ISOLATED ANTIBODY, USE OF AN ANTIBODY, NUCLEIC ACID, TRANSFORMED CELL AND PHARMACEUTICAL COMPOSITION

专利摘要:
isolated antibody, use of an antibody, nucleic acid, transformed cell and pharmaceutical composition the present invention relates to antibodies, immunoglobulin constructs or igg4 immunoglobulin fusion proteins whose half-lives in vivo are increased by the combination of: (i) a modified igg4 fc region or fcrn binding domain thereof and (ii) a sequence of the modified igg4 hinge region.
公开号:BR112013016235B1
申请号:R112013016235-0
申请日:2011-12-22
公开日:2020-03-31
发明作者:David Wilson；Tetsuya Taura
申请人:Cephalon Australia Pty Ltd；
IPC主号:

专利说明:

ISOLATED ANTIBODY, USE OF AN ANTIBODY, NUCLEIC ACID, TRANSFORMED CELL AND PHARMACEUTICAL COMPOSITION
RELATED REQUESTS
This document claims priority of USSN 61 / 425,858, the total content of which is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins whose half-lives in vivo are increased by the combination of: (i) a modified IgG4 Fc region or FcRn binding domain thereof and (ii) a sequence of the modified IgG4 hinge region.
BACKGROUND OF THE INVENTION
IgG is the most prevalent immunoglobulin class in humans and other mammals and is used in several types of immunotherapies and diagnostic procedures. A crucial issue in these therapies is the period of persistence of immunoglobulins in the circulation. The clearance rate of an immunoglobulin administered directly affects the amount and frequency of dosing of the immunoglobulin. Studies of IgG catabolism in the circulation identified the portions of the IgG constant domain that control IgG metabolism, including the rate of degradation of IgG in serum through interactions with FcRn (neonate Fc receptor). The increased affinity of binding for FcRn increases the circulating (or serum) half-life of an IgG (see, for example, Kim et al., Eur. J. Immunol., 24: 2.429 (1994)). Methods for obtaining physiologically active molecules whose half-lives are modified by introducing a polypeptide from
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2/103 binding of FcRn in the molecules are described, for example, in WO 97/43316, US 5,869,046, US 5,747,035, WO 96/32478. Methods for fusing the molecules to the antibodies or fragments of the FcRn binding domain thereof are described, for example, in WO 99/43713. However, the documents above do not reveal specific mutants in the IgG constant domain that affect half-life.
The modification of IgG molecules by substitution, addition or deletion of amino acid to increase or reduce affinity for FcRn is disclosed in WO 98/23289; however, that document does not list any specific mutants that exhibit longer or shorter half-lives in vivo.
A mouse IgG1 mutant that increases the circulating half-life has been shown to be the triple mutation Thr252Ala, Thr254Ser and Thr256Phe described, for example, in WO 97/34631. MedImmune (US 7,083,784) demonstrated that, in the context of human IgG1, modifications of one or more of amino acid residues 251-256, 285-290 and 308-314, within the CH2 domain, and amino acid residues 385-389 and 428-436, within the CH3 domain, can increase the affinity of the constant domains for FcRn and, thus, increase the circulating half-life. In particular, they demonstrated that a triple M252Y, S254T and T256E mutation, designated YTE in the Fc of a human IgG1 isotype antibody, can increase the circulating antibody half-life about 2-3 times in non-human primates.
Characteristics of IgG4 isotype antibodies
IgG4 differs from other human IgG isotypes in that, using SDS-PAGE under non-reducing conditions, two protein species are observed, the main species
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3/103 being tetrameric IgG (H2L2, that is, two heavy chains and two light chains) and a second smaller species being a half-immunoglobulin that contains a single heavy chain and a single light chain (HL). These findings indicate heterogeneity in the disulfide bond formation between the two heavy chains in the hinge region. In addition, when different human IgG4s with different antigen binding specificities are mixed together, the individual IgG4 molecules are able to dissociate into half-immunoglobulins (HL) and then re-associate to form tetrameric IgG (H2L2) that binds to two different antigens (bispecific antibodies). The HL species is believed to be an important intermediate in the assembly of IgG4. Analysis of human IgG heavy chain hinge sequences suggests that the presence of serine in residue 228 (also referred to in some publications residue 241; for the avoidance of doubt, this refers to serine at the center of the sequence of the CPSCP IgG4 hinge region (SEQ ID. N °: 1)) of
IgG4 (according to the Kabat numbering system et al, Sequences of Proteins of Immunological Interest, 4th Edition, Washington DC -. United States Department of Health and Human Services) may be the cause of heterogeneity. When this residue in IgG4 is changed from serine to proline (the residue found naturally in that position in IgG1 and IgG2), it leads to the production of homogeneous antibody with prolonged serum half-life (Angal S. et al, Molecular Immunology volume 30, No. 1: 105-108 (1993); Labrijn et al, Nature Biotechnology volume 27, No. 8: 767-771; Schuurman J. et al, Molecular Immunology 38
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4/103 (2001) 1-8).
There is a growing need for the generation of antibodies for therapeutic purposes with improved properties, for example, an increased circulating half-life.
SUMMARY OF THE INVENTION
The present invention relates to molecules, in particular antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins, whose half-lives in vivo are increased by the combination of: (i) an Fc region or region of the FcRn binding domain that comprises a sequence of the modified IgG4 isotype, and (ii) a sequence of the modified IgG4 hinge region. Specifically, these molecules have amino acid modifications, for example, mutations, which increase the affinity of the Fc or constant regions CH2 and CH3 of the heavy chain for the FcRn and, thus, their circulating half-life in an individual. In addition, the molecules include a sequence of the modified IgG4 hinge region that cancels the formation of mixed heterodimers that is typical of IgG4 isotype antibodies.
The invention is based on the surprising discovery that the combination of the above modifications increases the circulating half-life of the molecule in relation to its unmodified wild-type counterpart substantially longer than the modification (or modifications) of the Fc region or the modification hinge region in isolation. In addition, the combination of modifications results in a supra-additive (synergistic) extension of the half-life. Like any modification to a protein-based drug
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Human 5/103 (including a protein comprising a human antibody constant region) increases the risk of inducing an anti-drug immune response in a patient, it is generally advisable to limit the number of these mutations to limit the presumably additively increased risk of each of these mutations regarding the induction of these immune responses against the drug. However, the surprising results described here demonstrate that the combination of the two classes of substitutions (Fc modifications and hinge modifications) results in a supra-additive effect in increasing the circulating half-life of IgG4 antibodies. Consequently, this combination provides an unexpected advantage that can overcome a theoretical disadvantage in relation to the increased incidence of promoting anti-drug immune reactions. The advantages of increasing the half-life of a molecule will be immediately evident to those skilled in the art. These benefits include reduced dosage and / or frequency of administration, which reduces the risk of adverse events in an individual and reduces costs. Consequently, these immunoglobulins with an increased half-life are of significant pharmaceutical importance.
In addition, as the molecules comprise domains constant for the IgG4 isotype and an IgG4 hinge region, the molecules do not exhibit effector function or exhibit minimal effector function in vivo.
Consequently, in one embodiment, the present invention provides an isolated antibody, an immunoglobulin construct or an IgG4 immunoglobulin fusion protein with an increased in vivo half-life, comprising:
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6/103 (i) a human IgG4 Fc region or its modified FcRn binding domain with respect to a corresponding unmodified IgG4 Fc region or FcRn binding domain thereof to comprise substitutions in one or more amino acid residues 251-256 numbered according to the EU index as in Kabat; and (ii) a sequence of the central hinge region of
IgG4 human which comprises an replacement of the residue in serine dent of the sequence in amino acids CPSCP (ID. IN SEQ. No. : 1) for proline;in what the half-life in vivo antibody, construction in
immunoglobulin or modified IgG4 immunoglobulin fusion protein is increased compared to the corresponding antibody, immunoglobulin construct or unmodified IgG4 immunoglobulin fusion protein.
The increased in vivo half-life of the antibody, immunoglobulin construction or IgG4 immunoglobulin fusion protein is determined by reference to the half-life of an IgG4 antibody, immunoglobulin construction or corresponding human IgG4 immunoglobulin fusion protein that does not have the substitutions above.
The present invention also provides an isolated antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein with increased in vivo half-life, which comprises:
(i) a human IgG4 Fc region or the modified FcRn binding domain thereof with respect to a corresponding unmodified IgG4 Fc region or FcRn binding domain thereof to comprise M252Y, S254T and T256E substitutions numbered according to the index I as in Kabat; and
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7/103 (ii) a sequence of the central hinge region of human IgG4 comprising the substitution of amino acid S228P according to the EU index as in Kabat;
wherein the in vivo half-life of the antibody, immunoglobulin construction or modified IgG4 immunoglobulin fusion protein is increased, compared to the corresponding antibody, immunoglobulin construction or unmodified IgG4 immunoglobulin fusion protein.
The antibody according to the invention can be a chimeric antibody, human antibody, humanized antibody, a Super-Humanized® antibody, a de-immunized antibody or a varnished (veneered) antibody.
In one example, the present invention provides an isolated antibody with an increased in vivo half-life, comprising:
(i) a human or humanized Fab, (ii) a human IgG4 Fc region or its modified FcRn binding domain with respect to a corresponding unmodified IgG4 Fc region or FcRn binding domain thereof to comprise substitutions in one or more of amino acid residues 251-256 numbered according to the EU index as in Kabat, and (iii) a sequence of the central hinge region of human IgG4 comprising a substitution of the serine residue within the amino acid sequence CPSCP (ID SEQ NO: 1) for proline, also described as an S228P replacement according to the EU index as in Kabat;
where the in vivo half-life of the antibody, immunoglobulin construct or modified IgG4 immunoglobulin fusion protein is increased compared to the antibody,
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8/103 corresponding unmodified immunoglobulin or IgG4 immunoglobulin fusion protein construct.
Throughout the specification, the residue numbering in a immunoglobulin heavy chain is that of the EU index or Kabat numbering system (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Edition, Washington DC United States Department of Health and Human Services ", 1991, National Institutes of Health", Bethesda. The EU index as Kabat "refers to a numbering of the EU antibody of human IgG1 (Edelman et al., Proc. Natl. Acad. USA, 63 , 78-85, 1969) .The amino acid sequences of the IgG2, IgG3 and IgG4 isotypes are aligned with the IgG1 sequence by placing the first and last cysteine residues of the respective hinge regions, which form the heavy interchain SS bonds amino acid residues 251-256 according to the EU index as in Kabat are located within the CH2 domain of the immunoglobulin heavy chain of the Fc region. These residues have been implicated in the alloy from the Fc region to the FcRn and, therefore, are involved in altering the antibody half-life.
In another example, the invention provides an isolated immunoglobulin construct with an increased in vivo half-life, comprising:
(i) an antibody fragment;
(ii) a CH2 domain of human IgG4 modified with respect to a corresponding unmodified CH2 domain to comprise substitutions in one or more of amino acid residues 251-256 numbered according to the EU index as in Kabat, and
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9/103 (iii) a sequence of the central hinge region of human IgG4 comprising a substitution of the serine residue within the CPSCP amino acid sequence (SEQ ID NO: 1) for proline, also described as an S228P substitution according to the EU index as in Kabat;
wherein the in vivo half-life of the modified immunoglobulin construct is increased, compared to the corresponding unmodified immunoglobulin construct.
In one embodiment, the isolated immunoglobulin construct comprises a human IgG4 Fc region or FcRn binding domain thereof.
Preferably, the isolated immunoglobulin construct comprises substitutions M252Y, S254T and T256E numbered according to the EU index as in Kabat.
Specific antibody fragments include, without limitation, (i) a Fab fragment (ii) an Fd fragment, (iii) an Fv fragment, (iv) an dAb fragment, (v) isolated CDR regions, (vi) F ( ab ') 2, (vii) single chain Fv molecules (scFv), (viii) bispecific single chain Fv, and (ix) diabody (x) triabody and (xi) tetrabody.
The invention also provides IgG4 immunoglobulin fusion proteins with increased in vivo half-life which comprise a recombinantly fused or chemically conjugated or genetically modified bioactive molecule to contain: (i) a human IgG4 Fc region or the FcRn binding domain thereof. modified with respect to a corresponding unmodified IgG4 Fc region or FcRn binding domain thereof to comprise M252Y, S254T and T256E substitutions numbered according to EU index as in Kabat, and (ii) a human IgG4 comprising the substitution of
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10/103 amino acid S228P following the central hinge region according to the EU index as in Kabat.
The bioactive molecule can include proteinaceous agents or
non-protein or proteins no immunoglobulin. In a modality, The bioactive molecule is a polypeptide. In another example, the present invention provides a
IgG4 immunoglobulin fusion protein with increased in vivo half-life, comprising:
(i) a bioactive molecule;
(ii) a human IgG4 CH2 domain modified in relation to an IgG4 CH2 domain to comprise substitutions in one or more of amino acid residues 251256 numbered according to the EU index as in Kabat, and (iii) a sequence from the region of central hinge of human IgG4 comprising a substitution of the serine residue within the amino acid sequence CPSCP (SEQ ID NO: 1) for proline, also described as an S228P substitution according to the EU index as in Kabat;
wherein the in vivo half-life of the modified IgG4 immunoglobulin fusion protein is increased compared to the corresponding unmodified IgG4 immunoglobulin fusion protein.
Preferably, the isolated IgG4 immunoglobulin fusion protein comprises a human IgG4 Fc region or FcRn binding domain thereof.
Preferably, the isolated IgG4 immunoglobulin fusion protein comprises M252Y, S254T and T256E substitutions numbered according to the EU index as in Kabat.
The sequence of the central IgG4 hinge region
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Human 11/103 according to the invention preferably comprises an S228P substitution according to the EU index as in
Kabat. This substitution was also called S241P according to Kabat et al. (1987 “Sequences of Proteins of Immunological Interest”, United States Department of Health and Human Services ”, Washington DC). The substitution has the effect of producing a sequence of the center of the hinge region equal to that of an isotype antibody
IgG1 or IgG2 of the wild type. With regard to the IgG4 isotype antibody, it results in the production of the homogeneous form of the IgG4 antibody and, thus, cancels the dissociation and reassociation of heavy chains, which often leads to the production of heterodimeric IgG4 antibodies.
The antibody, immunoglobulin construct or IgG4 immunoglobulin fusion molecule of the invention comprises a human IgG4 Fc region or FcRn binding domain thereof which comprises a substitution in one or more of the amino acid residues 252, 254 and 256 according to EU index as in Kabat. In certain examples, the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein according to the invention comprises a single substitution of any of the amino acid residues 252, 254 or 256 of the Fc region. In other examples, the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein comprises substitutions of residues 252 and 254, or residues 254 and 256 or residues 252 and 256 of the Fc region. In a particular example, the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein comprises substitutions for each of residues 252, 254 and 256 of the
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12/103 sequence of the human IgG4 Fc region.
In particular examples of the invention, residue 252 is replaced with tyrosine, phenylalanine, serine, tryptophan or threonine, residue 254 is replaced with threonine or serine and residue 256 is replaced with serine, arginine, glutamine, glutamic acid, aspartic acid, alanine, asparagine or threonine. In a particular example, residue 252 is replaced with tyrosine (M252Y), residue 254 is replaced with threonine (S254T) and residue 256 is replaced with glutamic acid (T256E). These substitutions are collectively referred to as the “YTE modification”.
In another embodiment, the antibody or immunoglobulin construct according to the invention can be further recombinantly fused, chemically conjugated or genetically modified to contain a portion. The portion according to the invention can be selected, without limitation, from a therapeutic agent that is linked, directly or indirectly, to the antibody, a cytotoxin, a radioisotope, an immunomodulatory agent, an anti-angiogenic agent, an anti-vascular agent and / or other vascularizing agent, a toxin, an antiproliferative agent, a pro-apoptotic agent, a chemotherapeutic agent and a therapeutic nucleic acid.
In one example, the modified antibody according to the present invention is an antibody that specifically binds to human IL-5. In another example, the modified antibody according to the present invention is an antibody that specifically binds to human CD33.
Consequently, in one example, the present invention
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13/103 also provides an isolated antibody that specifically binds to IL-5 which comprises:
(i) a human IgG4 Fc region or the modified FcRn binding domain thereof relative to a corresponding unmodified human IgG4 Fc region or FcRn binding domain thereof to comprise amino acid substitutions M252Y, S254T and T256E accordingly numbered with the EU index as in Kabat, and (ii) a sequence of the central hinge region of human IgG4 comprising the replacement of amino acid S228P according to the EU index as in Kabat, in which the in vivo half-life of the modified antibody is increased compared to the corresponding unmodified antibody half-life.
In a particular example, the corresponding unmodified anti-IL-5 antibody is hu39D10. In another embodiment, the Fab sequence of the isolated antibody may correspond to the sequence of the variable region of the light and heavy chains of mepolizumab.
In another example, the present invention provides an antibody that specifically binds to IL-5, the antibody comprising a constant heavy chain sequence shown in ID. SEQ. No. 6 and a sequence of the variable region of the heavy chain shown in the ID. SEQ. No.: 7. In another example, the antibody that specifically binds to IL-5 still comprises a light chain that comprises the sequences of the variable and constant region shown in the ID. SEQ. N °: 8.
In another example, the present invention provides an isolated antibody that specifically binds to CD33 that
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10/14 comprises:
(i) a human IgG4 Fc region or the modified FcRn binding domain thereof relative to a corresponding unmodified human IgG4 Fc region or FcRn binding domain thereof to comprise amino acid substitutions M252Y, S254T and T256E accordingly numbered with the EU index as in Kabat, and (ii) a sequence of the central hinge region of human IgG4 comprising the replacement of amino acid S228P according to the EU index as in Kabat, in which the in vivo half-life of the modified antibody is increased compared to the corresponding unmodified antibody half-life.
In a particular example, the modified anti-CD33 antibody according to the invention is the huMab195 antibody.
In another example, the present invention provides an antibody that specifically binds to CD33, the antibody comprising a heavy chain sequence shown in ID. SEQ. No. 11 and a light chain sequence shown in the ID. SEQ. N °: 12.
The present invention also provides for the use of an isolated antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein with increased in vivo half-life, which comprises:
(i) a human IgG4 Fc region or the modified FcRn binding domain thereof relative to a corresponding unmodified IgG4 Fc region or unmodified FcRn binding domain thereof to comprise substitutions at one or more 251- amino acid residues 256 numbered according to the EU index as in Kabat, and
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15/103 (ii) a sequence of the central hinge region of human IgG4 comprising a substitution of the serine residue within the amino acid sequence CPSCP for proline (SEQ ID NO: 1), also described as an S228P substitution according to the EU index as in Kabat;
in medicine.
Preferably, the isolated antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein comprises M252Y, S254T and T256E substitutions numbered according to the EU index as in Kabat.
The present invention also provides for use of an isolated antibody, immunoglobulin construct or modified IgG4 immunoglobulin fusion protein according to the invention in the manufacture of a medicament for the treatment or prevention of a disorder.
The invention provides use of an isolated antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein with an increased in vivo half-life comprising:
(i) a human IgG4 Fc region or the modified FcRn binding domain thereof relative to a corresponding unmodified human IgG4 Fc region or FcRn binding domain thereof to comprise amino acid substitutions M252Y, S254T and T256E accordingly numbered with the EU index as in Kabat, and (ii) a sequence of the central hinge region of human IgG4 which comprises the replacement of amino acid S228P according to the EU index as in Kabat, in the manufacture of a drug for the treatment or prevention of a disorder in an individual. This
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10/163
invention also provides a method in treatment or prevention of a disorder in an individual characterized per production excessive eosinophils what comprises The administration of an anti- IL-5 modified gives
invention.
The present invention also provides a method of treating a disorder in an individual characterized by excessive eosinophil production, which comprises administering to the individual an isolated antibody that specifically binds IL-5 which comprises:
(i) a human IgG4 Fc region or its modified FcRn binding domain with respect to a corresponding unmodified IgG4 Fc region or unmodified FcRn binding domain thereof to comprise M252Y, S254T and T256E substitutions numbered according to the EU index as in Kabat, and (ii) a sequence of the central hinge region of human IgG4 comprising the replacement of amino acid S228P according to the EU index as in Kabat, where the in vivo half-life of the modified antibody is compared to the half-life of the corresponding unmodified antibody.
The present invention also extends to the use of these modified antibodies in the treatment or prevention of a disorder in an individual characterized by excessive production of eosinophils and to the use of the modified antibodies in the manufacture of a medicament for the treatment or prevention of a disorder characterized by excessive production of eosinophils.
In one example, the invention provides use of an antibody
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isolated with half-life in alive increased bind specifi specifically to IL-5, what comprises:(i) an Fc region in IgG4 human or the domain of
this modified FcRn binding to a corresponding unmodified human IgG4 Fc region or FcRn binding domain thereof to comprise amino acid substitutions M252Y, S254T and T256E numbered according to the EU index as in Kabat, and (ii) one sequence of the central hinge region of human IgG4 comprising the replacement of amino acid S228P according to the EU index as in Kabat, in the manufacture of a drug for the treatment or prevention of a disorder characterized by excessive production of eosinophils in an individual.
In one example, the disorder is characterized by excessive production of eosinophils (eosinophilia).
A disorder characterized by excessive eosinophil production can be selected from the group consisting of atopic asthma, atopic dermatitis, allergic rhinitis, non-allergic rhinitis, asthma, severe asthma, chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome , eosinophilic myalgia syndrome, hypereosinophilic syndrome, edematous reactions, including episodic angioedema, helminth infections, onchocerciasis dermatitis, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis, nasal polyosclerosis, nasal polyposis, polyposis, polyposis, nasal polyps obstructive sleep apnea, chronic asthma, Crohn's disease, scleroderma and endomyocardial fibrosis.
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In another example, the disorder is an autoimmune disease.
It will be appreciated that the anti-IL-5 antibody modified according to the invention can be used in methods of prophylaxis or diagnosis in relation to a disorder characterized by excessive production of eosinophils.
The invention also provides a method of treating a cancerous disorder in an individual, which comprises administering an anti-CD33 antibody modified according to the invention to the individual.
The present invention also provides a method of treating a cancerous disorder in an individual, which comprises administering to the individual an isolated antibody that specifically binds to CD33, which comprises:
(i) a human IgG4 Fc region or its modified FcRn binding domain with respect to a corresponding unmodified IgG4 Fc region or unmodified FcRn binding domain thereof to comprise M252Y, S254T and T256E substitutions numbered according to the EU index as in Kabat, and (ii) a sequence of the central hinge region of human IgG4 comprising the replacement of amino acid S228P according to the EU index as in Kabat, where the in vivo half-life of the modified antibody is increased compared to the corresponding unmodified antibody half-life.
The present invention also extends to the use of these modified antibodies in the treatment or prevention of a cancerous disorder and to the use of the modified antibodies in the manufacture of a medicament for the treatment or prevention
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19/103 of a cancerous disorder.
In one particular example, the cancerous disorder is acute myeloid leukemia.
In another example, the invention provides use of an isolated antibody with an increased in vivo half-life that specifically binds to CD33, which comprises:
(i) a human IgG4 Fc region or its modified FcRn binding domain relative to a corresponding unmodified human IgG4 Fc region or an unmodified FcRn binding domain thereof to comprise numbered M252Y, S254T and T256E amino acid substitutions according to the EU index as in Kabat; and (ii) a sequence of the central hinge region of human IgG4 comprising the substitution of amino acid S228P according to the EU index as in Kabat;
in the manufacture of a drug for the treatment or prevention of a cancerous disorder in an individual.
In one particular example, the cancerous disorder is acute myeloid leukemia. The present invention also provides a method for increasing the in vivo half-life of a human or humanized antibody of the IgG4 isotype or immunoglobulin construct that comprises an IgG4 Fc region or FcRn binding domain thereof and the IgG4 hinge region, the method comprising:
(i) introduction of amino acid substitutions M252Y, S254T and T256E numbered according to the EU index as in Kabat following the Fc region or FcRn binding domain thereof, and (ii) introduction of the amino acid substitution S228P according to EU index as in Kabat following the region
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20/103 center hinge.
In a particular example, the above method can be used to increase the half-life of an anti-IL-5 antibody, in particular hu39D10.
In a further particular example, the above method can be used to increase the half-life of an anti-CD33 antibody.
The present invention also provides a method for increasing the in vivo half-life of an IgG4 immunoglobulin fusion protein that comprises an IgG4 Fc region or FcRn binding domain therefrom and hinge region of
IgG4, the method comprising:
(i) introduction into a human IgG4 Fc region or FcRn binding domain thereof of amino acid substitutions M252Y, S254T and T256E numbered according to the EU index as in Kabat; and (ii) introduction of the substitution of amino acid S228P according to the EU index as in Kabat in a sequence of the central hinge region of human IgG4.
The invention also provides a method for increasing the in vivo half-life of a protein by its genetic modification as a fusion protein comprising ID. SEQ. N °: 14.
The invention also provides a fusion protein that comprises ID. SEQ. N °: 14.
In one example, the fusion protein still comprises a single lysine attached immediately at the C-terminus with respect to the ID. SEQ. N °: 14.
The invention also provides a method for reducing the effector function of a non-IgG4 antibody, the method
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21/103 comprising:
(i) replacement of the non-IgG4 antibody heavy chain constant region with a human IgG4 constant region or FcRn binding domain thereof, modified to comprise amino acid substitutions M252Y, S254T and T256E numbered according to the EU index as in Kabat ; and (ii) replacement of the hinge region of the non-IgG4 antibody with an IgG4 hinge region that has the substitution of amino acid S228P according to the EU index as in Kabat.
The invention also provides a method for increasing the in vivo half-life of a non-human IgG4 antibody, the method comprising:
(i) replacement of the non-human IgG4 antibody heavy chain constant region with a human IgG4 constant region or FcRn binding domain thereof, modified to comprise amino acid substitutions M252Y, S254T and T256E numbered according to the EU index as in Kabat;
(ii) replacement of the hinge region of the non-human IgG4 antibody with an IgG4 hinge region that has the substitution of amino acid S228P according to the EU index as in Kabat.
The present invention also provides a nucleic acid that encodes an antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein, as described herein according to any embodiment.
The present invention also provides a transformed cell that expresses an antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein,
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22/103 as described herein according to any modality.
The present invention also provides a transformed cell that comprises a nucleic acid encoding an antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein as described herein.
In another embodiment, the invention provides a pharmaceutical composition comprising the isolated antibody, immunoglobulin construct or immunoglobulin IgG4 fusion protein according to the invention, together with a pharmaceutically acceptable excipient. Preferably, the composition comprises a therapeutically effective amount of the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein.
DESCRIPTION OF THE FIGURES
Figure 1 shows the relizumab heavy chain sequence, including the IgG4 variable and constant domains, sequences of the constant domain (native IgG4 isotype) or the IgG4 constant domain with the S228P mutation, YTE mutations or a combination of S228P and YTE mutations; the sequence of the variable domain of the hu39D10 heavy chain and the hu39D10 light chain is also shown.
Figure 2 shows the sequence of the human FcRn extracellular domain.
Figure 3 shows the sequence of the mature portion of human beta-2 microglobulin.
Figure 4 shows an ELISA-based assay to measure the affinity of human FcRn for hu39D10 containing a native IgG4 Fc domain, or one that carries the S228P mutation, the YTE mutations, or both the S228P and YTE mutations.
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Figure 5 shows a PK study in mice with humanized FcRn, that is, mice that had deleted the endogenous FcRn gene, but have ectopic expression from the human counterpart. On day 0, the mice received hu39D10 or variants that contain the YTE substitutions alone, the hinge replacement (S228P) alone, or both types of substitutions. Each mouse was bled from the retro-orbital sinus at 2, 12, 24 hours and 2, 4, 7, 10, 14, 18, 21 and 28 days. Plasma samples were analyzed for humanized antibody concentration. Antibody levels are expressed as a percentage of the level at the 24-hour time point in the same mouse.
Figure 6 shows the sequence of the CD33 antibody huMab195 in which the Fc domain is an IgG4 isotype that contains the S228P and TYE modifications; the huMab195 light chain is also shown.
Figure 7 shows the sequence of the human CD33-Fc extracellular domain fusion protein.
Figure 8 shows a PK study in mice with humanized FcRn, that is, mice that had deleted the endogenous FcRn gene, but have ectopic expression from the human counterpart. On day 0, the mice received huMab195 with a native IgG4 Fc domain or variants that contain the YTE substitutions alone, the hinge replacement (S228P) alone, or both types of substitutions. Each mouse was bled from the retroorbital sinus at 2, 12, 24 hours and 2, 4, 7, 10 and 14 days.
Plasma samples were analyzed for humanized antibody concentration. Antibody levels are
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24/103 expressed as a percentage of the level at the 24-hour time point in the same mouse.
Figure 9 shows the sequence of the Fc hinge portion of the native human IgG4 heavy chain and the sequence of the Fc hinge portion of the human IgG4 heavy chain with S228P and YTE mutations and devoid of C-terminal lysine.
DETAILED DESCRIPTION OF THE INVENTION
General
The term and / or, for example, X and / or Y should be understood to mean X and Y or X or Y and should be considered to provide explicit support for either or both meanings.
Throughout this specification, unless specifically stated differently or the context requires differently, reference to a single step, material composition, group of steps or group of material compositions should be considered as encompassing one and several ( that is, one or more) of those stages, compositions of matter, groups of stages or groups of compositions of matter. Thus, as used here, the singular forms one, one, o and a include aspects in the plural, unless the context clearly determines differently. For example, reference to one or one includes a single, as well as two or more; reference to or includes a single, as well as two or more, and so on. Each example of the disclosure must be applied, with the necessary modifications, to each and all other modalities, unless specifically stated differently.
Those skilled in the art will note that the
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25/103 The disclosure presented here is susceptible to variations and modifications in addition to those specifically described. It must be understood that the disclosure encompasses all of these variations and modifications. The disclosure also includes all the steps, characteristics, compositions and compounds cited or indicated in that specification, individually or collectively, and any and all combinations or any two or more of said steps or characteristics.
The present disclosure is not limited in scope by the specific modalities described here, which have the sole purpose of exemplification. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure.
The compositions of matter and methods described herein are produced or performed without unnecessary experimentation using, unless otherwise stated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide solution synthesis, solid phase peptide synthesis and immunology. These procedures are described, for example, in Sambrook, Fritsch & Maniatis, “Molecular Cloning: A
Laboratory Manual ”, Cold Spring Harbor Laboratories, New York, Second Edition (1989), all Volumes I, II and III; Benny K.C. Lo, “Antibody Engineering: Methods and
Protocols ”, (2004) Humana Press, Vol. 248; “DNA Cloning: A Practical Approach”, Volumes I and II (D.N. Glover, ed., 1985), IRL Press, Oxford, the entire text; “Oligonucleotide Synthesis: A Practical Approach” (M.J. Gait, ed, 1984) IRL
Press, Oxford, all the text and, particularly, the articles
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26/103 in it by Gait, ppl-22; Atkinson et al, pages 35-81; Sproat et al, pages 83-115; and Wu et al., pages 135-151; “4. Nucleic Acid Hybridization: A
Practical Approach ”(B.D. Hames & S.J. Higgins, eds., 1985) IRL Press, Oxford, all text; Immobilized Cells and Enzymes: A Practical Approach ”(1986) IRL Press, Oxford, all text; Perbal, B., A Practical Guide to Molecular Cloning ”(1984); Methods In Enzymology ”(S. Colowick and N. Kaplan, eds., Academic Press, Inc.), the entire series; J.F. Ramalho Ortigao, The Chemistry of Peptide Synthesis ”In:
Knowledge Database of Access to Virtual Laboratory Website ”(Interactive, Germany); Sakakibara, D., Teichman,
J., Lien, E. Land Fenichel, R.L. (1976). Biochem. Biophys. Commun. 73 336-342; Merrifield, R.B. (1963). J. Am.
Chem. Soc. 85, 2.149-2.154; Barany, G. and Merrifield, R.B.
(1979) in The Peptides ”(Gross, E. and Meienhofer, J. eds.), Vol. 2, pages 1-284, Academic Press, New York. 12. Wunsch, E., ed. (1974) Synthesis von Peptiden in HoubenWeyls Metoden der Organischen Chemie ”(Muller, E., ed.), Vol. 15, 4th Edition, Parts 1 and 2, Thieme, Stuttgart;
Bodanszky, M. (1984) Principles of Peptide Synthesis ”, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis ”, SpringerVerlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental
Immunology ”, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach ”, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, all text.
Throughout this specification the word
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27/103 comprise, or variations such as, for example, comprise or comprise, will implicitly include the inclusion of an element, integer or step established, or group of elements, integers or steps, but not the exclusion of any other element, whole number or step, or group of elements, whole numbers or steps.
Definitions
As used herein, the corresponding unmodified antibody term means an antibody of the same sequence
that the modified antibody, but without at changes The amino acid sequence here described, in private The region Fc and hinge. THE epitope aims refer The part of a
antigenic molecule to which an antibody is produced and to which the antibody will bind. The term epitope, as used herein, refers to a portion (or portions) of a peptide that has antigenic or immunogenic activity in an animal, preferably a vertebrate, more preferably a mammal and, more preferably still, in a human or an transgenic animal that expresses relevant components of the human immune system. Epitopes may comprise proteins, protein fragments, peptides, carbohydrates, lipids and other molecules, but, for the purposes of the present invention, they are more commonly short oligopeptides. The term epitope is intended to encompass an immunogenic epitope, an antigenic epitope, or antigen epitope.
The term antibody, as used herein, refers to a molecule that is capable of binding to a target through at least one epitope recognition site, located in the
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28/103 variable region of the immunoglobulin molecule. The terms immunoglobulin and antibody can be used interchangeably throughout the specification. The immunoglobulin or antibody molecule includes four-chain antibodies (for example, two light chains and two heavy chains), recombinant or modified antibodies (for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies with grafted CDR, primatized antibodies, antibodies de-immunized, Super-Humanized® antibodies, half-antibodies, bispecific antibodies). An antibody generally comprises constant domains, which can be arranged in a constant region or constant fragment or crystallizable fragment (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (approximately 50-70 kD) covalently linked and two light chains (approximately 23 kD each). Each heavy and light chain comprises variable regions and constant domains. A light chain usually comprises a variable region (if present) and a constant domain and, in mammals, it is a κ light chain or a λ light chain. A heavy chain generally comprises a variable region and one or two constant domains connected by a hinge region to additional constant domains. Heavy mammal chains are one of the following types: α, δ, ε, γ or μ. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by interchain disulfide bonds and by
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29/103 non-covalent interactions. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has a variable N-terminal region (VH or VL, each of which is approximately 110 amino acids in length) and one or more constant domains at the C-terminal. The light chain constant domain (CL, which has approximately 110 amino acids of length) is aligned and disulfide bonded to the first constant domain of the heavy chain (Ch1, which is 330-440 amino acids in length). The variable region of the light chain is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional Ch domains (e.g., Ch2, Ch3 and the like) and can comprise a hinge region between the Ch1 and Ch2 constant domains. Unmodified antibodies can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA ₂ ) or subclass.
The term "immunoglobulin construct", as used herein, refers to a construct that comprises at least one constant domain and hinge region of the CH2 heavy chain of a primate or human IgG4 antibody. Preferably, the term is intended to refer to a construct that comprises at least constant domains and hinge region of the primate or human IgG4 antibody.
The term constant region ”or constant fragment” refers to the portion of an immunoglobulin or antibody molecule that has a conserved amino acid sequence central to another portion of the
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30/103 immunoglobulin or antibody, the so-called variable region, which contains the antigen binding site. In the heavy chain, the constant region contains the CH1, CH2 and CH3 domains.
The term "Fc region, as used herein, refers to that portion of an antibody or immunoglobulin molecule that is correlated with a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region consists of the C-terminal region of an IgG heavy chain made up of the C-terminal and approximately half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. Although the limits may vary slightly (in some cases it includes part of the hinge), as numbered according to the EU Kabat index, the Fc region extends from amino acid 231 to amino acid 447. The Fc region of an IgG comprises two constant domains , CH2 and CH3. The CH2 domain of a human IgG Fc region normally extends from amino acid 231 to amino acid 341 according to the EU index of Kabat. The CH3 domain of a human IgG Fc region normally extends from amino acids 342 to 447 according to the index
I from Kabat. The Fc region has no antigen binding activity, but contains the carbohydrate portion and the binding site for the Fc receptor, including the neonatal Fc receptor (FcRn).
The term "FcRn binding domain thereof", as used herein, refers to a portion of the Fc region that is capable of binding to FcRn. In the present context, it also aims to refer to a fragment of the Fc region sequence that includes at least the CH2 domain. The term “FcRn receptor, as used herein, refers to an Fc receptor (“ n indicating
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31/103 neonatal) that is involved in the transfer of maternal IgGs to a fetus via the human or primate placenta and to a newborn by colostrum through the small intestine. FcRn is also involved in maintaining constant serum IgG levels by binding the IgG molecules and recycling them in the serum. The binding of FcRn to IgG molecules is strictly pH-dependent with optimal binding at pH 6.0. FcRn is typically complexed with beta-2 microglobulin.
The term "hinge region", as used herein, refers to a proline-rich portion of an immunoglobulin heavy chain between the Fc and Fab regions that confers mobility on both Fab arms of the antibody molecule. It is located between the first and second domains in the heavy chain. The hinge region includes cysteine residues that are involved in heavy interchain disulfide bonds. It is generally defined as stretching from Glu216 to Pro230 of human IgG1 according to the EU numbering system of Kabat (or Glu226 to Pro243 according to the Kabat numbering system). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form heavy interchain disulfide (S-S) bonds in the same positions (see, for example, WO 2010/080538). The hinge region includes cysteine residues that are involved in heavy interchain disulfide bonds.
The term “central hinge region sequence”, as used here, is intended to refer to the sequence of amino acids
CPSCP (SEQ ID. N °: 1) present in IgG4 and extending
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32/103 of amino acid 226 to 230 according to the EU index of Kabat (often called the lower hinge). The central hinge region is distinguished from the upper hinge region, which, in a human IgG4, is the ESKYGPP sequence.
The term "variable region", as used herein, refers to the portions of the light and / or heavy chains of an antibody, as defined herein, that are capable of specifically binding to an antigen, and includes amino acid sequences from determining regions of complementarity (CDRs); that is, CDR1, CDR2 and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (for example, FR1, FR2, FR3 and, optionally, FR4) along with three CDRs. In the case of an IgNAR-derived protein, the protein may not have a CDR2. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.
The term "Fab", as used herein, is intended to refer to a region of an antibody composed of a constant domain and a variable domain of each of the heavy and light chains (monovalent antigen binding fragment), but in which the chain heavy is truncated in such a way that it does not have the CH2 and CH3 domain (ie, VH, CH1, VL and CL), and may also not have a part or all of the hinge region. It can be produced by digesting an entire antibody with the enzyme papain. Fab can refer to that region alone or to that region in the context of a full-length antibody, an immunoglobulin construct or a Fab fusion protein. The term Fab ', as used herein, can be obtained by treating an antibody whole with pepsin, followed by reduction, to generate a
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33/103 molecule consisting of an intact light chain and a portion of a heavy chain comprising a Vh and a single constant domain. Two Fab 'fragments are obtained by antibodies treated in this way.
By scFv means an antibody fragment that comprises the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. See, e.g., US Patent ^Nos 4,946,778, 5,260,203, 5,455,030 and 5,856,456. Generally, the Fv polypeptide still comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, volume 113 ed. Rosenburg and Moore (Springer-Verlag, New York) pages 269315. The complex of FV fragments of the VH and VL domain can also be stabilized by a disulfide bond (US Patent No. 5,747,654).
By absent or minimal effector function, it means that certain activities normally attributable to IgG1 antibodies, such as complement fixation or stimulation of antibody-dependent cell mediated cytotoxicity (ADCC), are reduced or eliminated.
The term isolated, as used herein, refers to an antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein removed from its native environment. Accordingly, an antibody, immunoglobulin construct or fusion protein produced by a recombinant host is considered to be isolated for the purposes of the present invention. Preferably, the
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34/103 antibody, immunoglobulin construct, or isolated IgG4 immunoglobulin fusion protein is substantially purified.
By substantially purified ”means that the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived, or is substantially free of chemical precursors or other substances when synthesized chemically. The term includes preparations of an antibody, immunoglobulin construct or fusion protein that is separated from cellular components of the cells from which it is isolated or recombinantly produced. Accordingly, an antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein that is substantially free of cellular material includes preparations that contain less than about 30%, 20%, 10% or 5% (by dry weight) of proteins and contaminating culture medium.
The term IgG4 immunoglobulin fusion protein ”refers to a bioactive molecule that is linked to or attached to a modified human IgG4 hinge region and modified human IgG4 Fc region and / or FcRn binding domain thereof. Fusion proteins will be discussed in more detail later.
The term in vivo half-life ”, as used herein, refers to a circulating half-life of a particular IgG4 antibody, immunoglobulin construction or immunoglobulin fusion protein that contains an Fc region and / or FcRn binding domain thereof. in the circulation of
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35/103 certain animal and is represented by the time necessary for half the amount administered to the animal to be cleared from circulation. When a clearance curve for a certain antibody, immunoglobulin construction or IgG4 immunoglobulin fusion protein according to the invention is constructed as a function of time, the curve is normally biphasic, with a rapid alpha phase that represents a balance of IgG molecules injected between the intra and extravascular spaces and that is, in part, determined by the size of the molecules, and a longer beta phase that represents the catabolism of the IgG molecules in the intravascular space. The term "in vivo half-life" practically corresponds to the half-life of immunoglobulins or IgG4 fusion proteins modified or unmodified in the beta phase.
The term "increased in vivo half-life", as used herein, means that the antibody, immunoglobulin construct or immunoglobulin fusion protein modified according to the invention has a longer persistence in serum or plasma and / or takes a period longer time to halve the maximum serum or plasma concentration measured in relation to the same antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein that does not contain the same substitutions.
The term "recombinant" should be understood as meaning the product of artificial genetic recombination. Consequently, in the context of a recombinant protein that comprises an antibody antigen binding domain, that term does not encompass an antibody that occurs naturally within an individual's body which is the natural recombination product that occurs during
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36/103 B cell maturation. However, if such an antibody is isolated, it should be considered an isolated protein that comprises an antibody variable region. Similarly, if a nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein that comprises an antigen binding domain of the antibody. The term recombinant also encompasses an antibody, immunoglobulin or fusion protein expressed by artificial recombinant means when it is within a cell, tissue or individual, for example, in which it is expressed.
The term specifically bind refers to a molecule (eg, antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein) that specifically or preferentially binds to an antigen (eg, epitope or immune complex) and does not specifically bind (that is, cross-reacts with) antigens such as, for example, other structurally or functionally related proteins, or proteins with sequence homology. A molecule that specifically binds to an antigen can bind to other peptides or polypeptides with lesser affinity, as determined, for example, by immunoassays, BIAcore, or other assays known in the art. Preferably, molecules that specifically bind to an antigen do not cross-react with other proteins. Molecules that bind specifically to an antigen can be identified, for example, by immunoassays, BIAcore, or other methodologies known to those skilled in the art. Just as a non-limiting example, an antibody can be considered
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37/103 as binding to an antigen preferably if it binds to that antigen with a dissociation constant (KD) that is less than the KD of the antibody to another antigen. In another non-limiting example, an antibody can be considered to bind preferentially to a first antigen if it binds to said first antigen with an affinity that is at least an order of magnitude less than the KD of the antibody to the second antigen. In another non-limiting embodiment, an antibody can be considered to bind preferentially to a first antigen if it binds to said first antigen with an affinity that is at least two orders of magnitude less than the KD of the antibody to the second antigen.
The term "treating" or "treating", as used herein, refers to the administration of a "therapeutically effective amount" of the antibody, immunoglobulin construction or fusion protein according to the invention sufficient to reduce or eliminate at least one symptom of a specified disease or condition.
The term "avoid" or "avoid", as used herein, refers to the administration of a therapeutically effective amount of an antibody, immunoglobulin construction or IgG4 immunoglobulin fusion protein sufficient to stop or halt the development of a disorder or condition specified.
As used herein, the term "therapeutically effective amount" means a sufficient amount of an antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein to reduce or inhibit one or
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38/103 more symptoms of a clinical disease to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that disease. Those skilled in the art will be aware that this amount will vary depending, for example, on the specific antibody (or antibodies), immunoglobulin construct (or constructs) and / or IgG4 immunoglobulin fusion protein (or proteins) administered and / or the particular individual and / or the type or severity or level of illness. Consequently, that term should not be considered as limiting the invention to a specific amount, for example, weight or amount, and instead the present invention encompasses any amount of the antibody (antibodies), immunoglobulin construct (or constructs) and / or sufficient IgG4 immunoglobulin fusion protein (proteins) to obtain the result established in an individual.
The term pharmaceutically acceptable ", as used herein, means approved by a regulatory agency of the federal or state government or listed in the US Pharmacopeia" or in another pharmacopoeia generally recognized for use in humans.
As used herein, the term "individual" means a human or non-human primate or non-primate mammal with a human FcRn.
Amino acid substitutions
Amino acid substitution methods are known in the art. For example, amino acid substitutions can be made by site-directed mutagenesis (for example, Zoller and Smith Nucl. Acids Res. 10: 6,487 (1982)). THE
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39/103 mutagenesis can be performed by synthesis of an oligonucleotide that has one or more modifications within the sequence of the constant domain of an antibody to be modified. Site-directed mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent oligonucleotides to provide a primer of sufficient length and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered. Several of these primers that introduce several different mutations at one or more positions can be used to generate a library of mutants.
The methodology of site-directed mutagenesis is well known in the art, (see, for example, Kunkel et al., Methods Enzymol., 154: 367-82, 1987). In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector or by melting two strands of a double-stranded vector that includes a DNA sequence encoding the desired peptide within its sequence. An oligonucleotide primer that houses the desired mutated sequence is prepared, usually synthetically. This primer is then annealed with the single ribbon vector and subjected to the polymerization enzymes of
DNA, such as T7 DNA polymerase, in order to
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40/103 complete the synthesis of the ribbon that houses the mutation. In this way, a heteroduplex is formed in which one strand encodes the original unmuted sequence and the second strand houses the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, for example, E. call cells, and clones are selected that include recombinant vectors that house the mutated sequence arrangement. As will be noted, the technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as, for example, the M13 phage. These phages are readily available commercially and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from a plasmid to a phage. Site-directed mutagenesis has also been used to identify amino acid residues that influence plasma clearance of murine IgG1 Fc hinge fragments, as described in Kim Jin-Kyoo et al., (1994) Eur. J. Immunol. 24: 542-548).
Alternatively, PCR with commercially available thermostable enzymes such as, for example, Taq DNA polymerase, can be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. See, for example, Tomic et al., Nucleic Acids Res., 18 (6): 1,656, 1987, and Upender et al., Biotechniques, 18 (1): 29-30, 32, 1995,
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41/103 for PCR-mediated mutagenesis procedures. PCR using a thermostable ligase in addition to a thermostable polymerase can also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector (see, for example, Michael , Biotechniques, 16 (3): 410-2, 1994).
Other methods known to those skilled in the art of producing sequence variants of the Fc region of an antibody or an FcRn binding domain thereof can be used. For example, recombinant vectors that encode the amino acid sequence of the constant domain of an antibody or a fragment thereof can be treated with mutagens, for example, hydroxylamine, to obtain sequence variants.
Mutants that result in increased FcRn affinity and increased in vivo half-life can be selected using routine assays such as those described later.
Exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to Kabat's EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.
Antibodies of the invention
The antibody or immunoglobulin according to the invention includes any immunoglobulin molecule or antibody that binds (as determined by immunoassays known in the art for the evaluation of specific antigen binding
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42/103 antibody) to an antigen and contains an Fc region or FcRn binding domain. Antibodies can be polyclonal, monoclonal or monospecific, bispecific (in the context of multimeric forms of the antibody), human, humanized, chimeric, Super-Humanized®, primatized or de-immunized. In another example, the antibodies of the present invention can be monospecific (or bispecific, triespecific or of greater multispecificity, if present in a multimeric form).
In particular, the antibody is a monospecific tetramer.
The antibody (and other immunoglobulin or fusion protein described herein) can be of any animal origin. Preferably, the antibody is human or humanized. As used herein, the term human antibody includes antibodies that have the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic to one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described, for example. for example, in US 5,939,598.
The antibodies of the invention comprise a stabilized IgG4 hinge region. The term stabilized IgG4 hinge region will be understood to mean an IgG4 hinge region that has been modified to reduce the Fab arm exchange or the propensity to undergo Fab arm exchange or the formation of a half-antibody or a propensity to form a half-antibody. The term Fab arm switch refers to a type of protein modification for human IgG4, in which
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43/103 an IgG4 heavy chain and attached light chain (meiamolecule) is exchanged for a heavy-light chain pair from another IgG4 molecule. In this way, IgG4 molecules can acquire two distinct Fab arms that recognize two distinct antigens (which result in bispecific molecules). The Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as, for example, reduced glutathione. A half-antibody forms when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.
The stabilized IgG4 hinge region comprises a substitution of serine for proline at position 228 according to the EU numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969), which corresponds to a substitution of serine for proline in position 241 according to the Kabat numbering system (Kabat et al. , Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). For the avoidance of doubt, this refers to the serine at the center of the IgG4 CPSCP hinge region sequence (SEQ ID. NO: 1). After replacing serine for proline, the IgG4 hinge region comprises a CPPCP sequence. In this regard, those skilled in the art will be aware that the hinge region is a proline rich portion of a region
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44/103 antibody heavy chain that links the Fc and Fab regions that gives mobility to the two Fab arms of an antibody.
In one example, the antibody of the invention can be in a multimeric form. For example, the antibody can take the form of a dimer, antibody trimer, or higher order multimer of immunomeric monomer molecules. Dimers of whole immunoglobulin molecules or F (ab ') 2 fragments are tetravalent, while dimers of Fab fragments or scFv molecules are divalent. The individual monomers within an antibody multimer can be identical or different, that is, they can be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer can have the same or different binding specificities.
The multimerization of antibodies can be achieved through natural antibody aggregation or through chemical or recombinant binding methodologies known to those skilled in the art. For example, a percentage of purified antibody preparations spontaneously form protein clusters that contain antibody homodimers and other higher order antibody multimers. Alternatively, homodimers of antibodies can be formed using chemical binding methodologies known to those skilled in the art. As a non-limiting example, heterobifunctional cross-linking agents that include, without limitation, SMCC [succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acetylthioacetate] (available, for example, from Pierce Biotechnology , Inc. (Rockford, 111))
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45/103 can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie M.A. et al. Antibody homodimers can be converted to F (ab ') 2 homodimers by digestion with pepsin. Another way to form homodimers of antibodies is through the use of the autophilic peptide TI 5 described in Zhao Y & Kohler H., J. Immunother. (1997) 25 (5): 396-404.
Alternatively, antibodies can be made to undergo multimerization naturally or using recombinant DNA techniques. ScFv dimers can also be formed using recombinant methodologies known in the art; an example of the construction of scFv dimers is given in Goel A. et al. Cancer Research 60 (24): 6,9646,971. Multimers of antibodies can be purified by any suitable method known in the art, for example, size exclusion chromatography.
Antibody derivatives
The present invention also provides antibodies that comprise or, alternatively, consist of variants (including derivatives) of the antibody molecules (for example, the VH domains and / or VL domains) described herein, and those antibodies specifically bind to antigenic peptides (for example, example, the IL-5 antigen or the CD33 antigen). Standardized methodologies known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions.
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46/103
Antibody derivatives according to the invention also encompass conservative amino acid substitutions in the immunoglobulin V1 and / or Vh region. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue that has a side chain with a similar charge. Families of amino acid residues that have side chains with similar charges have been defined in the technique. 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, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and side chains aromatic (eg tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly over all or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be evaluated for biological activity to identify mutants that retain activity (for example, the ability to bind to the antigenic peptides of the invention (for example, the ability to bind to the antigenic peptides of the invention).
The term "conservative substitution" means amino acid substitutions shown in Table 1.
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47/103
Table 1 - Exemplary substitutions.
Original waste Exemplary replacements Wing (A) val; read; ile; gly Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) to be Gln (Q) asn; his Glu (E) asp Gly (G) pro; Allah His (H) asn; gln Ile (I) read; val; Allah Leu (L) ile; val; met; Allah; phe Lys (K) arg Met (M) read; phe Phe (F) read; val; Allah Pro (P) gly Being (S) thr Thr (T) to be Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; read; met; phe; Allah
For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. The introduced mutations can be 5 mutations with silent or neutral sense changes, that is, they have little or no effect on the antibody's ability to bind to the antigen. These types of mutations can be useful to optimize the use of codons, or
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48/103 to increase antibody production by a cell line.
Alternatively, non-neutral sense-switching mutations can alter an antibody's ability to bind to the antigen. Those skilled in the art would be able to design and test mutant molecules with desired properties, for example, no change in antigen binding activity or any change in binding activity (for example, improvements in antigen binding activity or change in the specificity of antibody). After mutagenesis, the encoded protein can be expressed routinely and the functional and / or biological activity of the encoded protein (eg, ability to specifically bind to the antigenic peptides of the invention) can be determined using techniques described herein or by routine modification methodologies known in the art.
The antibodies of the invention include derivatives that are otherwise modified by covalent adhesion of any type of molecule to the antibody, such that covalent adhesion does not prevent the antibody from binding to the antigen. For example, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to a cell linker or other protein, etc. Any of several chemical modifications can be performed by methodologies known in the art, including specific chemical cleavage, acetylation, formylation, metabolic synthesis of
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49/103 tunicamycin etc. In addition, the derivative may contain one or more non-classical amino acids.
In addition, the antibodies of the invention can be chemically synthesized. For example, a peptide that corresponds to a portion of a protein can be synthesized using a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as substitutions and / or additionals following one, either, both, several or all of the complex polypeptides.
Non-classic amino acids include, without limitation, the D-isomers of common amino acids, fluorine-amino acids, planned amino acids such as, for example, beta-methyl amino acids, C gamma-methyl amino acids, N gamma-methyl amino acids and amino acid analogs in general.
The present invention also provides immunoconjugates that comprise an antibody or immunoglobulin construct of the present invention conjugated to a distinct portion, for example, a therapeutic agent, which is directly or indirectly bound to the antibody. Examples of other portions include, without limitation, a cytotoxin, a radioisotope (e.g., iodine-131, yttrium-90 or indium-111), an immunomodulatory agent, an antiangiogenic agent, an antineovascularization agent and / or other vascularizing agent, a toxin, an antiproliferative agent, a pro-apoptotic agent, a chemotherapeutic agent and a therapeutic nucleic acid.
A cytotoxin includes any agent that is harmful (for example, mate) to cells. For a description of these classes of drugs that are known in the
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50/103 art, and their mechanisms of action, see Goodman et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics", 8th Edition, Macmillan Publishing Co., 1990. Additional techniques relevant to the preparation of antibody immunotoxins are provided, for example, in Vitetta (1993) and US 5,194,594. Exemplary toxins include diphtheria A chain, active non-binding fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantina proteins, proteins of American Phytolaca (PAPI, PAPII, and PAP-S), inhibitor of Momordica charantia, curcine, crotine, inhibitor of Saponaria officinalis, gelonin, mitogiline, restrictocine, phenomycin, enomycin and trichothecenes. See, for example, WO 93/21232.
Therapeutic agents suitable for the formation of immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracine dione, mitoxantrone, mitoxantrone D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, antimetabolites (for example, methotrexate, 6mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5fluoruracil, decarbazine, hydroquinone, hydroxyabine, hydroxyabine, hydroxyabine, hydroxyabine, (for example, mecloretamine, thiope, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, dacarbazine (DTIC),
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51/103 procarbazine, mitomycin C, cisplatin and other platinum derivatives, eg carboplatin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)).
Several radionuclides are available for the production of radioconjugate antibodies. Examples include,
without limitation, ²¹² Bi, ¹³¹ I, ⁹⁰ Y and ¹⁸⁶ Re. Conjugated of antibody and agents therapeutic are made with use of several agents bifunctional in
protein coupling such as, without limitation, 4- (4 ^, acetylphenoxy) butanoic acid (AcBut), 3acetylphenyl acidic acid (AcPac), 4-mercapto-4-methylpentanoic acid (Amide), N-succinimidyl-3- ( 2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (for example, dimethyl adipimidate HCl), active esters (for example, disuccinimidyl suberate), aldehydes (for example, glutaraldehyde), bis-azide compounds ( bis (pazidobenzoyl) hexanediamine), bis-diazonium derivatives (eg, bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (eg, toluene 2,6-diisocyanate) and fluorine bis-active compounds ( for example, 1,5-difluoro2,4-dinitrobenzene), and derivatives thereof. For example, a ricin immunotoxin can be prepared as described by Vitetta et al. (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3methyldiethylene triaminapentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radionucleotide to the antibody (WO 94/1 1026).
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Immunoglobulin constructs of the invention
As used herein, the term "immunoglobulin construct" is intended to refer to constructs in which an antibody antigen binding fragment is linked to a modified human IgG4 hinge region and to a modified human IgG4 Fc region or binding domain. of FcRn thereof according to the present invention. Of particular interest are immunoglobulin constructs that comprise Fc regions, Fc fusions and the heavy chain constant region (CH1-hinge-CH2-CH3).
Specific antibody fragments include, without
limitation , i) the fragment Fab that consists in domains VL, VH, CL and CH1; (ii) The Fd fragment what consists of Domains VH and CH1, (iii The Fv fragment what consists of Domains VL and VH of a only co antibody; (iv) the fragment
dAb (Ward et al., (1989) Nature 341: 544-546) which consists of a single variable region; (v) isolated CDR regions;
(vi) F (ab ') 2 fragments, a divalent fragment comprising two linked Fab fragments; (vii) single-chain Fv (scFv) molecules, in which a VH domain and a VL domain are linked by a linker peptide, which allows the two domains to join to form an antigen binding site (Bird et al. , (1988) Science 242: 423426, Huston et al., (1989) Proc. Natl. Acad. Sci. USA 85:
5,879-5,883); (viii) Bispecific single chain Fv (WO 03/11 161); and (ix) diabodies and triabodies or tetrabodies (Tomlinson et al., (2000) Methods Enzymol. 326: 461-479;
WO 94/13804; Hollinger et al., (1993) Proc. Natl. Acad.
Sci. USA 90: 6.444-6.448). The molecules can be stabilized by incorporating disulfide bridges that
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53/103 link the VH and VL domains (Reiter et al, (1996) Nature
Biotech. 14: 1,239-1,245).
It will be noted that the fragments described above (which do not contain a hinge region) can be joined to a hinge-Fc region, where the hinge serves as a linker.
In another example, the antibody fragment may be a flex minibody consisting of scFV-CH3 and hinge region sequence (as described in Hu, Shi-zhen et al., (1996) Cancer Research 56: 3.055-3.061).
Preparation of antibodies
Antibodies can be prepared using a wide variety of methodologies known in the art, including the use of hybridoma, recombinant and phage display technologies, or a combination of these. For example, monoclonal antibodies can be produced using hybridoma methodologies, including those known in the art and taught, for example, by Harlow et al., “Antibodies: A laboratory Manual” (Cold Spring Harbor
Laboratory Press, 2nd edition 1988).
The term "monoclonal antibody", as used herein, is not limited to antibodies produced using hybridoma technology. The term refers to any antibody that is derived from a single clone, including any prokaryotic, eukaryotic or phage clone, and not the method by which it is produced.
Methods for producing and evaluating specific antibodies using hybridoma technology are routine in the art. For example, mice can be immunized with an antigen of interest or a cell that expresses
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54/103 this antigen. After detecting an immune response, the spleen of the mouse is collected and the splenocytes isolated. The splenocytes are then fused to myeloma cells. Hybridomas are selected and cloned by limiting dilution. The clones are then tested by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascitic fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.
Antibody fragments that recognize specific epitopes can be generated by routine techniques. For example, Fab and F (ab ') 2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab') 2 fragments)) . F (ab ') 2 fragments contain the complete light chain, and the variable region, the CH1 region and the hinge region of the heavy chain.
Antibodies can also be generated using a variety of phage display methods. In phage display methods, functional domains of the antibody are displayed on the surface of phage particles that carry the polynucleotide sequences that encode them. In a particular embodiment, this phage can be used to exhibit antigen binding domains, for example, Fab and Fv or Fv disulfide stabilized, expressed by a combinatorial antibody repertoire or library (for example, human or murine). Phages that express an antigen binding domain that binds to the antigen of
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55/103 interest can be selected or identified with antigen, for example, using labeled antigen or antigen bound or captured on a solid surface or globule. The phages used in these methods are typically filamentous phages, including fd and M13. The antigen binding domains are expressed as a protein recombinantly fused to the phage III or VIII gene protein. Alternatively, an immunoglobulin-modified FcRn binding portion of the present invention can also be expressed in a phage display system. Examples of phage display methods that can be used to produce the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkman et al, J. Immunol. Methods, 182: 41-50, 1995; Ames et al, J. Immunol. Methods, 184: 177-186, 1995; Kettleborough et al, Eur. J. Immunol., 24: 952-958, 1994; Persic et al, Gene, 187: 9-18, 1997; Burton et al, Advances in Immunology, 57: 191-280, 1994; PCT Application No. PCT / GB91 / 01134; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401; and US Patents ^Nos 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,
5,571,698, 5,427. 908, 5,516 .637, 5,780,225, 5,658,727, 5,733,743 and 5,969. 108. After selection of phage, coding regions in antibody of the phage can be isolated and used to generate antibodies whole, including human antibodies, or any others fragments desired, and expressed in
any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria.
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For example, techniques can also be employed to recombinantly produce Fab, Fab 'and F (ab') 2 fragments using methods known in the art such as, for example, those disclosed in PCT Publication WO 92/22324; Mullinax et al., BioTechniques, 12 (6): 864-869, 1992; and
Sawai et al, AJRI, 34: 26-34, 1995; and Better et al.,
Science, 240: 1.041-1.043, 1988; Examples of techniques that can be used to produce single - chain Fvs and antibodies include those described in US Patents ^Nos 4,946,778 and 5,258,498; Huston et al, Methods in Enzymology, 203: 46-88, 1991; Shu et al, PNAS, 90: 7,9957,999, 1993; and Skerra et al., Science, 240: 1,038-1,040,
1988.
Recombinant production of antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins
The antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins of the present invention can be produced recombinantly. For example, DNA encoding an antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein of the invention is easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that are able to specifically bind to the genes encoding heavy and light chains of antibodies). A hybridoma cell serves as a preferred source of that DNA for antibodies. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, COS simian cells, Chinese hamster ovary (CHO) cells or cells from
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57/103 myeloma that otherwise do not produce antibody protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al, Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs.,
730: 151-188 (1992). Molecular cloning methodologies to achieve these objectives are known in the art. A wide variety of in vitro cloning and amplification methods are suitable for the construction of recombinant nucleic acids. Examples of these techniques and sufficient instructions to direct those skilled in the technique through many cloning exercises are found in Berger and Kimmel, “Guide to Molecular Cloning Techniques”, “Methods in Enzymology”, volume 152, Academic Press, Inc., San Diego, California (Berger); Sambrook et al. (1989) "Molecular Cloning : A Laboratory Manual" (2nd Edition), Volume 13, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and “Current Protocols in Molecular Biology”, FM Ausubel et al., eds., “Current Protocols”, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (Supplement 1994) (Ausubel). Methods of producing recombinant immunoglobulins are also known in the art. See, Cabilly, US Patent No. 4,816,567; and Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10.029-10.033.
For recombinant production, the nucleic acid encoding the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein is preferably isolated and inserted into a replicable vector for cloning
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58/103 posterior (amplification of DNA) or for expression. DNA encoding the antibody or fusion protein is easily isolated or synthesized using conventional procedures (for example, by using oligonucleotide probes that are able to specifically bind to the DNAs that encode the antibody's heavy and light chains). Many vectors are available. The components of the vector generally include, without limitation, one or more of the following: a signal sequence, a sequence encoding an antibody of the present invention or a fragment thereof (for example, derived from the information provided herein), an enhancer element, a promoter and the transcription termination sequence.
(i) Signal sequence component. The antibody of this invention can be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide that has a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the leader group of alkaline phosphatase, penicillinase, Ipp or thermostable enterotoxin II. For yeast secretion, the native signal sequence can be replaced, for example, by
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59/103 yeast invertase leader, α factor leader or acid phosphatase leader, C. albicans glycoamylase leader or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences are available, as well as viral secretory leaders, for example, the herpes simplex gD signal. The DNA for this precursor region is linked in a reading frame to the DNA encoding the antibody.
(ii) Promoter component. Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operationally linked to the antibody's nucleic acid. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody.
Promoters for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases above the site where transcription is initiated. Another sequence found 70 to 80 bases above the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence that can be the signal for adding the poly A tail to the 3' end of the coding sequence. All of these strings are
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60/103 properly inserted into eukaryotic expression vectors. Examples of promoter sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, for example, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isase 3 phosphoglycerate mutase, pyruvate kinase, triosphosphate isomerase, phosphoglycosis isomerase and glycokinase. Other yeast promoters, which are inducible promoters that have the added advantage of transcription controlled by growing conditions, are the promoter regions for alcohol dehydrogenase 2, iso-cytochrome C, acid phosphatase, degradation enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose. Vectors and promoters suitable for use in yeast expression are further described in EP 73,657. Yeast enhancers are also used advantageously with yeast promoters.
The transcription of antibody by vectors in mammalian host cells is controlled, for example, by promoters obtained from virus genomes such as, for example, the polyoma virus, avian yaws virus, adenovirus (eg Adenovirus 2), CMV, virus bovine papilloma, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and, more preferably, simian virus 40 (SV40), by heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, by heat shock promoters, provided that those promoters
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61/103 are compatible with the host cell systems.
(iii) Intensifying element component. Transcription of a DNA encoding the antibody of that invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known for mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, an eukaryotic cell virus enhancer will be used. Examples include the SV40 enhancer on the late side of the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication and adenovirus enhancers. See also Yaniv (1982) Nature 297: 17-18 on enhancement elements for activation of eukaryotic promoters. The enhancer can be joined to the vector at a position 5 'or 3' to the antibody coding sequence, but is preferably located at a site 5 'to the promoter.
(iv) Transcription termination component. The expression vectors used in eukaryotic host cells (yeasts, fungi, insects, plants, animals, humans or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and stabilization of mRNA. These sequences are commonly available through the 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the
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62/103 untranslated portion of the mRNA encoding the antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/1 1026 and the expression vector disclosed therein.
(v) Selection and transformation of host cells. Host cells suitable for cloning or expressing DNA in the vectors presented here are the prokaryotic, yeast or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, for example, Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, for example, Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example , Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, in addition to bacilli such as, for example, B. subtilis and B. licheniformis, Pseudomonas, for example, P. aeruginosa, and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X 1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are adequate. These examples are illustrative, not limiting.
In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for vectors that encode antibodies. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, several other genera, species and strains are commonly available and useful in this application, for example,
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Schizosaccharomyces pombe; hosts of Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;
Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as, for example, Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as, for example, A. nidulans and A. niger.
Host cells suitable for glycosylated antibody expression are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Several strains and baculoviral variants and corresponding permissive insect host cells from hosts such as, for example, Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. Several viral strains for transfection are publicly available, for example, the LI variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses can be used here as the virus according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
Examples of useful mammalian host cell lines are the monkey kidney line CVI transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lineage (293 or 293 cells subcloned for growth in suspension culture, Graham et al (1977)
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Gen. Virol. 36:59); hamster cub kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO, Urlaub et al (1980) Proc. Natl. Acad. Sci USA 77: 4,216); mouse Sertoli cells (TM4, Mather (1980) Biol. Repro ^ d. 23: 243-251); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al (1982) Annals N.Y. Acad. Sci. 383: 44-68);
MRC 5 cells; FS4 cells; and PER.C6 ™ (Crucell NV).
The host cells are transformed with the expression or cloning vectors described above for the production of IgG4 immunoglobulin antibody or fusion protein and cultured in conventional nutrient media modified accordingly to induce promoters, select transformants or amplify the genes that encode the desired strings.
(vii) Cultivation of host cells. The host cells used to produce the antibody of this invention can be grown in a variety of media. Commercially available media such as Ham FI0 medium (Sigma), minimal essential medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's modified Eagle medium ((DMEM), Sigma) are suitable for culturing host cells, in addition, any of the media described in Ham et al (1979) Meth. Enz. 58: 44, Barnes et al (1980) Anal. Biochem.
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102: 255, U.S. Patent ^Nos 4,767,704; 4,657,866;
4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO
87/00195; or U.S. Patent Re. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as necessary with hormones and / or other growth factors (eg, insulin, transferrin or epidermal growth factor), salts (eg, sodium chloride, calcium, magnesium and phosphate), buffers (for example, HEPES), nucleotides (for example, adenosine and thymidine), antibiotics (for example, the drug GENTAMICINA ™), microelements (defined as inorganic compounds normally present in final concentrations in the micromolar range) and glucose or an energy source equivalent. Any other necessary supplements can also be included in appropriate concentrations that would be known to those skilled in the art. Culture conditions, for example, temperature, pH and the like, are those previously used with the host cell selected for expression, and will be evident to those skilled in the art.
Chimeric antibodies
The antibody according to the invention can be a chimeric antibody. Chimeric antibodies are made by recombinant means by combining the variable regions of the light and heavy chain (VL and VH) obtained from antibody-producing cells of one species with the constant regions of the light and heavy chain of another. Typically, chimeric antibodies use variable regions of rodent or rabbit and human constant regions in order to produce an antibody with predominantly human domains. Per
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66/103 example, a chimeric antibody comprises a variable region of a mouse antibody fused to a human constant region. The production of these chimeric antibodies is known in the art, and can be achieved by standard means (as described, for example, in Morrison, Science 229: 1,202 (1985); Oi et al., BioTechniques 4: 214 (1986);
Gillies et al., (1989) J. Immunol. Methods 725: 191-202;
US Patents ^Nos 5,807,715, 4,816,567 and 4,816,397).
Primatized antibody
The term "primatized antibody" refers to an antibody that comprises monkey variable regions and human constant regions. Methods for producing primatized antibodies are known in the art. See, e.g., US Patent ^Nos 5,658,570, 5,681,722 and
5,693,780.
Humanized and human antibodies
Included within the scope of the invention deimmunized antibodies that have sequence variations produced using methods described in , for example, in Patent Publications ^Nos EP 0983303, WO 00/34317 and WO
98/52976.
The term "human" antibodies includes antibodies that have the amino acid sequence of a human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or from transgenic animals to one or more human immunoglobulins as described, for example, in US Patent No. 5,939 .598. Human antibodies can be made by a variety of methods known in the art, including phage display using libraries of antibodies derived from human immunoglobulin sequences. Look
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67/103 also WO 98/46645, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741.
The antibodies of the present invention can be humanized antibodies. Humanized forms of non-human antibodies (eg, murines) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ', F (ab') 2 or other antibody antigen binding subsequences) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibody) in which residues from a recipient complementarity-determining region (CDR) are replaced by residues from a CDR of a non-human species (donor antibody) such as, for example, mouse, rat or rabbit , which have the desired specificity, affinity and capacity. In some cases, one or more residues
Human immunoglobulin frameworks are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, where all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a consensus sequence of human immunoglobulin. The optimally humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin
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68/103 (Jones et al (1986) Nature, 321: 522-525; Riechmann et al (1988) Nature, 332: 323-329; and Presta (1992) Curr.
Op. Struct. Biol., 2: 593-59).
Methods for humanizing non-human antibodies can be carried out basically according to the method of Winter et al. (Jones P.T. et al (1986) Nature 321 (6069): 522; Riechmann L et al (1988) Nature
332 (6162): 323-327; Verhoeyen M. et al. (1988) Science 239 (4847): 1.534-1.536. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. Such non-human amino acid residues are often called imported residues, which are typically collected from an imported variable domain. Consequently, these humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some RF residues are replaced by residues from analogous sites in rodent antibodies.
Antibodies can be humanized using a variety of methodologies known in the art, including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; US Patent ^Nos 5,225,539, 5,530,101 and 5,585,089), varnishing (veneering) or reconditioning (EP 592106; EP519596; Padlan EA et al (1991) Mol. Immunol. 28 (4-5): 489; Studnicka GM et al (1994) Protein Eng. 7 (6): 80514 ) and chain shuffling (US Patent 5,565,332).
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In some cases, residues within the framework regions of one or more variable regions of human immunoglobulin are replaced with corresponding non-human residues (see, for example, US Patent 5,585,089; US Patents 5,693,761, 5,693,762 and 6,180 .370.
The invention also extends to humanized antibodies according to the methods called SuperHumanization® described in US 6,881,557 and 7,732,578. Briefly, these methods for humanized antibodies are based on the selection of framework sequences from the variable region of human antibody genes by comparing canonical CDR structure types to CDR sequences from the variable region of a non-human antibody with canonical CDR structure types. for corresponding CDRs from a human antibody sequence library. Human antibody variable regions that have similar types of canonical CDR structure to non-human CDRs form a subset of human member antibody sequences from which human framework sequences are selected.
Also included within the scope of the invention are "varnished antibodies". The term varnished antibody refers to a selective substitution of residues from the framework region with residues from the human framework region in order to provide a xenogenic molecule that comprises an antigen binding site that substantially retains the entire folding structure native to the framework region. Varnishing techniques are based on the understanding that the ligand-binding characteristics of an antigen-binding site are determined primarily by
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70/103 structure and relative arrangement of heavy and light chain CDR assemblies within the antigen binding surface. Using varnishing techniques, the outer residues (for example, accessible to solvents) of the framework region, which are easily found by the immune system, are selectively replaced with human residues to provide a hybrid molecule that comprises a weakly immunogenic varnished surface, or substantially non-immunogenic.
Human antibodies can also be produced using a variety of methodologies known in the art, including phage display libraries (Hoogenboom and Winter (1991) J. Mol. Biol., 227: 381; Marks et al (1991)
J. Mol. Biol., 222: 581). The techniques of Cole et al
Boerner et al. They are also suitable for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 77 (1985) and Boerner et al. (1991) J. Immunol., 147: 86-95 ).
Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After attack, the production of human antibody is observed, which is very similar to that observed in humans in all aspects, including rearrangement of genes, assembly and repertoire of antibodies. This approach is described, for example, in US Patents ^Nos 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016.
In another embodiment, fully human antibodies are obtained by immunizing transgenic mice. a
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71/103 this type mouse is obtained using XenoMouse ™ technology (Abgenix; Fremont, California) and is disclosed in US Patents ^Nos 6,075,181, 6,091,001 and 6,114,598. Fully human antibodies are expected to minimize immunogenic and allergic responses intrinsic to mouse monoclonal or mouse derivatized antibodies and thereby increase the efficacy and safety of administered antibodies.
Completely human antibodies that recognize a selected epitope can also be generated using a technique called "guided selection". In this approach, a selected non-human monoclonal antibody, for example, a mouse antibody, is used to guide the selection of a completely human antibody that recognizes the same epitope (Jespers et al, Bio / technology 72: 899-903 (1988 )).
Antibodies can also be matured in terms of affinity using known selection and / or mutagenesis methods that are known in the art. Preferred affinity matured antibodies have an affinity that is five times, more preferably 10 times,
further preferably 20 or 30 times bigger than The antibody starting (usually murine humanized or human) from which the antibody matured is prepared. An antibody matured in terms of affinity ” is
one with one or more changes in one or more CDRs of this that result in an increase in the affinity of the antibody for IL-5, compared to a relative antibody that does not have those changes. Marks et al (1991) J. Mol. Biol. 222:
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581-597 describe maturity of affinity by shuffling the VH and VL domains.
Antibody binding
The antibodies of the invention can be evaluated for specific binding by any method known in the art. Immunoassays that can be used include, without limitation, competitive and non-competitive assay systems that use techniques such as, for example, BIAcore analysis, FACS analysis (fluorescence activated cell classifier), immunofluorescence, immunocytochemistry, western blots, radioimmunoassays, ELISA , sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, etc.
IgG4 immunoglobulin fusion proteins
The present invention also provides fusion proteins that comprise a recombinantly fused or chemically conjugated bioactive molecule (including covalent or non-covalent conjugations) to a modified human IgG4 Fc region or FcRn binding domain thereof and sequence of the central IgG4 hinge region modified human form of the invention. The term "fusion protein" is often synonymous with the term "immunoadhesin". Without being bound by theory, mutations of the IgG4 immunoglobulin fusion protein are believed to stabilize the hinge region, and mutations in the Fc region increase affinity for human FcRn. In a particular embodiment, the protein of
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73/103 fusion comprises the amino acid sequence in the ID. SEQ. No. 14, or a variant of this devoid of the C-terminal amino acid (lysine).
The bioactive molecule that is fused can be any polypeptide or synthetic drug known to those skilled in the art. Examples of suitable polypeptides include cytokines, cell adhesion molecules (for example, CTLA4, CD2 and CD28), ligands (for example, TNFalpha, TNF-beta and antiangiogenic factor), receptors and growth factors (for example, PDGF, EGF, NGF and KGF), an enzyme, a chemokine.
The bioactive molecule that can be fused can also be a non-proteinaceous polymer, for example, polyethylene glycol or polypropylene glycol.
Methods for producing the bioactive molecule or IgG4 immunoglobulin fusion proteins of the invention include standard recombinant techniques or synthetic protein techniques, for example, by using an automated protein synthesizer. For example, a nucleic acid molecule encoding the bioactive molecule of the invention can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchoring primers that give rise to complementary protuberances between two consecutive gene fragments that can subsequently be annealed and re-amplified to generate a chimeric gene sequence. In addition, a nucleic acid sequence that encodes a molecule can be cloned into an expression vector that contains the Fc region or domain of
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74/103 FcRn binding in such a way that the molecule is bound in frame to the Fc region or FcRn binding domain of this.
Methods for fusing or conjugating polypeptides to antibody constant regions are known in the art
(see, for example, US 5. 336,603, US 5,622,929, US 5,359,046, US 5,349,053, US 5,447,851, US 5,723,125, US 5,783,181, US 5,908,626, US 5,844,096, US 5,112,946, US
7,955,590).
The nucleotide sequence encoding the bioactive molecule can be obtained, for example, from Genbank, and the nucleotide sequence encoding a constant domain can be determined by analyzing the sequence of mutants produced using the techniques described herein. The nucleotide sequence encoding the fusion protein can be inserted into an appropriate expression vector.
Polynucleotides
The present invention also provides polynucleotides that comprise a nucleotide sequence that encodes the antibody, immunoglobulin construct or modified IgG4 immunoglobulin fusion protein of the invention and polynucleotide sequences that hybridize under high stringency thereto.
Assays for evaluating the half-life of antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins of the invention
The half-life of the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein of the invention can be measured by pharmacokinetic studies (PK) according to the method described by Kim et al, Eur. J. of Immunol.
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24: 542 (1994). According to this method, radiolabeled modified immunoglobulin is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, in 3 minutes up to 72 hours after injection. The clearance curve thus obtained must be biphasic, that is, an alpha phase and a beta phase. For the determination of the in vivo half-life of the modified immunoglobulin or fusion protein of the invention, the beta-phase clearance rate is calculated and compared to that of the antibody, immunoglobulin construct or wild-type or unmodified IgG4 fusion protein.
PK studies such as those described above can be performed in a mouse model of humanized FcRn in which endogenous murine FcRn is knocked out and human FcRn knocked in as described in Petkova S.B. et al., (2006)
International Immunology 18 (12): 1,759-1,769.
It has recently been reported that the increased half-life of the antibody may be correlated with increased in vivo activity (Zalevsky J. et al., (2010) Nature Biotechnology
28 (2): 157-159).
In order to compare the ability of the antibody, immunoglobulin construct or modified IgG4 immunoglobulin fusion protein to bind FcRn with that of wild-type IgG4, the modified IgG4 antibody, immunoglobulin construction or IgG4 immunoglobulin fusion protein comprising modification of the IgG4 hinge region and modifications of the heavy chain constant region and wild-type IgG4 can be radiolabeled and reacted with cells that express FcRn in vitro. The radioactivity of fractions linked to the cell
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76/103 can then be counted and compared. The cells expressing FcRn used in this assay are preferably endothelial cell lines, including mouse lung capillary endothelial cells (B10,
D2.PCE) derived from lungs of mice B10.DBA / 2 and cells endothelial transformed SV40 (SVEC) (Kim and cols., J. Immunol., 40: 457-465 , (1994)) derived in
C3H / HeJ mice. However, other cell types, such as intestinal brush borders isolated from 10 to 14-day-old infant mice, which express a sufficient number of FcRn, can also be used. Alternatively, mammalian cells that express recombinant FcRn of a species of choice can also be used. After counting the radioactivity of the bound fraction of modified immunoglobulin or fusion protein or that of wild-type IgG4, the bound molecules can then be extracted with detergent and the percentage of release per unit number of cells can be calculated and compared.
The affinity of the modified antibody, immunoglobulin construct or IgG4 immunoglobulin fusion by FcRn can be measured by surface plasmon resonance (SPR) measurement using, for example, a BIAcore 2000 (BIAcore, Inc.) as described (Popov et al ., Mol. Immunol., 33: 493-502 (1996); Karlsson et al., J. Immunol.
Methods, 145: 229-240 (1991), which are incorporated by reference). In these methods, FcRn molecules are coupled to a BIAcore sensor chip (eg, Cm5 chip by Pharmacia) and the binding of the modified immunoglobulin or fusion protein to the immobilized FcRn is measured to a certain extent.
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77/103 flow rate to obtain sensorgrams using the BIA 2.1 evaluation software, on the basis of which the on and off rates of the antibody, immunoglobulin construction or FcRn-modified IgG4 immunoglobulin fusion protein can be calculated.
The relative affinities of the antibody, immunoglobulin construct or modified IgG4 immunoglobulin and wild-type IgG4 fusion protein by FcRn can also be measured by a simple competition binding assay. Modified antibody / immunoglobulin construction / immunoglobulin fusion protein IgG4 or unlabeled wild-type IgG4 is added in different amounts to the wells of a 96-well plate in which FcRn is immobilized. A constant amount of radiolabeled wild-type IgG is then added to each well. The percentage of radioactivity of the bound fraction is tabulated against the amount of modified immunoglobulin / fusion protein or unmarked wild-type IgG4 and the relative affinity of the modified antibody / immunoglobulin construction / IgG4 immunoglobulin fusion protein can be calculated from the slope of the curve.
In addition, the affinities of the antibody / immunoglobulin construction / modified IgG4 immunoglobulin fusion protein and wild-type IgG4 by FcRn can also be measured by a saturation study and Scatchard analysis.
The transfer of antibody / immunoglobulin construction / modified IgG4 and wild-type IgG4 fusion protein to FcRn can be
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78/103 measured by an in vitro transfer assay using radiolabeled IgG4 and cells expressing FcRn and comparing the radioactivity on one side of the cell monolayer with that on the other side. Alternatively, this transfer can be measured in vivo by feeding lactating mice 10 to 14 days old with radiolabeled modified antibody / immunoglobulin / immunoglobulin fusion protein and periodically counting the radioactivity in blood samples, which indicates the transfer of IgG4 through the intestine into the circulation (or any other target tissue). To test dose-dependent inhibition of IgG transfer through the intestine, a mixture of labeled and unlabeled IgG4 in a certain proportion is given to the mice, and plasma radioactivity can be measured periodically (Kim et al., Eur. J. of Immunol 24: 542 (1994)).
Pharmaceutical compositions and modes of administration
The antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins of the present invention are useful for parenteral, topical, oral or local administration, aerosol administration or transdermal administration, for prophylactic or therapeutic treatment. The pharmaceutical compositions can be administered in several unit dosage forms, depending on the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recognized that the pharmaceutical compositions of that invention, when administered orally, must be protected from digestion. This is typically achieved by forming a complex of
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79/103 protein with a composition to make it resistant to acidic and enzymatic hydrolysis or packing the protein in a suitably resistant vehicle such as a liposome. Means for protecting digestion proteins are known in the art.
The pharmaceutical compositions of that invention are particularly useful for parenteral administration, for example, intravenous administration or administration into a body cavity or lumen of an organ or joint. Compositions for administration will commonly comprise a solution of the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein of the present invention dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous vehicles can be used, for example, buffered saline and the like. These solutions are sterile and generally free of unwanted matter. Such compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances, as necessary to approach physiological conditions such as, for example, pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight
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80/103 and the like, according to the particular mode of administration selected and the individual's needs.
For example, for parenteral administration, the antibodies in question can be formulated in an injectable unit dosage form (solution, suspension, emulsion) in combination with a pharmaceutically acceptable parenteral vehicle. Examples of these vehicles are water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Non-aqueous vehicles, for example, mixed oils and ethyl oleate, can also be used. Liposomes can also be used as vehicles. Vehicles may contain smaller amounts of additives that increase isotonicity and chemical stability, for example, buffers and preservatives.
The antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins of the present invention can be formulated for parenteral administration, for example, formulated for injection via intravenous, intramuscular, subcutaneous, transdermal or other such routes, including peristaltic and direct instillation into a tumor or disease site (intracavity administration). Typically, these compositions can be prepared as injectables, as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions after adding a liquid before injection can also be prepared; and the preparations can also be emulsified.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions;
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81/103 formulations that include sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to a degree that there is a possibility of introduction into syringes. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, for example, bacteria and fungi.
The compositions can be formulated in a sterile aqueous composition in a neutral or salt form. Solutions of the antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins of the present invention as a free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, for example, hydroxypropylcellulose. Pharmaceutically acceptable salts, include acid addition salts (formed with the free amino groups of the protein), and those that are formed with inorganic acids, such as hydrochloric or phosphoric acid, or organic acids, such as acetic acid, trifluoroacetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as, for example, isopropylamine, trimethylamine, histidine, procaine and the like.
Suitable vehicles include solvents and dispersion media that contain, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and polyethylene
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82/103 liquid glycol, and the like), suitable mixtures thereof, and vegetable oils. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Adequate fluidity can be maintained, for example, by using a coating, for example, lecithin, by maintaining the required particle size, in the case of dispersion, and / or by using surfactants.
Under common conditions of storage and use, all of these preparations may contain a preservative to prevent the growth of microorganisms. The prevention of the action of microorganisms can be carried out by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of injectable compositions can be created by using absorption retarding agents in compositions, for example, aluminum monostearate and gelatin.
Before or by formulation, the antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins of the present invention can be intensively dialysed to remove unwanted small molecular weight molecules, and / or lyophilized for easier formulation in a desired vehicle, where appropriate . Sterile injectable solutions are prepared by incorporating the active ingredients in the desired amount in the appropriate solvent with several of the other ingredients listed above, as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle that contains the basic dispersion medium
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83/103 and the other necessary ingredients among those listed above.
In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques that generate a powder of the active ingredients, plus any desired additional ingredient from a previously sterile-filtered solution of these.
Suitable pharmaceutical compositions according to the invention will generally include an amount of the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein of the present invention mixed with an acceptable pharmaceutical diluent or excipient, for example, a sterile aqueous solution, to generate a range of final concentrations, depending on the intended use. Preparation methods are generally known in the art, as exemplified by "Remington's Pharmaceutical Sciences", 16th Edition Mack Publishing Company, 1980 here
incorporated by reference. Must be observed that The contamination endotoxin should be maintained going minimally in a safe level, for example, less than than 0.5 ng / mg in protein. After formulation, the antibody, construction in
immunoglobulin or IgG4 immunoglobulin fusion protein of the present invention will be administered in a manner compatible with the dosage formulation and in an amount that is therapeutically / prophylactically effective. The formulations are easily administered in various dosage forms, for example, the type of injectable solutions described above, but other pharmaceutically
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84/103 acceptable are also contemplated, for example, tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomal forms and the like. “Slow-release” pharmaceutical capsules or compositions can also be used. Slow release formulations are generally designed to generate a constant drug level over an extended period and can be used to release antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins of the present invention.
In some embodiments, liposomes and / or nanoparticles can also be used with the active ingredients. The formation and use of liposomes are generally known to those skilled in the art. Liposomes can be formed by phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs). MLVs can generally have diameters from 25 nm to 4 pm. formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms, which contain an aqueous solution in the nucleus.Phospholipids can form several structures in addition to liposomes when dispersed in water, depending on the molar ratio of lipid to water. the liposome is the preferred structure. The physical characteristics of the liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes may exhibit low permeability to ionic and polar substances, but at high temperatures they pass
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85/103 by a phase transition that markedly alters its permeability. The phase transition involves a change from an ordered, intensely compacted structure, known as the gel state, to a less ordered, poorly compacted structure, known as the fluid state. This occurs at a characteristic phase transition temperature and results in an increase in permeability to ions, sugars and drugs.
Nanocapsules can generally capture compounds in a stable and reproducible way. To avoid side effects due to the intracellular polymeric overload, these ultrafine particles (with dimensions around 0.1 pm) must be designed with the use of polymers capable of being degraded in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and these particles can be made easily.
International Publication No. WO / 2002/080967 describes compositions and methods for administering aerosolized compositions comprising antibodies for the treatment, for example, of asthma, which are also suitable for administering an antibody of the present invention.
The dosage of the antibody, immunoglobulin construct or IgG4 immunoglobulin fusion protein of the invention can be determined by those skilled in the art. The dosage, however, will depend on the extent to which the in vivo half-life of the modified immunoglobulin or fusion protein has been increased. In addition, the dosage and frequency of administration of antibodies or fusion proteins according to the invention can also be reduced by increasing the
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86/103 uptake and penetration into tissue (for example, into the lungs) by changes such as, for example, lipidation Treatment with the antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins of the invention includes single treatment or a series of treatments. The pharmaceutical composition of the invention can be administered once a week, twice a week, once every two weeks, once a month or once every six weeks.
Use of modified antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins of the invention
The modified antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins in the present invention can be used for various non-therapeutic purposes. They can be used as an affinity purifying agent. They can also be useful in diagnostic assays, for example, in detecting the expression of an antigen of interest in specific cells, tissues or serum. For diagnostic applications, antibodies will typically be labeled with a detectable portion, including radioisotopes, fluorescent labels and various enzyme substrate labels. Antibodies can also be used in any known test method, such as competitive binding assays, direct and indirect sandwich assays and immunoprecipitation assays. Antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion protein can also be used for in vivo diagnostic assays. Generally, antibodies,
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87/103 immunoglobulin constructs and IgG4 immunoglobulin fusion protein are labeled with a radionucleotide, so that the antigen or cell that expresses it can be located using immunoscintigraphy.
Use of anti-IL-5 antibodies in therapy
A general feature in the pathogenesis of asthma and other chronic allergic diseases has been shown to be high numbers of eosinophils, especially in the bronchial mucosa of the lungs. After activation, eosinophils secrete several mediators that are actively involved in the inflammatory response of the airway. In the activation of eosinophils, interleukin 5 (IL-5) plays an important role.
IL-5 is a cytokine found in many mammalian species and, among others, both the human and murine genes for IL-5 have been cloned. The human gene consists of four exons with three introns positioned on chromosome 5 and encodes an N-terminal leader sequence of 134 amino acids. Active IL-5 is a homodimer and the three-dimensional structure of recombinant hIL-5 was determined by X-ray crystallography. The IL-5 receptor is present primarily in eosinophils and is composed of an alpha chain and a beta chain. The alpha chain of the receptor is specific for IL-5 and the beta chain, which ensures high affinity binding and signal transduction, is shared with the heterodimer receptors for IL-3 and GM-CSF.
IL-5 is secreted mainly by completely differentiated Th2 cells, mast cells and eosinophils. It has been shown to act on eosinophils, basophils, lymphocytes
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Cytotoxic T and murine B cells.
IL-5's actions on eosinophils include chemotaxis, increased adhesion to endothelial cells, activation of terminal cell differentiation. In addition, IL-5 has been shown to prevent mature eosinophils from undergoing apoptosis. These findings contributed to the concept that IL-5 is the most important cytokine for differentiating eosinophils.
Although current treatment for asthma involves corticosteroids, it is anticipated that future treatment for asthma, as well as other eosinophil-mediated conditions, will include anti-IL-5 antibodies. Inadequate secretion of cytokines and other effector molecules by eosinophils causes damage and dysfunction to the surrounding tissue. The damage to the final organ that results from infiltration and activation of eosinophils represents a common pathogenic component of several disease states, including atopic diseases and hypereosinophilic syndromes (HES). There is clearly a need for therapies that reduce eosinophil numbers in humans.
Antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins of the invention in the treatment or prevention of disorders
The modified antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins have several therapeutic applications. The modified antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins can be used to treat an individual suffering from, or predisposed to, a disease or disorder, who could benefit from administering the
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89/103 modified antibodies. Conditions that can be treated with antibodies include cancer; inflammatory conditions such as asthma; autoimmune diseases; and viral infections etc.
The cancers that can be treated by the antibodies, immunoglobulin constructs, and IgG4 immunoglobulin fusion proteins described herein include, without limitation, breast cancer, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, colon cancer, colon-rectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, cancer of the vulva, thyroid cancer, liver carcinoma, and various types of cancer of the head and neck.
Autoimmune diseases include, without limitation, Addison's disease, autoimmune diseases of the ear, autoimmune diseases of the eye such as uveitis, autoimmune hepatitis, Crohn's disease, diabetes (Type I), epididymitis, glomerulonephritis, Graves' disease, syndrome de GuillainBarre, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, thyroiditis and thyroiditis.
The present invention also includes methods for the treatment of diseases characterized by eosinophilia. Asthma is a key target for the method of the invention, but also other chronic conditions like, for example, allergy,
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90/103 allergic rhinitis and eosinophilic esophagitis are suitable targets for treatment. Thus, a modality of the method of the invention comprises the treatment and / or prevention and / or amelioration of asthma or other chronic allergic conditions characterized by eosinophilia, which comprises the administration of an anti-IL-5 antibody that infra-regulates the activity of IL-5 to such an extent that the number of eosinophil cells is significantly reduced.
In the present context, a significant reduction in the number of eosinophil cells is at least 20%, compared to the number of eosinophils from the treatment of the established technique, but higher percentages are contemplated, for example, at least 30%, at least 40% , at least 50%, at least 60%, at least 70%, at least 80% and even at least 90%. The reduction can be systemic or, more often, locally, for example, in the lungs.
Eosinophil numbers are determined by methods known in the art, typically using microscopy of an appropriate bronchoalveolar lavage fluid (BAL) sample and counting the number of eosinophil cells manually under a microscope. Alternatively, eosinophil numbers can be counted using flow cytometry capable of distinguishing eosinophils.
The modified anti-CD33 antibodies of the invention are particularly useful in the treatment of cancer, more particularly myeloid leukemia. The present invention also encompasses an anti-CD33 that has been modified according to the invention to increase its half-life, conjugated to calicheamicin (Hamann PR et al., (2002) Bioconj. Chem.
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13 (1): 40-6). The anti-CD33-calicheamicin conjugate can be used to treat acute myeloid leukemia.
Usefulness of non-immunostimulating antibodies
The antibodies, immunoglobulin constructs and IgG4 immunoglobulin fusion proteins of the invention comprise a modified human IgG4 Fc region or FcRn binding domain therefrom and a sequence of the modified central human IgG4 hinge region. It is known in the art that the isotype of the antibody constant domain influences the effector functions of the antibody. Of the various classes of human immunoglobulins, only human IgG1, IgG2, IgG3 and IgM are known to activate complement; and human IgG1 and IgG3 mediate anti-body dependent cell cytotoxicity (ADCC) more effectively than IgG2 and IgG4. As the immunoglobulins and fusion proteins of the present invention comprise sequences of the IgG4 constant region, they are unable to activate the complement cascade or ADCC activity and, therefore, any unwanted NK or T cell activation. Consequently, they are particularly suitable for allergic conditions such as, for example, asthma, in which it is not desirable to trigger the activation of cells that can only exacerbate the condition. IgG4 antibodies functionally differ from other IgG subclasses in their anti-inflammatory activity, which includes a low ability to induce complement and cell activation because of the low affinity for C1q (the q fragment of the first complement component) and Fc gamma receptors. Consequently, IgG4 has become the preferred subclass for immunotherapy, in which recruitment of the host's effective function is undesirable.
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Anti-IL-5 antibodies
The present invention extends to antibodies, immunoglobulin constructs or IgG4 immunoglobulin fusion proteins that comprise sequences of the known IL-5 antibody heavy and light chain variable regions, joined to a modified human IgG4 Fc region and hinge region of human IgG4 modified according to the present invention. Various examples of anti-IL-5 antibodies are described in US 5,683,892, US 5,693,323, US 5,783,184, US 5,851,525, US 6,129,913, US 5,096,071, US 6,056,957 and US 6,451. 982. In addition, humanized anti-IL-5 antibodies CTIL-5-10gH / -gL6 (as described in US RE39,548E), hereinafter referred to as 39D10 or humanized hu39D10) and mepolizumab are particularly suitable for modification according to the present invention.
It will be noted by those skilled in the art that various variations and / or modifications can be made to the invention, as shown in the specific modalities, without departing from the scope of the widely described invention. The present modalities, therefore, should be considered in all aspects as illustrative, and not restrictive.
Synergistic effect of substitutions
The present inventors have found that YTE substitutions (i.e., M252Y, S254T and T256E mutations) in an immunoglobulin or IgG4 antibody Fc region, when combined with the hinge region S228P mutation in an IgG4 antibody, synergistically increase the half-life of the modified IgG4 antibody in vivo. This has been demonstrated for two different antibodies that bind to
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93/103 two different, unrelated antigens, as described in the examples.
In particular, the inventors found that, while YTE substitutions increased the affinity of antibodies modified by human FcRn, the additional inclusion of the S228P modification to the hinge region did not produce additional effects on the antibody's affinity for FcRn. This is not entirely unexpected, considering that this region does not interact with FcRn. Therefore, it would be predicted that there would be no synergy with respect to the S228P substitution and YTE substitutions. Since any modification to a human protein-based drug (including a protein that comprises a human antibody constant region) increases the risk of inducing an anti-drug immune response in a patient, the general practice is to limit the number of these mutations to limit the presumably additively increased risk of each of these mutations with respect to inducing these immune responses against the drug. However, due to the surprising results described here that the combination of the two classes of modifications (Fc modifications and hinge modifications) results in a supra-additive effect on increasing the circulating half-life of IgG4 antibodies, the benefits of The combination of these two classes of mutations may override the theoretical disadvantages in relation to the increased incidence of promoting anti-drug immune reactions. The advantages of increasing the half-life of a molecule will be immediately evident to those skilled in the art. These benefits include reduced dosage and / or frequency of
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94/103 administration, which reduces the risk of adverse events in an individual and reduces costs. Consequently, these immunoglobulins with an increased half-life are of significant pharmaceutical importance.
All references or documents cited herein are considered to be incorporated by reference in their entirety.
Throughout this specification, the word comprise, or variations such as, for example, understand or understand, will implicitly include the inclusion of an element, whole number or step established, or group of elements, whole numbers or steps, but not exclusion any other element, integer or step, or group of elements, integer or step.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification should not be considered as an admission that any or all of these matters form part of the basis of the established technique or that is general common knowledge in the field. relevant to the present invention as if it existed prior to the priority date of each claim in that application.
EXAMPLE 1
Materials and methods
Generation of hu39D10 and its variants
The genes encoding the human IgG4 heavy chain constant region were isolated from the Quickclone cDNA library (Clontech, Mountain View, CA) and cloned into the pTT5 expression vector (Durocher et al., Nucleic Acids Research volume 30, No. 2 , page e9). To enter the
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95/103 mutations described above in the Fc domain, a set of two complementary primers with one or more mutations was synthesized and used for PCR-based site-directed mutagenesis. The Ig kappa expression vector was constructed using a similar means. The DNA fragments encoding the hu39D10 variable regions (Figure 1) were reverse-designated from the published protein sequences (US RE39,548E), using 18 (heavy chain) or 16 (light chain) oligonucleotides by mounting PCR-based gene. The fragments were cloned into the expression vectors using restriction sites integrated into the vectors for cloning. The final amino acid sequence for the hu39D10 heavy and light chains is shown in Figure 1. The figure also shows the hu39D10 sequence with the 4 amino acid substitutions (YTE + S228P, SEQ ID NO: 6).
Expression and purification of hu39D10 and variants
PTT5 expression vectors for hu39D10 and their variants were transfected into HEK293 6E cells according to Durocher et al, Nucleic Acids Research volume 30, No. 2, page e9. After 6 days of transfection, the culture medium was isolated and then subjected to affinity purification using Protein G-agarose globules (GE Healthcare Life Sciences, Piscataway, NJ).
Generation of construçõesοΕη / β2 microglobulin complex expression constructs
The DNA fragments encoding human FcRn and β2 microglobulin were isolated from cDNA synthesized with Universal human RNA (BioChain, Hayward, CA), a pool of total human RNA, using a First-Strip Synthesis kit
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Superscript III (Invitrogen, Carlsbad, CA). The extracellular domain of FcRn (amino acids 24-290) and the mature part of β2 microglobulin (amino acids 21-1 19) were cloned into the pTT5 expression vector individually. The sequences of the extracellular domain of human FcRn and microglobulin
β2 are shown in Figure 2 and in the figure 3, respectively. Expression and purification of complex in FcRn / microglobulin β2 The vectors of pTT5 expression for production of FcRn
and β2 microglobulin were co-transfected into HEK293 6E cells. Six days after transfection, the culture medium was isolated and then subjected to affinity purification using IgG-Sepharose globules (GE Healthcare Life Sciences, Piscataway, NJ).
ELISA to measure the affinity of hu39D10 and variants to the FcRn / β2 microglobulin complex
Maxisorp 96-well plates (Thermo Fisher Scientific,
Rochester, NY) were coated with 5 pg / ml anti-microglobulin β2 monoclonal antibody. The wells were then washed with PBS and treated with Superblock blocking solution (Thermo Fisher Scientific, Rockford, IL). Then, the FcRn / β2 microglobulin complex was diluted to 5 pg / ml in SPBS6T (50 mM sodium phosphate buffer pH 6.0, 150 mM NaCl, 0.05% Tween-20) and added to allow capture by anti-microglobulin β2 antibody coated for 60 min at room temperature. The wells were then washed by SPBS6T and then exposed to hu39D10 or its variants in SPBS6T and incubated for 60 min at room temperature. The hu39D10 / FcRn complex formed
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97/103 by incubation was probed with an F (ab ') 2 fragment of a human anti-kappa conjugated to HRP (Sourthern Biotechnology, Birmingham, AL; dilution of 1 / 5,000 in SPBS6T) for 30 min at room temperature. After washing with SPBS6T, 100 μΐ of TMB (Sigma) was loaded in each well for signal detection. Next, 50 μl of 2 N sulfuric acid was added to stop color development, and then A450 was measured in a Vmax plate reader (Molecular Devices, Sunnyvale, CA). The affinity of IgG variants to FcRn was calculated and tabulated using the Prism software by GraphPad Software (La Jolla, CA).
Results and conclusions
As shown in Figure 4, the affinity of hu39D10 for human FcRn (EC50 = 3.8 nM) was increased by 4.7 times by making YTE mutations (for EC50 = 0.81 nM). The subsequent addition of the S228P mutation had no effect on the affinity for FcRn (EC50 = 0.81 nM, the same as for hu39D10 with YTE mutations). This result was not unexpected, as the S228P mutation is far from the Fc region that interacts with FcRn. Based on this result, no synergy between YTE and S228P mutations over the circulating half-life would be expected.
EXAMPLE 2
PK Study
The mouse PK study was carried out by the Jackson Laboratory - West (Sacramento, CA) with mice that have their endogenous knocked out FcRn, but that have the knocked in human FcRn (the 4919 Tg276 mouse model described in Petkova et al., (2006)
International Immunology vol. 18, N ° 12, pages 1,759 Petition 870190126762, of 12/02/2019, p. 111/122
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1,769). On day 0, seven mice in each group received hu39D10 or its variants intraperitoneally (IP) (200 pg). Each mouse was bled from the retro-orbital sinus at 2, 12, 24 hours and 2, 4, 7, 10, 14, 18, 21 and 28 days to prepare plasma samples.
ELISA to measure hu39D10 and variants in plasma samples
Maxisorp 96-well plates (Thermo Fisher Scientific, Rochester, NY) were coated with recombinant IL-5 (The hu39D10 antigen; R&D Systems, Minneapolis, MN) at 2 pg / ml in PBS overnight in a refrigerator . The wells were then washed with PBS and blocked with 200 µl of Superblock blocking solution (Thermo Fisher Scientific, Rockford, IL) for 30 min to minimize non-specific binding. The plasma samples were then diluted to 1/50 in PBS-Tween 20 (PBST) and loaded into IL-5 coated wells. In addition, a recombinant hu39D10 standard was diluted in PBST with 2% mouse serum (Sigma-Aldrich, St. Louis, MO) and loaded into coated wells to make a standard curve for quantification. After a 60 min incubation at room temperature, the wells were washed with PBST, and then an HRP-conjugated human anti-Fc (Sigma-Aldrich, St. Louis, MO, 1/1000 dilution in PBST) was added and incubated for min at room temperature. The wells were then washed with PBST. For signal detection, 100 pl of TMB (Sigma-Aldrich, St. Louis, MO) was loaded into each well. Next, 50 µl of 2 N sulfuric acid was added to stop color development, and then A450 was measured in a Vmax plate reader
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99/103 (Molecular Devices, Sunnyvale, CA). The concentration of hu39D10 and the variants in the plasma was calculated using the Prism software (GraphPad Software, La Jolla, CA) and using the standard curve of hu39D10. For each mouse, the relative concentration of hu39D10 compared to the concentration measured on Day 1 (defined as 100%) was tabulated as a function of time. The half-life of hu39D10 or its variants in individual animals was also calculated using the software, assuming an exponential drop and an asymptote of zero. For the calculation of the half-life of the variant with combined mutations (S228P + YTE), two results outside the curve (ie the results of two of the mice), which increase the calculated half-life, were excluded for the calculation of the half - average life.
Results and conclusions
As shown in Figure 5, the serum half-life of hu39D10 with the S228P mutation (t1 / 2 = 6.5 days) was increased by 42%, compared with hu39D10 with unmodified Fc (t1 / 2 = 4.6 days) . YTE mutations also show a significant increase in serum half-life by 75% (t1 / 2 = 8.0 days). Surprisingly, when combined, the S228P and YTE mutations further extended the circulating synergistic half-life (up to t1 / 2 = 13.3 days). The addition of the S228P mutation to the YTE mutations increased the circulating half-life by 66% (or 5.3 days) compared to YTE alone, while the increase in the S228P half-life, in the context of non-mutated IgG4 with YTE, resulted in an increase of only 42% (1.9 days). Without the synergy between the S228P and YTE mutations, the S228P mutation would cause the same increase or a proportionately reduced increase in half-life in the context of
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100/103 hu39D10 mutated with YTE, as in the context of hu39D10 not mutated with YTE, generating a half-life of maximum 11 days for hu39D10 with both mutations, YTE and S228P.
Due to this synergy, it was concluded that it is beneficial to combine the YTE and S228P mutations in the same molecule in order to obtain a very long half-life for an antibody-based drug (or Fc fusion protein), despite the increased probability of promotion of anti-drug immune responses as a result of an increased number of mutations in the Fc constant region.
EXAMPLE 3
Materials and methods
Generation of huMab195 and its Fc variants
The DNA fragments encoding the variable regions of the CD33-binding huMab195 antibody (Figure 6), were created in reverse from the published protein sequences (US 5,693,761), using 18 (heavy chain) and 18 (heavy chain) light) oligonucleotides by PCR-based gene assembly. The fragments of the variable region of the heavy and light chain were then cloned into the human IgG4 expression vectors (native and variant) described in the previous section to create an IgG4 / kappa version of huMab195, with native IgG4 constant domain sequence, or versions containing the S228P mutation, the YTE mutations, or both the S228P and YTE mutations. The sequence of the IgG4 heavy chain huMab195 with both the S228P and YTE mutations is shown in Figure 6, as well as the sequence of the light chain.
Expression and purification of huMab195 and variants
The expression vectors pTT5 for huMab195 and their
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101/103 variants were transfected into HEK293 6E cells to produce the various IgG4 proteins, as described in Example 2. These proteins were then purified using Protein G-agarose globules as in Example 2.
Generation of extracellular domain of human CD33
A fragment of DNA encoding the human CD33 extracellular domain (hCD33 ECD, amino acids 1-258, including leader sequence) was amplified from the Quickclone human cDNA library (Clontech, Mountain View, CA). DNA encoding a tag (His) and, followed by a thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser), was added to the 3 'end of the hCD33 ECD fragment by PCR using a primer that carries these sequences. The DNA fragment encoding hCD33 ECD with his6 tag was then ligated to a DNA fragment encoding human IgG1 Fc by PCR (hCD33 ECD-Fc) and cloned into the pTT5 expression vector. The protein sequence of hCD33 ECD-Fc is shown in Figure 7.
Expression and purification of the extracellular domain of human CD33
The pTT5 expression vector encoding the hCD33 ECD-Fc fusion protein was transfected into HEK293 6E cells, and then the culture medium was isolated and subjected to affinity purification using Gagarose Protein globules (GE Healthcare Sciences, Piscataway, NY ). To isolate hCD33 ECD, the purified fusion protein was treated with thrombin (EMD Chemicals, San Diego, CA) to remove the Fc portion. Next, hCD33 ECD was isolated by affinity chromatography on NiNTA-agarose (Qiagen GmbH, Hilden, Germany).
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102/103
PK Study
The mouse PK study was carried out by the Jackson Laboratory - West (Sacramento, CA) with mice that have their endogenous knocked out FcRn, but that have the knocked in human FcRn (the 4919 Tg276 mouse model described in Petkova et al., International
Immunology vol. 18, No. 12, pages 1,759-1,769). On day 0, seven mice in each group received huMab195 or its variants intraperitoneally (IP) (200 pg). At 2, 12 and hours and 2, 4, 7, 10, 14 days after administration, each mouse was bled to prepare plasma samples.
ELISA to measure huMAb195 and variants in plasma samples
Maxisorp 96-well plates (Thermo Fisher Scientific, Rochester, NY) were coated with recombinant hCD33 ECD at 2 pg / ml in PBS solution. The wells were then washed with PBS and blocked with Superblock blocking solution (Thermo Fisher Scientific, Rockford, IL). Plasma samples were diluted to 1/50 in PBS-Tween 20 (PBST), and then loaded into wells coated with hCD33 ECD. In parallel, known concentrations of recombinant huMab195 standards were diluted in PBST with 2% mouse serum (Sigma-Aldrich, St. Louis, MO) and loaded into coated wells to make a standard curve for quantification. After a 60 min incubation at room temperature, the wells were washed with PBST, and then a human antikappa fragment conjugated to HRP (Invitrogen, Carlsbad, CA; 1/2000 dilution in PBST) was added and incubated for 30 min. After washing the wells, the signals were developed, measured and analyzed as described in Example 2.
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Results and conclusions
As shown in Figure 8, the serum half-life of huMab195 with the S228P mutation (t1 / 2 = 2.0 days) was increased by 26%, compared to that of huMAb195 with unmodified Fc (t1 / 2 = 1.6 day). YTE mutations also show a significant increase in serum half-life BY 110% (t1 / 2 = 3.4 days).
When the S228P and YTE mutations were combined, the half-life was further increased to 14 days, an increase of 312% compared to that of the YTE variant. Without the synergy, the maximum increase by adding the S228P mutation to the huMab195 containing YTE would be 26%, resulting in a 4.3-day half-life, which is significantly shorter than the observed 14 days. This second example confirms the observation that the S228P and YTE modifications to human IgG4 antibodies are synergistic with respect to their effect on increasing the circulating half-lives of IgG4 antibodies in individuals with a human FcRn. This synergy may justify the use of these two modifications in the same protein, despite the potential for increased immunogenicity.

权利要求:
Claims (9)
[1]
1. Isolated antibody, characterized by specifically binding to interleukin (IL) -5 and comprising:
(i) a human IgG4 Fc region or the modified FcRn binding domain thereof with respect to a corresponding unmodified IgG4 Fc region or FcRn binding domain thereof to comprise amino acid substitutions M252Y, S254T and T256E numbered according to the EU index as in Kabat; and (ii) a sequence of the central hinge region of human IgG4 modified to comprise the substitution of amino acid S228P according to the EU index as in Kabat;
wherein the combination of modifications results in an increased in vivo half-life of the antibody to modified (IL) -5 compared to the corresponding antibody having only (i) or (ii).
[2]
2. Use of an isolated antibody that specifically binds to interleukin (IL) -5, comprising:
(i) a human IgG4 Fc region or its modified FcRn binding domain in relation to an unmodified IgG4 Fc region or unmodified FcRn binding domain thereof to comprise numbered M252Y, S254T and T256E amino acid substitutions according to the EU index as in Kabat; and (ii) a sequence of the central hinge region of human IgG4 modified to comprise the substitution of amino acid S228P according to the EU index as in Kabat;
characterized by being in the preparation of a medicine to treat or prevent a disorder defined by excessive production of eosinophils
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2/3 where the combination of modifications results in an increased in vivo half-life of the antibody to modified (IL) -5 compared to the corresponding antibody having only (i) or (ii).
[3]
3. Antibody according to claim 1 or use according to claim 2, characterized in that the antibody is chimeric, human, humanized, grafted, primatized, de-immunized, varnished or bispecific CDR.
[4]
Antibody according to claim 1 or use according to claim 2, characterized in that it comprises a constant region sequence of the heavy chain shown in the ID. SEQ. No. 6 and a sequence of the variable region of the heavy chain shown in the ID. SEQ. N °: 7.
[5]
Antibody or use according to claim 4, characterized in that it further comprises a light chain comprising sequences of variable and constant region shown in the ID. SEQ. N °: 8.
[6]
6. Use of an antibody according to the claim
2, characterized by the fact that the disorder is selected from the group consisting of atopic asthma, atopic dermatitis, allergic rhinitis, non-allergic rhinitis, asthma, severe asthma, chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome, syndrome eosinophilic myalgia, hypereosinophilic syndrome, edematous reactions including episodic angioedema, helminth infections, onchocerciasis dermatitis, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, eosinophilic colitis, nasal micropolyposis, nasal polyps, nasal polyposis
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3/3 aspirin intolerance, obstructive sleep apnea, chronic asthma, Crohn's disease, scleroderma, endomyocardial fibrosis and autoimmune disease.
[7]
7. Nucleic Acid characterized by the fact that it encodes
An antibody as defined in any one of claims 1 and 3-5.
[8]
8. Transformed Cell characterized by the fact that it comprises nucleic acid encoding an antibody, as defined in claim 7 and is limited to one
10 transgenic microorganism.
[9]
Antibody according to any one of claims 1 and 3-5, characterized in that it is supplied in a pharmaceutical composition together with a pharmaceutically acceptable excipient.

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

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-19| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-09-03| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-02-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-03-31| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2020-04-22| B25D| Requested change of name of applicant approved|Owner name: TEVA PHARMACEUTICALS AUSTRALIA PTY LTD. (AU) |

优先权:

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申请号 | 申请日 | 专利标题

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US201061425858P| true| 2010-12-22|2010-12-22|

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US61/425,858|2010-12-22|

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PCT/AU2011/001662|WO2012083370A1|2010-12-22|2011-12-22|Modified antibody with improved half-life|

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