conservative sequence modifications thereof; (v) a heavy chain variable region CDR1 comprising SEQ ID NO: 26; a heavy chain variable region CDR2 comprising SEQ ID NO: 27; a heavy chain variable region CDR3 comprising SEQ ID NO: 28; a light chain variable region CDR1 comprising SEQ ID NO: 32; a light chain variable region CDR2 comprising SEQ ID NO: 33; a light chain variable region CDR3 comprising SEQ ID NO: 34; or conservative sequence modifications thereof; (vi) a heavy chain variable region CDR1 comprising SEQ ID NO: 74; a heavy chain variable region CDR2 comprising
SEQ ID NO: 75; a heavy chain variable region CDR3 comprising SEQ ID NO: 76; a light chain variable region CDR1 comprising 5 SEQ ID NO: 80; a light chain variable region CDR2 comprising SEQ ID NO: 81; a light chain variable region CDR3 comprising SEQ ID NO: 82; or conservative sequence modifications thereof; (vii) a heavy chain variable region CDR1 comprising SEQ ID NO: 8; a heavy chain variable region CDR2 comprising SEQ ID NO: 9; a heavy chain variable region CDR3 comprising SEQ ID NO: 10; a light chain variable region CDR1 comprising SEQ ID NO: 14 or 20; a light chain variable region CDR2 comprising SEQ ID NO: 15 or 21; a CDR3 light chain variable region comprising SEQ ID NO: 16 or 22, or conservative sequence modifications thereof; and (viii) a heavy chain variable region CDR1 comprising SEQ ID NO: 62; a heavy chain variable region CDR2 comprising SEQ ID NO: 63; a heavy chain variable region CDR3 comprising SEQ ID NO: 64;
a light chain variable region CDR1 comprising SEQ ID NO: 68; a light chain variable region CDR2 comprising SEQ ID NO: 69; 5 a CDR3 light chain variable region comprising SEQ ID NO: 70; or conservative sequence modifications thereof.
In another embodiment, the variable region sequence of the heavy chain CDR3 comprises an amino acid sequence selected from the consensus sequence: R (G, E, D) (S, L, G, -) (G, L, T, W, -) (N, A, T, H, -) (V, T, -) (M, P, -) (G, V, -) (R, -) (G, M, -) (D , H, L, T, W) (A, G, N, W) (D, F, V, Y) (F, L) (D, E) (H, I, L, Y) (SEQ ID NO : 113), where “-” denotes the option of no amino acid residue being present in the consensus position.
The antibodies may further include a CD3 light chain variable region sequence comprising an amino acid sequence selected from the consensus sequence: Q (F, R, Y) (N, S) (N, T, S) (Y, W) P (F, L, P, R) T (SEQ ID NO: 114), where “-” denotes the option of no amino acid residue being present in the consensus position.
In another embodiment, the variable region sequence of the heavy chain CDR2 comprises an amino acid sequence selected from the consensus sequence: I (K, W) (Y, N, Q) DGS (E, N) (K, Q) (SEQ ID NO: 115), where “-” denotes the option of no amino acid residue being present in the consensus position, and the light chain variable region sequence CDR2 comprises an amino acid sequence selected from the consensus sequence: (A, D) AS (SEQ ID NO: 116). In another embodiment, the variable region sequence of the heavy chain CDR1 comprises an amino acid sequence selected from the consensus sequence: GF (T, S) (F, L) (S, N) (I, S, H) ( Y, H) (SEQ ID NO: 117); and the CDR1 light chain variable region sequence comprises an amino acid sequence selected from the consensus sequence: Q (D, G, S) (I, V)
(D, S) (R, S) (A, W, Y) (SEQ ID NO: 118). In another embodiment, isolated antibodies of the invention bind to human CD27 and include a heavy chain variable region including an amino acid sequence selected from the group consisting of 5 SEQ ID NOs: 6, 7, 24, 25, 36, 37, 48 , 49, 60, 61, 72, 73, 84, 85, 102, 103, and conservative sequence modifications thereof.
The antibody may further include a light chain variable region including an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 18, 19, 30, 31, 42, 43, 54, 55, 66, 67, 78, 79, 90, 91, 96, 97, 108, 109, and conservative sequence modifications thereof.
In yet another embodiment, isolated antibodies of the invention bind to human CD27 and include a heavy chain variable region and a light chain variable region including an amino acid sequence selected from the group consisting of SEQ ID NOs: (a) SEQ ID NOs : 37 and / or 43, respectively, and conservative sequence modifications thereof; (b) SEQ ID NOs: 49 and / or 55, respectively, and conservative sequence modifications thereof; (c) SEQ ID NOs: 103 and / or 109, respectively, and conservative sequence modifications thereof; (d) SEQ ID NOs: 85 and / or 91 and / or 97, respectively, and conservative sequence modifications thereof; (e) SEQ ID NOs: 25 and / or 31, respectively, and conservative sequence modifications thereof; (f) SEQ ID NOs: 73 and / or 79, respectively, and conservative sequence modifications thereof; (g) SEQ ID NOs: 7 and / or 13 and / or 19, respectively, and conservative sequence modifications thereof; and (h) SEQ ID NOs: 61 and / or 67, respectively, and conservative sequence modifications thereof; Isolated antibodies that include heavy and light chain variable regions containing at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 5 97%, or at least 98%, or at least 99%, or more of sequence identity with any of the above sequences are also included in the present invention.
Intermediate ranges of the aforementioned values, for example, heavy and light chain variable regions with at least 80-85%, 85-90%, 90-95% or 95-100% sequence identity with any of the above sequences said are also intended to be included by the present invention.
Also encompassed by the present invention are isolated antibodies that compete for binding to CD27 with the antibodies of the present invention.
In a particular embodiment, the antibody competes for binding to CD27 with an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 37 and 43, SEQ ID NOs: 49 and 55, SEQ ID NOs: 103 and 109, SEQ ID NOs: 85 and 91, SEQ ID NOs: 85 and 97, SEQ ID NOs: 25 and 31, SEQ ID NOs: 73 and 79, SEQ ID NOs: 7 and 13, SEQ ID NOs: 7 and 19, SEQ ID NOs: 61 to 67, respectively, or amino acid sequences of at least 80% identical to the same.
In another embodiment, the antibody competes for binding to CD27 with an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 37 and 43 (1F5), SEQ ID NOs: 49 and 55 (1H8) or SEQ ID NOs: 103 and 109 (3H12). In another embodiment, the antibody competes for binding to CD27 with an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 25 and 31 (2C2), SEQ ID NOs: 7 and 13 (3H8), SEQ ID NOs: 7 and 19 (3H8), SEQ ID NOs: 61 to 67 (1G5) or SEQ ID NOs: 73 and 79 (2G9). In yet another embodiment, the antibody competes for binding to CD27 with an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 85 and 91 (3A10) or SEQ ID NOs: 85 and 97 (3A10). 5 Other antibodies of the invention bind to an epitope on CD27 recognized by the antibodies described herein.
In another particular embodiment, the antibody binds to an epitope on CD27 recognized by an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 37 and 43, SEQ ID NOs: 49 and 55 , SEQ ID NOs: 103 and 109, SEQ ID NOs: 85 and 91, SEQ ID NOs: 85 and 97, SEQ ID NOs: 25 and 31, SEQ ID NOs: 73 and 79, SEQ ID NOs: 7 and 13, SEQ ID NOs: 7 and 19, SEQ ID NOs: 61 to 67, respectively, or amino acid sequences of at least 80% identical to the same.
In another embodiment, the antibody binds to an epitope on CD27 recognized by an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 37 and 43 (1F5), SEQ ID NOs: 49 and 55 (1H8) or SEQ ID NOs: 103 and 109 (3H12). In another embodiment, the antibody binds to an epitope on CD27 recognized by an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 25 and 31 (2C2), SEQ ID NOs: 7 and 13 (3H8), SEQ ID NOs: 7 and 19 (3H8), SEQ ID NOs: 61 to 67 (1G5) or SEQ ID NOs: 73 and 79 (2G9). In yet another embodiment, the antibody binds to an epitope on CD27 recognized by an antibody comprising variable regions of heavy and / or light chain comprising the amino acid sequences set out in SEQ ID NOs: 85 and 91 (3A10) or SEQ ID NOs: 85 and 97 (3A10). The antibodies of the invention can be full-length, for example, any of the following isotypes: IgG1, IgG2, IgG3, IgG4,
IgM, IgA1, IgA2, IgAsec, IgD, and IgE.
Alternatively, the antibodies can be fragments such as an antigen-binding portion or a single chain antibody (for example, a Fab, F (ab ') 2, Fv fragment, a single chain Fv fragment, an isolated complementarity determining region 5 (CDR) or a combination of two or more isolated CDRs). The antibodies can be any type of antibody, including, but not limited to, human, humanized, and chimeric antibodies.
Tumor antigens employed by the present invention (for example, in a vaccine, used in combination with an anti-CD27 antibody of the invention) includes any antigen or determinant that is present in (or associated with) a tumor cell and not typically in normal cells , or an antigen or antigenic determinant that is present in or associated with tumor cells in larger quantities than normal (non-tumor) cells, or an antigen or antigenic determinant that is present in tumor cells in a different way than that found in cells normal (non-tumor). Said antigens include tumor-specific antigens, including tumor-specific membrane antigens, tumor-associated antigens, including tumor-associated membrane antigens, embryonic antigens in tumors, growth factor receptors, growth factor ligands and any type of antigen that is associated with cancer.
A tumor antigen can be, for example, an epithelial cancer antigen (for example, breast, gastrointestinal, lung), a prostate-specific cancer antigen (PSA), or a prostate-specific membrane antigen (PSMA), a bladder cancer, lung cancer antigen (e.g., small cell lung), colon cancer antigen, ovarian cancer antigen, brain cancer antigen, gastric cancer antigen, cell cell carcinoma antigen kidney, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, or a colorectal cancer antigen.
For example, the antigen may include a tumor antigen, such as βhCG, gp100 or Pmel17, CEA, gp100, TRP-2, NY-BR-1, NY-CO-58, MN (gp250), idiotype, tyrosinase, telomerase, SSX2, MUC-1, MAGE-A3, and the antigen associated with high molecular weight melanoma 5 (HMW-MAA) MART1, melan-A, EGFRvIII, NY-ESO-1, MAGE-1, MAGE-3, WT1, Her2, or mesothelin.
Other antigens used in the present invention (for example, in a vaccine, used in combination with an anti-CD27 antibody of the invention) include antigens from infectious disease pathogens, such as viruses, bacteria, parasites and fungi, examples of which are disclosed here .
The invention also provides a bispecific molecule comprising an antibody of the invention attached to a second functional fraction, containing a different binding specificity than said antibody.
For example, in one embodiment, the second molecule can bind to a T cell receptor (for example, CD3, CD40). Compositions including an antibody or bispecific molecule described herein, formulated with a pharmaceutically acceptable carrier, are also provided.
The compositions can also include an adjuvant, immunostimulatory agent (for example, CD40 linker, FLT 3 linker, cytokines, colony stimulating factors, an anti-CTLA-4 antibody, anti-PD1 antibody, anti-41BB antibody, anti OX- 40, LPS (endotoxin), ssRNA, dsRNA, Calmette-Guerin bacillus (BCG), Levamisol hydrochloride, intravenous immunoglobulins and a Toll-like receptor agonist (TLR) (for example, a TLR3 agonist such as Poly IC, a TLR4 agonist, a agonist TLR5, agonist TLR7, agonist TLR8, and agonist TLR 9)), immunosuppressive agent, other antibody or antigen.
Exemplary antigens include, but are not limited to, a component of a pathogen, a tumor antigen (for example, βhCG, gp100 or Pmel17, HER2 / neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan-A, NY -ESO-1, NY-
BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE -3, MAGE -A3, tyrosinase, Telomerase, SSX2 antigens, MUC-1 antigens and germ cell-derived tumor antigens), an infectious disease antigen (for example, viral, bacterial and parasitic antigens) an allergen, or an autoantigen.
Any of the antigens disclosed herein can be included in a composition of the invention.
Nucleic acid molecules encoding the antibodies of the invention are further included by the invention, as well as expression vectors comprising said nucleic acids and host cells comprising said expression vectors.
For example, in one embodiment, the invention provides an isolated monoclonal antibody that binds to human CD27, wherein the antibody comprises a variable region of the heavy chain and a variable region of the light chain encoded by nucleic acid sequences selected from the group consisting of in: (a) SEQ ID NOs: 5 and 11, respectively; (b) SEQ ID NOs: 5 and 17, respectively; (c) SEQ ID NOs: 23 and 29, respectively; (d) SEQ ID NOs: 35 and 41, respectively; (e) SEQ ID NOs: 47 and 53, respectively; (f) SEQ ID NOs: 59 and 65, respectively; (g) SEQ ID NOs: 71 and 77, respectively; (h) SEQ ID NOs: 83 and 89, (i) SEQ ID NOs: 83 and 95; (j) SEQ ID NOs: 101 and 107, respectively or nucleic acid sequences containing at least 90% identity to the (a) - (h) sequences. In another embodiment, the present invention provides methods for inducing or improving an immune response (for example, a T cell-mediated immune response, and / or an NK-mediated response and / or B cell-mediated immune response) against an antigen in an individual by administering an effective amount of an antibody (for example, a full length antibody), composition or bispecific molecule described herein.
Said methods are particularly well adapted for use in vaccine therapies.
The antibodies and other compositions of the present invention can also be used to inhibit the growth of cells that express CD27 by contacting the cells with an antibody or composition of an effective amount to inhibit the growth of cells that express CD27 (for example, in the treatment of cancer). Antibodies useful in inhibiting the growth of cells that express CD27 include full-length antibodies and fragments thereof, as well as antibodies that contain a second binding specificity for an Fc receptor.
In one embodiment, cells expressing CD27 are contacted with an antibody in the presence of effector cells under conditions sufficient to induce antibody dependent cell cytotoxicity (ADCC) of target cells (eg, the antibody induces at least about 40% specific lysis) of cells expressing CD27 at a concentration of 10 µg / ml and comprises SEQ ID NOs: 61, 67, 85, 91, 97, 37, and / or 43). In another embodiment, cells are contacted with an antibody in conditions sufficient to induce complement-mediated cytotoxicity (CDC) of cells (for example, the antibody induces at least about 40% complement-mediated cytotoxicity (CDC) of cells that express CD27 at a concentration of 10 µg / ml and comprises SEQ ID NOs: 7, 13, 19, 49, 55, 103, and / or 109). In another embodiment, the antibody used to inhibit the growth of CD27-expressing cells may still have (or be devoid of) additional functional characteristics.
For example, the antibody can also inhibit the binding of CD70 to CD27 in cells expressing these proteins (for example, an mAb comprising sequences of variable regions of heavy and / or light chain comprising SEQ ID NOs: 37 and / or 43 (mAb 1F5), SEQ ID NOs: 49 and / or 55 (mAb 1H8), or SEQ ID NOs: 103 and / or 109 (mAb 3H12). Alternatively, the antibody may not inhibit the binding of CD70 to CD27 in said cells (for example for example, an mAb comprising sequences of heavy and / or light chain variable regions comprising SEQ ID NOs: 61 and / or 67 (mAb 1G5), SEQ ID NOs: 85 and / or 91, 85 and / or 97 (mAb 3A10) , or SEQ ID NOs: 7 and / or 13, 7 and / or 19 (mAb 3H8) .5 Cells expressing CD27 include any and all cells expressing CD27, including but not limited to B cells, NK cells and T cells .
In a particular embodiment, cells that express CD27 include cell lines such as Jurkat cells, Raji cells, Ramos cells and Daudi cells.
In another embodiment, the cells that express CD27 are tumor cells or cancer cells.
In another embodiment, cells that express CD27 include B cells, NK cells, T cells that are found in infiltrating tumors, also called tumor infiltrating lymphocytes.
The methods of inhibiting the growth of CD27-expressing cells that are described here can be used to treat and prevent a wide variety of diseases and disorders.
For example, in one embodiment, methods can be used to treat or prevent cancer (for example, a cancer selected from the group consisting of leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblast promyelocytic monocytic erythroleukemia, chronic leukemia, myelocytic leukemia chronic (granulocytic), chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, macroglobulinemia Waldenstrom's disease, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangio-
leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat cell carcinoma, papillary gland carcinoma, sebaceous gland carcinoma , papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, lung carcinoma small cell, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, neuroblastoma, neuroblastoma, neuroblastoma basal cell carcinoma, biliary tract cancer, cancer bladder cancer, bone cancer, central nervous system and brain cancer (CNS), cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer and neck, gastric cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; cancer of the oral cavity (for example, lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancer). Preferred cancers include CD27-expressing tumors selected from the group consisting of chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B cell lymphoma.
In another embodiment, the methods can be used to treat or prevent a bacterial, fungal,
viral or parasitic.
The present invention further provides methods for inhibiting the binding of CD70 to CD27 in cells in an individual containing a disorder by administering antibodies to the individual or compositions as described herein, as well as methods for down-regulating a T cell response in an individual containing a disorder by administering to an individual the antibodies or compositions described herein.
These methods are ideal for use in the treatment of immune disorders, such as graft rejection, autoimmune diseases and allergy.
Antibodies useful in these methods include Fab fragments, as well as a mutant Fc region so that the antibody does not bind, or has significantly reduced binding to Fc receptors.
In a particular embodiment, the antibody comprises heavy and / or light chain variable regions comprising SEQ ID NOs: 37 and / or 43 (mAb 1F5), SEQ ID NOs: 49 and / or 55 (mAb 1H8), or SEQ ID NOs: 103 and / or 109 (mAb 3H12). The methods described here for inhibiting the binding of CD70 to CD27 in cells and for down regulation of a T cell response can be used to treat a wide variety of diseases and disorders, including, but not limited to, graft rejection, allergy and autoimmune diseases.
In a particular embodiment, the disease is an autoimmune disease (for example, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease and other inflammatory bowel diseases such as ulcerative colitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polyposis, pernicious anemia, idiopathic Addison's disease, infertility associated with autoimmune, glomerulonephritis, growing glomerulonephritis, proliferative glomerulonephritis, bullous pemphigus,
Sjogren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes mellitus, autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome, autoimmune uveoretinitis, 5 Guillainter's syndrome, and Arilliosclerosis Alzheimer's disease). The present invention further provides for bispecific antibodies, compositions and bispecific molecules described herein.
For example, in one embodiment, the invention provides for the use of a bispecific antibody, composition or molecule in the production of a drug to induce or improve an immune response against an antigen in an individual.
In other embodiments, the invention provides for the use of an antibody or composition in the production of a medicament to inhibit the growth of CD27-expressing cells, the use of an antibody or composition in the production of a medicament to inhibit CD70 binding the CD27 in cells in an individual containing a disorder and the use of an antibody or composition in the production of a drug to down-regulate a T cell response in an individual containing a disorder.
The present invention further includes a bispecific antibody, composition or molecule for use in inducing or improving an immune response against an antigen in an individual, an antibody or composition for use in inhibiting the growth of cells that express CD27, an antibody or composition for use in inhibiting the binding of CD70 to CD27 in cells in an individual containing a disorder and an antibody or composition for use in down-regulating a T cell response in an individual containing a disorder.
The present invention also provides methods for detecting the presence or absence of CD27 in a biological sample by (1) contacting a biological sample with an antibody described here (where the antibody is labeled with a detectable substance) and (2) detecting the bound antibody The
CD27. Also within the scope of the invention are kits comprising the compositions (for example, antibodies and / or bispecific molecules) of the invention and, optionally, instructions for use.
The kit can still contain at least one additional reagent, such as a cytokine or complement to one or more additional antibodies of the invention.
Other features and advantages of the present invention will be apparent from the following detailed description and claims.
Brief Description of the Drawings Figure 1 describes the affinity and kinetic parameters for mAbs 1G5, 1H8, 3H12, 3H8, 2G9, 1F5, 3A10, 2C2, ms 1A4, ms 9F4 and ms M-T271 as determined by Biacore ™ software BiaEvaluation (Biacore AB) with human recombinant CD27 immobilized on the chip.
Figure 2 is a graph showing the binding of anti-human CD27 antibodies (2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) to purified human recombinant CD27 using ELISA.
Figure 3 is a graph showing ELISA binding of 1F5 to purified recombinant human or monkey (monkey) CD27. Figure 4 is a graph showing the effect of anti-human CD27 antibodies (2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) and MsIgG (1A4, 9F4 and M-T271) on the binding of soluble CD70 (sCD70 ) the CD27 protein (shown as% block) by ELISA.
Figure 5 is a flow cytometric analysis of the 1F5 binding to human lymphoblastoid cell lines, and blocking the binding to sCD70. Figures 6A-D are graphs showing the binding of human anti-CD27 antibodies (2C2, 3H8, and 1F5) to CD27 in Jurkat cells (Figure 4A), Raji cells (Figure 4B), Ramos cell (Figure 4C), and Daudi cells (Figure 4D) as assessed by flow cytometry.
Figure 7 is a graph showing the binding of human anti-CD27 antibodies (2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) to CD27 in Daudi cells as assessed by flow cytometry.
Figure 8 is a bar graph showing the results of an anti-CD27 cross-blocking ELISA experiment, demonstrating that antibodies 1F5, 1H8 and 3H12 are capable of cross-blocking each other and thus bind to the same epitope.
Figure 9 is a bar graph showing the results of an anti-CD27 cross-blocking ELISA experiment, demonstrating that antibodies 2C2, 3H8, 1G5 and 2G9 are capable of cross-blocking each other and thus bind to the same epitope.
Figure 10 is a bar graph showing the results of an anti-CD27 cross-blocking ELISA experiment, demonstrating that the binding of antibody 3A10 to CD27 is not entirely cross-blocking by any of the other anti-CD27 antibodies tested, thus, indicating that 3A10 binds to a single epitope, but binding to 3A10 is partially blocked by 1F5, 1H8 and 3H12 antibodies, indicating that the epitope for 3A10 may be close to the epitope bound by 1F5, 1H8 and 3H12 antibodies. Figure 11 is a graph depicting the results of a complement dependent cell cytotoxicity (CDCC) assay using mAbs 1F5, 2C2, 3H8, 1G5, 1H8, 2G9, 3A10 and 3H12. Figure 12 is a graph describing the results of another complement dependent cell cytotoxicity (CDCC) assay using mAb 1F5 Figure 13 is a graph describing the results of an antibody dependent cell mediated cytotoxicity (ADCC) assay using mAbs 2C2, 1F5, 3H8, 1G5, 1H8, 2G9, 3A10, 3H12, Rituxan and HuIgG.
Figure 14 is a graph depicting the results of another antibody dependent cell mediated cytotoxicity (ADCC) using 1F5 mAb. Figure 15 is a VH sequence alignment of 5 human anti-CD27 antibodies (1F5, 1G5, 1H8, 2C2, 2G9, 3A10, 3H12 and 3H8). Figure 16 is an alignment of the VL sequences of human anti-CD27 antibodies (1F5, 1G5, 1H8, 2C2, 2G9, 3A10, 3H12 and 3H8). Figures 17 and 18 show the results of an in vivo study in a non-human primate using mAb 1F5. In particular, Figure 17 shows 1F5 in circulating lymphocytes after a single dose.
Figure 18 shows 1F5 not significantly depleted in circulating lymphocytes.
Figure 19 describes the result of a pentamer staining assay in peripheral mouse blood cells and splenocytes.
Figure 20 describes the results of an ELISPOT assay and increased IFNγ production using anti-CD27 antibodies. Figure 21 shows improvement by anti-CD27 mAbs of T cell responses to a vaccine antigen in a transgenic mouse model by pentamer and IFN ELISPOT staining.
Figures 22A-C are the protocol for and results of an experiment showing that anti-CD27 improves T cell responses to an APC-directed vaccine (α-DEC205-OVA). Figure 22A shows the protocol for the experiment.
Figure 22B shows the results of a tetramer staining experiment to measure antigen-specific T cells.
Figure 22C shows the results of an IFN-gamma ELISPOT assay to measure antigen-specific T cells.
Figures 23A-D are the results of an experiment showing that anti-CD27 in combination with the TLR3 agonist PolyIC (at 25 µg, 50 µg or 100 µg) improves T cell responses to an APC-directed vaccine (α- DEC205-OVA). Figure 23A is a graph showing the% of IFN-gamma positive cells among CD8 + T cells for wild type 5 mice treated with poly IC and anti-CD27 mAb 1F5, transgenic huCD27 mouse with poly IC and a human IgG1 antibody control or transgenic mouse huCD27 treated with poly IC and anti-CD27 mAb 1F5. Figures 24 and 25 show results of a study of administration of an anti-CD27 mAb before the vaccine in the presence or absence of T: LR agonist and show the significance of time of administration of the antibody in relation to the vaccine.
Figures 26 and 27 show results of administration of anti-CD27 mAb in combination with TCR activation in T cells of transgenic human CD27 mice, as shown by both proliferation and cytokine production.
Figures 28A-D are the protocol for and results of an experiment that shows that anti-CD27 improves the effectiveness of an α-DEC205-OVA vaccine in a MO4 melanoma challenge model (B16-OVA). Figure 24A shows the protocol for the experiment.
Figure 24B is a graph plotting the tumor size (in mm2) versus number of days after tumor inoculation in untreated mice.
Figure 24C is a graph plotting the tumor size (in mm2) against the number of days after tumor inoculation in mice treated with the vaccine alone.
Figure 24D is a graph plotting the tumor size (in mm2) against the number of days after tumor inoculation in mice treated with the vaccine in combination with an anti-CD27 antibody. Figures 29A and B are graphs showing prolonged survival of transgenic mice with human CD27 (tumor models) after challenge with a syngene lymphoma and administration of several doses of anti-CD27 mAb 1F5. Figures 30 to 32 show the results of an experiment that tests the effect of anti-CD27 treatment in a Raji 5 xenograft model in SCID mice.
Figure 30A plots the tumor size (in mm3) against the number of days after tumor inoculation in untreated mice, treated with a control human IgG1 antibody or treated with anti-CD27 antibodies 1F5 and 3H8. The arrows indicate the days when the treatment was administered by i.p.
Figure 30B shows survival on a Kaplan-Meier curve.
Figure 31A plots the tumor size (in mm3) against the number of days after tumor inoculation in untreated mice, treated with a control human IgG1 antibody or treated with the anti-CD27 1F5 antibody. The arrows indicate the days when the treatment was administered by i.p.
Figure 31B shows survival on a Kaplan-Meier curve.
Figure 32 shows the results of another experiment that tests the effect of anti-CD27 treatment in a Raji xenograft model in SCID mice on a Kaplan-Meier curve.
Figure 33 shows the results of an experiment that tests the effect of anti-CD27 treatment in a Daudi xenograft model in SCID mice.
Figure 33 plots the tumor size (in mm3) against the number of days after tumor inoculation in mice treated with a control human IgG1 antibody or treated with anti-CD27 1F5 antibody (0.1 mg or 0.3 mg ). The arrows indicate the days when the treatment was administered by i.p.
Figure 33B shows survival on a Kaplan-Meier curve.
Figure 34 shows the results of an ELISPOT assay and that production of enhanced antigen-specific IFNg using anti-CD27 antibody is stopped when the Fc portion of IgG is unable to engage Fc receptors. Detailed description of the invention The present invention provides anti-CD27 antibodies that exhibit particular functional properties correlating with significant therapeutic benefits, including up-regulation of immune function (e.g., T cell-mediated immune responses such as vaccine therapies, NK activation in cancer therapies), inhibition of cell growth (for example, in cancer therapy) and down regulation of T cell-mediated immune responses (for example, in autoimmune therapy). These functional characteristics include, for example: (1) inhibition of (for example, completely or partially blocks) binding of soluble CD70 to cells that express CD27 by at least about 70%, yet, for example, by at least 80% or at least 90% (2) binding to a human CD27 with a KD of 1 x 10-9 M or less, (3) induction of at least about 40% complement-mediated cytotoxicity (CDC) of cells expressing CD27 in a concentration of 10 μg / ml, (4) induction of at least about 40% of specific lysis of cells expressing CD27 by ADCC in a concentration of 10 μg / ml, (still, for example, at least about 50% , at least about 60% or at least about 70% of specific lysis) (5) induction or improvement of immune responses, especially TH1 responses and / or (6) induction or improvement of T cell activity, especially numbers and / or CD8 + T cell activity. In other embodiments, antibodies include heavy and light chain variable regions and / or CDR sequences.
In order that the present invention can be more easily understood, certain terms are first defined.
Additional definitions are established throughout the detailed description.
The term “CD27” (also referred to as “CD27 molecule”, “CD27L receptor”, “S1521”, “T cell activation CD27”, “TNFRSF7,” “MGC20393,” “5-member tumor necrosis factor superfamily, 7 "," T cell activation S152 antigen "" Tp55 "," tumor necrosis factor receptor superfamily member 7 "," CD27 antigen ", and" T cell activation CD27 ") refer to a receptor which is a member of the TNF receptor superfamily, which binds to the CD70 ligand. CD27 is necessary for the generation and long-term maintenance of T cell immunity and plays a key role in regulating B cell activation and immunoglobulin synthesis.
The term "CD27" includes any variant or isoforms of CD27 that are naturally expressed by cells (for example, human CD27 deposited with GENBANK® containing accession number AAH12160.1). Thus, the antibodies of the invention may cross-react with CD27 from species other than humans.
Alternatively, antibodies may be specific for human CD27 and may not cross-react with other species.
CD27 or any variants and their isoforms, can either be isolated from cells or tissues that naturally express them or be recombinantly produced using techniques well known in the art and / or those described herein.
Preferably, the antibodies are directed to hCD27 which has a normal glycosylation pattern.
Genbank ® (accession number AAH12160.1) reports the human CD27 amino acid sequence as follows (SEQ ID NO: 1): 1 marphpwwlc vlgtlvglsa tpapkscper hywaqgklcc qmcepgtflv kdcdqhrkaa 61 qcdpcipgvs fspdhhtrph cescrhcnsg llvrnctita naecacrngw qcrdkectec 121 dplpnpslta rssqalsphp qpthlpyvse mleartaghm qtladfrqlp 181 artlsthwpp qrslcssdfi rilvifsgmf lvftlagalf lhqrrkyrsn kgespvepae pcryscpree 241 egstipiqed yrkpepacsp
The term “CD70” (also referred to as “CD70 molecule”, “CD27L”, “CD27LG”, “TNFSF7,” “member 7 of the tumor necrosis factor (ligand) superfamily,” “CD27 ligand,” “CD70 antigen, ”“ CD70 surface antigen, ”“ tumor necrosis factor ligand superfamily, 5 limb 7, ”“ Ki-24 antigen, ”and“ CD27-L ”) refer to the CD27 ligand (see, for example, Bowman MR et al., J.
Immunol. 1994 Feb 15; 152 (4): 1756-61). CD70 is a type II transmembrane protein that belongs to the tumor necrosis factor (TNF) ligand family. It is a surface antigen on activated T and B lymphocytes that induces the proliferation of co-stimulated T cells, increases the generation of cytolytic T cells and contributes to the activation of T cells.
It has also been suggested that CD70 plays a role in regulating B cell activation, cytotoxic function of natural killer cells and immunoglobulin synthesis (Hintzen RQ et al., J.
Immunol. 1994 Feb 15; 152 (4): 1762-73). GenBank® (accession number NP_001243) reports the human CD70 amino acid sequence as follows (SEQ ID NO: 2): 1 mpeegsgcsv rrrpygcvlr aalvplvagl viclvvciqr faqaqqqlpl eslgwdvael 61 qlnhtgpqqd prlywqggpa lgrsflhgpe ldkgqlrihr dgiymvhiqv tlaicsstta 121 srhhpttlav gicspasrsi sllrlsfhqg ctiasqrltp largdtlctn ltgtllpsrn 181 tdetffgvqw vrp The term "antibody" as referenced herein includes total antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single strand thereof.
An "antibody" refers, in a preferred embodiment, to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen-binding portion thereof.
Each heavy chain is composed of a variable region of the heavy chain (abbreviated here as VH) and a constant region of the heavy chain.
The heavy chain constant region is composed of three domains, CH1, CH2 and CH3.
Each light chain is composed of a variable region of the light chain (abbreviated here as VL) and a constant region of the light chain.
The light chain constant region is composed of a domain, CL.
The VH and VL regions can also be subdivided into regions of hypervariability, called 5 complementarity determining regions (CDR), interspersed with regions that are more conserved, called structural regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminals to carboxy-terminals in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
Constant regions of the antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system.
The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used here, refers to one or more fragments of an antibody to specifically bind to an antigen (for example, human CD27 ). Said "fragments" are, for example between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to about 300, for example, about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length.
It has been shown that the antigen-binding function of an antibody can be performed by fragments of a complete antibody.
Examples of binding fragments included within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of 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 consists of a VH domain ; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs that can optionally be joined by a synthetic linker.
In addition, although the two domains of the 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 pair of regions VL and VH form monovalent molecules (known as single Fv chain (scFv); see for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc.
Natl.
Acad.
Sci.
USA 85: 5879-5883). Said single chain antibodies are further intended to be included within the term "antigen binding portion" of an antibody.
These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same way as they are intact antibodies.
Antigen-binding moieties can be produced by recombinant DNA techniques, or by chemical or enzymatic cleavage of intact immunoglobulins.
A "bifunctional antibody" or "bispecific" is an artificial hybrid antibody containing two heavy / light chain pairs and two different binding sites.
Bispecific antibodies can be produced by a variety of methods, including the fusion of hybridomas or ligation of Fab 'fragments. See, for example, Songsivilai & Lachmann, Clin.
Exp.
Immunol. 79: 315-321 (1990); Kostelny et al., J.
Immunol. 148, 1547-1553 (1992). The term "monoclonal antibody," as used herein, refers to an antibody that has unique binding specificity and affinity for a particular epitope.
Thus, the term "human monoclonal antibody" refers to an antibody that has a unique binding specificity and that has optional constant and variable regions derived from human germline immunoglobulin sequences.
In one embodiment, the 5 human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, such as a transgenic mouse, comprising a human heavy chain transgene and a light chain transgene fused with an immortalized cell.
The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transcromosomal to human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell to express the antibody, for example, from a transfectoma, (c) antibodies isolated from a recombinant human combinatorial antibody library, and (d) antibodies prepared, raised or isolated by any other means that involves processing human immunoglobulin gene sequences to other DNA sequences.
Said recombinant antibodies comprise variable and constant regions that use human germline immunoglobulin sequences are encoded by the germline genes, but include the subsequent rearrangements and mutations that occur, for example, during antibody maturation.
As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23 (9): 1117-1125), the variable region contains the antigen-binding domain, which is encoded by several genes that rearrange to form an antibody specific to a foreign antigen.
In addition to rearrangement, the variable region can be further modified by several unique amino acid changes (referred to as somatic mutation or hypermutation) to increase the antibody's affinity for the foreign antigen.
The constant region will change in response to another antigen (that is, isotype switching). Therefore, the rearranged and somatically mutated 5 nucleic acid molecules encoding the light and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecules, but will instead be substantially identical. or similar (that is, they have at least 80% identity). The term "human antibody" includes antibodies containing variable and constant regions (if present) of human germline immunoglobulin sequences.
Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random mutagenesis, either site-specific in vitro or by somatic mutation in vivo) (see, Lonberg , N. et al. (1994) Nature 368 (6474): 856-859); Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern.
Rev.
Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad.
Sci 764: 536-546). However, the term "human antibody" does not include antibodies in which the CDR sequences derived from germline of other mammalian species, such as a mouse, have been grafted onto human structure sequences (ie, humanized antibodies). As used here, a "heterologous antibody" is defined in relation to the transgenic non-human organism that produces such an antibody.
This term refers to an antibody containing an amino acid sequence or a coding nucleic acid sequence corresponding to that found in an organism that does not consist of a transgenic non-human animal, and generally from a species other than that of the non-human animal transgenic.
An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies containing different antigen specificities (for example, an isolated antibody that specifically binds to human CD27 is substantially free of antibodies that specifically binds to antigens other than human CD27). An isolated antibody that specifically binds to an epitope may, however, cross-react with other CD27 proteins of different species.
However, the antibody preferentially always binds to human CD27.
In addition, an isolated antibody is typically substantially free of other cellular materials, and / or chemicals.
In one embodiment of the invention, a combination of "isolated" antibodies containing different specificities and CD27 is combined into a well-defined composition.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds.
Epitopes can be formed either from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.
Epitopes formed from contiguous amino acids are typically maintained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost in treatment with denaturing solvents.
An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a single spatial conformation.
Methods for determining which epitopes are linked by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which the CD27 overlap or contiguous peptides are tested for reactivity with a given anti-CD27 antibody. Methods for determining the spatial conformation of epitopes include techniques in practice and those described herein, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G . AND.
Morris, Ed. (1996)). 5 Also encompassed by the present invention are antibodies that bind to an epitope on CD27 that comprises all or a portion of an epitope recognized by the particular antibodies described herein (for example, the same or an overlapping region or a region between or covering the region). Also encompassed by the present invention are antibodies that bind to the same epitope, and / or antibodies that compete for binding to human CD27 with the antibodies described herein.
Antibodies that recognize the same epitope or compete for binding can be identified using routine techniques.
Said techniques include, for example, an immunoassay, which shows the ability of an antibody to block the binding of another antibody to a target antigen, that is, a competitive binding assay.
Competitive binding is determined in an assay in which the test immunoglobulin inhibits specific binding of a reference antibody to a common antigen, such as CD27. Several types of competitive binding assays are known, for example: direct or indirect solid phase (RIA) radioimmunoassay, direct or indirect solid phase (EIA) immunoassay, sandwich competition assay (see Stahli et al., Methods in Enzymology 9: 242 (1983)); EIA of direct biotin-avidin in solid phase (see Kirkland et al., J.
Immunol. 137: 3614 (1986)); direct solid-phase labeling assay, solid-phase direct-label sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); Direct-phase solid-phase RIA using marker I-125 (see Morel et al., Mol.
Immunol. 25 (1): 7 (1988)); Direct solid-phase biotin-avidin EIA (Cheung et al., Virology 176: 546 (1990)); and direct dialing RIA. (Moldenhauer et al., Scand. J.
Immunol. 32:77 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells carrying any of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin.
Competitive inhibition is measured by determining the amount of marker attached to the solid surface or cells in the presence of the test immunoglobulin.
The test immunoglobulin is usually present in excess.
Generally, when a competing antibody is present in excess, it will inhibit the specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70- 75% or more.
Other techniques include, for example, epitope mapping methods, such as X-ray analysis of antigen crystals: antibody complexes that provide atomic resolution of the epitope.
Other methods monitor the binding of the antibody to antigen fragments or mutated variants of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered to be an indication of an epitope component.
In addition, computational combinatorial methods for epitope mapping can also be used.
These methods are based on the ability of the antibody of interest to isolate short peptides of specific affinities from phage display peptide libraries.
The peptides are then considered as clues for the definition of the epitope corresponding to the antibody used to screen the peptide library.
For epitope mapping, computational algorithms have also been developed, which have shown to map discontinuous conformational epitopes.
As used here, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds," refer to the binding of antibody to an epitope on a predetermined antigen.
Typically, the antibody binds with an equilibrium dissociation constant (KD) of approximately less than 10-7 M, such as approximately less than 10 -8 M, 10-9 M or 10-10 M or even less when determined by surface plasmon resonance technology 5 (SPR) in a BIACORE 2000 instrument using recombinant human CD27 as the analyte and antibody as the ligand and binds to the predetermined antigen with an affinity that is at least twice as high as its affinity for binding to a non-specific antigen (eg, BSA, casein) in addition to the predetermined antigen or a closely related antigen.
The phrases "an antibody that recognizes an antigen" and "an antibody specific to an antigen" are used interchangeably with the term "an antibody that specifically binds an antigen". The term "KD," as used here, is intended to refer to the dissociation equilibrium constant of a particular antigen-antibody interaction.
Typically, the human antibodies of the invention bind to CD27 with a dissociation equilibrium constant (KD) of approximately 10-8 M or less, as less than 10-9 M or 10-10 M or even less when determined by technology of surface plasmon resonance (SPR) in a BIACORE 2000 instrument using recombinant human CD27 as the analyte and the antibody as the ligand.
The term "kd" as used here, is intended to refer to an off rate constant for the dissociation of an antibody from the antibody / antigen complex.
The term “ka” as used here, is intended to refer to an on rate constant for the association of an antibody with an antigen.
The term “EC50,” as used here, refers to the concentration of an antibody or an antigen-binding potion, which induces a response, whether in an in vitro or in vivo assay, which is 50% of the maximum response , that is, half between the maximum response and the baseline.
As used herein, "isotype" refers to the class of antibody (for example, IgM or IgG1) that is encoded by heavy chain constant region genes.
In one embodiment, a human monoclonal antibody of the invention is the IgG1 isotype. In another embodiment, a human monoclonal antibody of the invention is the IgG2 isotype. The term "binds to immobilized CD27," refers to the ability of a human antibody of the invention to bind to CD27, for example, expression on the surface of a cell or that is attached to a solid support.
The term "cross reaction", as used here, refers to the ability of an antibody of the invention to bind to CD27 from a different species.
For example, an antibody of the present invention that binds to human CD27 can further bind to other CD27 species. As used here, cross-reactivity is measured by detecting a specific reactivity with purified antigen in binding assays (for example, SPR, ELISA) or binding to, or otherwise functionally interacting with, cells that physiologically express CD27. Methods for determining cross-reactivity include standard binding assays as described herein, for example, by BiacoreTM surface plasmon resonance (SPR) analysis using a BiacoreTM 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or cytometry techniques flow.
As used here, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from an Ig class to one of the other Ig classes.
As used herein, "non-switched isotype" refers to the heavy chain isotypic class that is produced when no isotype switching has occurred; the CH gene encoding the non-switched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene.
Isotype switching was classified as classic and non-classic isotype switching.
Classical isotype switching occurs by recombination events that involve at least one region of switching sequence in the transgene.
Non-classical isotype switching can occur, for example, by homologous recombination between human σµ and 5 µ human (deletion associated with δ). Alternative non-classical switching mechanisms, such as intertransgene and / or intercromosomal recombination, among others, can occur and effect isotype switching.
As used here, the term "switching sequence" refers to those DNA sequences responsible for switching recombination.
A "switching donor" sequence, typically a µ switching region, will be 5 '(ie upstream) of the construct region to be deleted during switching recombination.
The “switching acceptor” region will be between the construct region to be erased and the constant replacement region (for example, γ, ε, etc.). As there is no specific site where recombination always occurs, the sequence of the final gene will typically not be predictable from the construct.
As used here, "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently linked to a protein, more specifically to an immunoglobulin protein.
A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to the glycosylation patterns that occur naturally in antibodies produced by the species of the transgenic non-human animal, when one skilled in the art recognizes the glycosylation pattern of the heterologous antibody as being more similar the said pattern of glycosylation of the transgenic non-human animal species than the species from which the transgene CH genes were derived.
The term "naturally occurring" as used here as applied to an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that was not intentionally modified by man in the laboratory is naturally occurring.
The term "rearranged" as used here refers to a configuration of a heavy chain or light chain immunoglobulin locus in which the V segment is positioned immediately adjacent to a DJ segment or J segment in a conformation that essentially encodes a domain complete VH or VL, respectively.
A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus has at least one recombined heptamer / nonomer homology element.
The term "not rearranged" or "germline configuration" as used here in reference to segment V refers to the configuration in which segment V is not recombined so as to be immediately adjacent to a segment D or J.
The term "nucleic acid molecule," as used here, is intended to include DNA molecules and RNA molecules.
A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
The term "isolated nucleic acid molecule," as used herein in reference to nucleic acids encoding antibodies or portions of antibody (for example, VH, VL, CDR3) that bind to CD27, is intended to refer to an acid molecule nucleic acid in which the nucleotide sequences encoding the antibody or portion of the antibody are exempt from other nucleotide sequences encoding antibodies or portions of antibody that bind antigens other than CD27, which other sequences can naturally flank the nucleic acid in a DNA human genomics.
For example, SEQ ID NOs: 35 and 41, 47 and 53, 101 and 107, 83 and 89, 83 and 95, 23 and 29, 71 and 77 correspond, respectively, to nucleotide sequences comprising the heavy chain variable regions ( VH) and light chain (VL) of anti-CD27 monoclonal antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The present invention further includes "conserved sequence modifications 5" of the sequences set out in SEQ ID NOs: 5-112, that is, nucleotide and amino acid sequence modifications that do not disrupt the binding of the antibody encoded by the nucleotide sequence or containing the sequence amino acid, to the antigen.
Said conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions.
For example, modifications can be introduced in SEQ ID NOs: 5- 112 by means of conventional techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue with a similar side chain.
Families of amino acid residues containing similar side chains have been defined in the art.
These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, tyrosine, threonine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and side chains aromatic (eg tyrosine, histidine, phenylalanine, tryptophan). Thus, a non-essential amino acid residue predicted on a human anti-CD27 antibody is preferably replaced with another amino acid residue from the same family of side chains.
Methods of identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al.
Protein Eng. 12 (10): 879-884 (1999); and Burks et al.
Proc.
Natl.
Acad.
Sci.
USA 94: 412-417 (1997)) 5 Alternatively, in another embodiment, mutations can be introduced randomly over all or part of an anti-CD27 antibody coding sequence, for example, by saturation mutagenesis, and the Resulting modified anti-CD27 antibodies can be screened for binding activity.
For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when ideally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of nucleotides.
Alternatively, substantial homology exists when segments will hybridize under selective hybridization conditions, to complement the strand.
The percentage of identity between two strings is a function of the number of identical positions shared by the strings (ie% homology = # identical positions / # total positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of the percentage of identity between two sequences can be performed using a mathematical algorithm, as described in the following non-limiting examples.
The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using an NWSgapdna.CMP matrix and a gap weight of 40, 50 , 60, 70, or 80 and a weight of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the E algorithm.
Meyers and W.
Miller (CABIOS, 4: 11-17 (1989)) that was incorporated into the ALIGN program (version 2.0), using a table of 5 weight of PAM120 residues, a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J.
Mol.Biol. 444-453 (1970)) that was incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 1, 14, 12, 10, 8, 6 or 4 and a weight of length 1, 2, 3, 4, 5, or 6. The nucleic acid and protein sequences of the present invention can still be used as a "query sequence" to perform a search against public databases to, for example, identify related strings.
These searches can be carried out using the programs NBLAST and XBLAST (version 2.0) by Altschul, et al. (1990) J.
Mol.Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, punctuation = 100 word length, = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention.
Searches for BLAST proteins can be performed with the XBLAST program, punctuation = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention.
To obtain gapped alignments for comparison purposes, BLAST Gapped can be used as described in Altschul et al. (1997) Nucleic Acids Res 25 (17): 3389-3402. When using BLAST and BLAST Gapped programs, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used.
See http://www.ncbi.nlm.nih.gov.
Nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
A nucleic acid is "isolated" or "generated substantially pure" when purified out of other cellular components or other contaminants, for example, other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, chromatography on column, agarose gel electrophoresis and others well known in the art.
See, F.
Ausubel, et al., Ed.
Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). The nucleic acid compositions of the present invention, although often in a native sequence (except for modified and similar restriction sites), cDNA, genomic, or mixtures thereof can be mutated according to standard techniques to provide gene sequences .
For coding sequences, these mutations can affect the amino acid sequence, as desired.
In particular, DNA sequences substantially homologous to or derived from V, D, J, native, constants, switching and other said sequences described herein are contemplated (where "derivative" indicates that a sequence is identical or modified from another sequence ). A nucleic acid is "operationally linked" when it is placed in a functional relationship with another nucleic acid sequence.
For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
With respect to regulatory transcription sequences, operably linked means that the DNA sequences to be linked are contiguous and, when necessary to join two protein coding regions, contiguous and in reading region.
For switching sequences, operationally connected indicates that the sequences are capable of performing switching recombination.
The term "vector," as used herein, is intended to refer to a nucleic acid capable of transporting other nucleic acids to which it has been attached.
One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be linked.
Another type of vector is a viral vector, in which additional DNA segments can be linked in the viral genome.
Certain vectors are capable of autonomous replication in a host cell that are introduced (for example, bacterial vectors containing an origin of replication and episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated together with the host's genome.
In addition, certain vectors are capable of directing the expression of genes to which they are operationally linked.
Said vectors are referred to here as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are generally in the form of plasmid.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of the vector.
However, the invention is intended to include said other forms of expression vectors, such as viral vectors (for example, defective replication of retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term "recombinant host cell" (or simply "host cell"), as used here, is intended to refer to a cell in which a recombinant expression vector has been introduced.
It should be understood that such terms are intended to refer not only to the cell of the particular individual, but to the progeny of said cell.
Because certain modifications may occur in successor generations due to mutation or environmental influences, said progeny may not, in fact, be identical to the parental cell, but are still included in the scope of the term "host cell" as used here.
As used here, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide or hapten.
Antigens suitable for use in the present invention (for example, in a vaccine in combination with an anti-CD27 antibody of the invention) include, for example, infectious disease antigens and tumor antigens, against which protective or therapeutic responses are desired immune responses. , for example, antigens expressed by a tumor cell or a pathogenic organism or antigens from infectious diseases.
For example, suitable antigens include tumor-associated antigens for the prevention or treatment of cancers.
Examples of tumor-associated antigens include, but are not limited to, sequences comprising all or part of the sequences of βhCG, gp100 or Pmel17, HER2 / neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan- A, NY-ESO-1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3, tyrosinase, telomerase, SSX2 and MUC-1, antigens, and tumor antigens derived from germ cells.
Tumor-associated antigens also include blood group antigens, for example, Lea, Leb, LeX, LeY, H-2, B-1, B-2 antigens. Alternatively, more than one antigen can be included within the antibody antigen constructs of the present invention.
For example, a MAGE antigen can be combined with other antigens, such as melanin A tyrosinase, gp100 and, together with adjuvants, such as GM-CSF or IL-12, and linked to an anti-APC antibody.
Other suitable antigens include viral antigens for the prevention or treatment of viral diseases.
Examples of viral antigens include, but are not limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV (surface or core antigens), HPV, FAS, HSV-1, HSV-2, p17, ORF2 antigens and ORF3. Examples of bacterial antigens include, but are not limited to, Toxoplasma gondii or Treponema pallidum. The bacterial antibody-antigen conjugates of the invention can be in the treatment or prevention of various bacterial diseases, such as Antrax, botulism, tetanus, chlamydia, cholera, diphtheria, Lyme disease, tuberculosis and syphilis. Other appropriate antigens 5 of infectious disease pathogens such as viruses, bacteria, parasites and fungi are disclosed below. Sequences of prior antigens are well known in the art. For example, an example of a MAGE-3 cDNA sequence is provided in US 6,235,525 (Ludwig Institute for Cancer Research); examples of NY-ESO-1 nucleic acid and protein sequences are provided in US
5,804,381 and US 6,069,233 (Ludwig Institute for Cancer Research); examples of nucleic acid and Melan-A protein sequences are provided in US
5,620,886 and US 5,854,203 (Ludwig Institute for Cancer Research); examples of NY-BR-1 nucleic acid and protein sequences are provided in US
6,774,226 and US 6,911,529 (Ludwig Institute for Cancer Research) and NY-CO-58 nucleic acid and protein sequence examples are provided in WO 02090986 (Ludwig Institute for Cancer Research); an example of an amino acid sequence for the HER-2 / neu protein is available from GENBANK® Accession No. AAA58637; and a nucleotide sequence (mRNA) for human carcinoembryonic antigen type 1 (CEA-1) is available from GENBANK® Accession No. NM__020219. An HPV antigen that can be used in the compositions and methods of the invention can include, for example, an HPV-16 antigen, an HPV-18 antigen, an HPV-31 antigen, an HPV-33 antigen and / or an HPV-35 antigen ; and is suitably an HPV-16 antigen and / or an HPV-18 antigen. An HPV-16 genome is described in Virology, 145: 181- 185 (1985) and DNA sequences encoding HPV-18 are described in US patent 5,840,306, the disclosures of which are incorporated by reference here in their entirety. HPV-16 antigens (for example, HPV-16 E1 and / or E2 seroreactive regions) are described in the US Patent
6,531,127, and HPV-18 antigens (for example, HPV-18 L1 and / or L2 seroreactive regions) are described in US Patent 5,840,306, the disclosures of which are incorporated by reference here. Likewise, 5 a complete HBV genome is available from GENBANK ® Accession No. NC_003977, the disclosure of which is incorporated herein. The HCV genome is described in European Patent Application 318 216, the disclosure of which is incorporated herein. PCT / US90 / 01348, incorporated herein by reference, discloses information about the sequence of the HCV genome clones, the amino acid sequences of the HCV viral proteins and the methods of producing and using said HCV vaccine compositions comprising the proteins of the HCV HCV and derived peptides. Antigenic protein peptides (i.e., these epitopes containing T cells) can be identified in a variety of ways well known in the art. For example, T cell epitopes can be predicted through web-based protein sequence analysis using prediction algorithms (BIMAS & SYFPEITHI) to generate potential MHC class I and II binding peptides that correspond to a base of internal data of 10,000 well-characterized MHC-binding peptides previously defined by CTLs. High-score peptides can be classified and selected as "interesting" based on the high affinity of a certain MHC molecule. Another method for identifying antigenic peptides that contain T cell epitopes is to divide the protein into non-overlapping peptides of desired length or overlapping peptides of desired lengths that can be produced recombinantly, synthetically, or in certain limited situations, by chemical cleavage. of the protein and tested for immunogenic properties, for example, inducing a T cell response (ie, lymphocin proliferation or secretion).
To determine accurate protein T cell epitopes in, for example, fine mapping techniques, a peptide containing T cell stimulation activity and thus comprising at least one T cell epitope, as determined by T cell biology techniques, can 5 be modified by adding or deleting amino acid residues at either amino or carboxy terminus of the peptide and tested to determine a change in T cell reactivity to the modified peptide.
If two or more peptides that share an overlapping area in the sequence of the native protein have been shown to have stimulating human T cell activity, as determined by T cell biology techniques, additional peptides can be produced comprising all or a portion of such peptides and these additional peptides can be tested by a similar process.
Following this technique, the peptides are selected and produced recombinantly or synthetically.
Peptides are selected based on several factors, including the strength of the T cell response to the peptide (for example, stimulation index). The physical and chemical properties of these selected peptides (for example, solubility, stability), can then be analyzed to determine whether the peptides are suitable for use in therapeutic compositions or whether the peptides require modification.
The term "antigen presenting cell" or "APC" is a cell that has foreign antigen complexed with MHC on its surface.
T cells recognize this complex using a T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), monocytes (such as THP-1), lymphoblastoid B cells (such as C1R.
A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens either by phagocytosis or by receptor-mediated endocytosis.
Examples of APC receptors include, but are not limited to, type C lecithins, such as,
human dendritic cell receptor and epithelial cells 205 (CD27) and the human macrophage mannose receptor.
The term "antigen presentation" refers to the process by which APCs capture antigens and allow their recognition by T cells, for example, as a component of an MHC-I and / or MHC-II conjugate. “MHC molecules” includes two types of molecules, MHC class I and MHC class II.
Class I MHC molecules present antigen to specific CD8 + T cells and class II MHC molecules present antigen to specific CD4 + T cells.
Antigens supplied exogenously to APCs are processed primarily for association with MHC class II.
In contrast, antigens supplied exogenously to APCs are processed primarily for association with MHC class I.
As used here, the term "immunostimulatory agent" includes, among others, compounds capable of stimulating APCs, such as DCs and macrophages.
For example, immunostimulating agents suitable for use in the present invention are able to stimulate APCs, so that the process of APC maturation is accelerated, the proliferation of APCs is increased, and / or the recruitment or release of costimulatory molecules (for example, for example, CD80, CD86, ICAM-1, MHC and CCR7 molecules) and pro-inflammatory cytokines (for example, IL-1β, IL-6, IL-12, IL-15, and IFN-γ) are regulated upwards.
Suitable immunostimulating agents are also able to increase the proliferation of T cells.
Said immunostimulating agents include, among others, CD40 ligand; FLT linker 3; cytokines, such as IFN-α, IFN-β, IFN-γ and IL-2; colony stimulating factors, such as G-CSF (granulocyte colony stimulating factor) and GM-CSF (granulocyte macrophage colony stimulating factor); an anti-CTLA-4 antibody, anti-PD1 antibody, anti-41BB antibody, or anti-OX-40 antibody; LPS (endotoxin); ssRNA; dsRNA; Calmette-Guerin bacillus (BCG); Levamisol hydrochloride; and intravenous immune globulins.
In one embodiment, an immunostimulating agent can be a Toll-like receptor agonist (TLR). For example, the immunostimulating agent can be a TLR3 agonist, such as double-stranded inosine: polynucleotide cytosine (Poly I: C, for example, available as AmpligenTM from Hemispherx Bipharma, PA, US or Poly 5 IC: LC from Oncovir) or Poly A: U; a TLR4 agonist such as a monophosphoryl A (MPL) or RC-529 lipid (for example, as available from GSK, UK); a TLR5 agonist as flagellin; a TLR7 or TLR8 agonist as an imidazoquinoline TLR7 or TLR 8 agonist, for example imiquimod (for example, AldaraTM) or resiquimod and related imidazoquinoline agents (for example, as available from 3M Corporation); or a TLR 9 agonist as a deoxynucleotide with demethylated CpG motifs (so called "CpGs", for example, as available from Coley Pharmaceutical). A preferred immunostimulating agent is a TLR3 agonist, preferably Poly I: C.
Said immunostimulating agents can be administered simultaneously, separately or sequentially with the antibodies and constructs of the present invention and can also be physically linked to the antibodies and constructs.
As used here, the term "linked" refers to the association of two or more molecules.
The bond can be covalent or non-covalent.
The link can also be genetic (that is, recombinantly fused). These bonds can be achieved using a wide variety of techniques recognized in practice, such as chemical conjugation and the production of recombinant protein.
As used here, the term "cross-presentation" antigen refers to the presentation of exogenous protein antigens to T cells via MHC class I and class II molecules in APCs.
As used here, the term "T cell-mediated response" refers to any T-cell-mediated response, including effector T cells (for example, CD8 + cells) and T helper cells (for example, cells
CD4 +). The T cell mediated response includes, for example, T cell cytotoxicity and proliferation.
As used herein, the term "cytotoxic T lymphocyte response (CTL)" refers to an immune response induced by cytotoxic T cells. 5 CTL responses are mediated mainly by CD8 + T cells. As used here, the terms "inhibits" or "blocks" (for example, referring to inhibition / blocking of binding of CD70 to CD27 in cells) are used interchangeably and includes both partial and complete inhibition / blocking.
CD70 inhibition / blocking preferably reduces or alters the normal level or type of activity that occurs when binding to CD70 occurs without inhibition or blocking.
Inhibition and blocking are still intended to include any measurable reduction in the binding affinity of CD70 when in contact with an anti-CD27 antibody as compared to CD70 not in contact with an anti-CD27 antibody, for example, inhibits CD70 binding in at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In a preferred embodiment, the anti-CD27 antibody inhibits CD70 binding by at least about 70%. In another embodiment, the anti-CD27 antibody inhibits CD70 binding by at least about 80%. As used here, the term "inhibits growth" (for example, referring to cells) is intended to include any measurable reduction in the growth of a cell, for example, inhibiting the growth of a cell by at least about 10%, 20% , 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. The terms "inducing an immune response" and "increasing an immune response" are used interchangeably and refer to the stimulation of an immune response (that is, passive or adaptive) to a particular antigen.
The terms “induces” as used in connection with the induction of CDC or ADCC refer to the stimulation of particular mechanisms of direct cell death.
For example, in one embodiment, the antibody induces at least about 20, 25, 30, 35, 40, 45, 50, 55 or 60% of lysis, through CDC of cells expressing CD27, to a concentration of 10μg / ml.
In a preferred embodiment, the antibody induces at least about 40% of lysis 5 through CDC of cells expressing CD27 at a concentration of 10µg / ml.
In another embodiment, the antibody induces at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80 or 85% of lysis, through ADCC (i.e., lysis of cells expressing CD27, at a concentration of 10μg / ml.
In a preferred embodiment, the antibody induces at least about 40% lysis through ADCC of cells expressing CD27 at a concentration of 10 µg / ml.
The terms "treat," "treating," and "treatment," as used here, refer to the therapeutic or preventive measures described here.
The "treatment" methods employ administration to an individual, in need of said treatment, a human antibody of the present invention, for example, an individual in need of an improved immune response against a particular antigen or an individual who can ultimately acquire said one. disorder, to prevent, cure, delay, reduce the severity of, or alleviate one or more symptoms of the disorder or recurrent disorder, or to prolong an individual's survival beyond that expected in the absence of said treatment.
The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect.
The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially stop the disease and its complications in a patient who is already suffering from the disease.
Effective amounts for this use will depend on the severity of the disorder being treated and the general condition of the patient's own immune system.
The term "patient" includes a human subject and other mammals receiving prophylactic or therapeutic treatment.
As used here, the term "individual" includes any human or non-human animal.
For example, the methods and compositions of the present invention can be used to treat an individual with an immune disorder.
The term "non-human animal" includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
Various aspects of the invention are described in more detail in the following subsections. I.
Production of Antibodies to CD27 The present invention comprises antibodies, for example, fully human antibodies, which bind CD27, for example, human CD27.
Exemplary monoclonal antibodies that bind to CD27 include 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. Monoclonal antibodies of the invention can be produced using a variety of known techniques, such as the standard somatic hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975). Although somatic hybridization procedures are preferred, in principle, other techniques for the production of monoclonal antibodies can also be employed, for example, viral or oncogenic transformation of B lymphocytes, a phage presentation technique using libraries of human antibody genes.
Thus, in one embodiment, a hybridoma method is used to produce an antibody that binds to human CD27.
In this method, a mouse or other appropriate host animal can be immunized with an appropriate antigen to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization.
Alternatively, lymphocytes can be immunized in vitro.
Lymphocytes can then be fused with myeloma cells, using an appropriate fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The culture medium in which the hybridoma cells are growing is analyzed for the production of monoclonal antibodies directed against the antigen. After hybridoma 5 cells are identified that produce antibodies of the desired specificity, affinity, and / or activity, clones can be subcloned, limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo as ascites tumors in an animal. Monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as, for example, protein A sepharose, hydroxylapatite chromatography, electrophoresis gel, dialysis or affinity chromatography. In another embodiment, antibodies and antibody portions that bind to human CD27 can be isolated from phage antibody libraries generated using the techniques described, for example, McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991), Marks et al., J. Mol. Biol., 222: 581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23, 344-348 ; US patent 5,223,409; 5,403,484; and 5,571,698 for Ladner et al .; US patents 5,427,908 and 5,580,717 to Dower et al .; US patents 5,969,108 and 6,172,197 to McCafferty et al .; and US Patents
5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 for Griffiths et al .. In addition, the production of high-affinity human antibodies (nM range) by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992 )), as well as combinatorial infection and in vivo recombination as a strategy for building very large phage libraries (Waterhouse et al., Nuc.
Acids.
Res., 21: 2265-2266 (1993)) can still be used.
In a particular embodiment, the antibody that binds to human CD27 is produced using the phage exposure technique described by Hoet 5 et al., Supra.
This technique involves the generation of a human Fab library containing a unique combination of immunoglobulin sequences isolated from human donors and containing synthetic diversity in the heavy chain CDRs that is generated.
The library is then selected for Fabs that bind to human CD27.
The preferred animal system for the generation of hybridomas that produce antibodies of the invention is the murine system.
The production of hybridoma in mice is well known in the art, including immunization protocols and techniques for isolating and fusing immunized splenocytes.
In one embodiment, antibodies directed against CD27 are generated using transgenic or trans-chromosomal mice containing parts of the human immune system, rather than the mouse system.
In one embodiment, the invention employs transgenic mice referred to here as “HuMAb mice” that contain a human immunoglobulin monolocyte gene that encodes the heavy chain (µ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous µ and κ chain loci (Lonberg, N. et al. (1994) Nature 368 (6474): 856-859). As a result, mice have reduced expression of mouse IgM or κ, and in response to immunization, introduced human light and heavy chain transgenes undergo somatic mutation and class switching to generate high affinity IgGκ monoclonal antibodies (Lonberg, s. et al. (1994), supra; revised in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern.
Rev.
Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad.
Sci 764: 536-546).
The preparation of HuMAb mice is described in detail in section II below and in Taylor, L. et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90: 3720-3724; Choi et al. (1993) Nature 5 Genetics 4: 117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 2912-2920; Lonberg et al., (1994) Nature 368 (6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764: 536- 546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851. See also, US Patents 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;
5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all for Lonberg and Kay, and GenPharm International; US patent 5,545,807 to Surani et al .; International publication Nos. WO 98/24884, published on June 11, 1998; WO 94/25585, published on November 10, 1994; WO 93/1227, published on June 24, 1993; WO 92/22645, published on December 23, 1992; WO 92/03918, published on March 10, 1992. Immunizations To generate fully human antibodies to CD27, transgenic trans-genomic mice that contain human immunoglobulin genes (for example, HCo12, HCo7 or KM from mice) can be immunized with a purified preparation or enriched with CD27 antigen and / or cells that express CD27, as, for example, described by Lonberg et al. (1994) Nature 368 (6474): 856-859; Fishwild et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. As described here, HuMAb mice are immunized with recombinant CD27 proteins or cell lines that express CD27 as immunogens. Alternatively, mice can be immunized with DNA encoding human CD27.
Preferably, the mice will be 6-16 weeks old after the first infusion.
For example, a purified or enriched (5-50 µg) preparation of recombinant CD27 antigen can be used to immunize HuMAb 5 mice intraperitoneally.
In the case that immunizations using a purified or enriched preparation of the CD27 antigen do not result in antibodies, mice can also be immunized with cells expressing CD27, for example, a cell line, to promote the immune response.
Exemplary cell lines include stable CHO and Raji cell lines that overexpress CD27. Accumulated experience with various antigens has shown that transgenic HuMAb mice respond best when initially immunized intraperitoneally (IP) or subcutaneously (SC) with antigen in the complete Freund's adjuvant, followed by immunizations at interval intervals of IP / SC (up to a total of 10 ) with antigen in incomplete Freund's adjuvant.
The immune response can be monitored throughout the immunization protocol with plasma samples, being obtained by retroorbital bleeding.
Plasma can be screened by ELISA (as described below), and mice with sufficient titers of human anti-CD27 immunoglobulin can be used for fusions.
Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen.
Generation of hybridomas producing monoclonal antibodies to CD27 To generate hybridomas producing monoclonal antibodies to CD27, splenocytes and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse multiple myeloma cell line.
The resulting hybridomas can then be screened for the production of antigen-specific antibodies.
For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to SP2 / 0-Ag8. 653 non-secreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG (w / v). Cells can be plated in approximately 1 x 105 on the 5-flat microtiter plate, followed by a two-week incubation containing selective medium in addition to usual reagents 10% fetal serum from the Clone, 5-10% hybridoma of origin cloning factor ( IG) and 1 X HAT (Sigma). After approximately two weeks, the cells can be grown in the medium in which HAT is replaced with HT.
The individual wells can then be screened by ELISA for anti-human CD27 monoclonal IgM and IgG antibodies, or for binding to the surface of cells expressing CD27, for example, a CHO cell line expressing CD27, by FLISA fluorescence immunoadsorption). Once extensive hybridoma growth occurs, the medium can generally be seen after 10-14 days.
The hybridoma-secreting antibody can be replaced, presented again, and if still positive for IgG, anti-CD27 monoclonal antibodies can be subcloned at least twice limiting dilution.
The stable subclones can then be cultured in vitro to generate antibodies in tissue culture medium for characterization.
Generation of transfectomas producing monoclonal antibodies to CD27 The antibodies of the invention can also be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S (1985) Science 229: 1202). For example, in one embodiment, the genes of interest, for example, human antibody genes, can be linked in an expression vector as a eukaryotic expression plasmid as used by the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art.
The plasmid purified with the cloned antibody genes can be introduced into eukaryotic cells such as CHO cells or NSO cells or alternatively other eukaryotic cells, such as cells derived from plants, fungi or yeasts.
The method used to introduce these genes could be methods described in the art such as electroporation, lipofectin, lipofectamine or the like.
After introducing these antibody genes into host cells, cells expressing the antibody can be identified and selected.
These cells represent the transfectomas that can be amplified for their levels of expression and with scale increase to produce antibodies.
Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.
Alternatively, these cloned antibody genes can be expressed in other expression systems such as E. coli or in whole organisms or can be synthetically expressed.
Use of partial antibody sequences to express intact antibodies Antibodies interact with target antigens predominantly through amino acid residues that are located in six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside CDRs.
Because CDR sequences are responsible for most antigen-antibody interactions, it is possible to express recombinant antibodies that mimic the specific properties of naturally occurring antibodies by building expression vectors that include CDR sequences from specific naturally occurring antibodies grafted onto structural sequences a from a different antibody with different properties (see, for example, Riechmann, L. et al., 1998, Nature 332: 323-327; Jones, P. et al., 1986, Nature 321: 522-525; and Queen , C. et al., 1989, Proc.
Natl.
Acad.
See. U.S.
A. 86: 10029-10033). Said structure sequences can be obtained from public DNA databases that include germline antibody gene sequences.
These germline sequences differ from mature antibody gene sequences in that they do not include fully assembled variable genes, which are formed by 5 V (D) J coupling during B cell maturation.
Sequences of germline genes will also differ from the sequences of a secondary high-affinity repertoire antibody in the individual uniformly across the variable region.
For example, somatic mutations are relatively uncommon in the amino-terminal portion of the region of the structure.
For example, somatic mutations are relatively uncommon in the amino-terminal portion of the 1st region of the structure and in the carboxyl-terminal portion of the structural region 4. In addition, many somatic mutations do not significantly alter the binding properties of the antibody.
For this reason, it is not necessary to obtain the entire DNA sequence of a specific antibody to recreate an intact recombinant antibody, having binding properties similar to the original antibody (see PCT / US99 / 05535 deposited on March 12, 1999). Partial light and heavy chain sequence, spanning the CDR regions is usually sufficient for this purpose.
The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombinant antibody variable genes.
The germline sequence is then used to fill in missing parts of the variable regions.
Leading heavy and light chain sequences are modified during protein maturation and do not contribute to the properties of the final antibody.
To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification.
Alternatively, the entire variable region can be synthesized as a set of short, overlapping oligonucleotides and combined by PCR amplification to create a fully synthetic variable region clone.
This process has some advantages such as the elimination or inclusion or particular restriction sites or optimization of particular codons.
The nucleotide sequences of heavy and light chain transcripts from a hybridoma are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capabilities as natural sequences.
Synthetic heavy chain and kappa sequences can differ from natural sequences in three ways: repeated nucleotide chains are disrupted to facilitate oligonucleotide synthesis and PCR amplification; translation start sites are incorporated according to Kozak rules (Kozak, 1991, J.
Biol.
Chem. 266: 19867-19870); and, HindIII sites are designed upstream of the translation start sites.
For both light and heavy chain regions, optimized coding and corresponding non-coding, strand sequences are broken into 30-50 nucleotides approximately the midpoint of the corresponding non-coding oligonucleotide.
Thus, for each strand, the oligonucleotides can be mounted in overlapping sets of double strips that span segments of 150-400 nucleotides.
The pools are then used as models to produce 150-400 nucleotide PCR amplification products.
Typically, a set of single variable region oligonucleotides will be divided into two pools that are amplified separately to generate two overlapping PCR products.
These overlapping products are then combined by PCR amplification to form the complete variable region.
It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the kappa light chain BbsI site, or the AgeI site if the heavy chain is gamma) in the PCR amplification to generate fragments that can be easily cloned in the constructs of the expression vector.
The reconstructed light and heavy chain variable regions are then combined with cloned promoter, leader sequence, translation start, leader sequence, constant region, 3 'untranslated, polyadenylation and transcription termination, sequences to form expression vector constructs.
The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected or separately transfected into host cells that are then fused to form a host cell that expresses both chains.
Plasmids for use in the construction of expression vectors were constructed so that PCR amplified light chain V and heavy chain cDNA sequences could not be used to reconstruct complete elevated heavy chain minigenes.
These plasmids can be used to fully express human IgG1κ or IgG4κ antibodies.
Fully human and chimeric antibodies of the present invention also include IgG2, IgG3, IgE, IgA, IgM and IgD antibodies.
Similar plasmids can be constructed for expression of other heavy chain isotypes or for expression of antibodies comprising lambda light chains.
Thus, in another aspect of the invention, structural features of anti-CD27 antibodies of the invention are used to create structurally related anti-CD27 antibodies that maintain at least one functional property of the antibodies of the invention, such as, for example, (1) inhibiting (for example, example, completely or partially blocking CD70 binding to CD27-expressing cells by at least about 70% (for example, at least about 70%, or at least about 70%, at an antibody concentration of 10 µg / ml); (2) binds to a human CD27 with a Kd equilibrium dissociation constant of 10-9 M or less, or alternatively, a Ka equilibrium association constant of 10 + 9 M-1 or more
(3) induces at least about 30% complement-mediated cytotoxicity (CDC) of cells expressing CD27 at a concentration of 10 μg / ml (or induces at least 30%, or at least about 40% or at least 40% CDC of cells expressing CD27 at a concentration of 5 10 g / ml); (4) induces at least about 30% specific ADCC-mediated lysis of cells expressing CD27 at a concentration of 10 μg / ml (or induces at least 30%, or at least about 40% or at least 40% of specific ADCC-mediated lysis of cells expressing CD27 at a concentration of 10 µg / ml); (5) preventing or inhibiting the growth of tumor cells that express CD27e in a xenograft model (for example, it reduces the tumor size in several severe combined immunodeficiency (SCID) mice by at least about 50% 20 days after inoculation of tumor cell in vivo at 0.5 mg ip in at least 6 days); (6) induces or improves antigen-specific immune responses when combined with a vaccine or other antigen; (7) induce or increase immune responses, in particular, among others, the TH1 immune response; (8) induces or increases T cell activity, in particular, among others, specific CD8 + T cell numbers or functional activity or T cell proliferation or activation; and / or (9) reduces or inhibits T cell proliferation or activation.
In one embodiment, one or more CDR regions of antibodies of the invention can be recombinantly combined with known framework regions and CDRs to create additional recombinantly engineered anti-CD27 antibodies of the invention.
The variable heavy and light chain variable regions can be derived from the same or different antibody sequences.
The antibody sequences can be the sequences of naturally occurring antibodies or they can be consensus sequences of various antibodies.
See Kettleborough et al., Protein Engineering 4: 773 (1991); Kolbinger et al., Protein Engineering 6: 971 (1993) and Carter et al., WO 92/22653. Thus, in another embodiment, the invention provides a method 5 for preparing an anti-CD27 antibody including: preparing an antibody including (1) heavy chain framework regions and heavy chain CDRs, where at least one of the heavy chain CDRs includes a selected amino acid sequence from CDR amino acid sequences shown in SEQ ID NOs: 8, 9, 10, 26, 27, 28, 38, 39, 40, 50, 51, 52, 62, 63, 64, 74, 75, 76 , 86, 87, 88, 104, 105, 106; and (2) light chain structure regions and light chain CDRs, where at least one of the light chain CDRs includes an amino acid sequence selected from CDR amino acid sequences shown in SEQ ID NOs: SEQ ID NOs: 14, 15, 16, 20, 21, 22, 32, 33, 34, 44, 45, 46, 56, 57, 58, 68, 69, 70, 80, 81, 82, 92, 93, 94, 98, 99, 100, 110, 111, 112; where the antibody retains the ability to bind to CD27. The ability of the antibody to bind to CD27 can be determined using standard binding assays, such as those set out in the Examples (for example, ELISA or FLISA). It is well known in the art that heavy and light antibody CDR3 domains play a particularly important role in the binding / affinity specificity of an antibody to an antigen (see, Hall et al., J.
Immunol., 149: 1605-1612 (1992); Polymenis et al., J.
Immunol., 152: 5318-5329 (1994); Jahn et al., Immunobiol., 193: 400-419 (1995); Klimka et al., Brit. J.
Cancer, 83: 252-260 (2000); Beiboer et al., J.
Mol.
Biol, 296: 833-849 (2000); Rader et al., Proc.
Natl.
Acad.
Sci.
USA, 95: 8910-8915 (1998); Barbas et al., J.
Am.
Chem.
Soc., 116: 2161-2162 (1994); Ditzel et al., J.
Immunol., 157: 739-749 (1996)). Thus, the recombinant antibodies of the invention prepared as set forth above preferably comprise the heavy and / or light chain CDR3s of antibodies 1F5, 1H8,
3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibodies may further comprise the CDR2s of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibodies may further comprise the CDR1s of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibodies can further comprise any combination of CDRs.
Thus, in one embodiment, the invention further provides anti-CD27 antibodies comprising: (1) heavy chain framework regions, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region, wherein the heavy chain CDR3 region is selected from 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5 CDR3s and (2) light chain framework regions, a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region, where the light chain CDR3 region is selected from CDR3s of 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5, in which the antibody binds to CD27. The antibody further may include the CDR2 heavy chain and / or the CDR2 light chain of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibody further may include the CDR1 heavy chain and / or the CDR1 light chain of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. Generation of antibodies containing modified sequences In another embodiment, the variable region sequences or parts thereof, of the anti-CD27 antibodies of the invention are modified to create structurally related anti-CD27 antibodies that retain binding (that is, for the same epitope as the unmodified antibody) and are thus functionally equivalent.
Methods for identifying residues that can be altered without removing the antigen binding are well known in the art (see, for example, Marks et al. (Biotechnology (1992) 10 (7): 779- 83 (diversification of monoclonal antibodies by shuffling of light chain variable regions, then heavy chain variable regions with fixed CDR3 sequence changes), Jespers et al. (1994) Biotechnology
12 (9): 899-903 (selection of human antibodies from phage presentation repertoires to a simple antigen epitope), Sharon et al. (1986) PNAS USA 83 (8): 2628-31 (site-directed mutagenesis of an invariant amino acid residue at the junction of variable diversity segments of an antibody); Casson et al. (1995) J.
Immunol. 155 (12): 5647-54 (evolution of loss and change in specificity resulting from random mutagenesis of a variable region of heavy chain). Thus, in one aspect of the invention, the CDR1, 2, and / or 3 regions of the projected antibodies described above may include the exact amino acid sequences of those antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5 disclosed here .
However, in other aspects of the invention, antibodies comprise derivatives of exact CDR sequences of 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5 still retain the ability to bind to CD27 effectively.
Said sequence modifications may include one or more amino acid additions, deletions or substitutions, for example, conservative sequence modifications as described above.
The sequence modifications may further be based on the consensus sequences described above for the particular sequences CDR1, CDR2, and CDR3 of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. Thus, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98% or 99.5% identical to one or more CDRs of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. Intermediate ranges from the values mentioned above, for example, CDRs that are 90-95%, 95-98%, or 98-100% identical in identity to one or more of the above sequences are still intended to be included by the present invention.
In another embodiment, one or more residues from a CDR can be altered to modify the bond to obtain a favored bond on-rate, a more favored bond off-rate, or both, so that an idealized bond constant is obtained .
Using this strategy, an antibody containing an ultra high binding affinity of, for example, 1010 M-1 or more, can be reached.
The affinity maturation techniques, well known in practice and those described here, can be used to alter the CDR regions followed by screening the resulting binding molecules for the desired change in binding.
Thus, as CDR (s) are altered, changes in binding affinity as well as immunogenicity can be monitored and classified so that an antibody optimized for the best combined binding and low immunogenicity is obtained.
In addition, or on the other hand, modifications within CDRs, modifications can still be prepared within one or more of the structural regions, FR1, FR2, FR3 and FR4, of the variable heavy chain and / or light chain regions of an antibody, so that these modifications do not eliminate the binding affinity of the antibody.
For example, one or more amino acid residues of non-germline lineage in the structural regions of the variable region of heavy and / or light chain of an antibody of the invention, is replaced with an amino acid residue of germline lineage, that is, the corresponding residue of amino acid in the human germline sequence for the heavy or light chain variable region, whose antibody has significant sequence identity with.
For example, an antibody chain can be aligned with a germline antibody chain that it shares sequence identity with, and amino acid residues that do not match between antibody structural sequence and the germline chain structure can be replaced with corresponding residues from the germline sequence.
When an amino acid differs between an antibody variable structure region and an equivalent human germline sequence variable structure region, the antibody structure amino acid could be replaced by the equivalent human germline amino acid sequence if it is reasonably expected that the amino acid falls within one of the following characteristics: (1) an amino acid residue that non-covalently binds directly to the antigen, 5 (2) an amino acid residue that is adjacent to a CDR region, (3) an amino acid residue that , otherwise, interacts with a CDR region (for example, it is within about 3-6 Å of a CDR region as determined by computer modeling), or (4) an amino acid residue that participates in the VL-VH interface .
Residues with "not covalently binds to antigen directly" include amino acids at positions in structural regions that have a good chance of interacting directly with amino acids in the antigen according to established chemical forces, for example, by hydrogen bonding, Van der forces Waals, hydrophobic interactions, and the like.
Thus, in one embodiment, an amino acid residue in the structural region of an antibody of the invention is replaced with the corresponding germline amino acid residue that binds non-covalently to the antigen directly.
Residues that are "adjacent to a CDR region" include amino acid residues at positions immediately adjacent to one or more CDRs in the primary antibody sequence, for example, at positions immediately adjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia (see, for example, Chothia and Lesk J.
Mol.
Biol. 196: 901 (1987)). Thus, in one embodiment, an amino acid residue within the structural region of an antibody of the invention is replaced with a germline amino acid residue that is adjacent to a CDR region.
Residues that "otherwise interact with a CDR region" include those that are determined by secondary structural analysis because they are in special orientation sufficient to affect a CDR region.
Said amino acids will generally have a side chain atom within about 5 of 3 angstrom units (Å) of some atoms in the CDRs and must contain an atom that could interact with the CDR atoms according to established chemical forces, such as those above lists.
Thus, in one embodiment, an amino acid residue within the structural region of an antibody of the invention is replaced with a corresponding germline amino acid residue that otherwise interacts with a CDR region.
Amino acids at various positions in the structure are known to be important for determining CDR confirmation (for example, capable of interacting with CDRs) in many antibodies (Chothia and Lesk, supra, Chothia et al., Supra and Tramontano et al ., J.
Mol.
Biol. 215: 175 (1990), all of which are incorporated by reference). These authors identified conserved structural residues important for CDR conformation by analyzing the structures of several known antibodies.
The analyzed antibodies are within a limited number of structural or “canonical” classes based on the conformation of CDRs.
Structural residues conserved within members of a canonical class as referred to as a “canonical” residue. Canonical residues include residues 2, 25, 29, 30, 33, 48, 64, 71, 90, 94 and 95 of the light chain and residues 24, 26, 29, 34, 54, 55, 71 and 94 of the heavy chain.
Additional residues (for example, CDR structure determining residues) can be identified according to the methodology of Martin and Thorton (1996) J.
Mol.
Biol. 263: 800. Notably, the amino acids at positions 2, 48, 64 and 71 of the light chain and 26-30, 71 and 94 of the heavy chain (numbering according to Kabat) are known to be able to interact with CDRs in many antibodies.
Amino acids at positions 35 in the light chain and 93 and 103 in the heavy chain are also likely to interact with CDRs.
Additional residues that can affect the conformation of CDRs can be identified according to the methodology of Foote and Winter (1992) J.
Mol.
Biol. 224: 487. 5 Such residues are called “vernier” residues and those residues in the structural region intimately underlying (that is, forming a “platform” under) the CDRs.
Residues that "participate in the VL-VH interface" or "packaging waste" include those residues at the interface between VL and VH as defined, for example, by Novotny and Haber, Proc.
Natl.
Acad.
Sci.
USA, 82: 4592-66 (1985) or Chothia et al, supra.
Occasionally, there is some ambiguity about whether a particular amino acid falls within one or more of the categories mentioned above.
In said cases, alternative variant antibodies are produced, one of which has this particular substitution, the other of which does not.
Alternative variant antibodies so produced can be tested in any of the assays described for the desired activity and the preferred antibody selected.
Additional candidates for substitution within the structural region are amino acids that are unusual or "rare" for an antibody in that position.
These amino acids can be replaced with amino acids from the equivalent position of the human germline sequence or equivalent positions of more typical antibodies.
For example, substitution may be desirable when the amino acid in a structural region of the antibody is rare for that position and the corresponding amino acid in the germline sequence is common for that position in immunoglobulin sequences; or when the amino acid in the antibody is rare for that position and the corresponding amino acid in the germline sequence is also rare, in relation to other sequences.
It is contemplated that by replacing an unusual amino acid with an amino acid of the germline sequence that happens to be typical for antibodies, the antibody can be prepared less immunogenic.
The term "rare", as used here, indicates an amino acid 5 occurring in that position in less than about 20%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than that about 3%, even more preferably less than about 2% and even more preferably less than about 1% of sequences in a representative sample of sequences, and the term "common", as used here, indicates an occurrence amino acid in more than about 25% but generally more than about 50% of sequences in a representative sample.
For example, all light and heavy chain variable region sequences are respectively grouped into “subgroups” of sequences that are especially homologous to each other and have the same amino acids at certain critical positions (Kabat et al., Supra). When deciding whether an amino acid in an antibody sequence is "rare" or "common" between sequences, it will generally be preferred to consider only those sequences in the same subgroup as the antibody sequence.
In general, the antibody framework regions are generally substantially identical, and more generally, the framework regions of the human germline sequences from which they are derived.
Of course, many of the amino acids in the structural region make little or no direct contribution to the specificity or affinity of an antibody.
Thus, many individual conservative substitutions of structural residues can be tolerated without appreciably altering the specificity or affinity of the resulting immunoglobulin.
Thus, in one embodiment, the region of variable structure of the antibody shares at least 85% sequence identity to a sequence of variable structural region of human germline or consensus of said sequences.
In another embodiment, the variable structure region of the antibody shares at least 90%, 95%, 96%, 97%, 98% or 99% of sequence identity to a sequence of variable structural region of human germline or consensus of 5 said sequences.
In addition to simply binding to CD27, an antibody can be selected to retain other functional properties of antibodies of the invention, such as: (1) inhibits (for example, completely or partially blocks) CD70 binding to cells that express CD27 by at least about 70%; (2) binds to human CD27 with a Kd equilibrium dissociation constant of 10-9 M or less, or alternatively, a Ka equilibrium association constant of 10 + 9 M-1 or more (3) induces at least about 40% complement-mediated cytotoxicity (CDC) of cells expressing CD27 at a concentration of 10 μg / ml; and / or (4) induces at least about 40% specific ADCC-mediated lysis of cells expressing CD27 at a concentration of 10 μg / ml.
Characterization of monoclonal antibodies to CD27 The monoclonal antibodies of the invention can be characterized by binding to CD27, using a variety of known techniques.
Generally, antibodies are initially characterized by ELISA.
Briefly, microtiter plates can be coated with CD27 purified in PBS, and then blocked with irrelevant proteins, such as bovine serum albumin (BSA) diluted in PBS.
Dilutions of plasma from mice immunized with CD27 are added to each well and incubated for 1-2 hours at 37ºC.
The plates are washed with PBS / Tween
20 and then incubated with a goat anti-human IgG polyclonal reagent specific for Fc conjugated with alkaline phosphatase, for 1 hour at 37ºC.
After washing, the plates were developed with ABTS substrate, and analyzed in OD of 405. Preferably, the mice that develop the highest titers will be used for the fusions.
An ELISA assay, as described above, can be used to screen antibodies and thus hybridomas that produce antibodies that show positive reactivity with the CD27 immunogen. Hybridomas that bind, preferably with a high affinity, to CD27 can then be subcloned and further characterized.
One clone of each hybridoma, which retains the reactivity of the parental cells (by ELISA), can then be chosen to make a cell bank, and for the purification of antibodies.
To purify anti-CD27 antibodies, selected hybridomas can be grown in roller bottles, two-liter roller bottles or other culture systems.
Supernatants can be filtered and concentrated before protein A-Sepharose affinity chromatography (Pharmacia, Piscataway, NJ) to purify the protein.
After changing PBS buffer, the concentration can be determined by OD280 using 1.43 extinction coefficient or preferably by nephelometric analysis.
IgG can be verified by gel electrophoresis and by specific antigen method.
To determine whether the selected anti-CD27 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding to biotinylated MAb can be detected with a labeled streptavidin probe.
To determine the isotype of purified antibodies, isotype ELISAs can be performed using techniques recognized in practice.
For example, microtiter plate wells can be coated with 10 µg / ml of anti-Ig antibodies overnight at 4 ° C.
After blocking with 5% BSA, the plates are reacted with 10 µg / ml of monoclonal antibodies or purified isotype controls, at room temperature for two hours.
The wells can then be reacted with IgGl or other isotype-specific conjugate probes.
The plates are developed 5 and analyzed as described above.
To test the binding of monoclonal antibodies to living cells that express CD27, flow cytometry can be used.
Briefly, cell lines, and / or PBMCs that express membrane-bound CD27 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4 ° C for 1 hour.
After washing, the cells are reacted with fluorescein-labeled anti-IgG antibody according to the same conditions as the staining of the primary antibody.
The samples can be analyzed by a FACScan instrument using light and side scattering properties to record on individual cells and the binding of the labeled antibodies is determined.
An alternative assay using fluorescence microscopy can be used (in addition to or instead of) the flow cytometry assay.
The cells can be stained exactly as described above and examined by fluorescence microscopy.
This method allows the visualization of individual cells, but can decrease the sensitivity, depending on the density of the antigen.
Anti-CD27 IgGs can also be tested for reactivity with the CD27 antigen by Western blotting.
Briefly, cell extracts from CD27-expressing cells can be prepared and electrophoresed on polyacrylamide dodecyl sulfate gel.
After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested.
IgG binding can be detected using anti-alkaline IgG phosphatase antibody and developed with BCIP / NBT substrate tablets (Sigma Chem.
Co., St.
Louis, MO). Methods for the analysis of binding affinity, cross-reactivity, and binding kinetics of various anti-CD27 antibodies include standard assays known in the art, for example, BiacoreTM surface plasmon resonance analysis (SPR) using an BiacoreTM 2000 SPR (Biacore AB, Uppsala, Sweden), as described in Example 2 here. II.
Immunotoxins In another embodiment, the antibodies of the present invention are linked to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope.
When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins". A cytotoxin, or cytotoxic agent, includes any agent that is harmful to (for example, kills) cells.
Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracinadione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrochloride, 1- , procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues therefor.
Therapeutic agents include, but are not limited to, anti-metabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mecloretamine, chlorambucil thiope, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (for example, daunorubicin (antigen daunomycin) and doxorubicin), for example, antibiotics, dactinomycin (actinomycin antigen), bleomycin, mitramycin, and anthramycin (AMC)), and anti-mitotic agents (for example, vincristine and vinblastine). An antibody of the present invention can be conjugated to a radioisotope, for example, radioactive iodine, to generate cytotoxic radiopharmaceuticals for the treatment of a dendritic-related disorder, such as an autoimmune or inflammatory disease, or graft versus host disease. The conjugated antibody of the invention can be used to modify a given biological response, and the drug fraction should not be interpreted as limited to classical chemical therapeutic agents.
For example, the drug fraction can be a protein or polypeptide having a desired biological activity.
Said proteins can include, for example, an enzymatically active toxin or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein as a tumor necrosis factor or interferon-γ; or, biological response modifiers such as lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), colony stimulating factors macrogranulocyte macrophage (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
The techniques for conjugating said therapeutic antibody fraction are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243- 56 (Alan R.
Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic
Properties Of Antibody-Toxin Conjugates ”, Immunol.
Rev., 62: 119-58 (1982). III.
Compositions In another embodiment, the present invention provides a composition, for example, a composition, which contains one or a combination of monoclonal antibodies of the present invention, formulated together with a carrier (for example, a pharmaceutically acceptable carrier). Compositions containing bispecific molecules that comprise an antibody of the present invention are also provided.
In one embodiment, the compositions include a combination of several (e.g., two or more) antibodies isolated from the present invention.
Preferably, each of the antibodies in the composition binds to a different preselected epitope of CD27. The pharmaceutical compositions of the invention can also be administered in combination therapy, i.e., combined with other agents.
For example, combination therapy may include a composition of the present invention with at least one or more additional therapeutic agents, such as anti-inflammatory agents, DMARDs (disease-modifying antirheumatic drugs), immunosuppressive agents, and chemotherapeutic agents.
The pharmaceutical compositions of the present invention can also be administered in conjunction with radiation therapy.
Co-administration with other antibodies is also included by the present invention.
As used herein, the terms "carrier" and "pharmaceutically acceptable carrier" include any and all solvents, dispersion, medium, coatings, antibacterial and antifungal agents, isotonic agents and absorption retardants, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral spinal or epidermal administration
(for example, by injection or infusion). Depending on the route of administration, the active compound, that is, antibody, bispecific and multispecific molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound. Examples of adjuvants that can be used with the antibodies and constructs of the present invention include: Freund's Incomplete Adjuvant and Complete Adjuvant e (Difco Laboratories, Detroit, Michigan); Merck 65 Adjuvant (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts, such as aluminum hydroxide gel (alum) or aluminum phosphate, iron, calcium or zinc salts, an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres, cytokines, such as GM-CSF, interleukin-2, -7, -12 and other similar factors, 3D-MPL; oligonucleotide CpG, and monophosphoryl lipid A, for example, 3-de-O-acylated monophosphoryl lipid A. MPL adjuvants are available from Corixa Corporation (Seattle, Wash, see, for example, US Patents 4,436,727;
4,877,611; 4,866,034 and 4,912,094). Oligonucleotides containing CpG (in which the CpG dinucleotide is unmethylated) are well known and are described, for example, in WO 96/02555, WO 99/33488 and US Patents.
6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273: 352, 1996. Other alternative adjuvants include, for example, saponins, such as Quil A, or their derivatives, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Escina; digitonin, or Gypsophila or Chenopodium quinoa saponins; Montanide ISA 720 (Seppic, France); SAF (Chiron, California, United States); ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (for example, SBAS-2 or
SBAS-4, available from SmithKline Beecham, Rixensart, Belgium); Detox (EnhanzynTM) (Corixa, Hamilton, Mont) .; RC-529 (Corixa, Hamilton, Mont.) And other aminoalkyl glucosaminide 4-phosphates (AGP); polyoxyethylene ether adjuvants, such as those described in WO 99 / 52549A1, 5 synthetic imidazoquinolines, such as imiquimod [S-26308, R-837], (Harrison, et al., Vaccine 19: 1820-1826, 2001, and resiquimod [ S-28463, R-848] (Vasilakos, et al., Cellular immunology 204: 64-74, 2000; Schiff bases of carbonyls and amines that are constitutively expressed in antigen presenting cells and T cell surfaces, such as tucaresol ( Rhodes, J. et al., Nature 377: 71-75, 1995); cytokine, chemokine and costimulatory molecules such as protein or peptide, including, for example, pro-inflammatory cytokines such as Interferon, GM-CSF, IL-1 alpha, IL-1 beta, TGF-alpha and TGF-beta, Th1 inducers like gamma interferon, IL-2, IL-12, IL-15, IL-18 and IL-21, Th2 inducers like IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokine and costimulatory genes such as MCP-1, MIP-1 alpha, MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L; ligands targeted by immunostimulants like CTLA-4 and L-selectin, proteins apoptosis stimulants and peptides such as Fas; adjuvants based on synthetic lipids, such as vaxfectin, (Reyes et al., Vaccine 19: 3778-3786, 2001) squalene, alpha-tocopherol, polysorbate 80, DOPC and cholesterol; endotoxin, [LPS], (Beutler, B., Current Opinion in Microbiology 3: 23-30, 2000); ligands that trigger Toll receptors to produce Th1-inducing cytokines, such as synthetic mycobacterial lipoproteins, mycobacterial p19 protein, peptidoglycan, teicoic acid and lipid A, and CT (cholera toxin, subunits A and B) and LT (E thermolabile enterotoxin) coli, subunits A and B), heat shock protein family (HSPs), and LLO (listeriolisin O; WO 01/72329). These and several other Toll-like Receptor (TLR) agonists are described, for example, in Kanzler et al, Nature Medicine, May 2007, Vol. 13, No. 5. A preferred immunostimulating agent for use in combination with an anti -CD27 of the present invention is a TLR3 agonist, such as Poly IC.
A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not confer any undesired toxicological effects (see, for example, Berge, S.M., et al. (1977) J.
Pharm.
Sci. 66: 1-19). Examples of said salts include acid addition salts and base addition salts.
Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from non-toxic organic acids such as mono-and dicarboxylic aliphatic acids, acids phenyl-substituted alacanoics, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, as well as from non-toxic organic amines, such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
A composition of the present invention can be administered by a variety of methods known in the art.
As will be appreciated by a person skilled in the art, the route and / or mode of administration will vary depending on the desired results.
The active compounds can be prepared with vehicles that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyesters and polylactic acid.
Many methods for the preparation of said formulations are either patented or generally known to those skilled in the art.
See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978. To administer a compound of the present invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its 5 inactivation.
For example, the compound can be administered to an individual in an appropriate carrier, for example, liposomes, or a diluent.
Acceptable diluents include saline and aqueous buffer solutions.
Liposomes include CGF water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al. (1984) J.
Neuroimmunol. 7:27). Carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
The use of said means and agents for pharmaceutically active substances is known in the art.
Except to the extent that any conventional medium or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated.
Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions should typically be sterile and stable under conditions of production and storage.
The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
Adequate fluidity can be maintained, for example, through the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.
In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount, in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by microfiltration sterilization.
Generally, dispersions are prepared by incorporating the active compound 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 lyophilization (lyophilization), which produce a powder of the active ingredient plus any desired additional ingredient from a solution previously sterilized by filtration. Dosage regimens are adjusted to provide the desired optimal response (for example, a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased, as indicated by the requirements of the therapeutic situation.
For example, the antibodies of the invention can be administered once or twice a week by subcutaneous or intramuscular injection or once or twice a month by subcutaneous or intramuscular injection.
It is especially advantageous to formulate parenteral compositions in a unit dosage form for ease of administration and uniformity of dosage.
The unit dosage form as used herein refers to physically discrete units as unitary dosages for the individuals to be treated, each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the pharmaceutically required carrier.
The specification for the unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technique of handling said active compound for the treatment of sensitivity in individuals. 5 Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like, (2) oil-soluble antioxidants, such as ascorbyl palmitate , butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like, and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
For therapeutic compositions, the formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), vaginal rectal and / or parenteral administration.
The formulations can conveniently be presented in unit dosage form and can be prepared by any methods known in the pharmaceutical art.
The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the individual being treated, and the particular mode of administration.
The amount of active ingredient that can be combined with a support material to produce an individual dosage form will generally be the amount of the composition that produces a therapeutic effect.
Generally, out of one hundred percent, this amount will range from about 0.001 percent to about ninety percent of active ingredient, preferably from about 0.005 percent to about 70 percent, more preferably, between about 0, 01 percent to about 30 percent.
The formulations of the present invention that are suitable for vaginal administration also include pessaries, tampons, creams, gels,
pastes, foams or spray formulations containing said carriers as are known in the art to be suitable.
Dosage forms for topical or transdermal administration of compositions of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, adhesives and inhalants.
The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be needed.
The phrases “parenteral administration” and “administered parenterally” as used here mean modes of administration in addition to enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal and infusion.
Examples of aqueous or non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and their suitable mixtures, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
Adequate fluidity can be maintained, for example, through the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by using surfactants.
These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
The prevention of the presence of microorganisms can be guaranteed by the sterilization procedures, above, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, sorbic acid, phenol, and the like.
It may also be desirable to include isotonic agents, such as sugars, sodium chloride, similar in the compositions.
In addition, prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be administered alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Regardless of the route of administration selected, the compounds of the present invention, which can be used in a suitable hydrated form, and / or, the pharmaceutical compositions of the present invention, are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art .
The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic. for the patient.
The dosage level selected will depend on a variety of factors, including the pharmacokinetic activity of the particular compositions of the present invention used, or their ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound to be used, duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compositions employed, age, sex, weight, condition, general health and previous medical history of the patient to be treated, and factors similar well-known in the medical art.
A physician or veterinarian skilled in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the doctor or veterinarian could start with doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than required, in order to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the present invention will be the amount of the compound that is the lowest effective dose to produce a therapeutic effect. Said effective dose will generally depend on factors described above. It is preferred that the administration be intravenous, intramuscular, intraperitoneal or subcutaneous, preferably administered close to the target site. If desired, the effective daily dose of a therapeutic composition can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. Although it is possible for a compound of the present invention to be administered alone, it is preferred to administer the compound as a pharmaceutical formulation (composition). The therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the present invention can be administered with an injection device without a hypodermic needle, such as the devices described in US Patents 5,399,163,
5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present invention include: US patent 4,487,603, which discloses an implantable micro-infusion pump for delivering medication at a controlled rate; US patent 4,486,194, which discloses a therapeutic device for administering medications through the skin; US patent 4,447,233, which discloses a drug infusion pump for drug delivery, at an accurate infusion rate; US patent 4,447,224, which discloses an implantable variable flow infusion set for continuous drug delivery; US patent
4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments, and US Patent 4,475,196, which discloses an osmotic drug delivery system. Many other implants, delivery systems, and modules are well known to those skilled in the art. In certain embodiments, the antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, US patents 4,522,811; 5,374,548, and 5,399,331. Liposomes can comprise one or more units that are selectively transported in specific cells or organs, thereby improving the release of specific drugs (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Examples of targeting fractions include folate or biotin (see, for example, US Patent 5,416,016 to Low et al.); mannosids (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PG Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180), surfactant protein A receptor (Briscoe et al. (1995) Am J. Physiol. 1233: 134), different species from which you can understand the formulations of the inventions, as well as components of the invented molecules; p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4: 273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes, in a more preferred embodiment, liposomes include a targeting fraction. In a more preferred embodiment, therapeutic compounds in which liposomes are released by bolus injection to a site proximal to the tumor or infection.
The composition should be fluid to the extent that easy syringability exists.
It must be stable under the conditions of production and storage and must be preserved 5 against the contaminating action of microorganisms such as bacteria and fungi.
The ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors.
Alternatively, this property of a composition can be assessed by examining the ability of the compound to inhibit, said inhibition in vitro by assays known to those skilled in the art.
A therapeutically effective amount of a therapeutic compound can decrease the size of the tumor, or otherwise improve symptoms in an individual.
A person skilled in the art would be able to determine said amounts based on said factors such as the size of the individual, the severity of the individual's symptoms, and the particular composition or route of administration selected.
The composition must be sterile and fluid, as the composition is released by syringe.
In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and their suitable mixtures.
Adequate fluidity can be maintained, for example, through the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.
In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
When the active compound is adequately protected, as described above, the compound can be administered orally, for example,
with an inert diluent or an assimilable edible carrier. IV.
Uses and methods of the present invention In one embodiment, antibodies, bispecific molecules, and compositions of the present invention can be used to treat and / or prevent (e.g., immunization against) a variety of diseases and conditions.
One of the indications of the primary disease that can be treated is cancer.
In particular, an anti-CD27 antibody, which induces or enhances an immune response, can be used in the treatment of cancer.
Types of cancer include, but are not limited to, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblast promyelocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic leukemia (granulocytic), chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma Burkitt's lymphoma, marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, mycosarcoma, liposarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosoma, colon cancer, cancer of the colon, carcinoma, colon cancer, carcinoma squamous cell carcinoma, basal cell carcinoma, adenocarcinoma ma, sweat cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, kidney cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryo cancer cervical, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, oloma menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma 5, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, central nervous system and brain cancer (CNS), cervical cancer, choriocarcinoma, cancers colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, cancer esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; cancer of the oral cavity (for example, lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancer.
Preferred cancers include CD27-expressing tumors selected from the group consisting of chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B cell lymphoma.
Other disease indications for use of an anti-CD27 antibody, which induces or enhances an immune response include infectious bacterial, fungal, viral and parasitic diseases.
Other disease indications for using an anti-CD27 antibody that inhibits or reduces the immune response include graft rejection, autoimmune diseases and allergies.
Exemplary autoimmune diseases include, but are not limited to, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease and other inflammatory bowel diseases such as ulcerative colitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), thyroiditis of MG Hashimoto, Goodpasture's syndrome, pemphigus,
Serious, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polyposis, pernicious anemia, idiopathic Addison's disease, autoimmune associated infertility, glomerulonephritis, glomerulonephritis 5, growing, glomerulonephritis Sjogren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes mellitus, autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome, autoimmune uveoretinitis, Guillainter's disease, and Guillainter's disease Alzheimer's disease.
Examples of allergic disorders include, but are not limited to, allergic conjunctivitis, vernal conjunctivitis, vernal keratoconjunctivitis, and giant papillary conjunctivitis; allergic nasal disorders, including allergic rhinitis and sinusitis; optical allergic disorders, including itching Eustachian tube; allergic disorders of the upper and lower airways, including intrinsic and extrinsic asthma; allergic skin disorders, including eczema, dermatitis and hives, and allergic disorders of the gastrointestinal tract.
In another aspect, an antibody of the present invention is administered in combination with a vaccine, to increase the immune response against the vaccine antigen, for example, a tumor antigen (to thereby increase the immune response against the tumor), or a antigen of an infectious disease pathogen (to thereby increase the immune response against the infectious disease pathogen). Thus, in this embodiment, a vaccine antigen may comprise, for example, an antigen or antigenic composition capable of inducing an immune response against a tumor or an infectious disease pathogen such as a virus, bacterium, parasite or fungus.
The antigen or antigens can be, for example, peptides / proteins, polysaccharides, and / or lipids.
The tumor-derived antigen or antigens, such as the various tumor antigens described hereinabove.
Alternatively, the antigen or antigens can be derived from pathogens, such as viruses, bacteria, parasites and / or fungi, such as the antigens of various pathogens previously described herein.
Other examples of suitable pathogen antigens include, but are not limited to, the following: 5 Viral antigens or antigenic determinants can be derived from, for example: Cytomegalovirus (especially human, such as gB or its derivatives); Epstein Barr virus (such as gp350); flavivirus (eg yellow fever virus, Dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus); hepatitis viruses, such as hepatitis B virus (eg hepatitis B surface antigen such as PreS1, PreS2 and S antigens described in EP-A-414 374; 578 EP-A-0304 and 198474-EP-A), virus hepatitis A, hepatitis C virus and hepatitis E virus; HIV-1, (such as tat, nef, gpl20 or gpl60); human herpes virus, such as gD or its derivatives, or immediate-onset protein such as HSP1 or HSV2 ICP27; human papilloma virus (for example HPV6, 11, 16, 18); Influenza viruses (whole or inactivated viruses, split influenza viruses, cultured in eggs or MDCK cells, or Vero cells or complete influenza viruses (as described by Gluck, Vaccine, 1992,10, 915-920) or recombinant or purified proteins , such as proteins, NP, NA, HA or M); measles virus; mumps virus; parainfluenza virus; rabies virus; Respiratory syncytial virus (such as F and G proteins); rotavirus (including live attenuated virus); smallpox virus; Varicella Zoster virus (such as gpI, II and IE63); and the HPV viruses responsible for cervical cancer (for example, early E6 or E7 proteins fused with a protein D carrier to form HPV 16 D-E6 or E7 protein fusions, or combinations thereof; or combinations of E6 or E7 with L2 (see for example WO 96/26277). Bacterial antigens or antigenic determinants can be derived from, for example: Bacillus spp., Including B. anthracis (eg, botulinum toxin); Bordetella spp, including B. pertussis (e.g. pertactin, pertussis toxin, filamentous hemagglutinin, adenylate cyclase, fimbriae); Borrelia spp., including B. burgdorferi (e.g., OspA, OspC, DbpA, DbpB), B. garinii (e.g., OspA, OspC, DbpA , 5 DbpB), B. afzelii (for example, OspA, OspC, DbpA, DbpB), B. andersonii (for example, OspA, OspC, DbpA, DbpB), B. hermsii; Campylobacter spp, including C. jejuni (by toxins, adhesins and invasins) and C. coli; Chlamydia spp., including C. trachomatis, (for example, MOMP, lytic proteins heparin generation), C. pneumonie (for example, MOMP, heparin binding proteins), C. psittaci; Clostridium spp., Including C. tetani (as tetanus toxin), C. botulinum (for example botulinum toxin), C. difficile (for example, clostridium toxins A or B); Corynebacterium spp., Including C. diphtheriae (for example, diphtheria toxin); Ehrlichia spp., Including E. equi and the Human Granulocytic Ehrlichiosis agent; Rickettsia spp, including R.rickettsii; Enterococcus spp., Including E. faecalis, E. faecium; Escherichia spp, including enterotoxic E. coli (eg colonization factors, heat labile toxin or derivatives thereof, or a heat stable toxin), enterohemorrhagic E. coli, enteropathogenic E. coli (eg shiga toxin type toxin ); Haemophilus spp., Including H. influenzae type B (eg, PRP), H. influenzae without type, eg OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and peptides derived from fimbrin (see for example US 5,843,464); Helicobacter spp, including H. pylori (e.g., urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Legionella spp, including L. pneumophila; Leptospira spp., Including L. interrogans; Listeria spp., Including L. monocytogenes; Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis (for example, high and low molecular weight adhesins and invasins); Morexella Catarrhalis (including outer membrane vesicles, and OMP106 (see, for example
W097 / 41731)); Mycobacterium spp., Including M. tuberculosis (e.g., ESAT6, antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Neisseria spp, including N. gonorrhea and N. meningitidis (for example, capsular polysaccharides and conjugates of the same 5, transferrin binding proteins, lactoferrin binding proteins, PilC, adhesins); Neisseria mengitidis B (including outer membrane vesicles, and NspA (see for example WO 96/29412); Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Staphylococcus spp., Including S. aureus, S. epidermidis; Streptococcus spp, including S. pneumonie (e.g., capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline binding proteins ) and the Pneumolysin protein antigen (Biochem Biophys Acta, 1989,67,1007; Rubins et al., Microbial Pathogenesis, 25,337-342), and detoxified derivatives thereof (see, for example WO 90/06951; WO 99/03884 ); Treponema spp., Including T. pallidum (for example, outer membrane proteins), T. denticola, T. hyodysenteriae; Vibrio spp, including V. cholera (for example, cholera toxin); and Yersinia spp, including Y enterocolitica (for example, a Yop protein), Y. pestis, Y. pseudotuberculosis.
Parasitic / fungal antigens or antigenic determinants can be derived from, for example: Babesia spp., Including B. microti; Candida spp., Including C. albicans; Cryptococcus spp., Including C. neoformans; Entamoeba spp., Including E. histolytica; Giardia spp., Including; G. lamblia; Leshmania spp., Including L. major; Plasmodium. faciparum (MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27 / 25, Pfsl6, Pfs2 in Plasmodium spp.); Pneumocystis spp., Including P.; carinii; Schisostoma spp., Including S. mansoni; Trichomonas spp., Including T. vaginalis; Toxoplasma spp., Including T.
gondii (for example, SAG2, SAG3, Tg34); Trypanosoma spp., Including T. cruzi.
It will be appreciated that in accordance with that aspect of the present invention, antigens and antigenic determinants can be used in many different ways.
For example, antigens or antigenic determinants may be present as isolated proteins or peptides (for example, in so-called “vaccine subunits”) or, for example, as antigens or antigenic determinants associated with cells or associated with viruses (for example, in strains live or dead). Live pathogens will preferably be attenuated in a known manner.
Alternatively, antigens or antigenic determinants can be generated in situ in the individual by using a polynucleotide encoding an antigen or antigenic determinant (such as the so-called “DNA vaccination”), although it is appreciated that the polynucleotides that can be used with this approach are not limited to DNA, and may also include modified RNA and polynucleotides as discussed above.
In one embodiment, a vaccine antigen can also be targeted, for example, to specific cell types or specific tissues.
For example, the vaccine antigen can be targeted to antigen presenting cells (APCs), for example, by using agents such as antibodies to APC surface receptors such as DEC-205, for example, as discussed in WO 2009/061996 (Celldex Therapeutics, Inc) or the Mannose Receptor (CD206), for example, as discussed in WO 03040169 (Medarex Inc). For use in therapy, the antibodies of the invention can be administered to an individual directly (i.e., in vivo), alone or with other therapies such as an immunostimulating agent therapy, a vaccine, chemotherapy or radiation therapy.
In all cases, antibodies, bispecifics, immunostimulating compositions and agents and other therapies are administered in an effective amount to exert their desired therapeutic effect.
The term "effective amount" refers to that amount needed or sufficient to achieve a desired biological effect.
For example, an effective amount could be that amount needed to eliminate a tumor, cancer, or bacterial, viral, or fungal infection.
The effective amount for any specific application can vary depending on such factors as the disease or condition being treated, the specific antibody being administered, the size of the individual, or the severity of the disease or condition.
A person skilled in the art can empirically determine the effective amount of a particular molecule without requiring undue experimentation.
Preferred routes of administration for vaccines include, for example, injection (for example, subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal). The invention can be a bolus or a continuous infusion.
Other routes of administration include oral administration.
Bispecific antibodies and molecules of the invention can also be co-administered with adjuvants and other therapeutic agents.
It will be appreciated that the term "co-administered" as used herein includes any and all of simultaneous, separate, or sequential administration of the antibodies and conjugates of the present invention with adjuvants and other agents, including administration as part of a dosage regimen.
Antibodies are usually formulated in a carrier, either alone or in combination with said agents.
Examples of said carriers include solutions, solvents, dispersion medium, retarding agents, emulsions and the like.
The use of said means for pharmaceutically active substances is known in the art.
Any other conventional carrier suitable for use with the molecules is within the scope of the current invention.
Suitable agents for coadministration with the conjugated, bispecific antibodies and compositions include other antibodies, cytotoxins and / or drugs, such as adjuvants, immunostimulating agents and / or immunosuppressants.
In one embodiment, the agent is a chemotherapeutic agent.
Antibodies, bispecifics and compositions can be administered in combination with radiation.
Chemotherapeutic agents suitable for co-administration with the antibodies and conjugates of the present invention in the treatment of tumors include, for example: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracinadione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologues thereof.
Other agents include, for example, anti-metabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (for example, mecloretamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (for example, daunorubicin (daunomycin antigen) and doxorubicin (eg, antibiotics), antibiotics (actinomycin antigen), bleomycin, mitramycin, and anthramycin (AMC)), and anti-mitotic agents (eg, vincristine and vinblastine) and temozolomide.
Agents that eliminate or inhibit immunosuppressive activities, for example, by immune cells (for example, regulatory T cells, NKT cells, macrophages, myeloid-derived suppressor cells, immature or suppressive dendritic cells) or suppressive factors produced by the tumor or host cells in the local tumor microenvironment (e.g. TFGbeta, indoleamine 2,3 dioxigenase-IDO), can also be administered with antibodies and conjugates of the present invention. Said agents include antibodies and small molecule drugs such as IDO inhibitors such as 1 methyl tryptophan or derivatives. Suitable agents for coadministration with the antibodies and bispecifics of the present invention for the treatment of said immune disorders include, for example, immunosuppressive agents such as rapamycin, cyclosporine, and FK506; anti-TNFα agents such as etanercept, adalimumab and infliximab; and steroids. Examples of specific natural and synthetic steroids include, for example: aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazole, deoxycortone, desonide, deoxymethasone, dexamethasone, difluorocortolone, fluclorolone, flumetasone, fluorinone, fluorinone , fluorocortolone, fluorometolone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, parametasone, prednisolone, prednisone, thixocortol and triamcinolone. Suitable agents for co-administration with the antibodies and bispecifics of the present invention for inducing or ameliorating an immune response include, for example, adjuvants and / or immunostimulating agents, not limiting the examples that have been disclosed herein before. A preferred immunostimulating agent is a TLR3 agonist, such as Poly IC: The present invention is further illustrated by the following examples which are not to be construed as limiting. The contents of the Sequence listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated here by reference.
EXAMPLES Example 1 Generation of human monoclonal antibodies specific for CD27 Human monoclonal anti-CD27 antibodies were generated by immunizing the HC2 / KCo7 strain of transgenic HuMAb® mice (“HuMAb” is a registered trademark of Medarex, Inc., Princeton, New Jersey ) with a soluble human CD27 antigen.
HC2 / KCo7 HuMAb mice were generated as described in US patents 5,770,429 and 5,545,806, the 5 full disclosures are incorporated here by reference.
Antigen and immunization: The antigen was a soluble fusion protein comprising an extracellular CD27 domain fused to an antibody Fc domain (chimeric human recombinant Fc-CD27 proteins (R&D Systems). The antigen was mixed with complete Freund's adjuvant (Sigma) for the first immunization.
After that, the antigen was mixed with incomplete Freund (Sigma). Additional mice were immunized with the soluble protein CD27 in adjuvant system RIBI MPL plus TDM (Sigma). 5-25 micrograms of recombinant CD27 antigen soluble in PBS or 5 x 106 CHO cells transfected for surface expression of human CD27 in PBS were mixed 1: 1 with the adjuvant.
The mice were injected with 100 microliters of the antigen prepared in the peritoneal cavity every 14 days.
Animals that developed anti-CD27 titers received an iv injection of 10 micrograms of soluble recombinant CD27 antigen three to four days before fusion.
The spleens of the mice were harvested and the splenocytes isolated used to prepare the hybridoma.
Hybridoma preparation: The murine myeloma cell line P3x63Ag8.653 (ATCC CRL 1580) was used for fusions.
RPMI 1640 (Invitrogen) containing 10% FBS was used for culture of multiple myeloma cells.
Additional medium supplements were added to the hybridoma growth medium, which included: 3% Origen-Hybridoma cloning factor (Igen), 10% FBS (Sigma), L-glutamine (Gibco) 0.1% gentamicin (Gibco ), 2-mercaptoethanol (Gibco), HAT (Sigma; 1.0 x 104 M hypoxanthine, 4.0 x 10-7 M aminopterin, 1.6 x 10-5 M thymidine), or HT
(Sigma; 0 x 10-4 M hypoxanthine, 1.6 x 10-5 M thymidine). Spleen cells were mixed with myeloma cells P3x63Ag8.653 in a 6: 1 ratio and pelleted by centrifugation.
Polyethylene glycol was added dropwise with careful mixing to facilitate melting.
Hybridomas were allowed to grow for one to two weeks until visible colonies were established.
The supernatant was collected and used for initial screening for human IgG by ELISA using a specific capture of the human kappa chain and a specific detection of human Fc.
IgG positive supernatants were then evaluated for CD27 specificity by flow cytometry or ELISA for anti-CD27 detection. Hybridomas producing specific human monoclonal antibodies (human mAbs; IgG) have been subcloned and expanded.
The human mAbs produced were then purified by protein A column chromatography under standard conditions that led to the isolation of a number of antibodies of particular interest, which were designed as 4B7-1B3 (also called here as 4B7), 3H12-1C8 (also called here as 3H12), 1F5-1H5 (also called here as 1F5), 2C2-1A10 (also called here as 2C2), 2G9-1D11 (also called here as 2G9), 1H8-B4 (also called here as 1H8), 3H12-1E12 (also called here as 3H12), 3G1-1A11 (also called here as 3G1) (1B10), 4A2-B11 (also called here as 4A2), 3A10-G10 (also called here as 3A10), 2G11-B5 (also called here as 2G11), 4H11-G11 (also called here as 4H11), 2H3-E8 (also called here as 2H3), 4A7-B3 (also called here as 4A7), 3H8-1B11 (also called here as 3H8 ) and 1G5-1B9 (also called here as 1G5). Hybridomas were also analyzed for cross-reactivity with rhesus monkey CD27 and all were positive for binding.
Example 2
Determination of affinity and constant rates of human mAbs by surface plasmon resonance (SPR) The binding and affinity kinetics of various anti-human CD27 antibodies in Example 1 were examined by BiacoreTM surface plasmon resonance analysis (SPR) using a BiacoreTM 2000 SPR instrument (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines.
Purified recombinant human CD27 protein / TNFRSF7 / Fc chimera (R&D Systems catalog no. 382-CD) was covalently linked to a BiacoreTM CM5 sensor chip (carboxymethylated dextran covalently attached to a gold surface; Biacore product number BR- 1000-14) using standard amine coupling chemistry with an Amine Coupling Kit provided by Biacore according to the manufacturer's guidelines (BIAcore Product No. BR-1000-50, comprising N-hydroxysuccinimide coupling reagents (NHS) and 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)). Low levels of binder were immobilized to limit the mass transport effects of the analyte on kinetic parameters, as observed RMAX was in the range of 100-400 RU.
Binding was measured by flowing antibodies over the HBS-EP buffer sensor chip (HBS-EP buffer, Biacore Product No. BR-1001-88: 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) 0 , 01M, sodium chloride 0.15M ethylenediaminetetraacetic acid (EDTA) 3mM, Surfactant P20 0.005%), in concentrations ranging from 1.56 to 50 nM and with a flow rate of 30 µl / minute for 180 seconds.
Antigen-antibody dissociation and association kinetics were followed for 480 seconds or 1200 seconds for antibodies with slower dissociation rates.
The corresponding controls were performed in each case, using a blank cell flow with no immobilized protein for subtracting the background. Sequential injections of 10mM HCl during
10 seconds at 50 µl / min followed by 10mM glycine pH 2.0 for 30 seconds at 50 µl / min were used as the regeneration conditions throughout the study.
Biacore’s BIAevaluation software version 3.2 (Biacore AB, 5 Uppsala, Sweden) was used in each case to derive kinetic parameters from the diluted analyte concentration series in HBS-EP running buffer.
The association and dissociation curves were fitted for a Langmuir 1: 1 connection model using BiacoreTM BIAevaluation software (Biacore AB) according to the manufacturer's instructions.
The affinity and kinetic parameters (with subtracted background) as determined are shown in Figure 1. For each antibody, the values presented are the average of two distinct series of experiments, using flow cells prepared separately in each case, (where ka = association rate constant, kd = dissociation rate constant, KD = dissociation equilibrium constant (affinity measure), KA = association equilibrium constant, Rmax = maximum SPR response signal). Example 3 ELISA assay to determine binding characteristics to human mAb in CD27 Microtiter plates were coated with soluble or recombinant human or monkey CD27 in PBS, and then blocked with bovine serum albumin in PBS. Human mAbs purified in protein A and a control isotype were added in saturation concentrations and incubated at 37ºC.
The plates were washed with PBS / Tween and then incubated with a goat anti-human IgG polyclonal reagent specific for Fc conjugated with alkaline phosphatase at 37ºC.
After washing, the plates were developed with pNPP substrate (1 mg / ml) and analyzed on OD 405- 650 using a microtiter plate reader.
Representative binding curves are shown in Figure 2. The results were also used to estimate the 50% saturation concentration (C value in a 4-parameter adjustment curve) as shown in Table 1 below.
To establish that cynomolgus monkeys are a relevant model 5 for testing anti-CD27 mAbs, various concentrations of purified monkey CD27 or human CD27 were captured onto ELISA plates with anti-Flag antibody, followed by incubation with human anti-CD27 mAb. A goat anti-human IgG Fc-HRP antibody and Super Blue TMB substrate were used for detection.
The results are shown in Table 1, which indicate similar binding to monkey and human CD27.
Representative binding curves for 1F5 antibody are also shown in Figure 3. Table 1. Characterization of selected anti-CD27 mAb Half binding Half binding Maximum to maximum binding to CD27 maximum mAb to human CD27 human monkey CD27 (M) ** (µg / ml) ** (µg / ml) ** 1G5 4.9E-10 0.074 0.065 1H8 4.3E-10 0.064 0.105 3H12 5.7E-10 0.085 0.123 3H8 4.6E-10 0.069 0.065 2G9 4.3E- 10 0.064 0.069 1F5 3.9E-10 0.059 0.12 3A10 6.3E-10 0.094 No 2C2 binding 3.0E-10 0.045 0.034 ** estimated by binding to CD27-coated plates in an ELISA format In another experiment, for To establish a similar distribution of 1F5 binding to peripheral blood cells, PBMCs were isolated from the blood of 3 humans and 3 cynomolgus monkeys.
The cells were stained with 1F5 mAb along with markers to outline the main populations of T cells and B cells that express CD27. The following table (Table 2) summarizes the mean ± standard error of results for human and monkey cells in relation to the percentage of cells expressing CD27 and the intensity of expression (IFM). These data establish a similar distribution of 1F5 binding to peripheral blood cells from humans and monkeys.
Table 2 Analysis CD4 + T cells CD8 + T cells B (CD20 +) NK cells human monkey human monkey human monkey human monkey% CD27 + b 84 ± 5 81 ± 1 70 ± 12 90 ± 1 37 ± 4 15 ± 1 11 ± 4 88 ± 6 MFIc 1517 ± 123 461 ± 14 1415 ± 153 519 ± 11 893 ± 101 491 ± 113 667 ± 28 1050 ± 42
Example 4 4A: Blocking sCD70 binding by ELISA 5 The effect of the human mAbs from example 1 on binding of soluble CD70 (sCD70) to the CD27 protein was measured by ELISA.
A microtiter plate was coated with 1µg / ml of soluble recombinant human CD27 / Fc chimera from R&D Systems at 1µg / ml., Then blocked with 5% PBA.
Anti-CD27 antibodies ([final] = 25 µg / mL) were pre-mixed with recombinant human soluble CD70-biotin from US Biologicals ([final] = 0.5 µg / mL) and added to the plate. rCD70 captured by CD27 was detected with streptavidin-HRP and Super Azul TMB substrate.
The results (shown as% block) are shown in Figure 4 with controls as indicated.
These results show that several of the antibodies (including 1F5, 1H8, 3H12 and 1A4) had the blocking property or at least significantly inhibited the binding of sCD70. 4B: CD27 mAb binds to CD27 in human lymphoblastoid cell lines and blocks ligand (CD70) binding The binding of anti-human CD27 mAb 1F5 to human lymphoblastoid cell lines and sCD70 binding block was analyzed by flow cytometry , using a Becton Dickinson FACSCanto II flow cytometer.
The results are shown in Figure 5 and show that 1F5 effectively binds to a variety of cell lines and competitively inhibits sCD70 binding. Example 5 Binding of human mAbs to cells expressing human CD27 The ability of human anti-CD27 mAbs to bind CD27 in cells expressing human CD27 on their surface was investigated by flow cytometry, as follows. Antibodies were tested for binding to human cell lines expressing human D27 on its surface. Protein A purified 2C2, 3H8 5 and 1F5 mAbs were incubated with Jurkat, Raji, Ramos and Daudi cells expressing human CD27, as well as control cells at 4ºC. All antibodies were used at saturation concentrations. After 1 hour, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and bound antibodies were detected by incubating the cells with a goat anti-human IgG-specific probe labeled with PE, at 4º C. The excess probe was washed from the cells with PBA and the fluorescence of the associated cell was determined by analysis using an LSRTM instrument (BD Biosciences, NJ, USA) according to the manufacturer's instructions. As shown in figures 6 and 7, human mAbs demonstrated high level binding to cells expressing human CD27. These data demonstrate that these antibodies bind efficiently and specifically to human CD27 expressed in living cells, as compared to control cells. Example 6 Cross-blocking / competition of human mAbs determined by
ELISA A microtiter plate was coated with a recombinant human chimeric Fc-CD27 fusion protein, then blocked with 5% BSA in PBS. Unconjugated human mAbs (20 g / mL) were mixed with secondary antibodies labeled with horseradish peroxidase (0.5 g / mL), then added to the plate and incubated at 37ºC. The plates were washed with PBS / Tween and developed with TMB substrate and analyzed in OD 450 using a microtiter plate reader. The results,
shown in figures 8 to 10, indicate that a first set of human mAbs (including mAbs 1F5, 1H8 and 3H12) competed crosswise with each other (See Figure 8), that an additional set of human mAbs (including 2C2 mAbs, 3H8 and 1G5 and 2G9) also cross-competed about 5 with each other (See Figure 9), and that human mAb 3A10 binds to a single epitope but can bind in a distinct location, but possibly close to the 1F5 mAbs binding sites , 1H8 and 3H12, since these antibodies were able to partially cross-block the binding of 3A10 to CD27 (See Figure 10). Example 7 Complement-dependent cell cytotoxicity (CDCC or CDC) Target cells (Raji Lymphoma cells) were cultured (in AIM-V medium) for 1-2 hours at 37oC, 5% CO2 in the presence of anti-CD27 antibodies and complement of rabbit (final dilution of 1: 15) in a final volume of 150ul. Appropriate controls with a leak signal (targets only) and MAX signal (targets with 12% LysolTM detergent for a final concentration of 4%) were also included. The cells were adjusted to 1 x 106 / ml and 50 µl were added to each well (to generate
50,000 cells / well). The wells were then resuspended and 100 µl of cell suspension was transferred to an opaque, white plate. For each of these wells, 100uL of Promega CellTiter Glo reagent was added and the plate was mixed for 2 minutes at room temperature. The plate was left to incubate for 10 minutes to stabilize the luminescent signal. Luminescence was recorded on a Perkin Elmer Victor X4 plate reader. Cytotoxicity was determined with the following formula: (100 - ((sample - MAX) / (leak - MAX))) x 100. The results (shown as% of lysis) are shown in Figure 11, which can be seen that a number of anti-CD27 antibodies showed significant CDCC activity. In another experiment, target cells (Ramos) were washed and loaded with AM calcein (Molecular Probes). The loaded cells were then washed again and resuspended in 1 x 106 / ml, in culture media (RPMI + 10% FBS). The target cells were cultured for 2 hours at 37ºC, 5% CO2 in the presence of anti-CD27 antibodies and rabbit complement 5 (1:15 final dilution) in a final volume of 150ul.
Appropriate controls with a leak signal (targets only) and MAX signal (targets with 20% X-100 tritone for a final concentration of 2%) were also included.
After incubation, 75ul of supernatant from the wells was transferred to an opaque, black plate.
Fluorescence (Ex-485; In 535) was recorded on a Perkin Elmer Victor X4 plate reader. Specific cytotoxicity was determined with the following formula: (experimental - spontaneous lysis) / (maximum lysis - spontaneous lysis) x 100. The results, shown in Figure 12, indicate that anti-CD27 mAb (1F5) showed at least 10% of CDC activity in Ramos cells at an antibody concentration of 3 µg / ml.
Example 8 Antibody-dependent cell mediated cytotoxicity (ADCC) Target cells (Raji Lymphoma cells) were washed and loaded with BATDA reagent (Perkin Elmer). The loaded cells were then washed again and resuspended in 2 x 105 / ml in culture media (RPMI + 10% FBS). Effector cells were prepared and adjusted to the appropriate concentrations in culture media to generate desired ratios of effector: target (100: 1 - 50: 1). In a round-bottom plate, target cells, effector cells and antibody were added in a final volume of 150ul.
Appropriate controls were used, including a leak signal (targets only), a spontaneous lysis signal (targets + effectors) and a maximum lysis signal (targets + LysolTM detergent at 12% for a final concentration of 4%). The cells were pelleted on the plate and incubated for 2 hours at 37ºC, 5% CO2. After incubation, 20 µl of supernatant from the wells were transferred to an opaque, black plate.
For each of these wells, 200ul of the europium solution (Perkin Elmer) was added and the plate was mixed for 15 minutes.
Time-resolved fluorescence was recorded on a Perkin Elmer Victor X4 plate reader. Specific cytotoxicity was determined using the following formula: (experimental - spontaneous lysis) / (maximum lysis - spontaneous lysis) x 100. The results (shown as% lysis) are shown in Figure 13, which can be seen that a number of anti-CD27 antibodies showed significant ADCC activity.
In another experiment, target cells (Ramos and Daudi lymphoma cells) were washed and loaded with AM calcein (Molecular Probes). The loaded cells were then washed again and resuspended in 1 x 105 / ml in culture media (RPMI + 10% FBS). Effector cells were prepared and adjusted to the appropriate concentrations in culture media to generate desired effector: target ratios (75: 1). In a round-bottom plate, target cells, effector cells and antibody were added in a final volume of 150ul.
Appropriate controls were used, including a leak signal (targets only), a spontaneous lysis signal (targets + effectors) and a maximum lysis signal (targets + Triton X-100 at 20% for a final concentration of 2%) . The cells were pelleted on the plate and incubated for 4 hours at 37ºC, 5% CO2. After incubation, 75ul of supernatant from the wells was transferred to an opaque, black plate.
Fluorescence (Ex-485; In 535) was recorded on a Perkin Elmer Victor X4 plate reader. Specific cytotoxicity was determined with the following formula: (experimental - spontaneous lysis) / (maximum lysis - spontaneous lysis) x 100. The results, shown in Figure 14, indicate that anti-CD27 (1F5) mAb showed at least 10% of ADCC activity (measured as specific cytotoxicity) in Daudi and Ramos cells at an antibody concentration of 3µg / ml and proportion of effector cells:
75: 1 target. Example 9 Antibody Sequencing As described above in Example 1, human mAbs from 5 hybridomas producing specific human IgG mAbs were purified by protein A column chromatography, which led to the isolation of a panel of antibodies (human mAbs) of particular interest. The VH and VL coding regions of human mAbs 4B7, 3H12, 1F5, 2C2, 2G9, 1H8, 3H12, 3G1 (1B10) 4A2, 3A10, 2G11, 4H11, 2H3, 4A7, 3H8 and 1G5 were identified using RNA from the corresponding hybrids. The RNA was transcribed reverse to cDNA, the V coding regions were amplified by PCR and the PCR product was sequenced. The following are nucleic and amino acid sequences from the VH and VL regions of human mAbs (in the case of amino acid sequences, the complementarity determining regions (CDRs) are underlined). 3H8 VH (VH 3-7; D7-27, JH2) VH nucleic acid sequence (SEQ ID NO: 5) atggagttggggctgagctgggttttccttgttgctattttagaaggtgtccagtgtgaggtgcagctggtggagt ctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagtagtt attggatggcctgggtccgccaggctccagggaaagggctggagtggctgggcaatataaagcaagatgga agtgagaaatactatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgta tctacaaatgaacagcctgagagccgaggacacggctgtgtattactgtgtgagggaactggggatggactgg tacttcgatctctggggccgtggcaccctggtcactgtctcctca VH amino acid sequence (SEQ ID NO: 6) (including signal peptide, underlined italics ): MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMAWVRQAPGKGLEWLGNIKQDGSEKYYVDSVKGRFTISRDNA
KNSLYLQMNSLRAEDTAVYYCVRELGMDWYFDLWGRGTLVTVSS VH “mature” amino acid sequence (SEQ ID NO: 7)
excluding signal peptide: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMAWVRQAPGKGLE WLGNIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTA
VYYCVRELGMDWYFDLWGRGTLVTVSS VH CDR1 (SEQ ID NO: 8): GFTFSSYW VH CDR2 (SEQ ID NO: 9): IKQDGSEK VH CDR3 (SEQ ID NO: 10): VRELGMDWYFDL 3H8 VK # 2; VL (SEQ ID NO: 11) Atggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggagaaattgtgttgaca cagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttga cagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagg gccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcaacctag agcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctccgacgttcggccaagggaccaag gtggaaatcaaa VL amino acid sequence (SEQ ID NO: 12) (including signal peptide, underlined italics): MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVD SYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISNLE
PEDFAVYYCQQRSNWPPTFGQGTKVEIK VL amino acid sequence (SEQ ID NO: 13) excluding signal peptide: EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFTLTISNLEPEDFAVYYCQQRSNWPP
TFGQGTKVEIK VL CDR1 (SEQ ID NO: 14): QSVDSY VL CDR2 (SEQ ID NO: 15): DAS VL CDR3 (SEQ ID NO: 16): QQRSNWPPT 3H8 VK # 3 (VK 3-11; JK1)
Another light chain also shown to be active as follows (although only slightly above the chain (VK 3H8-1B11 # 2) was used in the above examples): VL nucleic acid sequence (SEQ ID NO: 17) Atggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggagaaattgtgttgaca cagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttag cagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccagcagg gccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactgg agcctgaagattttgcagtgtattactgtcagcagcgtagcaactggcctccgacgttcggccaagggaccaag gtggaaatcaaa 5 Amino acid sequence VL (SEQ ID NO: 18) (including underlined italic signal peptide): MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVS SYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQRSNWPPTFGQGTKVEIK VL amino acid sequence (SEQ ID NO: 19) excluding signal peptide: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPPTF
GQGTKVEIK VL CDR1 (SEQ ID NO: 20): QSVSSY VL CDR2 (SEQ ID NO: 21): DAS VL CDR3 (SEQ ID NO: 22): QQRSNWPPT 2C2 VH (VH 3-33; D1-7; JH4) VH nucleic acid (SEQ ID NO: 23) Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcaactggtggagt ctgggggaggcgtggtccagcctgggaggtccctgcgactctcctgtgcagcgtctggattcaccttcagtag ctatgacatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggaatgatgg aagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccacgaactcgctgtt tctgcaaatgaacagcctgagagccgaggacacggctgtgtattattgtgtgggaggaactgctgaccttgaac actgggaccagggaaccctggtcaccgtctcctca VH amino acid sequence (SEQ ID NO: 24) (including signal peptide, underlined italics): MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTF SSYDIHWVRQAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNS
TNSLFLQMNSLRAEDTAVYYCVGGTADLEHWDQGTLVTVSS VH amino acid sequence (SEQ ID NO: 25) excluding signal peptide: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVRQAPGKGLEW VAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVY
YCVGGTADLEHWDQGTLVTVSS VH CDR1 (SEQ ID NO: 26): GFTFSSYD VH CDR2 (SEQ ID NO: 27): IWNDGSNK VH CDR3 (SEQ ID NO: 28): VGGTADLEHWDQ 2C2 VK (VK 1D-16); SEQ ID NO: 29) Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac ccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattag cagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgca gcctgaagattttgcaacttattactgccaacagtataatagttaccctctcactttcggcggagggaccaaggtg gagatcaaa VL amino acid sequence (SEQ ID NO: 30) (including signal peptide, underlined italics): MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIS SWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQYNSYPLTFGGGTKVEIK VL amino acid sequence (SEQ ID NO: 31) excluding signal peptide: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLT
FGGGTKVEIK VL CDR1 (SEQ ID NO: 32): QGISSW VL CDR2 (SEQ ID NO: 33): AAS VL CDR3 (SEQ ID NO: 34): QQYNSYPLT 1F5 VH (VH 3-33; D7-27; JH4) VH nucleic acid (SEQ ID NO: 35) Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagt ctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagt tatgacatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaa gtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat ctccaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagaggtagtggtaactggggtt tctttgactactggggccagggaaccctggtcaccgtctcctca VH amino acid sequence (SEQ ID NO: 36) (including signal peptide, underlined italics): MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTF SSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSS VH amino acid sequence (SEQ ID NO: 37) excluding signal peptide: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLE WVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCARGSGNWGFFDYWGQGTLVTVSS VH CDR1 (SEQ ID NO: 38): GFTFSSYD VH CDR2 (SEQ ID NO: 39): IWYDGSNK VH CDR3 (SEQ ID NO: 40): ARGSGNWGFFDY 1F5 VK # 2;
VL nucleic acid sequence (SEQ ID NO: 41) Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac ccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattag caggtggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgca gcctgaagattttgcaacttattactgccaacagtataatacttaccctcggacgttcggccaagggaccaaggt ggaaatcaaa VL amino acid sequence (SEQ ID NO: 42) (including signal peptide, underlined italics): MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIS RWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYNTYPRTFGQGTKVEIK VL amino acid sequence (SEQ ID NO: 43) excluding signal peptide: DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRT
FGQGTKVEIK VL CDR1 (SEQ ID NO: 44): QGISRW VL CDR2 (SEQ ID NO: 45): AAS VL CDR3 (SEQ ID NO: 46): QQYNTYPRT 1H8 VH (VH 3-33; D7-27; JH4) Sequence of VH nucleic acid (SEQ ID NO: 47) Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagt ctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcaatatc tatgacatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaa gtaatcaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat ctgcaaatgaacattttgagagccgaggacacggctgtgtattactgtgcgagaggtactcactgggggtacttt gactactggggccagggaaccctggtcaccgtctcctca VH amino acid sequence (SEQ ID NO: 48) (including signal peptide, underlined italics): MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTF NIYDMHWVRQAPGKGLEWVAVIWYDGSNQYYADSVKGRFTISRDN
SKNTLYLQMNILRAEDTAVYYCARGTHWGYFDYWGQGTLVTVSS VH amino acid sequence (SEQ ID NO: 49) excluding signal peptide: QVQLVESGGGVVQPGRSLRLSCAASGFTFNIYDMHWVRQAPGKGLE WVAVIWYDGSNQYYADSVKGRFTISRDNSKNTLYLQMNILRAEDTA
VYYCARGTHWGYFDYWGQGTLVTVSS VH CDR1 (SEQ ID NO: 50): GFTFNIYD VH CDR2 (SEQ ID NO: 51): IWYDGSNQ VH CDR3 (SEQ ID NO: 52): ARGTHWGYFDY 1H8 VK (Sequence) (VK 1; SEQ ID NO: 53) Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac ccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattag cagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccaatttgca aagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcag cctgaagattttgcaacttattactgccaacagtataatagttaccctcggacgttcggccaagggaccaaggtg gaaatcaaa VL amino acid sequence (SEQ ID NO: 54) (including signal peptide, underlined italics): MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIS SWLAWYQQKPEKAPKSLIYAASNLQSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYNSYPRTFGQGTKVEIK VL amino acid sequence (SEQ ID NO: 55) excluding signal peptide: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLI YAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRT
FGQGTKVEIK VL CDR1 (SEQ ID NO: 56): QGISSW VL CDR2 (SEQ ID NO: 57): AAS VL CDR3 (SEQ ID NO: 58): QQYNSYPRT 1G5 VH (VH 3-33; D6-19; JH2) VH nucleic acid (SEQ ID NO: 59) 5 Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggt gcaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctgg attcagcttcagtagctatggcatgcactgggtccgccaggctccaggcaagggactggagtgggtggcactt ctatggtatgatggtagccataaagactttgcagactccgtgaagggccgattcaccatctccagagacaattcc aagaacacgctagatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagag ggtttagcagtacctggtcactggtacttcgatctctggggccgtggcaccctggtcactgtctcctca VH amino acid sequence (SEQ ID NO: 60) (including signal peptide, underlined italics): MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFSF SSYGMHWVRQAPGKGLEWVALLWYDGSHKDFADSVKGRFTISRDN SKNTLDLQMNSLRAEDTAVYYCAREGLAVPGHWYFDLWGRGTLVT
VSS VH amino acid sequence (SEQ ID NO: 61) excluding signal peptide: QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLE WVALLWYDGSHKDFADSVKGRFTISRDNSKNTLDLQMNSLRAEDTA
VYYCAREGLAVPGHWYFDLWGRGTLVTVSS VH CDR1 (SEQ ID NO: 62): GFSFSSYG VH CDR2 (SEQ ID NO: 63): LWYDGSHK VH CDR3 (SEQ ID NO: 64): AREGLAVPGHWYFDL 1G5 VK (Sequence) VK (VK; SEQ ID NO: 65) Atgagggtccccgctcagctcctggggcttctgctgctctggctcccaggtgccagatgtgccatccagttgac ccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcatta gcagtgctttagcctggtatcagcagaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttgg aaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgca gcctgaagattttgcaacttattactgtcaacagtttaatacttaccctcggacgttcggccaagggaccaaggtg gaaatcaaa VL amino acid sequence (SEQ ID NO: 66) (including signal peptide, underlined italics): MRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGIS SALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQFNTYPRTFGQGTKVEIK VL amino acid sequence (SEQ ID NO: 67) excluding signal peptide: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIY DASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNTYPRTF
GQGTKVEIK VL CDR1 (SEQ ID NO: 68): QGISSA VL CDR2 (SEQ ID NO: 69): DAS VL CDR3 (SEQ ID NO: 70): QQFNTYPRT 2G9 VH (VH 3-33; D1-7; JH4) VH nucleic acid (SEQ ID NO: 71) Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagttggtggagt ctgggggaggcgtggtccagcctgggaggtccctgcgactctcctgtgcagcgtctggattcaccctcagtag ccatgacatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggaatgatgg aagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccacgaactcgctgtt tctgcaaatgaacagcctgagagccgaggacacggctgtgtattattgtgtgagaggaactgctgaccttgaac actgggaccagggaaccctggtcaccgtctcctca VH amino acid sequence (SEQ ID NO: 72) (including signal peptide, underlined italics): MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTL SSHDIHWVRQAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNS
TNSLFLQMNSLRAEDTAVYYCVRGTADLEHWDQGTLVTVSS VH amino acid sequence (SEQ ID NO: 73) excluding signal peptide: QVQLVESGGGVVQPGRSLRLSCAASGFTLSSHDIHWVRQAPGKGLE WVAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTA
VYYCVRGTADLEHWDQGTLVTVSS VH CDR1 (SEQ ID NO: 74): GFTLSSHD VH CDR2 (SEQ ID NO: 75): IWNDGSNK VH CDR3 (SEQ ID NO: 76): VRGTADLEHWDQ 2G9 VK (VK 1D-16; JK nucleic acid); SEQ ID NO: 77) Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac ccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattag cagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgca gcctgaagattttgcaacttattactgccaacagtataatagttaccctctcactttcggcggagggaccaaggtg gagatcaaa VL amino acid sequence (SEQ ID NO: 78) (including signal peptide, underlined italics): MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIS SWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQYNSYPLTFGGGTKVEIK VL amino acid sequence (SEQ ID NO: 79) excluding signal peptide: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLT
FGGGTKVEIK VL CDR1 (SEQ ID NO: 80): QGISSW
VL CDR2 (SEQ ID NO: 81): AAS VL CDR3 (SEQ ID NO: 82): QQYNSYPLT 3A10 VH (VH 3-33, D3-10, JH3) VH nucleic acid sequence (SEQ ID NO: 83) Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagt ctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtcat tatggcatgcactgggtccgccaggctccaggcaaggggccggagtgggtggcaattatatggtatgatgga agtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgga tctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagatggatggactactatg gttcggggacttaatgtttttgatatctggggccaagggacaatggtcaccgtctcttca VH amino acid sequence (SEQ ID NO: 84) (including signal peptide, underlined italics): MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTF SHYGMHWVRQAPGKGPEWVAIIWYDGSNKYYADSVKGRFTISRDNS KNTLDLQMNSLRAEDTAVYYCARDGWTTMVRGLNVFDIWGQGTM
VTVSS VH amino acid sequence (SEQ ID NO: 85) excluding signal peptide: QVQLVESGGGVVQPGRSLRLSCAASGFTFSHYGMHWVRQAPGKGPE WVAIIWYDGSNKYYADSVKGRFTISRDNSKNTLDLQMNSLRAEDTA
VYYCARDGWTTMVRGLNVFDIWGQGTMVTVSS VH CDR1 (SEQ ID NO: 86): GFTFSHYG VH CDR2 (SEQ ID NO: 87): IWYDGSNK VH CDR3 (SEQ ID NO: 88): ARDGWTTMVRGLNVFDI-1K (1); VL (SEQ ID NO: 89) Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac ccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcaggatattag cagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgca gcctgaagattttgcaacttattactgccaacagtataatagttaccctcccaccttcggccaagggacacgactg gagattaaa VL amino acid sequence (SEQ ID NO: 90) (including signal peptide, underlined italics): MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQDIS SWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQYNSYPPTFGQGTRLEIK VL amino acid sequence (SEQ ID NO: 91) excluding signal peptide: DIQMTQSPSSLSASVGDRVTITCRASQDISSWLAWYQQKPEKAPKSLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPT
FGQGTRLEIK VL CDR1 (SEQ ID NO: 92): QDISSW VL CDR2 (SEQ ID NO: 93): AAS VL CDR3 (SEQ ID NO: 94): QQYNSYPPT 5 3A10 VK # 4 (VK 1-13; JK5) Another light chain also shown to be active as follows (although only slightly above the chain (VK 3H8-1B11 # 1) was used in the above examples): VL nucleic acid sequence (SEQ ID NO: 95) Atgagggtcctcgctcagctcctggggcttctgctgctctggctcccaggtgccagatgtgccatccagttgac ccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcatta gcagtgctttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgca gcctgaagattttgcaacttattactgccaacagtataatagttaccctcccaccttcggccaagggacacgactg gagattaaa VL amino acid sequence (SEQ ID NO: 96) (including underlined italic signal peptide): MRVLAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGIS SALAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQYNSYPPTFGQGTRLEIK VL amino acid sequence (SEQ ID NO: 97) excluding signal peptide: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPEKAPKSLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTF
GQGTRLEIK VL CDR1 (SEQ ID NO: 98): QGISSA VL CDR2 (SEQ ID NO: 99): AAS VL CDR3 (SEQ ID NO: 100): QQYNSYPPT 3H12 VH (VH 3-33; D7-27; JH4) VH nucleic acid (SEQ ID NO: 101) atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagt ctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcaacgtctggattcaccttcagtag ctatgacatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatttggtatgatgga agtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgta tctccaaatgaacagcctgggagacgaggacacggctgtgtattactgtgcgagaggtagtggtaactggggt ttctttgactactggggccagggaaccctggtcaccgtctcctca VH amino acid sequence (SEQ ID NO: 102) (including signal peptide, underlined italics): MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCATSGFTF SSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDN
SKNTLYLQMNSLGDEDTAVYYCARGSGNWGFFDYWGQGTLVTVSS VH amino acid sequence (SEQ ID NO: 103) excluding signal peptide: QVQLVESGGGVVQPGRSLRLSCATSGFTFSSYDMHWVRQAPGKGLE WVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLGDEDTA VYYCARGSGNWGFFDYWGQGTLVTVSS
VH CDR1 (SEQ ID NO: 104): GFTFSSYD VH CDR2 (SEQ ID NO: 105): IWYDGSNK VH CDR3 (SEQ ID NO: 106): ARGSGNWGFFDY 3H12 VK # 2 (VK 1D-16; JK1) VL nucleic acid sequence (SEQ ID NO: 107) Atgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgac ccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattag caggtggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgca gcctgaagattttgcaacttattactgccaacagtataatacttaccctcggacgttcggccaagggaccaaggt ggaaatcaaa VL amino acid sequence (SEQ ID NO: 108) (including signal peptide, underlined italics): MRVLAQLLGLLLLCFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIS RWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYNTYPRTFGQGTKVEIK 5 VL amino acid sequence (SEQ ID NO: 109) excluding signal peptide: DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRT
FGQGTKVEIK VL CDR1 (SEQ ID NO: 110): QGISRW VL CDR2 (SEQ ID NO: 111): AAS VL CDR3 (SEQ ID NO: 112): QQYNTYPRT ALIGNMENTS ARE SHOWN IN FIGS 15 AND 16 Example 10 Study of non-human primate in vivo To assess the tolerance of anti-human CD27 mAb 1F5 in non-human primates, 3 cynomolgus monkeys were treated with an iv dose of 1, 3 or 10 mg / kg of 1F5. The animals were followed for 29 days.
Total lymphocytes (based on the size of the front and side dispersion), memory B cells (CD20 + and clear CD95) and monocytes (based on the size of the front and side dispersion) were stained with anti-human IgG antibody 5 (line in bold) and compared to unstained controls (shaded histogram). The results are shown in Figure 17. These results show that the 1F5 mAb was bound to the surface of circulating lymphocytes known to express CD27 for the duration of the study.
Monocytes, cells that do not express CD27 had no 1F5 binding. In addition, to determine the effect of 1F5 on circulating lymphocyte populations, lymphocytes were stained with markers and% positive cells plotted vs time in Figure 17 for each animal treated at different doses (square points = 1 mg / kg; circular points = 3 mg / kg; triangular points = 10 mg / kg). The results are shown in Figure 18. Collectively, the results of these studies, shown in Figures 17 and 18, demonstrate that 1F5 was well tolerated and did not significantly deplete circulating lymphocytes (except that some transient depletion of NK cells) after a single dose of 1 -10 mg / kg.
In addition, there was no increase in body temperature and undetectable levels of TNF-α, IL-6 or IL-1β.
Example 11 Proliferation and activation of antigen-specific CD8 + T cell improved anti-CD27 mAbs Staining of pentamer in peripheral mouse blood cells and splenocytes.
Transgenic mice with human CD27 (Tg-huCD27) were injected intravenously with 5 mg of chicken ovalbumin and the panel of fully human antibodies recognizing human CD27 generated as in example 1 (human CD27 mAbs). AT124 anti-mouse CD27 clone and irrelevant human IgG1 were included as positive and negative controls, respectively.
Each antibody (250 g) was co-injected with ovalbumin on day 0 and an additional 250 µg of antibody alone on day 1. Peripheral blood and spleen cells were harvested on day 7. Splenocytes 5 (1 x 106) or whole blood (100 l) were used for staining.
After blocking the Fc receptor, cells were stained with 10 µl of H-2Kb / SIINFEKL, a mouse MHC tetrameric complex complexed with the ovalbumin peptide T cell epitope (Beckman Coulter), or a similar complex pentameric ( ProImmune), anti-CD8 (eBioscience) and anti-huCD27 mAb or anti-msCD27 mAb (BD Biosciences) at room temperature for 30 minutes.
The cells were then lysed by RBC, washed and fixed, and at least 100,000 events were acquired on Flow Cytometer LSR (BD Biosciences). The fraction of cells positive for tetramer or pentamer in the gated CD8 + or CD27 + population was determined.
The results are shown in Figures 19 and 21 from which it can be seen that anti-CD27 antibodies significantly improved immune responses.
Example 12 ELISPOT Assay Splenocytes (2.5 x 105 and 0.5 x 105) from the pentamer staining preparation of Example 8 above were placed on plates coated with anti-IFNγ monoclonal antibody (mAb) in triplicate wells after lysis RBC.
SIINFEKL peptide was added at a final concentration of 2 µg / ml.
A background control was adjusted for each sample in triplicate in the absence of peptide.
The stimulus was maintained overnight at 37 ° C in a tissue culture incubator.
ELISPOT detection was performed using an ELISPOT kit (BD Biosciences) following the manufacturer's protocol.
IFNγ-spot number was counted.
The results are shown in Figures 20 and 21, from which it can be seen that anti-CD27 mAbs significantly improved T cell activity.
Example 13 Anti-CD27 mAb improves T cell responses to a vaccine antigen HuCD27 transgenic mice were immunized with 5 µg (sc) of the APC-directed vaccine comprising an ovalbumin-fused mouse anti-DEC-205 IgG antibody (OVA) (referred to as α-mDEC-205-OVA), in combination with anti-human CD27 mAb 1F5 (ip) in different doses (25, 50, 100, 200 or 400 g). A week later, splenocytes were analyzed for CD8 + T cell reactivity to SIINFEKL OVA peptide (OVA peptide 257-264) by tetramer staining (% positive tetramer of all CD8 + shown) and IFN-ELISPOT by the procedure generally described as examples 8 and 9, respectively.
The results are shown in figures 22A-C, where Figure 22A shows the protocol used, Figure 22B shows the results of the tetramer staining experiment and Figure 22 C shows the results of the IFN-gamma ELISPOT experiment.
These results indicate that coadministered human mAb 1F significantly improved T cell responses to the administered vaccine component.
Example 14 Synergistic effect of anti-CD27 (1F5) mAb and TLR agonist (poly IC) on T cell responses to vaccine (anti-DEC205-OVA) Tg-huCD27 mice (transgenic) and wild type (WT) littermates were injected intraperitoneally with 1F5 anti-CD27 mAb (50 g) on day -3 and then subcutaneously via 4 legs injected with anti-mDec205-OVA (g 5) plus poly IC 0, 25, 50 or 100 µg on day 0. Spleens were collected on day 7 and evaluated by tetramer staining, IFNγ ELISPOT and IFNγ-ICS.
Mean ± SD of positive IFN-ICS between CD8 gated T cells from 3 mice per group were calculated and a representative Dot Plots panel was collected.
The results, shown in
Figures 23A-D, indicate that the human anti-CD27 mAb acted synergistically with the TLR3 Poly IC agonist to improve T cell responses to the administered vaccine component.
Example 15 5 Administration of anti-CD27 mAb prior to Dec205-directed vaccine in the absence or presence of TLR agonist HuCD27-Tg and wild-type (WT) mice were injected intraperitoneally with anti-CD27 mAb (50µg) over several days related to vaccine, as shown in Figure 28 and subcutaneously via 4 legs injected with anti-mDec205-OVA (5 µg) more or less TLR poly IC-LC agonist (20 µg) on day 0. Spleens were collected on day 7 and evaluated by tetramer staining and IFNγ ELISPOT.
A representative tetramer staining panel between gated CD8 T cells is shown in figures 24 and 25. IFNγ-ELISPOT showed a similar pattern.
These results show that, surprisingly, when the anti-CD27 antibody is administered in combination with a vaccine in the presence or absence of the TLR agonist, T cell activation is greater when the antibody is administered before the vaccine, for example, one day or more before the vaccine (antigen) is administered.
Example 16 anti-CD27 mAb combined with TCR activation activates transgenic mice T cells with human CD27 To assess the TF activation capacity of 1F5 mAb, T cells were purified from spleens of hCD27-Tg mice by negative selection with beads .
The cells were labeled with CFSE and incubated with 0.2µg / ml of human anti-CD27 mAb 1F5 or isotype control for 3 days.
The cross-linked anti-human IgG was passed through an endotoxin removal column prior to use.
IFN-ICS and CSFE dilution between CD8 and CD4 T cells are shown in Figures 26 and 27. TNF-ICS showed the same IFNg pattern.
As shown in Figures 26 and 27, when combined with TCR activation, mAb 1F5 effectively induces T cell proliferation and cytokine proliferation in vitro.
Data 5 shows that cross-linking with human anti-IgG and T cell receptor activation with anti-CD3 mAb were necessary for cytokine production and 1F5-induced proliferation. Example 17 anti-CD27 mAb increases vaccine efficacy in a Melanoma MO4 challenge model To assess antitumor activity in vivo, huCD27 Tg and WT control, 8 mice per group, were subcutaneously inoculated with 0.3 x 105 cells of MO4 on day 0. On days 5 and 12, these mice were injected intraperitoneally with anti-CD27 mAb 1F5 (50µg); on day 8 and 15, additional doses of CD27 HuMab (50 µg) and vaccinated with anti-mDec205-OVA (5 g). Tumor growth was measured with tweezers twice a week.
The results are shown in Figure 24. In another study, huCD27 Tg and WT control, 5 mice per group, were inoculated subcutaneously with 1 x 105 MO4 cells on day 0. On day 5 and 12, these mice were vaccinated with anti-mDec205-OVA (5µg) plus the TLR agonist poly IC-LC (10µg) intraperitoneally.
On days 6 and 13, the mice were injected with anti-CD27 mAb 1F5 (50µg). Tumor growth was measured with tweezers twice a week as indicated in the protocol illustrated in Figure 285A.
The results, shown in figures 28B, C and D, show the effect of no treatment (Figure 28B), vaccine treatment alone (Figure 28 C) or vaccine treatment in combination with anti-CD27 treatment (Figure 28 D) in size of tumor in mice as a function of the number of days after tumor inoculation.
The results demonstrate that the combination treatment with anti-CD27 (1F5) mAb significantly prolonged the survival of mice challenged with the tumor.
Example 18 1F5 shows potent anti-tumor activity in a 5 tumor challenge model in BCL1-B lymphoma transgenic mice To assess the antitumor activity of 1F5 in vivo, groups of 9-10 hCD27 transgenic mice (Balb / c background) were challenged with 107 BCL1 B lymphoma cells, administered intravenously on day 0. The animals were then treated with 5 doses of anti-human CD27 mAb 1F5 as indicated.
As shown in figures 29A and 29B, it significantly prolonged the survival of tumor challenged mice, in a dose dependent manner.
Example 19 Tumor death in a Raji Xenograft SCID mouse model CB.17 SCID mice (acquired from Taconic) were maintained in a pathogen-free mouse facility.
Raji Lymphoma cells (1 x 105) were injected subcutaneously into SCID mice, 4 mice per group.
On day 6, these mice were treated with human CD27 mAbs via intraperitoneal administration, 0.5 mg per dose and dosed twice a week for 3 weeks.
Tumor growth was measured with forceps 3 times a week.
The results of tumor growth and Kaplan-Meier analysis are shown in Figures 30A and 30B, from which it can be seen that anti-CD27 mAbs significantly prolonged the survival of tumor challenged mice.
In another experiment, CB.17 SCID mice (purchased from Taconic) were kept in a pathogen-free mouse facility.
Human Raji Lymphoma cells (5 x 105) were injected subcutaneously into SCID mice, on day 0, 6 mice per group.
On day 5, these mice were treated with anti-CD27 mAb 1F5 via intraperitoneal administration, 0.033, 0.1 or 0.3 mg per dose and dosed twice a week for 3 weeks.
Tumor growth was measured with tweezers twice a week. 5 The results, shown in Figure 31A, indicate that the anti-CD27 mAb (1F5) significantly inhibited tumor growth and thus significantly prolonged the survival of tumor challenged mice.
A Kaplan-Meier survival curve of data is also provided in Figure 31B, which shows that the median survival was increased by at least 10 days in mice in the treated group compared to the control group.
An additional xenograft experiment was conducted with 1G5 and the results are shown in Figure 32. Example 20 Tumor death in a SCID mouse model of Daudi's Xenograft CB.17 SCID mice (acquired from Taconic) were kept in a mouse-free facility of pathogens.
Human Daudi Lymphoma cells (1 x 106) were injected subcutaneously into SCID mice, on day 0, 6 mice per group.
On day 5, these mice were treated with anti-CD27 mAb 1F5 via intraperitoneal administration, 0.033, 0.1 or 0.3 mg per dose and dosed twice a week for 3 weeks.
Tumor growth was measured with tweezers twice a week.
The results, shown in Figure 33, indicate that the anti-CD27 mAb (1F5) significantly inhibited tumor growth (Figure 33A) and thus significantly prolonged the survival of tumor-challenged mice (Kaplan-Meier curve in Figure 33B) . Example 21 anti-CD27 mAb designed not to bind to Fc receptors does not increase the T cell response to a vaccine antigen
HuCD27 transgenic mice were immunized with 5 µg (sc) of the APC-directed vaccine comprising an anti-DEC-205 mouse IgG antibody fused to ovalbumin (OVA) (referred to as α-mDEC-205-OVA), in combination with mAb Anti-human CD27 5 1F5 (ip) or mutant 1F5 mAb (Fc portion mutated to prevent binding to the Fc receptor) or control mAb IgG.
One week later the splenocytes were analyzed for CD8 + T cell reactivity to the OVA SIINFEKL peptide (OVA peptide 257-264) by IFN- ߛ ELISPOT by the procedure as generally described.
The results, shown in Figure 34, demonstrate that the altered human mAb 1F5 does not improve T cell responses to the vaccine and thus would be an effective agent to block the CD70 / CD27 pathway. Equivalents Those skilled in the art will recognize, or are able to verify using, no more than routine experimentation, many equivalents of the specific embodiments of the invention described here.
Equivalent said are intended to be included by the following claims.
Sequence listing summary SEQ ID NO: DESCRIPTION 1 human CD27 (Gene Bank Accession No .: AAH12160,1) 2 Human CD70 (Gene Bank Accession No .: NP_001243) 3 VH sequence 3 -33 germline is provided (Gene Bank Accession No. AAP44382) 4 VH Sequence 3-7 germline is provided (Gene Bank Accession No. AAP44389) 5 3H8-1B11 VH nucleic acid 6 3H8- 1B11 VH amino acid including signal peptide 7 3H8-1B11 VH "mature" amino acid excluding signal peptide 8 3H8-1B11 VH CDR1 Amino acid 9 3H8-1B11 VH CDR2 Amino acid 10 3H8-1B11 VH CDR3 Amino acid 11 3H8-1B11 VL # 2 nucleic acid 12 3H8 -1B11 VL # 2 amino acid including signal peptide 13 3H8-1B11 VL # 2 "mature" amino acid excluding signal peptide 14 3H8-1B11 VL # 2 CDR1 amino acid 15 3H8-1B11 VL # 2 CDR2 amino acid 16 3H8-1B11 VL # 2 CDR3 amino acid 17 3H8-1B11 VL # 3 nucleic acid 18 3H8-1B11 VL # 3 amino acid including signal peptide 19 3H8-1B11 VL # 3 "mature" amino acid excluding signal peptide 20 3H8- 1B11 VL # 3 CDR1 amino acid 21 3H8-1B11 VL # 3 CDR2 amino acid 22 3H8-1B11 VL # 3 CDR3 amino acid 23 2C2-1A10 VH nucleic acid 24 2C2-1A10 VH amino acid including signal peptide 2 2C2-1A10 VH "mature" amino acid excluding signal peptide 26 2C2-1A10 VH CDR1 amino acid 27 2C2-1A10 VH CDR2 amino acid 28 2C2-1A10 VH CDR3 amino acid 29 2C2-1A10 VL nucleic acid 30 2C2-1A10 VL amino acid including signal peptide 2 2C2-1A10 VL "mature" amino acid excluding peptide signal 32 2C2-1A10 VL CDR1 amino acid 33 2C2-1A10 VL CDR2 amino acid
SEQ ID NO: DESCRIPTION 34 2C2-1A10 VL CDR3 amino acid 35 1F5-1H5 VH nucleic acid 36 1F5-1H5 VH amino acid including signal peptide 37 1F5-1H5 VH "mature" amino acid excluding signal peptide 38 1F5-1H5 VH CDR1 amino acid 39 1F5- 1H5 VH CDR2 amino acid 40 1F5-1H5 VH CDR3 amino acid 41 1F5-1H5 VL # 2 nucleic acid 42 1F5-1H5 VL # 2 amino acid including signal peptide 43 1F5-1H5 VL # 2 "mature" amino acid excluding signal peptide 44 1F5-1H5 VL # 1 CDR1 amino acid 45 1F5-1H5 VL # 2 CDR2 amino acid 46 1F5-1H5 VL # 2 CDR3 amino acid 47 1H8-B4 VH nucleic acid 48 1H8-B4 VH amino acid including signal peptide 49 1H8-B4 VH "mature" amino acid excluding signal peptide 50 1H8-B4 VH CDR1 amino acid 51 1H8-B4 VH CDR2 amino acid 52 1H8-B4 VH CDR3 amino acid 53 1H8-B4 VL nucleic acid 54 1H8-B4 VL amino acid including signal peptide 55 1H8-B4 VL "mature" amino acid excluding signal peptide 56 1H8-B4 VL CDR1 amino acid 57 1H8-B4 VL CDR2 amino acid 58 1H8-B4 VL CDR3 amino acid 59 1G5-1B9 VH nucleic acid 6 0 1G5-1B9 VH amino acid including signal peptide 61 1G5-1B9 VH "mature" amino acid excluding signal peptide 62 1G5-1B9 VH CDR1 amino acid 63 1G5-1B9 VH CDR2 amino acid 64 1G5-1B9 VH CDR3 amino acid 65 1G5-1B9 VL nucleic acid 66 1G5-1B9 VL amino acid including signal peptide
SEQ ID NO: DESCRIPTION 67 1G5-1B9 VL "mature" amino acid excluding signal peptide 68 1G5-1B9 VL CDR1 amino acid 69 1G5-1B9 VL CDR2 amino acid 70 1G5-1B9 VL CDR3 amino acid 71 2G9-1D11 VH nucleic acid 72 2G9-1D11 VH amino acid including signal peptide 73 2G9-1D11 VH "mature" amino acid excluding signal peptide 74 2G9-1D11 VH CDR1 amino acid 75 2G9-1D11 VH CDR2 amino acid 76 2G9-1D11 VH CDR3 amino acid 77 2G9-1D11 VL nucleic acid 78 2G9-1D11 VL amino acid including signal peptide 79 2G9-1D11 VL "mature" amino acid excluding signal peptide 80 2G9-1D11 VL CDR1 amino acid 81 2G9-1D11 VL CDR2 amino acid 82 2G9-1D11 VL CDR3 amino acid 83 3A10-1G10 VH nucleic acid 84 3A10-1G10 VH amino acid including signal peptide 85 3A10-1G10 VH "mature" amino acid excluding signal peptide 86 3A10-1G10 VH CDR1 amino acid 87 3A10-1G10 VH CDR2 amino acid 88 3A10-1G10 VH CDR3 amino acid 89 3A10-1G10 VL # 1 nucleic acid 90 3A10-1G10 VL # 1 amino acid including signal peptide 91 3A10-1G10 VL # 1 amino acid “ma hard ”excluding signal peptide 92 3A10-1G10 VL # 1 CDR1 amino acid 93 3A10-1G10 VL # 1 CDR2 amino acid 94 3A10-1G10 VL # 1 CDR3 amino acid 95 3A10-1G10 VL # 4 nucleic acid 96 3A10-1G10 VL # 4 amino acid including signal peptide 97 3A10-1G10 VL # 4 "mature" amino acid excluding signal peptide 98 3A10-1G10 VL # 4 CDR1 amino acid 99 3A10-1G10 VL # 4 CDR2 amino acid
SEQ ID NO: DESCRIPTION 100 3A10-1G10 VL # 4 CDR3 amino acid 101 3H12-1E12 VH nucleic acid 102 3H12-1E12 VH amino acid including signal peptide 103 3H12-1E12 VH "mature" amino acid excluding signal peptide 104 3H12-1E12 VH CDR1 amino acid 105 3H12-1E12 VH CDR2 amino acid 106 3H12-1E12 VH CDR3 amino acid 107 3H12-1E12 VL # 2 nucleic acid 108 3H12-1E12 VL # 2 amino acid including signal peptide 109 3H12-1E12 VL # 2 "mature" amino acid excluding signal peptide 3H12- 1E12 VL # 2 CDR1 amino acid 111 3H12-1E12 VL # 2 CDR2 amino acid 112 3H12-1E12 VL # 2 CDR3 amino acid 113 VH CDR3 consensus 114 VL CDR3 consensus 115 VH CDR2 consensus 116 VL CDR2 consensus 117 VH CDR1 consensus 118 VL CDR1 consensus

权利要求:
Claims (31)
[1]
1. Isolated human or humanized monoclonal antibody that binds to human CD27, characterized by the fact that the antibody has at least one of the following properties: 5 (a) blocks the binding of sCD70 to CD27 by at least about 70% in one antibody concentration of 10 µg / ml; (b) binds human CD27 with a Kd equilibrium dissociation constant of 10-9 M or less, or alternatively, a Ka equilibrium association constant of 10 + 9 M-1 or more; (c) induces specific complement-mediated cytotoxicity (CDC) of cells expressing at least 10% CD27 at an antibody concentration of 3 µg / ml and approximately 6% complement of rabbit serum; (d) induces specific antibody-dependent cell-mediated cytotoxicity (ADCC) lysis of cells expressing CD27 of at least 10% at an antibody concentration of 3 µg / ml and effector: target cells ratio of 75: 1; (e) improves mean survival by at least 20% in severe combined immunodeficiency (SCID) in mice after inoculation of tumor cell in vivo (5 x 105 Raji cells or 1 x 106 Daudi cells) when administered at 0.3mg (ip) at least twice a week for 3 weeks compared to mice to which the antibody is not administered; (f) induces or enhances antigen specific immune response in vivo in combination with a vaccine or endogenous antigen; (g) induces or enhances antigen-specific TH1 immune response in vivo in combination with a vaccine or endogenous antigen; (h) induces or intensifies antigen-specific T cell proliferation or activation in vivo in combination with a vaccine or endogenous antigen; (i) reduces or inhibits T cell proliferation or activation; (j) induces or intensifies T cell activity when combined with simultaneous, separate or sequential TCR activation; 5 (k) blocks sCD70 binding to CD27 by at least about 70% at an antibody concentration of 10 µg / ml and reduces or inhibits T cell activation when it is unable to bind to, or containing reduced binding to Fc receptors; (l) results in less than 50% depletion of CD3 + T cells (in addition to NK cells) in monkeys when administered at 3 mg / kg (i.v.) for the period of 29 days immediately after administration; or (m) results in less than 50% depletion of memory B cells in monkeys when administered at 3 mg / kg (i.v.) for the period of 29 days immediately after administration.
[2]
2. Isolated monoclonal antibody, characterized by the fact that it binds to human CD27 and induces or intensifies activation or proliferation of CD8 + T cells.
[3]
3. Isolated human or humanized monoclonal antibody that binds to human CD27, characterized by the fact that the antibody: (a) induces or enhances antigen-specific immune responses in vivo in combination with a vaccine or endogenous antigen; (b) induces or intensifies T cell activity when combined with simultaneous, separate or sequential TCR activation; (c) results in less than 50% depletion of CD3 + T cells (other than NK cells) in monkeys when administered at 3 mg / kg (i.v.) for the period of 29 days immediately following administration; and (d) results in less than 50% depletion of memory B cells in monkeys when administered at 3 mg / kg (i.v.) for the period of 29 days immediately following administration.
[4]
4. Isolated human or humanized monoclonal antibody that binds to human CD27, characterized by the fact that the antibody improves median survival by at least 20% in severe combined immunodeficiency (SCID) mice after inoculation into a tumor cell in vivo ( 5 x 105 Raji cells or 1 x 106 Daudi cells) when administered at 0.3 mg (ip) at least twice a week for 3 weeks compared to mice to which the antibody is not administered.
[5]
5. Isolated human or humanized monoclonal antibody that binds to human CD27, characterized by the fact that the antibody: (a) blocks the binding of sCD70 to CD27 by at least about 70% at an antibody concentration of 10 µg / ml ; (i) reduces or inhibits T cell proliferation or activation; and (k) reduces or inhibits T cell activity when not capable of binding to, having reduced binding to, Fc receptors.
[6]
Bispecific molecule, characterized in that it comprises the antibody of any one of claims 1 to 5 linked to a second molecule having a binding specificity that is different from the antibody.
[7]
7. Isolated monoclonal antibody that binds to human CD27 and inhibits the binding of CD70 to CD27 in cells, characterized by the fact that the antibody inhibits the binding of sCD70 to cells that express CD27 by at least about 70% at a concentration of antibody of 10 µg / ml and inhibits or reduces an immune response to an antigen.
[8]
8. Isolated monoclonal antibody that binds to human CD27 and induces or enhances effector cell function, characterized by the fact that the antibody induces at least about 40% ADCC specific lysis of cells expressing CD27 at an antibody concentration of 10 µg / ml or induces at least about 40% complement cytotoxicity (CDC) of cells expressing CD27 at a concentration of 10 µg / ml.
[9]
9. Isolated monoclonal antibody that binds to human CD27, characterized by the fact that the antibody comprises: (i) a heavy chain variable region CDR1 comprising SEQ ID NO: 38; 5 is a heavy chain variable region CDR2 comprising SEQ ID NO: 39; a heavy chain variable region CDR3 comprising SEQ ID NO: 40; a light chain variable region CDR1 comprising SEQ ID NO: 44; a light chain variable region CDR2 comprising SEQ ID NO: 45; and a light chain variable region CDR3 comprising SEQ ID NO: 46; (ii) a heavy chain variable region CDR1 comprising SEQ ID NO: 50; a heavy chain variable region CDR2 comprising SEQ ID NO: 51; a heavy chain variable region CDR3 comprising SEQ ID NO: 52; a light chain variable region CDR1 comprising SEQ ID NO: 56; a light chain variable region CDR2 comprising SEQ ID NO: 57; and a light chain variable region CDR3 comprising SEQ ID NO: 58; (iii) a heavy chain variable region CDR1 comprising SEQ ID NO: 104; a heavy chain variable region CDR2 comprising SEQ ID NO: 105; a heavy chain variable region CDR3 comprising SEQ ID NO: 106; a light chain variable region CDR1 comprising 5 SEQ ID NO: 110; a light chain variable region CDR2 comprising SEQ ID NO: 111; and a light chain variable region CDR3 comprising SEQ ID NO: 112; (iv) a heavy chain variable region CDR1 comprising SEQ ID NO: 86; a heavy chain variable region CDR2 comprising SEQ ID NO: 87; a heavy chain variable region CDR3 comprising SEQ ID NO: 88; a light chain variable region CDR1 comprising SEQ ID NO: 92 or 98; a light chain variable region CDR2 comprising SEQ ID NO: 93 or 99; and a light chain variable region CDR3 comprising SEQ ID NO: 94 or 100; (v) a heavy chain variable region CDR1 comprising SEQ ID NO: 26; a heavy chain variable region CDR2 comprising SEQ ID NO: 27; a heavy chain variable region CDR3 comprising SEQ ID NO: 28; a light chain variable region CDR1 comprising SEQ ID NO: 32;
a light chain variable region CDR2 comprising SEQ ID NO: 33; and a light chain variable region CDR3 comprising SEQ ID NO: 34; (Vi) a heavy chain variable region CDR1 comprising SEQ ID NO: 74; a heavy chain variable region CDR2 comprising SEQ ID NO: 75; a heavy chain variable region CDR3 comprising SEQ ID NO: 76; a light chain variable region CDR1 comprising SEQ ID NO: 80; a light chain variable region CDR2 comprising SEQ ID NO: 81; and a light chain variable region CDR3 comprising SEQ ID NO: 82; (vii) a heavy chain variable region CDR1 comprising SEQ ID NO: 8; a heavy chain variable region CDR2 comprising SEQ ID NO: 9; a heavy chain variable region CDR3 comprising SEQ ID NO: 10; a light chain variable region CDR1 comprising SEQ ID NO: 14 or 20; a light chain variable region CDR2 comprising SEQ ID NO: 15 or 21; and a light chain variable region CDR3 comprising SEQ ID NO: 16 or 22; or (viii) a heavy chain variable region CDR1 comprising SEQ ID NO: 62; a heavy chain variable region CDR2 comprising SEQ ID NO: 63; a heavy chain variable region CDR3 5 comprising SEQ ID NO: 64; a light chain variable region CDR1 comprising SEQ ID NO: 68; a light chain variable region CDR2 comprising SEQ ID NO: 69; and a light chain variable region CDR3 comprising SEQ ID NO: 70.
[10]
10. Isolated monoclonal antibody, characterized by the fact that it binds to human CD27 and comprises: (a) a variable region of heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 24, 25, 36, 37, 48, 49, 60, 61, 72, 73, 84, 85, 102 and 103; or (b) a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 18, 19, 30, 31, 42, 43, 54, 55, 66, 67, 78, 79, 90, 91, 96, 97, 108 and 109.
[11]
11. Isolated monoclonal antibody, characterized by the fact that it binds to human CD27 and comprises a heavy chain variable region and / or a light chain variable region comprising an amino acid sequence at least 90% identical to the amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 37 and / or 43, respectively; (b) SEQ ID NOs: 49 and / or 55, respectively; (c) SEQ ID NOs: 103 and / or 109, respectively;
(d) SEQ ID NOs: 85 and / or 91, respectively; (e) SEQ ID NOs: 85 and / or 97, respectively; (f) SEQ ID NOs: 25 and / or 31, respectively; (g) SEQ ID NOs: 73 and / or 79, respectively; (H) SEQ ID NOs: 7 and / or 13, respectively; (i) SEQ ID NOs: 7 and / or 19, respectively; and (j) SEQ ID NOs: 61 and / or 67, respectively.
[12]
12. Isolated antibody, characterized by the fact that it competes for binding to human CD27 or binds to the same epitope bound by the antibody of claim 11.
[13]
13. Isolated monoclonal antibody that binds to human CD27, characterized by the fact that the antibody comprises a heavy chain variable region and a light chain variable region encoded by nucleic acid sequences selected from the group consisting of: (a) SEQ ID NOs: 5 and 11, respectively; (b) SEQ ID NOs: 5 and 17, respectively; (c) SEQ ID NOs: 23 and 29, respectively; (d) SEQ ID NOs: 35 and 41, respectively; (e) SEQ ID NOs: 47 and 53, respectively; (f) SEQ ID NOs: 59 and 65, respectively; (g) SEQ ID NOs: 71 and 77, respectively; (h) SEQ ID NOs: 83 and 89, (i) SEQ ID NOs: 83 and 95; and (j) SEQ ID NOs: 101 and 107, respectively or nucleic acid sequences containing at least 90% identity to the nucleic acid sequences from (a) to (j).
[14]
14. Expression vector, characterized in that it comprises a nucleotide sequence that encodes the variable region of the heavy chain and / or light chain of the antibody as defined in any one of claims 1 to 13.
[15]
15. Transformed cell, characterized by the fact that it is with an expression vector as defined in claim 14.
[16]
16. Antibody according to any one of claims 51 to 5 or 7 to 13, characterized in that the antibody is a human, humanized or chimeric antibody.
[17]
17. Composition, characterized by the fact that it comprises the bispecific antibody or molecule as defined (a) in any one of claims 1 to 13.
[18]
18. Composition according to claim 17, characterized by the fact that it still comprises an adjuvant and / or an immunostimulatory agent.
[19]
19. Composition according to claim 18, characterized by the fact that the immunostimulatory agent is selected from the group consisting of CD40 ligand, FLT 3 ligand, cytokines, colony stimulating factors, an anti-CTLA-4 antibody, LPS (endotoxin ), ssRNA, dsRNA, Bacillus Calmette-Guerin (BCG), Levamisole hydrochloride, intravenous immunoglobulins and a Toll-like receptor agonist (TLR).
[20]
20. Composition according to claim 19, characterized in that the Toll-like receptor agonist is selected from the group consisting of a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist and a agonist to TLR9.
[21]
21. Composition according to claim 17, characterized in that it further comprises an immunosuppressive agent, another antibody and / or an antigen.
[22]
22. Composition according to claim 21, characterized in that the antigen comprises a component of a pathogen, a tumor antigen, allergen or an autoantigen.
[23]
23. Composition according to claim 22, characterized by the fact that the tumor antigen is selected from the group consisting of βhCG, gp100 or Pmel17, HER2 / neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan-A, NY-ESO-1, NY-BR-1, NY-CO58, 5 MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3, Tyrosinase, Telomerase, SSX2 antigens, MUC- antigens 1, and germ cell-derived tumor antigens.
[24]
24. Use of an antibody as defined in any of claims 1 to 5 or 7 to 13, characterized in that it is for the manufacture of a medicament to induce or enhance an immune response against an antigen in an individual.
[25]
25. Use of an antibody as defined in any of claims 1 to 5 or 7 to 13, characterized in that it is for the manufacture of a medicament to inhibit the growth of cells that express CD27.
[26]
26. Use of an antibody as defined in any of claims 1 to 5 or 7 to 13, characterized by the fact that it is for the manufacture of a medicament to treat a cancer selected from the group consisting of leukemia, acute lymphocytic leukemia, myelocytic leukemia acute myelomonocytic myelomonocytic erythroleukemia, chronic leukemia, chronic myelocytic leukemia (granulocytic), chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt lymphoma, marginal zone B cell lymphoma, Polycythemia lymphoma , Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosma, angiosoma lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, carcinoma of the sweat gland 5 , papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, lung carcinoma small cell, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngeal, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, neuroblastoma, neuroblastoma basal cell carcinoma, biliary tract cancer, c bladder cancer, bone cancer, central nervous system (CNS) and brain cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer and neck, gastric cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; cancer of the oral cavity (for example, lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancer.
[27]
27. Use of an antibody as defined in any of claims 1 to 5 or 7 to 13, characterized in that it is for the manufacture of a medicament to treat disorders consisting of bacterial, fungal, viral and parasitic infectious diseases.
[28]
28. Use of an antibody as defined in any one of claims 1 to 5 or 7 to 13, characterized in that it is for the manufacture of a medicament to inhibit the binding of CD70 to CD27 in the cells of an individual having a disorder.
[29]
29. Use of an antibody as defined in any of the 5 claims 1 to 58 or 7 to 13, characterized by the fact that it is for the manufacture of a medicament for down-regulation of a T cell response in an individual having a disorder.
[30]
30. Use of an antibody as defined in any one of claims 1 to 5 or 7 to 13, characterized in that it is for the manufacture of a medicament to treat a disorder selected from the group consisting of graft rejection, allergy and a disease autoimmune.
[31]
31. Use according to claim 30, characterized in that the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease and other inflammatory bowel diseases, such as ulcerative colitis , systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies mixed, polyposis, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis, crescent glomerulonephritis, proliferative glomerulonephritis, bullous pemphigus, Sjogren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes mellitus, autoimmune hemitis, autoimmune hemitis autoimmune lymphoproliferative (ALPS), autoimmune hepatitis, aut hemophilia oimune, autoimmune lymphoproliferative syndrome, autoimmune uveoretinitis, Guillain-Bare syndrome, arteriosclerosis and Alzheimer's disease.

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法律状态:
2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-03-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-04-06| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 10A ANUIDADE. |

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2021-07-06| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US32372010P| true| 2010-04-13|2010-04-13|

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US61/323720|2010-04-13|

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US201161471459P| true| 2011-04-04|2011-04-04|

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US61/471459|2011-04-04|

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PCT/US2011/032355|WO2011130434A2|2010-04-13|2011-04-13|Antibodies that bind human cd27 and uses thereof|

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