Bispecific T cell activating antigen binding molecules
阅读说明：本技术 双特异性t细胞活化抗原结合分子 (Bispecific T cell activating antigen binding molecules ) 是由 M·巴卡 P·布鲁恩克尔 C·耶格尔 C·克莱因 E·默斯纳 P·乌马纳 T·韦因策尔 于 2014-02-21 设计创作，主要内容包括：本发明总体上涉及用于T细胞活化和再指向特定靶细胞的新的双特异性抗原结合分子。此外,本发明涉及编码这类双特异性抗原结合分子的多核苷酸以及包含这类多核苷酸的载体和宿主细胞。本发明还涉及用于产生本发明的双特异性抗原结合分子的方法,以及涉及在治疗疾病时使用这些双特异性抗原结合分子的方法。(The present invention relates generally to novel bispecific antigen binding molecules for T cell activation and redirection to specific target cells. Furthermore, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules as well as vectors and host cells comprising such polynucleotides. The invention also relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.)
A T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to a T cell activating antigen and a second antigen binding moiety capable of specific binding to a target cell antigen,
wherein said one antigen binding portion is a Fab molecule or a crossover Fab molecule wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the other antigen binding portion comprises a single domain antigen binding molecule.
2. The T cell activating bispecific antigen binding molecule of claim 1, wherein the single domain antigen binding molecule is a single domain variable heavy chain.
3. The T cell activating bispecific antigen binding molecule of claim 2, wherein the first antigen binding moiety capable of specific binding to a T cell activating antigen is an exchange Fab molecule, wherein the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged and wherein the second antigen binding moiety capable of specific binding to a target cell antigen consists of a single domain variable heavy chain.
A T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to a T cell activating antigen and a second antigen binding moiety capable of specific binding to a target cell antigen,
wherein said one antigen binding portion is a Fab molecule or a crossover Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the other antigen binding portion is a binding protein comprising at least one ankyrin repeat motif.
5. The T cell activating bispecific antigen binding molecule of claim 4, wherein the first antigen binding moiety capable of specific binding to a T cell activating antigen is an exchange Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the second antigen binding moiety is a binding protein comprising at least one ankyrin repeat motif.
6. The T cell activating bispecific antigen binding molecule of claim 4 or 5, wherein the second antigen moiety comprises a binding protein comprising two, three, four or five ankyrin repeat motifs.
7. The T cell activating bispecific antigen binding molecule of any one of claims 1 to 6, further comprising an Fc domain comprising a first and a second subunit capable of stable association.
8. The T cell activating bispecific antigen binding molecule of any one of claims 1 to 6, comprising no more than one antigen binding moiety capable of specifically binding to a T cell activating antigen.
9. The T cell activating bispecific antigen binding molecule of any one of claims 1 to 6, wherein the first and second antigen binding moiety are fused to each other, optionally via a peptide linker.
10. The T cell activating bispecific antigen binding molecule of any one of claims 1 to 3, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety.
11. The T cell activating bispecific antigen binding molecule of any one of claims 1 to 10, comprising a third antigen binding moiety capable of specifically binding to a target cell antigen.
12. The T cell activating bispecific antigen binding molecule of claim 11, wherein the third antigen binding moiety is a single domain antigen binding molecule.
13. The T cell activating bispecific antigen binding molecule of claim 12, wherein single domain antigen binding molecule is a single domain variable heavy chain.
14. The T cell activating bispecific antigen binding molecule of claim 13, comprising
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion comprising an exchange Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the exchange Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding portion comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen binding portion comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of the Fab heavy chain of the first antigen binding portion.
15. The T cell activating bispecific antigen binding molecule of claim 11, wherein the third antigen binding moiety is a binding protein comprising at least one ankyrin repeat motif.
16. The T cell activating bispecific antigen binding molecule of claim 15, wherein the third antigen binding moiety is a binding protein comprising two, three, four or five ankyrin repeat motifs.
17. The T cell activating bispecific antigen binding molecule of claim 15 or 16, comprising
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion comprising an exchange Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the exchange Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding portion comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen-binding portion comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion.
18. The T cell activating bispecific antigen binding molecule of claim 14 or 17, wherein the first antigen binding moiety binds to a T cell activating antigen and the second and third antigen binding moieties bind to the same target cell antigen.
19. The T cell activating bispecific antigen binding molecule of any one of claims 7 to 19, wherein Fc domain is an IgG Fc domain, in particular an IgG1Or IgG4An Fc domain.
20. The T cell activating bispecific antigen binding molecule of claim 19, wherein Fc domain is a human Fc domain.
21. The T cell activating bispecific antigen binding molecule of any one of claims 7 to 20, wherein the Fc domain comprises a modification that facilitates association of the first and second subunits of the Fc domain.
22. The T cell activating bispecific antigen binding molecule of claim 21, wherein an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue having a larger side chain volume, thereby creating inside the CH3 domain of the first subunit a overhang that can be positioned in the lumen inside the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced by an amino acid residue having a smaller side chain volume, thereby creating inside the CH3 domain of the second subunit a lumen inside which an overhang inside the CH3 domain of the first subunit can be positioned.
23. The T cell activating bispecific antigen binding molecule of any one of claims 7 to 22, wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function compared to a native IgG1 Fc domain.
24. The T cell activating bispecific antigen binding molecule of any one of claims 7 to 23, wherein Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function.
25. The T cell activating bispecific antigen binding molecule of claim 24, wherein the one or more amino acid substitutions are at one or more positions selected from L234, L235 and P329.
26. The T cell activating bispecific antigen binding molecule of claim 25, wherein each subunit of an Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A, L235A and P329G.
27. The T cell activating bispecific antigen binding molecule of any one of claims 23 to 26, wherein Fc receptor is an fey receptor.
28. The T cell activating bispecific antigen binding molecule of any one of claims 20 to 23, wherein an effector function is antibody dependent cell mediated cytotoxicity (ADCC).
A T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to a T cell activating antigen and the other of which is a Fab molecule capable of specific binding to a target cell antigen;
wherein the first antigen binding moiety is
(a) A single chain Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker, or
(b) An exchanged Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged; and wherein the Fc domain comprises
And an Fc domain comprising a first and a second subunit capable of stable association,
wherein the first subunit and the second subunit have been modified to comprise one or more charged amino acids that electrostatically facilitate heterodimer formation.
30. The T cell activating bispecific antigen binding molecule of claim 29, wherein the first subunit comprises the amino acid mutations E356K, E357K and D399K and the second subunit comprises the amino acid mutations K370E, K409E and K439E.
31. The T cell activating bispecific antigen binding molecule of claim 29, wherein the first subunit comprises the amino acid mutations K392D, K409D and the second subunit comprises the amino acid mutations E356K, 399K (ddkk).
32. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 31, comprising no more than one antigen binding moiety capable of specifically binding to a T cell activating antigen.
33. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 32, wherein the first and second antigen binding moiety are fused to each other, optionally via a peptide linker.
34. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 33, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety.
35. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 33, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.
36. The T cell activating bispecific antigen binding molecule of claim 34 or 35, wherein the first antigen binding moiety is an crossover Fab molecule and the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a peptide linker.
37. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 33, wherein the second antigen binding portion of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding portion.
38. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 33, 35 or 37, wherein the second antigen binding portion of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
39. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 34, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
40. The T cell activating bispecific antigen binding molecule of claim 29 to 32, wherein the first and second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain.
41. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 40, comprising a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen.
42. The T cell activating bispecific antigen binding molecule of claim 41, wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
43. The T cell activating bispecific antigen binding molecule of claim 41 or 42, wherein the second and third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.
44. The T cell activating bispecific antigen binding molecule of claim 41 or 42, wherein the first and third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety.
45. The T cell activating bispecific antigen binding molecule of claim 43, wherein the second and third antigen binding portion and the Fc domain are parts of an immunoglobulin molecule, in particular of an IgG class immunoglobulin.
46. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 45, wherein Fc domainIs an IgG Fc domain, in particular an IgG1Or IgG4An Fc domain.
47. The T cell activating bispecific antigen binding molecule of claim 29 to 46, wherein the Fc domain is a human Fc domain.
48. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 47, wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function compared to a native IgG1 Fc domain.
49. The T cell activating bispecific antigen binding molecule of any one of claims 29 to 49, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function.
50. The T cell activating bispecific antigen binding molecule of claim 49, wherein said one or more amino acid substitutions is at one or more positions selected from L234, L235 and P329.
51. The T cell activating bispecific antigen binding molecule of claim 50, wherein each subunit of an Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A, L235A and P329G.
52. The T cell activating bispecific antigen binding molecule of any one of the preceding claims, wherein the T cell activating antigen is CD 3.
53. The T cell activating bispecific antigen binding molecule of any one of the preceding claims, wherein the target cell antigen is selected from the group consisting of: melanoma associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), CD19, CD20, CD33, carcinoembryonic antigen (CEA), and Fibroblast Activation Protein (FAP).
54. An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of claims 1 to 53, or a fragment thereof.
55. A polypeptide encoded by the isolated polynucleotide of claim 54.
56. A vector, particularly an expression vector, comprising the isolated polynucleotide of claim 54.
57. A host cell comprising the isolated polynucleotide of claim 54 or the expression vector of claim 56.
58. A method of producing the T cell activating bispecific antigen binding molecule of any one of claims 1 to 53, comprising the steps of a) culturing the host cell of claim 57 under conditions suitable for expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule.
A T cell activating bispecific antigen binding molecule produced by the method of claim 58.
60. A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of any one of claims 1 to 53 and a pharmaceutically acceptable carrier.
61. The T cell activating bispecific antigen binding molecule of any one of claims 1 to 53 or the pharmaceutical composition of claim 60 for use as a medicament.
62. The T cell activating bispecific antigen binding molecule of any one of claims 1 to 53 or the pharmaceutical composition of claim 60, for use in the treatment of a disease in an individual in need thereof.
63. The T cell activating bispecific antigen binding molecule or the pharmaceutical composition of claim 62, wherein the disease is cancer.
64. Use of the T cell activating bispecific antigen binding molecule of any one of claims 1 to 53 for the preparation of a medicament for the treatment of a disease in an individual in need thereof.
65. A method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of any one of claims 1 to 53 in a pharmaceutically acceptable form.
66. The use of claim 64 or the method of claim 65, wherein the disease is cancer.
67. A method for inducing lysis of a target cell, comprising contacting the target cell with the T cell activating bispecific antigen binding molecule of any one of claims 1-53 in the presence of a T cell.
68. The invention as described herein.
Technical Field
The present invention relates generally to bispecific antigen binding molecules for activating T cells. Furthermore, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules as well as vectors and host cells comprising such polynucleotides. The invention also relates to methods for producing the bispecific antigen binding molecules of the invention, and methods of using these bispecific antigen binding molecules in the treatment of disease.
Background
Selective destruction of individual cells or specific cell types is often desirable in a variety of clinical settings. For example, the main goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and intact undamaged tissue.
An attractive way to achieve this is to induce an immune response against the tumor, such that immune effector cells, such as Natural Killer (NK) cells or Cytotoxic T Lymphocytes (CTL), attack and destroy the tumor cells. CTLs constitute the most potent effector cells of the immune system, however they cannot be activated by effector cell mechanisms mediated by the Fc domain of conventional therapeutic antibodies.
In this regard, there has been increasing interest in recent years for bispecific antibodies designed to bind to an antigen on the surface of a target cell with one "arm" and to bind to the activating invariant component of the T Cell Receptor (TCR) complex with a second "arm". The simultaneous binding of this antibody to its two targets will force a transient interaction between the target cell and the T cell, resulting in the activation of any cytotoxic T cells and subsequent lysis of the target cell. Thus, the immune response is redirected to the target cell and is independent of the presentation of peptide antigens or T cell specificity by the target cell associated with general MHC-restricted CTL activation. It is crucial in this case that when the target cell presents the bispecific antibody to the CTL, only the CTL is activated, i.e. the immunological synapse is mimicked. Particularly desirable are: bispecific antibodies that do not require lymphocyte preconditioning or costimulation to elicit efficient lysis of target cells.
Several bispecific antibody formats have been developed and their applicability to T cell-mediated immunotherapy has been investigated. Among these antibody formats, the so-called BiTE (bispecific T cell engager) molecules have been very well characterized and they have shown some promise in clinical settings (reviewed in Nagorsen and nagarsen)
A variety of modalities are being developed that show a great potential for T cell redirection and activation attributed to immunotherapy. However, the task of generating bispecific antibodies suitable for this is not trivial anyway, which involves numerous challenges related to antibody potency, toxicity, applicability and producibility that have to be addressed.
Small constructs such as, for example, BiTE molecules-although capable of effectively cross-linking effector and target cells-have a very short serum half-life and require their administration to a patient by continuous infusion. On the other hand, IgG-like patterns-although of great benefit with long half-lives-suffer from toxicity associated with the natural effector functions inherent to IgG molecules. Their immunogenic potential constitutes another disadvantageous feature of IgG-like bispecific antibodies, especially in the non-human format, for successful therapeutic development. Finally, a significant challenge in the overall development of bispecific antibodies is to produce bispecific antibody constructs in clinically sufficient quantities and purities because, upon co-expression, the different specificity of the antibody heavy and light chains mis-pair, which reduces the yield of correctly assembled constructs and produces numerous non-functional byproducts that can be difficult to separate from the desired bispecific antibody.
In view of the difficulties and disadvantages associated with bispecific antibodies currently available for T cell mediated immunotherapy, there remains a need for novel and improved versions of such molecules. The present invention provides bispecific antigen binding molecules designed for T cell activation and redirection that combine good efficacy and producibility with low toxicity and favorable pharmacokinetic properties. In particular, novel bispecific antigen binding molecules are provided that comprise a binding protein having at least one ankyrin repeat motif. Novel bispecific antigen binding molecules comprising single domain variable heavy chains are also provided. These new molecules have the advantages: they can be produced with fewer by-products because there is no mis-pairing between the conjugate comprising the ankyrin motif or single domain variable heavy chain and the conjugate comprising the antibody heavy and light chains, respectively.
Also provided are novel bispecific antigen binding molecules comprising modifications that facilitate association of the first and second subunits of the Fc domain via electrostatic steering effects. Thereby promoting proper chain association of Fc domains and fewer undesirable by-products during production of these molecules.
Brief description of the invention
In one aspect the invention provides a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specifically binding to a T cell activating antigen and a second antigen binding moiety capable of specifically binding to a target cell antigen, wherein said one antigen binding moiety is a Fab molecule or an exchanged Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the other antigen binding moiety comprises a single domain antigen binding molecule.
In one embodiment, the single domain antigen binding molecule is a single domain variable heavy chain.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a first antigen binding portion capable of specific binding to a T cell activating antigen, wherein the first antigen binding portion comprises an exchange Fab molecule, wherein the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged, and a second antigen binding portion capable of specific binding to a target cell antigen, wherein the second antigen binding portion consists of a single domain variable heavy chain.
In one aspect the invention provides a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specifically binding to a T cell activating antigen and a second antigen binding moiety capable of specifically binding to a target cell antigen, wherein said one antigen binding moiety is a Fab molecule or an exchange Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the other antigen binding moiety is a binding protein comprising at least one ankyrin repeat motif.
In such an embodiment, the first antigen binding portion capable of specific binding to a T cell activation antigen is an exchange Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the second antigen binding portion is a binding protein comprising at least one ankyrin repeat motif.
In such an embodiment, the second antigenic moiety comprises a binding protein comprising two, three, four or five ankyrin repeat motifs.
In one embodiment, the T cell activating bispecific antigen binding molecule additionally comprises an Fc domain comprising a first and a second subunit capable of stable association.
In a specific embodiment, no more than one antigen binding moiety capable of specifically binding to a T cell activation antigen is present in the T cell activating bispecific antigen binding molecule (i.e. the T cell activating bispecific antigen binding molecule provides monovalent binding to the T cell activation antigen). In a specific embodiment, the first antigen binding moiety is a crossover Fab molecule.
In one embodiment, the first and second antigen-binding moieties are fused to each other, optionally via a peptide linker.
In one embodiment, the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion.
In one embodiment, the T cell activating bispecific antigen binding molecule additionally comprises a third antigen binding moiety capable of specifically binding to a target cell antigen.
In such an embodiment, the third antigen binding portion capable of specific binding to a target cell antigen is a single domain variable heavy chain.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion comprising an exchange Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the exchange Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding portion comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen binding portion comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of the Fab heavy chain of the first antigen binding portion.
In such an embodiment, the third antigen binding moiety capable of specifically binding to a target cell antigen is a binding protein comprising at least one ankyrin repeat motif.
In such an embodiment, the third antigen binding moiety capable of specifically binding to a target cell antigen is a binding protein comprising two, three, four or five ankyrin repeat motifs.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion comprising an exchange Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the exchange Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding portion comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen-binding portion comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion.
In one embodiment, the first antigen binding moiety binds to a T cell activation antigen and the second and third antigen binding moieties bind to the same target cell antigen.
In a specific embodiment, the Fc domain is an IgG Fc domain. In a specific embodimentThe Fc domain is IgG1An Fc domain. In another specific embodiment, the Fc domain is an IgG4An Fc domain. In a specific embodiment, the Fc domain is a human Fc domain.
In particular embodiments, the Fc domain comprises a modification that facilitates association of the first and second Fc domain subunits. In one such specific embodiment, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that can be positioned within the cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit can be positioned.
In a specific embodiment, the IgG is naturally associated with1Fc domains exhibit reduced binding affinity to Fc receptors and/or reduced effector function compared to Fc domains. In certain embodiments, the Fc domain is engineered to have reduced binding affinity for an Fc receptor and/or reduced effector function as compared to a non-engineered Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function. In one embodiment, one or more amino acid substitutions in the Fc domain that reduce Fc receptor binding and/or effector function are at one or more positions selected from L234, L235, and P329. In a specific embodiment, each subunit of the Fc domain comprises three amino acid substitutions that reduce Fc receptor binding and/or effector function, wherein the amino acid substitutions are L234A, L235A, and P329G. In one such embodiment, the Fc domain is an IgG1Fc domain, in particular human IgG1An Fc domain. In other embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding to an Fc receptor and/or effector function, wherein the amino acid substitutions are L235E and P329G. In such a caseIn embodiments, the Fc domain is an IgG4Fc domain, in particular human IgG4An Fc domain. In one such embodiment, the Fc domain is an IgG4Fc domain, in particular human IgG4An Fc domain and comprises the amino acid substitutions L235E and S228P (SPLE).
In one embodiment, the Fc receptor is an fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is human Fc γ RIIa, Fc γ RI and/or Fc γ RIIIa. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
In one aspect the invention provides a T cell activating bispecific antigen binding molecule comprising first and second antigen binding moieties, one of which is a Fab molecule capable of specific binding to a T cell activating antigen and the other of which is a Fab molecule capable of specific binding to a target cell antigen; wherein the first antigen binding portion is (a) a single chain Fab molecule in which the Fab light chain and Fab heavy chain are connected by a peptide linker or (b) an exchanged Fab molecule in which the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged; and comprising an Fc domain comprising first and second subunits capable of stable association, wherein said first subunit and said second subunit have been modified to comprise one or more charged amino acids that electrostatically facilitate heterodimer formation.
In one embodiment, the first subunit comprises the amino acid mutations E356K, E357K, and D399K and the second subunit comprises the amino acid mutations K370E, K409E, and K439E.
In one embodiment, the first subunit comprises the amino acid mutations K392D, K409D and the second subunit comprises the amino acid mutations E356K, D399K (DDKK).
In a specific embodiment, no more than one antigen binding moiety capable of specifically binding to a T cell activation antigen is present in the T cell activating bispecific antigen binding molecule (i.e. the T cell activating bispecific antigen binding molecule provides monovalent binding to the T cell activation antigen). In a specific embodiment, the first antigen binding moiety is a crossover Fab molecule. In an even more particular embodiment, the first antigen binding portion is an exchanged Fab molecule wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged.
In some embodiments, the first and second antigen-binding portions of the T cell activating bispecific antigen binding molecule are fused to each other, optionally through a peptide linker. In such an embodiment, the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In another such embodiment, the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion. In yet another such embodiment, the second antigen binding portion is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding portion. In such embodiments, wherein the first antigen-binding portion is an exchange Fab molecule and wherein (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion, the Fab light chain of the first antigen-binding portion and the Fab light chain of the second antigen-binding portion additionally may be fused to each other, optionally via a peptide linker.
In one embodiment, the second antigen binding portion of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In another embodiment, the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
In one embodiment, the second antigen-binding portions of the T cell activating bispecific antigen binding molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain.
In certain embodiments, the T cell activating bispecific antigen binding molecule comprises a third antigen binding moietyIn particular Fab molecules capable of specific binding to the target cell antigen. In such an embodiment, the third antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a specific embodiment, the second and third antigen-binding portions of the T cell activating antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In another specific embodiment, the first and third antigen binding portions of the T cell activating antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. The components of the T cell activating bispecific antigen binding molecule may be fused directly or through a suitable peptide linker. In one embodiment, the second and third antigen binding portions and the Fc domain are part of an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more specific embodiment, the immunoglobulin is an IgG1Subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4Subclass immunoglobulin.
In a specific embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is IgG1An Fc domain. In another specific embodiment, the Fc domain is an IgG4An Fc domain. In a specific embodiment, the Fc domain is a human Fc domain.
In a specific embodiment, the IgG is naturally associated with1Fc domains exhibit reduced binding affinity to Fc receptors and/or reduced effector function compared to Fc domains. In certain embodiments, the Fc domain is engineered to have reduced binding affinity for an Fc receptor and/or reduced effector function as compared to a non-engineered Fc domain. In one embodiment, the Fc domain comprises a decrease in binding to an Fc receptor and/or an effectorFunctional one or more amino acid substitutions. In one embodiment, one or more amino acid substitutions in the Fc domain that reduce Fc receptor binding and/or effector function are at one or more positions selected from L234, L235, and P329. In a specific embodiment, each subunit of the Fc domain comprises three amino acid substitutions that reduce Fc receptor binding and/or effector function, wherein the amino acid substitutions are L234A, L235A, and P329G. In one such embodiment, the Fc domain is an IgG1Fc domain, in particular human IgG1An Fc domain. In other embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding to an Fc receptor and/or effector function, wherein the amino acid substitutions are L235E and P329G. In one such embodiment, the Fc domain is an IgG4Fc domain, in particular human IgG4An Fc domain. In one such embodiment, the Fc domain is an IgG4Fc domain, in particular human IgG4An Fc domain and comprises the amino acid substitutions L235E and S228P (SPLE).
In one embodiment, the Fc receptor is an fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is human Fc γ RIIa, Fc γ RI and/or Fc γ RIIIa. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
In a specific embodiment, the T cell activation antigen to which the bispecific antigen binding molecule is capable of binding is
According to another aspect of the invention there is provided an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof. The invention also encompasses polypeptides encoded by the polynucleotides of the invention. The invention also provides expression vectors comprising the isolated polynucleotides of the invention, and host cells comprising the isolated polynucleotides or expression vectors of the invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell.
In another aspect, there is provided a method of producing a T cell activating bispecific antigen binding molecule of the invention, the method comprising the steps of a) culturing a host cell of the invention under conditions suitable for expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule. The invention also encompasses T cell activating bispecific antigen binding molecules produced by the methods of the invention.
The invention also provides a pharmaceutical composition comprising a T cell activating bispecific antigen binding molecule of the invention and a pharmaceutically acceptable carrier.
The invention also encompasses methods of using the T cell activating bispecific antigen binding molecules and pharmaceutical compositions of the invention. In one aspect, the invention provides a T cell activating bispecific antigen binding molecule or pharmaceutical composition of the invention for use as a medicament. In one aspect, the T cell activating bispecific antigen binding molecule or pharmaceutical composition of the invention is provided according to for use in the treatment of a disease in an individual in need thereof. In a specific embodiment, the disease is cancer.
Also provided is the use of a T cell activating bispecific antigen binding molecule of the invention for the manufacture of a medicament for the treatment of a disease in an individual in need thereof; and provides a method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising a T cell activating bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form. In a specific embodiment, the disease is cancer. In any of the above embodiments, the individual is preferably a mammal, especially a human.
The invention also provides a method for inducing lysis of a target cell, in particular a tumor cell, the method comprising contacting the target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, in particular a cytotoxic T cell.
Brief Description of Drawings
FIG. 1: exemplary configurations of the T cell activating bispecific antigen binding molecules of the invention (TCBs). Schematic representation of (a) "1 +1IgG scFab, single-arm", and (B) "1 +1IgG scFab, single-arm inverted" molecules. In the "1 +1IgG scFab, single-arm" molecule, the light chain of the T-cell-targeted Fab is fused to the heavy chain via a linker, while the "1 +1IgG scFab, single-arm inverted" molecule has a linker in the tumor-targeted Fab. (C) Illustration of a "2 +1IgG scFab" molecule. (D) Schematic representation of a "1 +1 IgGscFab" molecule. (E) Schematic representation of a "1 +1IgG Crossfab" molecule. (F) Schematic representation of a "2 +1IgG Crossfab" molecule. (G) Schematic representation of a "2 +1IgG Crossfab" molecule with alternative order ("inverted") of Crossfab and Fab components. (H) Schematic representation of a "1 +1IgG Crossfab Light Chain (LC) fusion molecule. (I) Schematic representation of a "1 +1 CrossMab" molecule. (J) Schematic representation of a "2 +1IgG Crossfab, linked light chain" molecule. (K) Schematic representation of a "1 +1IgG Crossfab, linked light chain" molecule. Schematic representation of the (L) "2 +1IgG Crossfab, inverted, linked light chain" molecule. Schematic representation of (M) "1 +1IgG Crossfab, inverted, ligated light chain" molecule. Black spot: optional modifications in the Fc domain to promote heterodimerization.
FIG. 2: SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) of non-reduced (A) and reduced (B) "1 +1IgG scFab, single-armed" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 1,3, 5) and non-reduced (C) and reduced (D) "1 +1IgG scFab, single-armed inverted" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 7, 9, 11).
FIG. 3: analytical size exclusion chromatography of "1 +1IgG scFab, single-arm" (anti-MCSP/anti-huCD 3) (see SEQ ID NO:1, 3, 5) (A) and "1 +1IgG scFab, single-arm inverted" (anti-MCSP/anti-huCD 3) (see SEQ ID NO:7, 9, 11) (B) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 4: non-reduced (A) and reduced (B) "1 +1IgG scFab, single-armed" (anti-EGFR/anti-huCD 3) (see SEQ ID NOS: 43, 45, 57) and non-reduced (C) and reduced (D) "1 +1IgG scFab, single-armed inverted" (anti-EGFR/anti-huCD 3) (see SEQ ID NOS: 11, 49, 51) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining).
FIG. 5: analytical size exclusion chromatography of "1 +1IgG scFab, single-arm" (anti-EGFR/anti-huCD 3) (see SEQ ID NO:43, 45, 47) (A) and "1 +1IgG scFab, single-arm inverted" (anti-EGFR/anti-huCD 3) (see SEQ ID NO:11, 49, 51) (B) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 6: (A, B) "1 +1IgG scFab, single-arm inverted" (anti-FAP/anti-huCD 3) (see SEQ ID NO:11, 51, 55) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) non-reduced (A) and reduced (B). (C) Analytical size exclusion chromatography of "1 +1IgG scFab, single arm inverted" (anti-FAP/anti-huCD 3) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 7: (A) non-reduced (lane 2) and reduced (lane 3) "2 +1IgG scFab, P329G LALA" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 5, 21, 23); (B) non-reduced (lane 2) and reduced (lane 3) "2 +1 IgGscFab, LALA" (anti-MCSP/anti-huCD 3) (see SEQ ID NOs: 5, 17, 19); (C) non-reduced (lane 2) and reduced (lane 3) "2 +1IgG scFab, wt" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 5, 13, 15); and (D) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) of non-reduced (lane 2) and reduced (lane 3) "2 +1IgG scFab, P329G LALA N297D" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 5, 25, 27).
FIG. 8: (A) "2 +1IgG scFab, P329G LALA" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 5, 21, 23); (B) "2 +1IgG scFab, LALA" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 5, 17, 19); (C) "2 +1IgG scFab, wt" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 5, 13, 15); and (D) "2 +1IgG scFab, P329G LALA N297D" (anti-MCSP/anti-huCD 3) (see SEQ ID NO:5, 25, 27) analytical size exclusion chromatography (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g of sample was injected).
FIG. 9: (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) of non-reduced (A) and reduced (B) "2 +1IgG scFab, P329G LALA" (anti-EGFR/anti-huCD 3) (see SEQ ID NOS: 45, 47, 53). (C) Analytical size exclusion chromatography of "2 +1IgG scFab, P329G LALA" (anti-EGFR/anti-huCD 3) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 10: (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) of non-reduced (A) and reduced (B) "2 +1IgG scFab, P329G LALA" (anti-FAP/anti-huCD 3) (see SEQ ID NOS: 57, 59, 61). (C) Analytical size exclusion chromatography of "2 +1IgG scFab, P329G LALA" (anti-FAP/anti-huCD 3) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 11: (A, B) "1 +1IgG Crossfab, Fc (deducted) P329G LALA/Fc (knot) wt" (anti-MCSP/anti-huCD 3) both non-reduced (A) and reduced (B) (see SDS PAGE of SEQ ID NO:5, 29, 31, 33) (4-12% Tris-acetate (A) or 4-12% Bis/Tris (B), NuPage Invitrogen, Coomassie staining). (C) Analytical size exclusion chromatography of "1 +1IgG Crossfab, Fc (Buckle) P329G LALA/Fc (knot) wt" (anti-MCSP/anti-huCD 3) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 12: (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) of non-reduced (A) and reduced (B) "2 +1IgG Crossfab" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 3, 5, 29, 33). (C) Analytical size exclusion chromatography of "2 +1IgG Crossfab" (anti-MCSP/anti-huCD 3) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 13: (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) of non-reduced (A) and reduced (B) "2 +1IgG Crossfab" (anti-MCSP/anti-cyCD 3) (see SEQ ID NOS: 3, 5, 35, 37). (C) Analytical size exclusion chromatography of "2 +1IgG Crossfab" (anti-MCSP/anti-cyCD 3) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 14: (A, B) SDS PAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining) of non-reduced (A) and reduced (B) "2 +1IgG Crossfab, inverted" (anti-CEA/anti-huCD 3) (see SEQ ID NOS: 33, 63, 65, 67). (C) Analytical size exclusion chromatography of "2 +1IgG Crossfab, inverted" (anti-CEA/anti-huCD 3) (Superdex 20010/300GL GE Healthcare; 2mM MOPS pH 7.3, 150mM NaCl, 0.02% (w/v) NaCl; 50. mu.g sample injected).
FIG. 15: (A) "(scFv)2-Fc "sum" (dsscFv)2Thermal stability of Fc "(anti-MCSP (LC 007)/anti-huCD 3 (V9)). Dynamic light scattering, measured at a temperature rate of 25-75 ℃ at 0.05 ℃/minute. Black curve: "(scFv)2-Fc "; gray curve: "(dsscFv)2-Fc ". (B) Thermal stability of "2 +1IgG scFab" (see SEQ ID NOS: 5, 21, 23) and "2 +1 IgGCrossfab" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 3, 5, 29, 33). Dynamic light scattering, measured at a temperature rate of 25-75 ℃ at 0.05 ℃/minute. Black curve: "2 +1IgG scFab"; gray curve: "2 +1 IgGCrossfab".
FIG. 16: for (A) determination of the interaction of various Fc mutants with human Fc γ RIIIa and (B) T-cell bispecific constructs with tumor target and human CD3 γ (G)4S)5Biacore assay setup for simultaneous binding of CD3 ε -AcTev-Fc (knot) -Avi/Fc (knot).
FIG. 17: t cell bispecific constructs with the D3 domain of human MCSP and human CD3 gamma (G)4S)5Simultaneous binding of CD3 ε -AcTev-Fc (knot) -Avi/Fc (knot). (A) "2 +1IgG Crossfab" (see SEQ ID NOS: 3, 5, 29, 33) and (B) "2 +1IgG scFab" (see SEQ ID NOS: 5, 21, 23).
FIG. 18: t cell bispecific constructs with human EGFR and human CD3 gamma (G)4S)5Simultaneous binding of CD3 ε -AcTev-Fc (knot) -Avi/Fc (knot). (A) "2 +1IgG scFab" (see SEQ ID NO:45, 47, 53), (B) "1 +1 IgGscFab, single arm" (see SEQ ID NO:43, SEQ ID NO:53,45. 47), (C) "1 +1IgG scFab, single-arm inverted" (see SEQ ID NOs: 11, 49, 51), and (D) "1 +1IgG scFab" (see SEQ ID NOs: 47, 53, 213).
FIG. 19: measured by FACS, "(scFv)2The "molecule (50nM) binds to CD3(A) expressed on Jurkat cells or to MCSP (B) on Colo-38 cells. The mean fluorescence intensity was plotted compared to untreated cells and cells stained with secondary antibody only.
FIG. 20: the "2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) construct (50nM) measured by FACS binds to CD3(A) expressed on Jurkat cells or MCSP (B) on Colo-38 cells. Mean fluorescence intensities were plotted compared to cells treated with reference anti-CD 3IgG (as shown), untreated cells, and cells stained with secondary antibody only.
FIG. 21: the "1 +1IgG scFab, single-arm" (see SEQ ID NO:1, 3, 5) and "1 +1IgG scFab, single-arm inversion" (see SEQ ID NO:7, 9, 11) constructs (50nM) bound to CD3(A) expressed on Jurkat cells or to MCSP (B) on Colo-38 cells. Mean fluorescence intensities were plotted compared to cells treated with
FIG. 22: the "2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) bispecific construct and corresponding anti-MCSP IgG dose-dependent binding to MCSP on Colo-38 cells as measured by FACS.
FIG. 23: surface expression levels of different activation markers on human T cells following incubation with 1nM of "2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) or "(scFv) 2" CD3-MCSP bispecific constructs in the presence or absence of Colo-38 tumor target cells as indicated (PBMC to tumor cell E: T ratio ═ 10: 1). Describe CD8 after 15 hours or 24 hours incubation, respectively+Expression level of early activation marker CD69(a) or late activation marker CD25(B) on T cells.
FIG. 24: presence or absence of the Colo-38 tumor target cells with 1nM of the "2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) or "(scFv) 2" CD3-MCSP bispecific constructSurface expression levels of the late activation marker CD25 on human T cells after incubation in the presence were as shown (E: T ratio 5: 1). Describe CD8 after 5 days incubation+On T cells (A) or CD4+Expression level of late activation marker CD25 on T cells (B).
FIG. 25: after incubation with the indicated concentrations of the "2 +1IgG Crossfab" bispecific construct (targeting cynomolgus monkey CD3 and human MCSP; see SEQ ID NOs: 3, 5, 35, 37) for 43 hours in the presence or absence of MV-3 tumor target cells expressing human MCSP (E: T ratio ═ 3:1), the late activation marker CD25 was compared to cynomolgus monkey CD8 from two different animals (cyno nester, cynoNobu)+Surface expression levels on T cells. As a control, a reference IgG (anti-cynomolgus CD3IgG, anti-human MCSP IgG) or a non-physiological stimulant PHA-M was used.
FIG. 26: levels of IFN- γ secreted by human pan T cells activated by a "2 +1IgG scFab, LALA" CD3-MCSP bispecific construct (see SEQ ID NOs: 5, 17, 19) in the presence of U87MG tumor cells (E: T ratio ═ 5:1) for 18.5 hours. As controls, corresponding anti-CD 3IgG and anti-MCSP IgG were administered.
FIG. 27 is a schematic view showing: co-cultured with human pan-T cells (E: T ratio ═ 5:1) and composed of various concentrations of "2 +1IgG scFab" (see SEQ ID NO:5, 21, 23), "2 +1IgG Crossfab" (see SEQ ID NO:3, 5, 29, 33) and "(scFv)2"killing of MDA-MB-435 tumor cells by bispecific molecules and corresponding IgG activated for 20 hours (as measured by LDH release).
FIG. 28: killing effect on MDA-MB-435 tumor cells (as measured by LDH release) in co-culture with human pan T cells (E: T ratio ═ 5:1) and activated for 20 hours by different concentrations of bispecific construct and corresponding IgG. The "2 +1IgG scFab" construct and the "2 +1IgG Crossfab" (see SEQ ID NOS: 3, 5, 29, 33) construct were compared that differed in their Fc domains (with either wild-type Fc domain (see SEQ ID NOS: 5, 13, 15), or an Fc domain mutated to abrogate (NK) effector function: P329G LALA (see SEQ ID NOS: 5, 21, 23), P329G LALAN297D (see SEQ ID NOS: 5, 25, 27)).
FIG. 29: and human pan T cells (E: T ratio)Rate 5:1) killing of Colo-38 tumor cells in coculture (as measured by LDH release) with CD3-MCSP bispecific "2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) constructs, "(scFv)2"molecules or corresponding IgG treatment for 18.5 hours.
FIG. 30: killing of Colo-38 tumor cells (as measured by LDH release) in coculture with human pan-T cells (E: T ratio ═ 5:1) with CD3-MCSP bispecific "2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) constructs, "(scFv)2"molecules or corresponding IgG treatment for 18 hours.
FIG. 31: co-cultured with human pan-T cells (E: T ratio ═ 5:1) and composed of CD3-MCSP bispecific "2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) constructs, "(scFv) at various concentrations2"killing of MDA-MB-435 tumor cells by molecules or corresponding IgG activated for 23.5 hours (as measured by LDH release).
FIG. 32: co-culture with human pan-T cells (E: T ratio ═ 5:1) and from various concentrations of CD3-MCSP with dual specificities "1 +1IgG scFab, single arm" (see SEQ ID NO:1, 3, 5), "1 +1IgG scFab, single arm inversion" (see SEQ ID NO:7, 9, 11) or "(scFv)2"killing of Colo-38 tumor cells (as measured by LDH release) with 19 hours of activation of the construct or corresponding IgG.
FIG. 33: killing of Colo-38 tumor cells (as measured by LDH release) in coculture with human pan-T cells (E: T ratio ═ 5:1) with "1 +1IgG scFab" CD3-MCSP bispecific constructs (see SEQ ID NO:5, 21, 213) or "(scFv)2"molecular treatment for 20 hours.
FIG. 34: killing effect on MDA-MB-435 tumor cells (as measured by LDH release) in coculture with human pan T cells (E: T ratio ═ 5:1) and activation by different concentrations of bispecific construct and corresponding IgG for 21 hours. The CD3-MCSP bispecific "2 +1IgG Crossfab" (see SEQ ID NOS: 3, 5, 29, 33) and "1 +1 IgGCrossfab" (see SEQ ID NOS: 5, 29, 31, 33) constructs, "" were compared "(scFv)2"molecule and corresponding IgG.
FIG. 35: killing (as measured by LDH release) of different target cells (MCSP positive Colo-38 tumor target cells, mesenchymal stem cells derived from bone marrow or adipose tissue, or pericytes from placenta; as shown) induced by activation of human T cells (E: T ratio 25:1) with 135ng/ml or 1.35ng/ml "2 +1IgG Crossfab" CD3-MCSP bispecific constructs (see SEQ ID NOs: 3, 5, 29, 33).
FIG. 36: killing of Colo-38 tumor target cells (as measured by LDH release) measured after 21 hours incubation overnight when co-cultured with human PBMC and different CD3-MCSP bispecific constructs ("2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) and "(scFv) 2") or glycoengineered anti-MCSP IgG (GlycoMab). The effector to target cell ratio was set at 25:1(A), or varied as described (B). PBMC were isolated from fresh blood (A) or from a dark yellow overlay (B).
FIG. 37: time-dependent cytotoxic effects of the "2 +1IgG Crossfab" construct targeting cynomolgus monkey CD3 and human MCSP (see SEQ ID NO:3, 5, 35, 37). Lactate dehydrogenase release from human MCSP-expressing MV-3 cells at 24 hours or 43 hours of co-culture with primary cynomolgus PBMC (E: T ratio ═ 3:1) is described. As a control, the same molarity of the volumes of reference IgGs (anti-cynoCD 3IgG and anti-human MCSP IgG) was used. PHA-M served as a control for T cell activation (non-physiologic).
FIG. 38: killing of huMCSP positive MV-3 melanoma cells (as measured by LDH release) in coculture with human PBMC (E: T ratio ═ 10:1) using different CD3-MCSP bispecific constructs ("2 +1IgG Crossfab" (see SEQ ID NOs: 3, 5, 29, 33) and "(scFv)2") for about 26 hours.
FIG. 39: killing of EGFR-positive LS-174T tumor cells (as measured by LDH release) in coculture with human pan-T cells (E: T ratio ═ 5:1) using different CD3-EGFR bispecific constructs ("2 +1IgG scFab" (see SEQ ID NO:45, 47, 53), "1 +1IgG scFab" (see SEQ ID NO:47, 53, 2)13) And "(scFv)2") or reference IgG for 18 hours.
FIG. 40: killing of EGFR-positive LS-174T tumor cells (as measured by LDH release) in coculture with human pan-T cells (E: T ratio ═ 5:1) using different CD3-EGFR bispecific constructs ("1 +1IgG scFab, single arm" (see SEQ ID NO:43, 45, 47), "1 +1IgG scFab, single arm inversion" (see SEQ ID NO:11, 49, 51), "1 +1IgG scFab" (see SEQ ID NO:47, 53, 213) and "(scFv)2") or reference IgG for 21 hours.
FIG. 41: killing of EGFR-positive LS-174T tumor cells (as measured by LDH release) in coculture with human pan-T cells (A) or human naive T cells (B) using different CD3-EGFR bispecific constructs ("1 +1IgG scFab, single arm" (see SEQ ID NO:43, 45, 47), "1 +1 IgGscFab, single arm inversion" (see SEQ ID NO:11, 49, 51) and "(scFv)2") or reference IgG for 16 hours. The effector to target cell ratio was 5: 1.
FIG. 42: killing of FAP-positive GM05389 fibroblasts (as measured by LDH release) in coculture with human pan-T cells (E: T ratio ═ 5:1) with different CD3-FAP bispecific constructs ("1 +1IgG scFab," single-arm inversion "(see SEQ ID NO:11, 51, 55)," 1+1IgG scFab "(see SEQ ID NO:57, 61, 213)," 2+1IgG scFab "(see SEQ ID NO:57, 59, 61) and" (scFv)2") for about 18 hours.
FIG. 43: CD8+Flow cytometric analysis of CD107a/b expression levels and perforin levels in T cells, wherein said CD8+T-cells have been treated with different CD3-MCSP bispecific constructs ("2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) and "(scFv) 2") or corresponding control IgG in the presence (A) or in the absence (B) of target cells for 6 hours. Human pan-T cells were incubated with 9.43nM of the different molecules in the presence or absence of Colo-38 tumor target cells at an effector to target cell ratio of 5: 1. Addition of monensin after 1 hour incubation to prevent eggWhite matter trafficking increases intracellular protein levels. All CD107a/b positive cells, perforin positive cells or double positive cells were gated as described.
FIG. 44: CD3-MCSP bispecific constructs ("2 +1IgG scFab, LALA" (see SEQ ID NO:5, 17, 19) or "(scFv) 2") other than 1nM, or corresponding control IgG, CD8 when incubated at an effector to target cell ratio of 5:1 in the presence or absence of Colo-38 tumor target cells+(A) Or CD4+(B) Relative proliferation of human T cells. CFSE labeled human pan T cells were characterized by FACS. The relative proliferation level is determined by gating the non-proliferating cells around and using the number of cells in this gated case relative to the measured total number of cells as a reference.
FIG. 45: different cytokine levels measured in supernatants of
FIG. 46: bispecific constructs with 1nM different CD3-MCSP bispecific constructs ("2 +1IgG scFab", "2 +1 IgGCrossfab" (see SEQ ID NO:3, 5, 29, 33) or "(scFv)2") or corresponding control IgG, measured in the supernatant of whole blood after 24 hours of treatment in the presence (A, B) or absence (C, D) of Colo-38 tumor cells. Among bispecific constructs there are different "2 +1IgG scFab" constructs with either wild-type Fc domains (see SEQ ID NOs: 5, 13, 15) or Fc domains mutated to abrogate (NK) effector functions (LALA (see SEQ ID NOs: 5, 17, 19), P329G LALA (see SEQ ID NOs: 5, 2, 23) and P329G LALA N297D (see SEQ ID NOs: 5, 25, 27)).
FIG. 47: CE-SDS analysis. SDS PAGE of the attached light chain (see SEQ ID NO:3, 5, 29, 179) as a 2+1IgG Crossfab shows an electrophoretogram. (Lane 1: reduced, Lane 2: non-reduced).
FIG. 48: analytical size exclusion chromatography of 2+1IgG Crossfab, linked light chain (see SEQ ID NOS: 3, 5, 29, 179) (final product). A20. mu.g sample was injected.
FIG. 49: killing of MCSP positive MV-3 tumor cells (as measured by LDH release) in coculture with human PBMC (E: T ratio ═ 10:1) treated with different CD3-MCSP bispecific constructs ("2 +1IgG Crossfab" (see SEQ ID NOs: 3, 5, 29, 33) and "2 +1IgG Crossfab, linked LC" (see SEQ ID NOs: 3, 5, 29, 179)) for about 44 hours. Human PBMCs were isolated from fresh blood of healthy volunteers.
FIG. 50: killing of MCSP positive Colo-38 tumor cells (as measured by LDH release) in coculture with human PBMC (E: T ratio ═ 10:1) treated with different CD3-MCSP bispecific constructs ("2 +1IgG Crossfab" (see SEQ ID NOs: 3, 5, 29, 33) and "2 +1IgG Crossfab, linked LC" (see SEQ ID NOs: 3, 5, 29, 179)) for about 22 hours. Human PBMCs were isolated from fresh blood of healthy volunteers.
FIG. 51: killing of MCSP positive Colo-38 tumor cells (as measured by LDH release) in coculture with human PBMC (E: T ratio ═ 10:1) treated with different CD3-MCSP bispecific constructs ("2 +1IgG Crossfab" (see SEQ ID NOs: 3, 5, 29, 33) and "2 +1IgG Crossfab, linked LC" (see SEQ ID NOs: 3, 5, 29, 179)) for about 22 hours. Human PBMCs were isolated from fresh blood of healthy volunteers.
FIG. 52: killing of MCSP positive WM266-4 cells (as measured by LDH release) in coculture with human PBMC (E: T ratio ═ 10:1) treated with different CD3-MCSP bispecific constructs ("2 +1IgG Crossfab" (see SEQ ID NOs: 3, 5, 29, 33) and "2 +1IgG Crossfab, linked LC" (see SEQ ID NOs: 3, 5, 29, 179)) for about 22 hours. Human PBMCs were isolated from fresh blood of healthy volunteers.
FIG. 53: CD3-MCSP bispecific constructs ("2 +1IgG Crossfab" (see SEQ ID NOS: 3, 5, 29, 33)) and "2 +1IgG Crossfab, linked LC" (see SEQ ID NOS: 3, 5, 29, 179)) that differ from 10nM, 80pM or 3pM, were present or absent in Colo-38 tumor target cells expressing human MCSPHuman CD8 after 22 hours incubation in the presence (E: T ratio 10:1)+Surface expression levels of the early activation marker CD69(a) and the late activation marker CD25(B) on T cells.
FIG. 54: CE-SDS analysis. (A) As 1+1IgG Crossfab; SDS-PAGE of VL/VH swapping (LC007/V9) (see SEQ ID NOS: 5, 29, 33, 181) shows the electrophoretogram: a) non-reduction, b) reduction. (B) As a 1+1 CrossMab; SDS-PAGE of the CL/CH1 exchange (LC007/V9) (see SEQ ID NOs: 5, 23, 183, 185) shows the electrophoretogram: a) reducing, b) non-reducing. (C) Inverted as 2+1IgG Crossfab; CL/CH1 exchange (LC007/V9) (see SEQ ID NOs: 5, 23, 183, 187): a) reduction, b) electrophoresis pattern shown by non-reduced SDS-PAGE. (D) As a 2+1IgG Crossfab; SDS-PAGE of VL/VH crossover (M4-3 ML2/V9) (see SEQ ID NOS: 33, 189, 191, 193) shows the electrophoretogram: a) reducing, b) non-reducing. (E) as a 2+1IgG Crossfab; SDS-PAGE of CL/CH1 exchanges (M4-3 ML2/V9) (see SEQ ID NOs: 183, 189, 193, 195) shows the electrophoretogram: a) reducing, b) non-reducing. (F) Inverted as 2+1 IgGCrossfab; SDS-PAGE of the CL/CH1 exchange (CH1A1A/V9) (see SEQ ID NOS: 65, 67, 183, 197) shows the electrophoretogram: a) reducing, b) non-reducing. (G) As a 2+1IgG Crossfab; SDS-PAGE of CL/CH1 exchange (M4-3 ML2/H2C) (see SEQ ID NOS: 189, 193, 199, 201) shows the electrophoretogram: a) reducing, b) non-reducing. (H) Inverted as 2+1IgG Crossfab; SDS-PAGE of the CL/CH1 exchange (431/26/V9) (see SEQ ID NOs: 183, 203, 205, 207) shows the electrophoretogram: a) reducing, b) non-reducing. (I) SDS-PAGE of the fusion as a "2 +1IgG Crossfab light chain" (CH1A1A/V9) (see SEQ ID NOS: 183, 209, 211, 213) shows the electrophoretogram: a) reducing, b) non-reducing. (J) SDSPAGE (4-12% Bis/Tris, NuPage Invitrogen, Coomassie staining), non-reduced (left) and reduced (right) of "2 +1IgG Crossfab" (anti-MCSP/anti-huCD 3) (see SEQ ID NOS: 5, 23, 215, 217). (K) SDS-PAGE as "2 +1IgG Crossfab, inverted" (anti-MCSP/anti-huCD 3) (see SEQ ID NO:5, 23, 215, 219) shows the electrophorogram: a) reducing, b) non-reducing. (L) "1 +1IgG Crossfab" (
FIG. 55: bispecific constructs (CEA/CD3 "2 +1IgG Crosfab, inversion (VL/VH)" (see SEQ ID NOs: 33, 63, 65, 67) and "2 +1IgG Crosfab, inversion (CL/CH1)
2 (see SEQ ID NOs: 65, 67, 183, 197)) binds to human CD3 expressed by Jurkat cells (a) or to human CEA expressed by LS-174T cells (B), as determined by FACS. As a control, the equivalent maximum concentration of reference IgG and the secondary antibody due to labeling were also assessed (goat anti-human FITC conjugated affinity pure F (ab')2Fragment, Fc gamma fragment specificity, Jackson immune Research Lab # 109-.
FIG. 56: the bispecific constructs (MCSP/CD3 "2 +1IgG Crosfab" (see SEQ ID NO:3, 5, 29, 33) and "2 +1IgG Crosfab, inverted" (see SEQ ID NO:5, 23, 183, 187)) bound to human CD3 expressed by Jurkat cells (A) or to human MCSP expressed by WM266-4 tumor cells (B), as determined by FACS.
FIG. 57: binding of "1 +1IgG Crossfab light chain fusions" (see SEQ ID NOS: 183, 209, 211, 213) to human CD3 expressed by Jurkat cells (A) or to human CEA expressed by LS-174T cells (B) as determined by FACS.
FIG. 58: binding of the "2 +1IgG Crossfab" (see SEQ ID NO:5, 23, 215, 217) and "2 +1 IgGCrossfab, inverted" (see SEQ ID NO:5, 23, 215, 219) constructs to human CD3 expressed by Jurkat cells (A) or human MCSP expressed by WM266-4 tumor cells (B), as determined by FACS.
FIG. 59: with the indicated concentrations of CD3/MCSP "1 +1 CrossMab" (see SEQ ID NO:5, 23, 183, 185), "1 +1IgG Crossfab" (see SEQ ID NO:5, 29, 33, 181) and "2 +1IgG CrossfabCrossfab "(see SEQ ID NOs: 3, 5, 29, 33) constructs after 24 hours incubation, human CD4+Or CD8+Surface expression levels of the early activation marker CD69(a) or the late activation marker CD25(B) on T cells. As shown, the assay is performed in the presence or absence of MV-3 target cells.
FIG. 60: with cynomolgus monkey PBMC in the presence or absence of huMCSP positive MV-3 tumor cells (E: T ratio ═ 3:1, normalized to CD3+Number) of cynomolgus monkeys (a and B), CD4 from two different cynomolgus monkeys (a and B)+Or CD8+Surface expression level of the early activation marker CD25 on T cells, wherein said cynomolgus monkey PBMCs were treated with "2 +1IgG Crossfab" (see SEQ ID NO:5, 23, 215, 217) and "2 +1IgG Crossfab, inverted" (see SEQ ID NO:5, 23, 215, 219) for about 41 hours.
FIG. 61: killing of tumor cells MKN-45(a) or LS-174T (b) (as measured by LDH release) with different concentrations of the "2 +1IgG Crossfab, inversion (VL/VH)" (see SEQ ID NOs: 33, 63, 65, 67) activated for 28 hours in co-culture with human PBMC (E: T ratio ═ 10:1) and the constructs inverted (CL/CH1) "(see SEQ ID NOs: 65, 67, 183, 197) relative to the" 2+1IgG Crossfab, as measured by LDH release.
FIG. 62: killing of the tumour cells WM266-4 (as measured by LDH release) was achieved by co-culturing with human PBMC (E: T ratio ═ 10:1) and activating 26 hours with different concentrations of "2 +1IgG Crossfab (VL/VH)" (see SEQ ID NOs: 33, 189, 191, 193) relative to the "2 +1IgG Crossfab (CL/CH 1)" (see SEQ ID NOs: 183, 189, 193, 195) construct.
FIG. 63: killing of tumor cells MV-3 (as measured by LDH release) was achieved by co-culturing with human PBMC (E: T ratio ═ 10:1) and activating 27 hours with different concentrations of "2 +1IgG Crossfab (VH/VL)" (see SEQ ID NOs: 33, 189, 191, 193) relative to the "2 +1IgG Crossfab (CL/CH 1)" (see SEQ ID NOs: 183, 189, 193, 195) construct.
FIG. 64: killing of human MCSP positive 266-4(a) or MV-3(B) tumor cells (as measured by LDH release) in coculture with human PBMC (E: T ratio ═ 10:1) and activated for 21 hours by various concentrations of "2 +1IgG Crossfab" (see SEQ ID NOs: 3, 5, 29, 33), "1 +1 CrossMab" (see SEQ ID NOs: 5, 23, 183, 185), and "1 +1IgG Crossfab" (see SEQ ID NOs: 5, 29, 33, 181) as shown.
FIG. 65: killing of tumor cells MKN-45(a) or LS-174T (b) (as measured by LDH release) in co-culture with human PBMC (E: T ratio ═ 10:1) and activated by different concentrations of "1 +1IgG CrossfabLC fusions" (see SEQ ID NOs: 183, 209, 211, 213) for 28 hours.
FIG. 66: killing of MC38-huCEA tumor cells (as measured by LDH release) in co-culture with human PBMC (E: T ratio ═ 10:1) and activated by different concentrations of "1 +1IgG Crossfab LC fusions" (see SEQ ID NOs: 183, 209, 211, 213) for 24 hours relative to the non-targeted reference "2 +1 IgGCrossfab".
FIG. 67: killing of human MCSP positive MV-3(a) or WM266-4(B) tumor cells (as measured by LDH release) in coculture with human PBMC (E: T ratio ═ 10:1) treated with "2 +1IgG Crossfab (V9)" (see SEQ ID NOs: 3, 5, 29, 33) and "2 +1IgG Crossfab, inverted (V9)" (see SEQ ID NOs: 5, 23, 183, 187), "2 +1IgG Crossfab (anti-CD 3)" (see SEQ ID NOs: 5, 23, 215, 217) and "2 +1 IgGCrossfab, inverted (anti-CD 3)" (see SEQ ID NOs: 5, 23, 215, 219).
FIG. 68: schematic representation of MCSP TCB (2+1Crossfab-IgG P329G LALA inverted) molecule.
FIG. 69: CE-SDS analysis of MCSP TCB (2+1Crossfab-IgG P329G LALA inverted, SEQ ID NO:278, 319, 320, 321). SDS-PAGE of MCSP TCB shows the electrophoretogram: A) non-reduction, B) reduction.
FIG. 70: analytical size exclusion chromatography of MCSP TCB (2+1Crossfab-IgG P329G LALA inverted SEQ ID NO:278, 319, 320, 321) chromatogram A280(TSKgel G3000 SW XL [ Tosoh)]；25mM K2HPO4125mM NaCl, 200mM L-arginine monohydrochloride, 0.02% (w/v) NaN3pH 6.7; a20. mu.g sample was injected.
FIG. 71: schematic representation of CEA TCB (2+1Crossfab-IgG P329G LALA inverted) molecule.
FIG. 72: CE-SDS analysis of CEA TCB (2+1Crossfab-IgG P329G LALA inverted SEQ ID NO:288, 322, 323, 324)) molecules. SDS-PAGE of CEA TCB shows the electrophoretogram: A) non-reduction, B) reduction.
FIG. 73: analytical size exclusion chromatography of CEA TCB (2+1Crossfab-IgG P329G LALA inverted SEQ ID NO:288, 322, 323, 324)) molecules, chromatogram A280(TSKgel G3000 SW XL [ Tosoh ]]； 25mMK2HPO4125mM NaCl, 200mM L-arginine monohydrochloride, 0.02% (w/v) NaN3pH 6.7; a20. mu.g sample was injected.
FIG. 74: MCSP TCB (SEQ ID NO:278, 319, 320, 321) with A375 cells (MCSP +) (A) and Jurkat (CD3+ cells) (B). "non-targeted TCB": a bispecific antibody that binds CD3 but not a second antigen.
FIG. 75: t cell killing induced by MCSP TCB antibody (SEQ ID NO:278, 319, 320, 321) on a375 (high MCSP) (a), MV-3 (medium MCSP) (B), HCT-116 (low MCSP) (C) and LS180(MCSP negative) (D) target cells (E: T ═ 10:1, effector cells human PBMC,
FIG. 76: CD25 and CD69 are upregulated on human CD8+ (A, B) and CD4+ (C, D) T cells after MCSP TCB antibodies (SEQ ID NOs: 278, 319, 320, 321) induce T cell mediated killing of MV3 melanoma cells (E: T ═ 10:1, 24 hour incubation). "non-targeted TCB": a bispecific antibody that binds CD3 but does not bind a second antigen.
Figure 77 human PBMCs secrete IL-2(a), IFN- γ (B), TNF α (C), IL-4(D), IL-10(E) and granzyme B (f) after MCSP TCB antibody (SEQ ID NOs: 278, 319, 320, 321) induces T cell mediated killing of MV3 melanoma cells (E: T ═ 10:1, 24 hour incubation): non-targeted TCB: bispecific antibody that binds CD3 but not a second antigen.
FIG. 78: CEA TCB (SEQ ID NO:288, 322, 323, 324) binding to LS180 (intermediate CEA tumor cells) (A) and Jurkat (CD3+ cells) (B).
FIG. 79: t cell killing induced by CEA TCB (SEQ ID NOs: 288, 322, 323, 324) on MKN45 (high CEA) (a), LS180 (medium CEA) (B), HT-29 (low CEA) (C) (E: T ═ 10:1, effector cells human PBMC,
FIG. 80: CD25 and CD69 on human CD8+ (A, B) and CD4+ (C, D) T cells were upregulated after CEA TCB (SEQ ID NO:288, 322, 323, 324) induced T cell mediated killing of LS180 colon adenocarcinoma cells (E: T ═ 10:1, 24 hour incubation). "non-targeted TCB": a bispecific antibody that binds CD3 but does not bind a second antigen.
Figure 81 secretion of IFN- γ (a), TNF α (B), granzyme B (c), IL-4(D), IL-10 (E). "non-targeted TCB" following induction of T cell mediated killing of LS180 colon adenocarcinoma cells by CEA TCB (SEQ ID NOs: 288, 322, 323, 324) (E: T ═ 10:1, 24 hours incubation): bispecific antibody conjugated to CD3 but not to a second antigen.
FIG. 82: CE-SDS analysis of DP47GS TCB (2+1Crossfab-IgG P329G LALA inverted ═ non-targeted TCB ", SEQ ID NO:325, 326, 327, 328) containing DP47GS as non-binding antibody and humanized CH2527 as anti-CD 3 antibody. SDS-PAGE of DP47GS TCB shows the electrophoretogram: A) non-reduction, B) reduction.
FIG. 83: analytical size exclusion chromatography of DP47GS TCB (2+1Crossfab-IgG P329G LALA inverted "non-targeted TCB", SEQ ID NOS: 325, 326, 327, 328) containing DP47GS as a non-binding antibody and humanized CH2527 as a CD3 antibody, chromatogram A280(TSKgel G3000 SW XL [ Tosoh)]；25mM K2HPO4125mM NaCl, 200mM L-arginine monohydrochloride, 0.02% (w/v) NaN3pH 6.7; a20. mu.g sample was injected.
FIG. 84: comparison of affinity matured anti-MCSP clones compared to the immature parental clone (M4-3 ML 2).
FIG. 85: schematic representation of aVH TCB molecules.
FIG. 86: CE-SDS analysis. As an electrophoretogram displayed by SDS-PAGE of aVH TCB (SEQ ID NO:369, 370, 371): A) non-reduction, B) reduction.
FIG. 87: binding of aVH TCB (SEQ ID NO:369, 370, 371) to MV-3 cells (MCSP +) (A) and Jurkat (CD3+ cells) (B).
FIG. 88: t cell killing induced by the ahh TCB antibody (SEQ ID NOs: 369, 370, 371) on MV-3 melanoma cells detected at 24 hours (a) and 48 hours (B) after incubation (E: T ═ 10:1, effector cells human PBMC).
FIG. 89: schematic representation of Darpin TCB molecules.
FIG. 90: CE-SDS analysis. An electrophoretogram shown as SDS-PAGE of DarpinTCB (SEQ ID NO:376, 377, 378): A) non-reduction, B) reduction.
FIG. 91: darpin TCB binds to KPL-4 cells (Her2+) (A) and Jurkat (CD3+ cells) (B).
FIG. 92: t cell killing induced by Darpin TCB antibody (SEQ ID NO:376, 377, 378) on KPL-4 cells detected at 24 hours (A) and 48 hours (B) after incubation (E: T ═ 10:1, effector cells human PBMC).
FIG. 93: schematic representation of hIgG1 DDKK TCB molecules.
FIG. 94: CE-SDS analysis. An electrophoretogram shown as SDS-PAGE of hIgG1 DDKK TCB (SEQ ID NOS: 372, 373, 374, 375): A) non-reduction, B) reduction.
FIG. 95: hIgG1 DDKK TCB (SEQ ID NO:372, 373, 374, 375) binding to MV-3 melanoma cells (MCSP +) (A) and Jurkat (CD3+ cells) (B).
FIG. 96: t cell killing induced by the hIgG1 DDKKTCB antibody (SEQ ID NO:372, 373, 374, 375) against MV-3 (moderate MCSP) detected at 24 hours (a) and 48 hours (B) after incubation (E: T ═ 10:1, effector cells human PBMC).
Detailed Description
Definition of
Unless defined otherwise below, terms are used herein as they are commonly used in the art.
As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives thereof, such as fragments thereof.
The term "bispecific" means that the antigen binding molecule is capable of specifically binding to at least two different antigenic determinants. In general, bispecific antigen binding molecules comprise two antigen binding sites, each antigen binding site being specific for a different antigenic determinant. In certain embodiments, a bispecific antigen binding molecule is capable of binding two antigenic determinants simultaneously, in particular two antigenic determinants expressed on two different cells.
The term "valency" as used herein refers to the presence of a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "binds monovalently to an antigen" means that there is one (and no more than one) antigen binding site in the antigen binding molecule that is specific for the antigen.
An "antigen binding site" relates to a site, i.e. one or more amino acid residues, in an antigen binding molecule that provides for interaction with an antigen. For example, the antigen binding site of an antibody comprises amino acid residues from Complementarity Determining Regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites and a Fab molecule typically has a single antigen binding site.
As used herein, the term "antigen-binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding moiety is capable of directing the entity (e.g., the second antigen-binding moiety) attached thereto to a target site, e.g., to a specific type of tumor cell or tumor stroma that carries an antigenic determinant. In another embodiment, the antigen binding portion is capable of activating signaling via a target antigen (e.g., a T cell receptor complex antigen). Antigen-binding portions include antibodies and fragments thereof, as well as binding proteins and scaffolds as further defined herein. A particular antigen-binding portion comprises an antigen-binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region. Other antigen binding portions comprise a binding protein comprising at least one ankyrin repeat motif and a Single Domain Antigen Binding (SDAB) molecule.
Useful heavy chain constant regions include any of the five isoforms α, delta, epsilon, gamma, or mu.useful light chain constant regions include any of the two isoforms kappa and lambda.
As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule to which an antigen-binding portion binds, forming an antibody antigen-binding portion-antigen complex (e.g., a contiguous stretch of amino acids or a conformational configuration composed of discrete regions of amino acids). Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and/or in the extracellular matrix (ECM). Unless otherwise indicated, a protein referred to herein as an antigen (e.g., MCSP, FAP, CEA, EGFR, CD33, CD3) can be any native form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a specific embodiment, the antigen is a human protein. In reference to a particular protein herein, the term encompasses "full-length," unprocessed protein as well as any form of protein that results from processing in a cell. The term also encompasses naturally occurring proteins, e.g., splice variants or allelic variants. Exemplary human proteins that can be used as antigens include, but are not limited to: melanoma-associated chondroitin sulfate proteoglycan (MCSP), also known as chondroitin sulfate proteoglycan 4(UniProt No. Q6UVK1 (70 th edition), NCBI RefSeq No. NP _ 001888.2); fibroblast Activation Protein (FAP), also known as Seprase (UniProt accession No. Q12884, Q86Z29, Q9999998, NCBI accession No. NP _ 004451); carcinoembryonic antigen (CEA), also known as carcinoembryonic antigen-associated cell adhesion molecule 5(UniProt No. P06731 (119 th edition), NCBI RefSeq No. np _ 004354.2); CD33, also known as gp67 or Siglec-3(UniProt number P20138, NCBI accession number NP-001076087, NP-001171079); epidermal Growth Factor Receptor (EGFR), also known as ErbB-1 or Her1(UniProt accession No. P0053, NCBI accession No. NP-958439, NP-958440), and CD3, in particular CD3 epsilon subunit (see UniProt accession No. P07766 (130 th edition), NCBINCBIRefSeq No. NP-000724.1, human sequence SEQ ID NO: 265; or UniProt accession No. Q95LI5 (49 th edition), NCBIGenBank No. BAB71849.1, Macaca fascicularis sequence SEQ ID NO: 266). In certain embodiments, the T cell activating bispecific antigen binding molecules of the invention bind to T cell activating epitopes or target cell epitopes that are conserved between T cell activating antigens or target antigens from different species.
As used herein, the term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding moiety to bind to a particular epitope can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (analysis on a BIAcore instrument) (Liljebllad et al, Glyco J17, 323-. In one embodiment, the extent of binding of the antigen-binding portion to an unrelated protein is less than about 10% of the extent of binding of the antigen-binding portion to the antigen, as measured by SPR. In certain embodiments, the antigen-binding portion that binds to an antigen or an antigen-binding molecule comprising the antigen-binding portion has a molecular weight of 1 μ M or less, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less (e.g., 10nM or less)-8M or less, e.g. from 10-8M to 10-13M, e.g. from 10-9M to 10-13M) dissociation constant (K)D)。
"affinity" refers to the sum of the strengths of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between the members of a binding pair (e.g., an antigen-binding portion and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)D) Typically, dissociation constants are the dissociation and association rate constants (k, respectively)offAnd kon) The ratio of (a) to (b). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein. A specific method for measuring affinity is Surface Plasmon Resonance (SPR).
"reduced binding", e.g. reduced Fc receptor binding, refers to a decrease in affinity of the corresponding interaction, as measured by e.g. SPR. For the sake of clarity, the term also includes a reduction of the affinity to zero (or below the detection limit of the analytical method), i.e.a complete elimination of the interaction. Conversely, "increased binding" refers to an increase in the binding affinity of the corresponding interaction.
As used herein, "T cell activating antigen" refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, wherein said antigenic determinant is capable of inducing T cell activation upon interaction with an antigen binding molecule. In particular, the interaction of the antigen binding molecule with a T cell activating antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In a specific embodiment, the T cell activating antigen is
As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity and activation marker expression. The bispecific antigen binding molecules of the invention that activate T cells are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art and described herein.
As used herein, "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell (e.g., a cell in a tumor, such as a cell of a cancer cell or tumor stroma).
As used herein, the terms "first" and "second" are used to facilitate differentiation with respect to an antigen-binding moiety, etc., when more than one moiety of each type is present. The use of these terms is not intended to confer a particular order or orientation to the T cell activating bispecific antigen binding molecule unless specifically stated as such.
"Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain ("Fab heavy chain") and the VL and CL domains of its light chain ("Fab light chain").
By "fused" is meant that the components (e.g., Fab molecule and Fc domain subunit) are linked by peptide bonds, either directly or through one or more peptide linkers.
By "exchanged Fab molecule (also referred to as" Crossfab ") is meant a Fab molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged, i.e., an exchanged Fab molecule comprises a peptide chain consisting of the light chain variable region and the heavy chain constant region, and a peptide chain consisting of the heavy chain variable region and the light chain constant region. For clarity, in an exchanged Fab molecule in which the variable regions of the Fab light chain and Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the exchanged Fab molecule. In contrast, in an exchanged Fab molecule in which the constant regions of the Fab light and Fab heavy chains are exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the exchanged Fab molecule.
Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain, also known as a light chain constant region1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1) And α2(IgA2). The light chains of immunoglobulins can be divided into one of two classes, called kappa and lambda, based on the amino acid sequence of their constant domains. An immunoglobulin essentially consists of two Fab molecules and one Fc domain connected by means of an immunoglobulin hinge region.
The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen binding activity.
An "antibody fragment" refers to a molecule other than an intact antibody, which molecule includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')2For reviews of certain antibody fragments see Hudson et al, Nat Med 9,129-2See U.S. Pat. No. 5,869,046 for a discussion of fragments. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al,
The term "antigen binding domain" refers to a portion of an antibody that comprises a region that specifically binds to and is complementary to a portion or the entire antigen. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
The term "binding protein comprising at least one ankyrin repeat motif" refers to a binding protein as described in WO2002/020565 and WO 2012069655, which are incorporated herein by reference. These binding proteins are also known as "DARPins" (acronym for designed ankyrin repeat proteins) and are genetically engineered antibodies that mimic proteins that typically display highly specific and high affinity target protein binding. They are derived from natural ankyrin and consist of at least one repeating motif. Exemplary binding proteins targeting HER2 comprising at least one ankyrin repeat motif are described in Zahnd, c. et al, j.mol.biol. (2007)369, 1015-. Other binding proteins such as fibronectin type III domain based adectin, lipocalin based anti-transporter (Anticalin), ubiquitin based Affilins, transferrin based Transbody, protein a domain based affibodies, tetranectin domain based TrimerX, Cys rich domain based MicroProtein, FynSH3 domain based Fynomer, EGFRA domain based high affinity multimers (Avimers), centryrin based Centyrin, Kuniz domain based kaliibortor and other scaffold proteins with randomized binding regions and antibody-like behavior are also encompassed by the present invention.
The term "single domain antigen binding molecule" refers to an antibody fragment consisting of a single, monomeric antibody variable domain, as described in EP 0656946 (incorporated herein in its entirety by reference). Like an intact antibody, it is capable of selectively binding to a particular antigen. The molecular weight is only 12-15 kDa, the single domain antibody is much smaller than the common antibody consisting of two protein heavy and two light chains (150-160 kDa) and even smaller than the Fab fragment (about 50kDa, one light and one half heavy chains) and the single chain variable fragment (about 25kDa, two variable domains, one from the light and one from the heavy chain). In particular, single domain antigen binding molecules are single domain variable heavy chains consisting of one variable domain (VH), also known as autovariable heavy chain (aVH) antibodies. These peptides have similar affinity for antigen as intact antibodies, but are more thermostable and more stable to detergents and high concentrations of urea. The lower molecular mass results in better permeability in the tissue and a short plasma half-life, since they are eliminated in a renal manner.
The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, for example, Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007). A single VH domain or VL domain may be sufficient to confer antigen binding specificity.
As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is highly variable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from Complementarity Determining Regions (CDRs) which have the highest sequence variability and/or are involved in antigen recognition. In addition to CDR1 in VH, the CDRs typically comprise amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used interchangeably herein in reference to the portions of the variable regions that form the antigen-binding regions. Such specific regions have been described by Kabat et al, U.S. department of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al, J.mol.biol.196:901-917(1987), wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. However, the use of any one of the definitions referring to the CDRs of an antibody or variant thereof is intended to be within the scope of the terms as defined and used herein. By way of comparison, suitable amino acid residues that make up the CDRs as defined in each of the references cited above are described below in table a. The exact number of residues that make up a particular CDR will vary depending on the sequence and size of that CDR. One skilled in the art can routinely determine which residues make up a particular CDR based on the variable region amino acid sequence of the antibody.
CDR definitions1
CDR
Kabat
Chothia
AbM2
VH CDR1
31-35
26-32
26-35
VH CDR2
50-65
52-58
50-58
VH CDR3
95-102
95-102
95-102
VL CDR1
24-34
26-32
24-34
VL CDR2
50-56
50-52
50-56
VL CDR3
89-97
91-96
89-97
1The numbering is defined for all CDRs in Table A according to the numbering convention described by Kabat et al (see below)
2"AbM" with lower case "b" as used in table a refers to the CDRs as defined by Oxford Molecular "AbM" antibody modeling software.
Kabat et al also define a variable region sequence numbering system suitable for use with any antibody. One of ordinary skill in the art can apply this Kabat numbering system unambiguously to any variable region sequence, independent of any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. department of Health and Human Services, "Sequences of Proteins of immunological Interest" (1983). Unless otherwise indicated, reference to the numbering of a particular amino acid residue position in a variable region of an antibody is according to the Kabat numbering system.
The polypeptide sequences of the sequence listing were not numbered according to the Kabat numbering system. However, it is well within the ability of one of ordinary skill in the art to convert the numbering of the sequences of the sequence listing to Kabat numbering.
"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following 4 FR domains: FR1, FR2, FR3 and
The "class" of antibodies or immunoglobulins refers to the type of constant domain or constant region that the heavy chain possesses. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes may be further divided into subclasses (isotypes), e.g., IgG1、IgG2、IgG3、IgG4、IgA1And IgA2The heavy chain constant domains corresponding to different classes of antibodies or immunoglobulins are designated α, δ, ε, γ, and μ, respectively.
The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. This definition includes native sequence Fc regions and variant Fc regions. Although the limits of the Fc region of IgG heavy chains may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system as described in Kabat et al, Sequences of Proteins of immunological Interes, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD,1991, also known as the EU index. As used herein, a "subunit" of an Fc domain refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide capable of stable self-association comprising the constant region of the C-terminus of an immunoglobulin heavy chain. For example, subunits of the IgG Fc domain comprise IgG CH2 and IgG CH3 constant domains.
A "modification that facilitates association of the first and second subunits of the Fc domain" is manipulation of the peptide backbone or post-translational modification of the Fc domain subunits that reduces or prevents association of a polypeptide comprising an Fc domain subunit with the same polypeptide to form a homodimer. As used herein, modifications that promote association specifically include independent modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that require association, wherein the modifications are complementary to each other, thereby promoting association of the two Fc domain subunits. For example, modifications that promote association can alter the structure or charge of one or both Fc domain subunits, thereby making their association sterically or electrostatically favorable, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may differ in the sense that the other components (e.g., antigen binding portions) fused to each subunit are not identical. In some embodiments, the modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a specific embodiment, the association-promoting modification comprises an independent amino acid mutation, in particular an amino acid substitution, in each of the two subunits of the Fc domain. In one embodiment, the modification that facilitates association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering effects, for example, as described in PCT publication WO 2009/089004. Typically, this approach involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues, such that homodimer formation becomes electrostatically unfavorable and heterodimerization is electrostatically favorable.
The term "effector function" refers to those biological activities attributed to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding interactions, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptors) and B cell activation.
As used herein, the term "engineered, engineered" is considered to encompass any manipulation of the peptide backbone or post-translational modification of naturally occurring or recombinant polypeptides or fragments thereof. Engineering includes modification of the amino acid sequence, glycosylation patterns, or side chain groups of individual amino acids, as well as combinations of these schemes.
The term "amino acid mutation" as used herein is intended to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor or increased association with another peptide. Amino acid sequence deletions and insertions include amino-terminal and/or carboxy-terminal deletions and insertions of amino acids. A particular amino acid mutation is an amino acid substitution. For the purpose of altering binding characteristics, e.g., the Fc region, non-conservative amino acid substitutions, i.e., the substitution of one amino acid for another with different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, may also be used. Various names may be used herein to indicate the same amino acid mutation. For example, substitutions from proline to glycine at position 329 of the Fc domain can be shown as 329G, G329, G329P329G or Pro329 Gly.
As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a product of a particular length. Thus, the definition of "polypeptide" includes internally peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to chains of two or more amino acids, and the term "polypeptide" may be used instead of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Polypeptides may be derived from a natural biological source or produced by recombinant techniques, but are not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis. The polypeptides of the invention may have a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded and polypeptides that do not have a defined three-dimensional structure, but instead can adopt a number of different conformations, are referred to as unfolded.
An "isolated" polypeptide or variant or derivative thereof means a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide may be removed from its native or natural environment. For the purposes of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides that have been isolated, fractionated or partially or substantially purified by any suitable technique.
"percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps, as necessary, to achieve the maximum percent sequence homology and not considering any conservative substitutions as part of the sequence identity, relative to the reference polypeptide sequence. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full-length sequences being compared. However, for purposes herein, the% amino acid sequence identity value was generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authorized by Genentech, inc, and the source code had been submitted with the user document to the U.S. copyright office of washington, dc 20559, where it was registered with U.S. copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif. or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are not changed. In the case of amino acid sequence comparison using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence a with a given amino acid sequence B, or for a given amino acid sequence B (which may alternatively be described as a given amino acid sequence a having or comprising a certain% amino acid sequence identity with a given amino acid sequence B, or for a given amino acid sequence B) is calculated as follows:
100 times a fraction X/Y
Where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the A and B alignments of that program, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A relative to B will not be equal to the% amino acid sequence identity of B relative to A. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mrna), virus-derived RNA, or plasmid dna (pdna). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
An "isolated" nucleic acid molecule or polynucleotide means a nucleic acid molecule, DNA or RNA that has been removed from its natural environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. An isolated polynucleotide includes a polynucleotide molecule contained within a cell that normally contains the polynucleotide molecule, but which is present extrachromosomally or at a chromosomal location different from its native chromosomal location. Isolated RNA molecules include RNA transcripts of the invention, either in vivo or in vitro, as well as both positive and negative strand forms, and double-stranded forms. The isolated polynucleotides or nucleic acids of the invention also include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may be or may comprise regulatory elements such as promoters, ribosome binding sites or transcription terminators.
By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5 'or 3' end positions of the reference nucleotide sequence or anywhere between these end positions, individually interspersed between residues in the reference sequence or interspersed in one or more contiguous groups within the reference sequence. Indeed, it is routinely determined whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).
As used herein, the term "expression cassette" refers to a polynucleotide, produced recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette may be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. In general, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter, among other sequences. In certain embodiments, the expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.
The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene to which it is operatively associated in a target cell and direct its expression. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which the vector has been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once inside the target cell at the expression vector, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding the bispecific antigen binding molecule of the invention or a fragment thereof.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may instead contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The host cell is any type of cellular system that can be used to produce the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, e.g., cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells and plant cells, to name just a few, which also include cells contained within transgenic animals, transgenic plants or cultured plant tissues or animal tissues.
Human activating Fc receptors include Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32), and Fc α RI (CD 89).
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in lysis of antibody-coated target cells by immune effector cells. The target cell is a cell that specifically binds to an antibody or derivative thereof comprising an Fc region (typically via a protein moiety that is N-terminal with respect to the Fc region). As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed by means of the ADCC mechanism defined above within a given time at a given antibody concentration in the medium surrounding the target cells and/or an increase in the antibody concentration required to achieve lysis of a given number of target cells by means of the ADCC mechanism within a given time in the medium surrounding the target cells. ADCC reduction is relative to ADCC mediated by the same antibody produced by the same type of host cell, but which has not been engineered, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art). For example, a reduction in ADCC mediated by an antibody comprising an amino acid substitution in its Fc domain that reduces ADCC is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT publication No. WO 2006/082515 or PCT patent application No. PCT/EP 2012/055393).
An "effective amount" of an agent refers to the amount required to produce a physiological change in the cell or tissue to which the drug is administered.
A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount effective to achieve a desired therapeutic or prophylactic result, a dosage to achieve the foregoing result, and a time period required to continue achieving the foregoing result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of the disease.
An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.
The term "pharmaceutical composition" refers to a preparation in such a form as to allow the biological activity of the active ingredient contained therein to be effective, and not containing additional components that are unacceptably toxic to a subject to whom the preparation will be administered.
"pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition that is not toxic to a subject other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
As used herein, the term "treatment" (and grammatical variations thereof, such as the verb "treat" or the word "treat") refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated, and may be intended to be prophylactic or to be performed during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating a disease state, and ameliorating or improving prognosis. In some embodiments, the T cell activating bispecific antigen binding molecules of the invention are used to delay disease progression or to slow disease progression.
The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
Detailed description of the embodiments
T cell activating bispecific antigen binding molecular patterns
Most antibodies consist of two heavy chains and two light chains. Both chains contribute to the antigen binding site, which is usually flat or concave. In addition to these conventional antibodies, alpacas, other camelids (camelids), and sharks also produce antibodies that consist only of heavy chains. The antigen binding site of these aberrant heavy chain antibodies is formed only by a single domain called VH (auto variable heavy chain) or single domain variable heavy chain. Single domain variable heavy chains are readily produced as recombinant proteins. Other advantageous features of single domain variable heavy chains include their small size, high solubility, thermal stability, ability to refold and good tissue penetration. Single domain antibodies are described, for example, in Wesolowski et al, Med Microbiol Immunol (2009)198: 157-174. Methods of producing single domain variable heavy chain antibodies are described, for example, in WO2012152823 and WO 2012056000, which are incorporated herein in their entirety by reference.
These single domain variable heavy chain antibodies lack the light chain and may also lack the CH1 domain. Thus, the antigen binding site of a single domain variable heavy chain antibody is formed by only a single domain.
Single Domain Antigen Binding (SDAB) molecules include molecules whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules that naturally lack a light chain, nanobodies, single domains derived from conventional 4-chain antibodies, engineered domains other than those derived from antibodies, and single domain scaffolds. The SDAB molecule may be any molecule of the prior art, or any single domain molecule in the future. SDAB molecules may be derived from any species, including but not limited to mouse, human, camel, alpaca, fish, shark, goat, rabbit, and cow. This term also includes naturally occurring single domain antibody molecules from species other than camels and sharks.
In one aspect, the SDAB molecule may be derived from the variable region of an immunoglobulin present in fish, for example, that derived from an immunoglobulin isotype known as neoantigen receptor (NAR) present in shark serum. Methods for generating single domain molecules ("IgNARs") derived from the NAR variable region are described in WO 03/014161 and Streltsov (2005) Protein Sci.14: 2901-2909.
According to another aspect, the SDAB molecule is a naturally occurring single domain antigen binding molecule, referred to as a heavy chain lacking a light chain. Such single domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al, (1993) Nature 363: 446-. For the sake of clarity, such a variable domain derived from a heavy chain molecule naturally lacking a light chain is referred to herein as a VHH or nanobody (TM) to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules may be derived from camelus (Camelidae) species, such as camel, alpaca, dromedary, camel and guanaco. Other species than camelids may produce heavy chain molecules that naturally lack a light chain; such VHHs are within the scope of the invention.
SDAB molecules have been described by, for example, EP 0656946, which is incorporated herein in its entirety by reference.
SDAB molecules can be recombinant, CDR grafted, humanized, camelized, de-immunized, and/or generated in vitro (e.g., by phage display selection). Single domain antibodies can be obtained by immunizing a camel, alpaca or shark with the desired antigen and subsequently isolating the mRNA encoding the heavy chain antibody. By reverse transcription and polymerase chain reaction, a gene library of single domain antibodies containing millions of clones is generated. Screening techniques such as phage display and ribosome display help identify clones that bind the antigen. A different approach uses a gene library from animals that have not been previously immunized. Such initial libraries typically contain only antibodies with low affinity for the desired antigen, which requires the use of affinity maturation via random mutagenesis as an additional step. When the most potent clones have been identified, their DNA sequences are optimized, for example to improve their stability towards enzymes. Another object is humanization to prevent the immune response of the human organism to the antibody. Humanization does not pose a problem because of the homology between camelid VHH and human VH fragments. The final step is the translation of the optimized single domain antibody in E.coli, Saccharomyces cerevisiae or other suitable organisms. Alternatively, single domain antibodies can be produced from common murine or human IgG with 4 chains. The process is similar, including gene libraries from immunized donors or naive donors and display techniques for identifying the most specific antigens.
In one embodiment, there is provided a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to a T cell activating antigen and a second antigen binding moiety capable of specific binding to a target cell antigen, wherein said one antigen binding moiety is an exchange Fab molecule, wherein the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged and wherein the other antigen binding moiety consists of a single domain antigen binding molecule.
In certain embodiments, the single domain antigen binding molecule is a human single domain binding molecule (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1).
In one embodiment, there is provided a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to a T cell activating antigen and a second antigen binding moiety capable of specific binding to a target cell antigen, wherein the antigen binding moieties are Fab molecules or exchanged Fab molecules, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the other antigen binding moiety consists of a single domain variable heavy chain.
In another embodiment, there is provided a T cell activating bispecific antigen binding molecule wherein the first antigen binding moiety capable of specific binding to a T cell activating antigen is a Fab molecule or an exchanged Fab molecule, wherein the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged and wherein the second antigen binding moiety capable of specific binding to a target cell antigen consists of a single domain variable heavy chain.
The bispecific antibodies of the invention may comprise one or more interchangeable Fab fragments. A crossover Fab fragment is a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. Bispecific antibody formats comprising exchanged Fab fragments have been described, for example, in WO 2009080252, WO2009080253, WO2009080251, WO 2009080254, WO 2010/136172, WO 2010/145792 and EP patent application No. 11178371.8, which are incorporated herein by reference.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a binding protein comprising a single domain antigen binding molecule and comprises no more than one antigen binding moiety capable of specifically binding to a T cell activating antigen.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises one antigen binding portion comprising a single domain antigen binding molecule fused to another antigen binding portion comprising a Fab molecule or a crossover Fab molecule, wherein the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged. Optionally, the antigen binding moieties are fused to each other by a peptide linker.
In one embodiment, the single domain antigen binding molecule is fused to the N-terminus of the heavy chain of the crossover Fab molecule.
In one embodiment, the single domain antigen binding molecule is fused to the N-terminus of the light chain of the crossover Fab molecule.
In one embodiment, the T cell activating bispecific antigen binding molecule additionally comprises a third antigen binding moiety capable of specifically binding to a target cell antigen.
In one embodiment, the third antigen binding portion capable of specific binding to a target cell antigen is a single domain antigen binding molecule. In one embodiment, the third antigen binding portion capable of specific binding to a target cell antigen is a single domain variable heavy chain as defined above.
In one embodiment of the invention, the T cell activating bispecific antigen binding molecule further comprises a peptide comprising a first and a second stably associated moleculeFc domains of subunits. In one embodiment, the Fc domain is an IgG, particularly an IgG1Or IgG4An Fc domain of (a). In particular embodiments, the Fc domain may further comprise modifications that facilitate association of the first and second subunits of the Fc domain, as outlined below. In other specific embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function, as outlined below.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion comprising a Fab molecule or an exchanged Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the Fab molecule or the exchanged Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding portion comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen binding portion comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of the Fab heavy chain of the first antigen binding portion.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion capable of specific binding to a T cell activation antigen comprising a Fab molecule or an exchanged Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the Fab molecule or the exchanged Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding portion capable of specific binding to a target cell antigen comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen binding portion capable of specific binding to a target cell antigen comprising a single domain variable heavy chain, wherein the single domain variable heavy chain is fused to the N-terminus of the Fab heavy chain of the first antigen binding portion.
In one embodiment, the second and third antigen binding portions bind to the same target cell antigen.
In one embodiment, the first and/or second antigen-binding portion is directly connected to the Fc domain via a hinge region. In another embodiment, the first and/or second antigen binding moiety is linked to the Fc domain by a peptide linker.
According to any of the above embodiments, the components of the T cell activating bispecific antigen binding molecule (e.g. antigen binding portion, Fc domain) may be fused directly or through various linkers described herein or known in the art, in particular peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. Suitably, the non-immunogenic peptide linker comprises, for example, (G)4S)n、(SG4)n、(G4S)nOr G4(SG4)nPeptide linkers, wherein n is generally a number between 1 and 10, typically between 2 and 4.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises one or more amino acid sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs 369, 370 and 371. In another embodiment, the T cell activating bispecific antigen binding molecule comprises SEQ ID NOS 369, 370 and 371.
In addition to antibodies, there are other binding proteins or binding domains that can be used to specifically bind to a target molecule (e.g., Binz, h.k., Amstutz, p. and Pluckthun, a., nat. biotechnol.23,1257-1268,2005). Such a new class of binding proteins or binding domains is based on designed repeat proteins or designed repeat domains (WO 2002/020565; Binz, H.K., Amstutz, P., Kohl, A., Stumpp, M.T., Briand, C, Forrer, P., Grutter, M.G., and Pluckthun, A., Nat.Biotechnol.22,575-582,2004; Stumpp, M.T., Binz, H.K and Amstutz, P., Drug Discov.Today 13,695-701, 2008).
Ankyrin repeat proteins have been identified in 1987 by sequence comparison between these four proteins in s.cerevisiae, Drosophila melanogaster (Drosophila melanogaster) and Caenorhabditis elegans. Breeden and Nasmyth reported multiple copies of an approximately 33 residue repeat unit in the sequence of swi6p, cdcl0p, notch and lin-12 (Breeden and Nasmyth, 1987). Subsequent discovery of 24 copies of this repeat unit in ankyrin resulted in the designation of this repeat unit as an ankyrin repeat (Lux et al, 1990). Later, such repeat units have been identified in hundreds of proteins from different organisms and viruses (Bork, 1993; SMART database, Schultz et al, 2000). These proteins are located in the nuclear, cytoplasmic or extracellular space. This is consistent with the fact that: these protein ankyrin repeat domains are independent of disulfide bonds and therefore independent of the oxidative state of the environment. The number of repeat units per protein varied from two to more than 20 (SMART database, Schultz et al, 2000). It appears that the lowest number of repeat units is required to form a stable folded domain (Zhang and Peng, 2000). On the other hand, there is some evidence of a maximum of six repeat units in one folding domain (Michaely and Bennet, 1993).
WO2002/020565 describes how a large ankyrin repeat protein library can be constructed and their general use. These designed repeat domains exploit the modular nature of repeat proteins and possess N-terminal and C-terminal capping modules to prevent aggregation of the designed repeat domains by shielding the hydrophobic core of the domain (Forrer, p., Stumpp, m.t., Binz, h.k. and Pluckthun, a., FEBS letters 539,2-6,2003). WO 2012069655 describes optimization of repeat sequence proteins by improving the C-terminal or N-terminal capping module or C-terminal or N-terminal capping repeat sequences of designed ankyrin repeat domains.
Other binding proteins such as fibronectin type III domain based adectin, lipocalin based anti-transporter (Anticalin), ubiquitin based Affilins, transferrin based Transbody, protein a domain based affibodies, tetranectin domain based TrimerX, Cys rich domain based MicroProtein, FynSH3 domain based Fynomer, EGFR A domain based high affinity multimers (Avimers), centryrin based centryrin, Kuniz domain based kalibitor and other scaffold proteins with randomized binding regions and antibody-like behavior are also encompassed by the present invention.
In one embodiment of the invention there is provided a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to a T cell activating antigen and a second antigen binding moiety capable of specific binding to a target cell antigen, wherein said one antigen binding moiety is a Fab molecule or a crossover Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the other antigen binding moiety is a binding protein comprising at least one ankyrin repeat motif.
In a preferred embodiment, the additional antigen binding moiety is a binding protein comprising two ankyrin repeat motifs. In another embodiment, the additional antigen binding portion is a binding protein comprising three, four or five ankyrin repeat motifs.
In one embodiment of the invention, there is provided a T cell activating bispecific antigen binding molecule comprising a first antigen binding moiety capable of specific binding to a T cell activating antigen and a second antigen binding moiety capable of specific binding to a target cell antigen, wherein the first antigen binding moiety is a Fab molecule or a crossover Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged and wherein the second antigen binding moiety is a binding protein comprising at least one ankyrin repeat motif.
In a preferred embodiment, the second antigen-binding portion is a binding protein comprising two ankyrin repeat motifs. In another embodiment, the second antigen-binding portion is a binding protein comprising three, four or five ankyrin repeat motifs.
Preferably said T cell activating bispecific antigen binding molecule comprises a binding protein comprising at least one ankyrin repeat domain, wherein said repeat domain comprises the ankyrin repeat consensus sequence dxxgxtplhlaaxxgapxpapvpllxpxgavnax, wherein "x" refers to any amino acid, "" refers to any amino acid or deletion, "a" refers to an amino acid with a non-polar side chain, and "p" refers to a residue with a polar side chain. In one embodiment, the repeat domain comprises the ankyrin repeat consensus sequence dxxgxtplhlaxxgxxxxvwxllllxgadvnax, where "x" refers to any amino acid. In one embodiment, the repeat domain comprises the ankyrin repeat motif D11G1TPLHLAA11GHLEIVEVLLK2GADVNA1, wherein 1 represents an amino acid residue selected from: A. d, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y; wherein 2 represents an amino acid residue selected from: H. n and Y.
The bispecific antibodies of the invention comprise one or more interchangeable Fab fragments. A crossover Fab fragment is a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. Bispecific antibody formats comprising exchanged Fab fragments have been described, for example, in WO 2009080252, WO2009080253, WO2009080251, WO 2009080254, WO 2010/136172, WO 2010/145792 and EP patent application No. 11178371.8, which are incorporated herein by reference.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a binding protein comprising at least one ankyrin repeat domain and comprises no more than one antigen binding moiety capable of specifically binding to a T cell activating antigen.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a binding protein comprising at least one ankyrin repeat domain, said binding protein comprising a fusion to another antigen binding portion comprising a Fab molecule or an exchange Fab molecule, wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged. Optionally, the antigen binding moieties are fused to each other by a peptide linker.
In one embodiment, the binding protein comprising at least one ankyrin repeat domain is fused to the N-terminus of the heavy chain of the crossover Fab molecule.
In one embodiment, the binding protein comprising at least one ankyrin repeat domain is fused to the N-terminus of the light chain of the crossover Fab molecule.
In one embodiment, the T cell activating bispecific antigen binding molecule additionally comprises a third antigen binding moiety capable of specifically binding to a target cell antigen.
In one embodiment, the third antigen binding moiety capable of specific binding to a target cell antigen is a binding protein comprising at least one ankyrin repeat motif. In one embodiment, said third antigen binding portion capable of specific binding to a target cell antigen is a binding protein comprising at least one ankyrin repeat motif as defined above. In one embodiment, the third antigen binding moiety capable of specific binding to a target cell antigen is a binding protein comprising two, three, four or five ankyrin repeat motifs.
In one embodiment of the invention, the T cell activating bispecific antigen binding molecule further comprises an Fc domain comprising a first and a second subunit capable of stable association. In one embodiment, the Fc domain is an IgG, particularly an IgG1Or IgG4An Fc domain of (a). In particular embodiments, the Fc domain may further comprise modifications that facilitate association of the first and second subunits of the Fc domain, as outlined below. In other specific embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function, as outlined below.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion comprising a Fab molecule or an exchanged Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the Fab molecule or the exchanged Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding portion comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen-binding portion comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises
a) An Fc domain comprising a first and a second subunit capable of stable association,
b) a first antigen binding portion capable of specific binding to a T cell activation antigen comprising a Fab molecule or an exchanged Fab molecule in which the variable or constant regions of the Fab light and Fab heavy chains are exchanged, wherein the Fab molecule or the exchanged Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;
c) a second antigen-binding moiety capable of specifically binding to a target cell antigen, comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of one of the subunits of the Fc domain, and
d) a third antigen-binding portion capable of specifically binding to a target cell antigen, comprising a binding protein comprising at least one ankyrin repeat motif, wherein the binding protein comprising at least one ankyrin repeat motif is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion.
In one embodiment, the second and third antigen binding portions bind to the same target cell antigen.
In one embodiment, the first and/or second antigen-binding portion is directly connected to the Fc domain via a hinge region. In another embodiment, the first and/or second antigen binding moiety is linked to the Fc domain by a peptide linker.
According to any of the above embodiments, the components of the T cell activating bispecific antigen binding molecule (e.g. antigen binding portion, Fc domain) may be fused directly or through various linkers described herein or known in the art, in particular peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. Suitably, the non-immunogenic peptide linker comprises, for example, (G)4S)n、(SG4)n、(G4S)nOr G4(SG4)nPeptide linkers, wherein n is generally a number between 1 and 10, typically between 2 and 4.
Fc domains
In some embodiments of the invention, the T cell activating bispecific antigen binding molecule comprises an Fc domain. The Fc domain of the T cell activating bispecific antigen binding molecule consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain dimer of an immunoglobulin g (IgG) molecule, each subunit of which comprises a CH2 and CH 3IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably associating with each other. In one embodiment, the T cell activating bispecific antigen binding molecule of the invention comprises no more than one Fc domain.
In one embodiment of the invention, the Fc domain of the T cell activating bispecific antigen binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is IgG1An Fc domain. In another embodiment, the Fc domain is an IgG4An Fc domain. In a more specific embodiment, the Fc domain is an IgG comprising an amino acid substitution at position S228(Kabat numbering), in particular comprising the amino acid substitution S228P4An Fc domain. This amino acid substitution reduces IgG4In vivo Fab arm exchange of antibodies (see Stubenrauch et al, Drug Metabolism and position 38,84-91 (2010)). In yet another specific embodiment, the Fc domain is human. Human IgG1Exemplary sequences of Fc regionsThe columns are given in SEQ ID NO: 149.
Fc domain modification to promote heterodimerization
The T cell activating bispecific antigen binding molecules of the invention comprise different antigen binding portions and in one embodiment are fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. In order to improve the yield and purity of the T cell activating bispecific antigen binding molecule in recombinant production, it is therefore advantageous to introduce a modification in the Fc domain of the T cell activating bispecific antigen binding molecule that facilitates the association of the desired polypeptide.
Thus, in a specific embodiment, the Fc domain of the T cell activating bispecific antigen binding molecule of the invention comprises a modification that facilitates the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.
In a particular embodiment, the modification is a so-called "knot-in-hole" modification, including a "knot modification" in one of the two subunits of the Fc domain and a "knot modification" in the other of the two subunits of the Fc domain.
Knot and stab techniques are described, for example, in US5,731,168; US7,695,936; ridgway et al, Prot Eng 9,617- & 621 (1996) and Carter, J Immunol Meth 248,7-15 (2001). In general, the method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("button") in the interface of a second polypeptide, such that the projection can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The overhang is constructed by replacing the side chain of a small amino acid derived from the first polypeptide interface with a larger side chain (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller side chain (e.g., alanine or threonine), a complementary "cavity" of the same or similar size as the overhang is created in the interface of the second polypeptide.
Thus, in a specific embodiment, an amino acid residue in the CH3 domain of the first subunit of the Fc domain of the T cell activating bispecific antigen binding molecule is replaced by an amino acid residue having a larger side chain volume, thereby creating within the CH3 domain of the first subunit a protuberance that can be positioned within the internal cavity of the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced by an amino acid residue having a smaller side chain volume, thereby creating within the CH3 domain of the second subunit a cavity within which the protuberance within the CH3 domain of the first subunit can be positioned.
The overhangs and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.
In a particular embodiment, the threonine residue at position 366 in the CH3 domain of the first subunit of the Fc domain is replaced with a tryptophan residue (T366W) and the tyrosine residue at position 407 in the CH3 domain of the second subunit of the Fc domain is replaced with a valine residue (Y407V). In one embodiment, the threonine residue at position 366 is additionally replaced by a serine residue in the second subunit of the Fc domain (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A).
In yet another embodiment, the serine residue at position 354 in the first subunit of the Fc domain is additionally replaced with a cysteine residue (S354C) and the tyrosine residue at position 349 in the second subunit of the Fc domain is additionally replaced with a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of disulfide bonds between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J immunological Methods 248,7-15 (2001)).
In a particular embodiment, an antigen binding moiety capable of binding to a T cell activation antigen is fused (optionally via an antigen binding moiety capable of binding to a target cell antigen) to the first subunit of the Fc domain (comprising a "knob" modification). Without wishing to be bound by theory, fusion of an antigen-binding moiety capable of binding to a T cell activation antigen with a junction-containing Fc domain subunit will (further) minimize the production of an antigen-binding molecule comprising two antigen-binding moieties capable of binding to a T cell activation antigen (steric hindrance of the two junction-containing polypeptides).
In one embodiment, the modification that facilitates association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering effects, for example, as described in PCT publication WO 2009/089004. Typically, this approach involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues, such that homodimer formation becomes electrostatically unfavorable and heterodimerization is electrostatically favorable.
In one aspect, the present invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to a T cell activating antigen and the other of which is a Fab molecule capable of specific binding to a target cell antigen;
wherein the first antigen binding moiety is
(a) A single chain Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker, or
(b) An exchanged Fab molecule wherein the variable or constant regions of the Fab light and Fab heavy chains are exchanged,
and an Fc domain comprising a first and a second subunit capable of stable association,
wherein the first subunit and the second subunit have been modified to comprise one or more charged amino acids that electrostatically facilitate heterodimer formation.
In one embodiment, said first subunit comprises the amino acid mutations E356K, E357K and D399K and said second subunit comprises the amino acid mutations K370E, K409E and K439E.
In another embodiment, the first subunit comprises the amino acid mutations K392D, K409D and the second subunit comprises the amino acid mutations E356K, D399K (DDKK).
The components of the T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. An exemplary configuration is depicted in fig. 1.
In some embodiments, the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
In one such specific embodiment, the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion. In one such specific embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding moiety, an Fc domain comprising a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In an even more specific embodiment, the first antigen binding portion is a single chain Fab molecule. Alternatively, in a specific embodiment, the first antigen binding moiety is an exchange Fab molecule. Optionally, if the first antigen binding portion is a crossover Fab molecule, the Fab light chain of the first antigen binding portion and the Fab light chain of the second antigen binding portion may additionally be fused to each other.
In one such alternative embodiment, the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In one such specific embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding portion, an Fc domain comprising a first and a second subunit, and optionally one or more peptide linkers, wherein each of the first and second antigen binding portions is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In an even more specific embodiment, the first antigen binding portion is a single chain Fab molecule. Alternatively, in a specific embodiment, the first antigen binding moiety is an exchange Fab molecule.
In yet another such embodiment, the second antigen-binding portion is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen-binding portion. In one such specific embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding moiety, an Fc domain comprising a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the N-terminus of the Fab light chain to the C-terminus of the Fab light chain of the second antigen binding moiety, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In an even more specific embodiment, the first antigen binding moiety is an exchange Fab molecule.
In other embodiments, the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
In one such specific embodiment, the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In one such specific embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a first and a second antigen binding moiety, an Fc domain comprising a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In an even more specific embodiment, the first antigen binding moiety is an exchange Fab molecule. Optionally, the Fab light chain of the first antigen-binding portion and the Fab light chain of the second antigen-binding portion may additionally be fused to each other.
In particular, in these embodiments, the first antigen binding moiety is capable of specifically binding to a T cell activation antigen. In other embodiments, the first antigen binding portion is capable of specifically binding to a target cell antigen.
The antigen binding portions may be fused to the Fc domain or to each otherEither directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitably, the non-immunogenic peptide linker comprises, for example, (G)4S)n、(SG4)n、(G4S)nOr G4(SG4)nPeptide linker, "n" is generally a number between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and second antigen-binding portions to each other is (G)4S)2. An exemplary peptide linker suitable for linking the Fab heavy chains of the first and second antigen-binding portions is EPKSC (D) - (G)4S)2(SEQ ID NOS: 150 and 151). In addition, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, when the antigen binding portion is fused to the N-terminus of an Fc domain subunit, it may be fused, with or without additional peptide linkers, through an immunoglobulin hinge region or portion thereof.
T cell activating bispecific antigen binding molecules having a single antigen binding moiety capable of specific binding to a target cell antigen (e.g., as shown in fig. 1A, 1B, 1D, 1E, 1H, 1I, 1K, or 1M) are useful, particularly where the target cell antigen is expected to be internalized following binding of the high affinity antigen binding moiety. In this case, the presence of more than one antigen-binding moiety specific for a target cell antigen may enhance internalization of the target cell antigen, thus reducing its availability.
However, in many other cases it is advantageous to have a T cell activating bispecific antigen binding molecule comprising two or more antigen binding moieties specific for a target cell antigen (see examples shown in fig. 1C, 1F, 1G, 1J or 1L), e.g. to optimize targeting to a target site or to allow for target cell antigen cross-linking.
Thus, in certain embodiments, the T cell activating bispecific antigen binding molecule of the invention further comprises a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen. In one embodiment, the third antigen binding portion is capable of specifically binding to the same target cell antigen as the first or second antigen binding portion. In a specific embodiment, the first antigen binding moiety is capable of specifically binding to a T cell activation antigen, and the second and third antigen binding moieties are capable of specifically binding to a target cell antigen.
In one embodiment, the third antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a specific embodiment, the second and third antigen binding portions are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion. In such an embodiment, the first antigen binding portion is a single chain Fab molecule. In one such specific embodiment, the first antigen binding moiety is an exchange Fab molecule. Optionally, if the first antigen binding portion is a crossover Fab molecule, the Fab light chain of the first antigen binding portion and the Fab light chain of the second antigen binding portion may additionally be fused to each other.
The second and third antigen binding portions may be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the second and third antigen binding portions are each fused to the Fc domain by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1A hinge region. In one embodiment, the second and third antigen binding portions and the Fc domain are part of an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more specific embodiment, the immunoglobulin is an IgG1Subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4Subclass immunoglobulin. In yet another specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a peptide capable of binding to a target cell antigenA specifically binding immunoglobulin molecule and an antigen binding moiety capable of specifically binding to a T cell activating antigen, wherein the antigen binding moiety is a single chain Fab molecule or an exchange Fab molecule, optionally fused to the N-terminus of one of the immunoglobulin heavy chains by a peptide linker.
In an alternative embodiment, the first and third antigen binding portions are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In one such specific embodiment, the T cell activating bispecific antigen binding molecule consists essentially of a first, a second and a third antigen binding portion, an Fc domain comprising a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In one such specific embodiment, the first antigen binding moiety is a crossover Fab molecule. Optionally, the Fab light chain of the first antigen-binding portion and the Fab light chain of the second antigen-binding portion may additionally be fused to each other.
In some T cell activating bispecific antigen binding molecules of the invention, the Fab light chain of the first antigen binding portion and the Fab light chain of the second antigen binding portion are fused to each other, optionally via a linker peptide. Depending on the configuration of the first and second antigen-binding portion, the Fab light chain of the first antigen-binding portion may be fused at its C-terminus to the N-terminus of the Fab light chain of the second antigen-binding portion, or the Fab light chain of the second antigen-binding portion may be fused at its C-terminus to the N-terminus of the Fab light chain of the first antigen-binding portion. The fusion of the Fab light chains of the first and second antigen binding portions further reduces unpaired Fab heavy and light chain mispairings and also reduces the number of plasmids required to express some T cell activating bispecific antigen binding molecules of the invention.
In certain embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the first Fab light chain shares a carboxy-terminal peptide bond with a peptide linker, which in turn shares a carboxy-terminal peptide bond with the first Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL-CL-linker-VH-CH 1-CH2-CH2(-CH4)), and a polypeptide wherein the second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH 4)). In some embodiments, the T cell activating bispecific antigen binding molecule further comprises a second Fab light chain polypeptide (VL-CL). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.
In some embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the first Fab light chain shares a carboxy-terminal peptide bond with a peptide linker which in turn shares a carboxy-terminal peptide bond with the first Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with the second Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VL-CL-linker-VH-CH 1-VH-CH1-CH2-CH3(-CH 4)). In one of these embodiments, the T cell activating bispecific antigen binding molecule further comprises a second Fab light chain polypeptide (VL-CL). According to these embodiments, the T cell activating bispecific antigen binding molecule may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide, wherein the third Fab heavy chain shares a carboxy terminal peptide bond with the Fc domain subunit (VH-CH1-CH2-CH3(-CH4)) and the third Fab light chain polypeptide (VL-CL). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.
In certain embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the first Fab light chain variable region shares a carboxy-terminal peptide bond with the first Fab heavy chain constant region (i.e., exchanges Fab heavy chains in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL-CH1-CH2-CH2(-CH4)), and a polypeptide wherein the second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH 4)). In some embodiments, the T cell activating bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region shares a carboxy terminal peptide bond with the Fab light chain constant region (VH-CL) and the Fab light chain polypeptide (VL-CL). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.
In alternative embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the first Fab heavy chain variable region shares a carboxy-terminal peptide bond with the first Fab light chain constant region (i.e. exchanges Fab heavy chains, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH-CL-CH2-CH2(-CH4)), and a polypeptide wherein the second Fab heavy chain shares a carboxy-terminal peptide bond with the Fc domain subunit (VH-CH1-CH2-CH3(-CH 4)). In some embodiments, the T cell activating bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region shares a carboxy terminal peptide bond with the Fab heavy chain constant region (VL-CH1) and the Fab light chain polypeptide (VL-CL). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.
In some embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the first Fab light chain variable region shares a carboxy-terminal peptide bond with a first Fab heavy chain constant region (i.e., exchanges Fab heavy chains, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL-CH1-VH-CH1-CH2-CH3(-CH 4)). In other embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide in which the first Fab heavy chain variable region shares a carboxy-terminal peptide bond with the first Fab light chain constant region (i.e., exchanges Fab heavy chains in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the second Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CL-VH-CH1-CH2-CH3(-CH 4)). In a further embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the second Fab heavy chain shares a carboxy-terminal peptide bond with the first Fab light chain variable region which in turn shares a carboxy-terminal peptide bond with the first Fab heavy chain constant region (i.e. exchanges the Fab heavy chain wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH-CH1-VL-CH1-CH2-CH3(-CH 4)). In other embodiments, the T cell activating bispecific antigen binding molecule comprises a polypeptide in which the second Fab heavy chain shares a carboxy-terminal peptide bond with the first Fab heavy chain variable region, which in turn shares a carboxy-terminal peptide bond with the first Fab light chain constant region (i.e., an exchange Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-VH-CL-CH2-CH3(-CH 4)).
In some of these embodiments, the T cell activating bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide, wherein the Fab heavy chain variable region shares a carboxy-terminal peptide bond with the Fab light chain constant region (VH-CL) and the Fab light chain polypeptide (VL-CL). In other of these embodiments, the T cell activating bispecific antigen binding molecule further comprises an crossover Fab light chain polypeptide, wherein the Fab light chain variable region shares a carboxy-terminal peptide bond with the Fab heavy chain constant region (VL-CH1) and the Fab light chain polypeptide (VL-CL). In still other embodiments of these embodiments, the T cell activating bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region shares a carboxy-terminal peptide bond with the Fab heavy chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide (VL-CH 1-VL-CL); comprises a polypeptide wherein the Fab heavy chain variable region shares a carboxy-terminal peptide bond with the Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide (VH-CL-VL-CL); comprises a polypeptide wherein a Fab light chain polypeptide shares a carboxy-terminal peptide bond with a Fab light chain variable region, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region (VL-CL-VL-CH 1); or comprises a polypeptide in which the Fab light chain polypeptide shares a carboxy-terminal peptide bond with the Fab heavy chain variable region, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region (VL-CL-VH-CL).
According to these embodiments, the T cell activating bispecific antigen binding molecule may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the third Fab heavy chain shares a carboxy terminal peptide bond with the Fc domain subunit (VH-CH1-CH2-CH3(-CH4)) and the third Fab light chain polypeptide (VL-CL). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the second Fab light chain shares a carboxy-terminal peptide bond with the first Fab light chain variable region, which in turn shares a carboxy-terminal peptide bond with the first Fab heavy chain constant region (i.e., exchanges the Fab light chain in which the light chain constant region is replaced by a heavy chain constant region) (VL-CL-VL-CH 1); comprises a polypeptide wherein the second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH 4)); and polypeptides comprising a first Fab light chain variable region sharing a carboxy-terminal peptide bond (VH-CL) with a first Fab light chain constant region. In another embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide wherein the second Fab light chain shares a carboxy-terminal peptide bond with the first Fab heavy chain variable region, which in turn shares a carboxy-terminal peptide bond with the first Fab light chain constant region (i.e. exchanges the Fab light chain wherein the light chain variable region is replaced by the heavy chain variable region) (VL-CL-VH-CL); comprises a polypeptide wherein the second Fab heavy chain shares a carboxy-terminal peptide bond with an Fc domain subunit (VH-CH1-CH2-CH3(-CH 4)); and polypeptides comprising a first Fab light chain variable region sharing a carboxy-terminal peptide bond (VL-CH1) with a first Fab heavy chain constant region. According to these embodiments, the T cell activating bispecific antigen binding molecule may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the third Fab heavy chain shares a carboxy terminal peptide bond with the Fc domain subunit (VH-CH1-CH2-CH3(-CH4)) and the third Fab light chain polypeptide (VL-CL). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.
According to any of the above embodiments, the components of the T cell activating bispecific antigen binding molecule (e.g. antigen binding portion, Fc domain) may be fused directly or through various linkers described herein or known in the art, in particular peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. Suitably, the non-immunogenic peptide linker comprises, for example, (G)4S)n、(SG4)n、(G4S)nOr G4(SG4)nPeptide linkers, wherein n is generally a number between 1 and 10, typically between 2 and 4.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises one or more amino acid sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs 372, 373, 374 and 375. In another embodiment, the T cell activating bispecific antigen binding molecule comprises SEQ ID NOs 372, 373, 374 and 375.
Fc domain modifications that reduce Fc receptor binding and/or effector function
The Fc domain confers advantageous pharmacokinetic properties to the T cell activating bispecific antigen binding molecule, including a long serum half-life and a favourable tissue-to-blood distribution ratio that contribute to good accumulation in the target tissue. At the same time, however, it may lead to the unwanted targeting of the T cell activating bispecific antigen binding molecule to Fc receptor expressing cells, rather than to preferred antigen carrying cells. In addition, co-activation of the Fc receptor signaling pathway can lead to cytokine release, along with the T cell activation properties and long half-life of the antigen binding molecules, which leads to over-activation of cytokine receptors and severe side effects upon systemic administration. Activating immune cells other than T cells (carrying Fc receptors) may even reduce the efficacy of T cell activating bispecific antigen binding molecules, since T cells may potentially be destroyed by e.g. NK cells.
Thus, in a specific embodiment, withNatural IgG1Fc domains the Fc domains of the T cell activating bispecific antigen binding molecules of the invention exhibit reduced binding affinity to Fc receptors and/or reduced effector function compared to Fc domains. In such an embodiment, the IgG is naturally associated with1Fc domain (or comprising native IgG)1Fc domain of a T cell activating bispecific antigen binding molecule) exhibits a binding affinity to an Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%, and/or to native IgG1Fc domain (or comprising native IgG)1Fc domain T cell activating bispecific antigen binding molecules) less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function. In one embodiment, the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or does not induce effector function. In a specific embodiment, the Fc receptor is an fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is a human activating Fc γ receptor, more specifically human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most specifically human Fc γ RIIIa. In one embodiment, the effector function is one or more selected from CDC, ADCC, ADCP and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the IgG is naturally associated with1Fc domains in contrast, Fc domains exhibit substantially similar binding affinities to neonatal Fc receptor (FcRn). When the Fc domain (or T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, more particularly more than about 90% native IgG1Fc domain (or comprising native IgG)1Fc domain T cell activating bispecific antigen binding molecules) binding affinity to FcRn, substantially similar binding to FcRn is achieved.
In certain embodiments, the Fc domain is engineered to have reduced binding affinity for an Fc receptor and/or reduced effector function as compared to a non-engineered Fc domain. In a specific embodiment, the Fc domain of the T cell activating bispecific antigen binding molecule comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, the T cell activating bispecific antigen binding molecule comprising an engineered Fc domain exhibits a binding affinity to an Fc receptor of less than 20%, particularly less than 10%, more particularly less than 5% compared to a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain. In a specific embodiment, the Fc receptor is an fey receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is a human activating Fc γ receptor, more specifically human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most specifically human Fc γ RIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity for complement components, particularly for C1q, is also reduced. In one embodiment, the binding affinity for neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved, i.e. the binding affinity of the Fc domain to the receptor is retained, when the Fc domain (or the T cell activating bispecific antigen binding molecule comprising the Fc domain) exhibits a binding affinity for FcRn of greater than about 70% in the un-engineered form of the Fc domain (or the T cell activating bispecific antigen binding molecule comprising the un-engineered form of the Fc domain). The Fc domain of the present invention or the T cell activating bispecific antigen binding molecule comprising said Fc domain may show such affinity of more than about 80% and even more than about 90%. In certain embodiments, the Fc domain of the T cell activating bispecific antigen binding molecule is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector functions may include, but are not limited to, one or more of the following: reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell-mediated cytotoxicity (ADCC), reduced antibody dependent endocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen presenting cells, reduced NK cell binding, reduced macrophage binding, reduced monocyte binding, reduced polymorphonuclear cell binding, reduced signal transduction-induced direct apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T-cell priming. In one embodiment, the reduced effector function is one or more selected from the group consisting of: reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by the non-engineered Fc domain (or the T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain).
In one embodiment, the amino acid mutation that reduces the binding affinity and/or effector function of the Fc domain to an Fc receptor is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331 and P329. In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329. In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG1Fc domains, in particularHuman IgG1An Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297 and P331. In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235. In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an IgG1Fc domain, in particular human IgG1An Fc domain. Combination of amino acid substitutions "P329G LALA" almost completely abolished human IgG1Fc domain Fc γ receptor binding as described in PCT patent application No. PCT/EP2012/055393, incorporated herein by reference in its entirety. PCT/EP2012/055393 also describes methods for making such mutant Fc domains and methods for determining properties such as Fc receptor binding or effector function.
And IgG1Antibody vs. IgG4Antibodies exhibit reduced Fc receptor binding affinity and reduced effector function. Thus, in some embodiments, the Fc domain of the T cell activating bispecific antigen binding molecules of the invention is an IgG4Fc domain, in particular human IgG4An Fc domain. In one embodiment, the IgG is4The Fc domain comprises the amino acid substitution at position S228, in particular the amino acid substitution S228P. To further reduce its Fc receptor binding affinity and/or its effector function, in one embodiment, IgG4The Fc domain comprises the amino acid substitution at position L235, in particular the amino acid substitution L235E. In another embodiment, the IgG is4The Fc domain comprises the amino acid substitution at position P329, in particular the amino acid substitution P329G. In a specific embodiment, the IgG4The Fc domain comprisesAmino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G. Such IgG' s4Fc domain mutants and their Fc γ receptor binding properties are described in PCT patent application No. PCT/EP2012/055393, which is incorporated herein by reference in its entirety.
In a specific embodiment, the IgG is naturally associated with1Fc domain in comparison to Fc domain showing reduced binding affinity to Fc receptor and/or reduced effector function is a human IgG comprising the amino acid substitutions L234A, L235A and optionally P329G1An Fc domain, or a human IgG comprising the amino acid substitutions S228P, L235E and optionally P329G4An Fc domain.
In certain embodiments, N-glycosylation of the Fc domain has been eliminated. In such an embodiment, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid substitution replacing asparagine to alanine (N297A) or aspartic acid (N297D).
In addition to the Fc domains described above and in PCT patent application No. PCT/EP2012/055393, Fc domains with reduced Fc receptor binding and/or effector function include those that replace one or more of
The mutant Fc domain may be prepared by amino acid deletion, substitution, insertion or modification using genetic methods or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of DNA coding sequences, PCR, gene synthesis, and the like. The correct nucleotide change can be verified, for example, by sequencing.
Binding to Fc receptors can be readily determined using standard instruments such as BIAcore instruments (GE Healthcare) and as Fc receptors that can be obtained by recombinant expression, for example by ELISA or by Surface Plasmon Resonance (SPR). One such suitable binding assay is described herein. Alternatively, the binding affinity of an Fc domain or Fc domain-containing bispecific antigen binding molecule of an activated cell to an Fc receptor can be evaluated using cell lines known to express specific Fc receptors, such as human NK cells expressing Fc γ IIIa receptors.
The effector function of an Fc domain or a T cell activating bispecific antigen binding molecule comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. nos. 5,500,362; hellstrom et al, Proc Natl Acad Sci USA 83, 7059-; U.S. Pat. nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-. Alternatively, nonradioactive analysis methods may be used (see, e.g., ACTI for flow cytometry)TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and CytotoxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells for use in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al, Proc Natl Acad Sci USA 95, 652-.
In some embodiments, the Fc domain has reduced binding to complement components, particularly to C1 q. Thus, in some embodiments in which the Fc domain is engineered to have reduced effector function, the reduced effector function comprises reduced CDC. A C1q binding assay may be performed to determine whether a T cell activating bispecific antigen binding molecule is capable of binding C1q and thus has CDC activity. See, for example, WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J Immunol Methods 202,163 (1996); Cragg et al,
Antigen binding moieties
The antigen binding molecule of the present invention is bispecific, i.e. it comprises at least two antigen binding portions capable of specifically binding to two different antigenic determinants. According to the present invention, the antigen binding portion is a Fab molecule (i.e., an antigen binding domain consisting of heavy and light chains each comprising a variable region and a constant region), a Single Domain Antigen Binding (SDAB) molecule, or a protein scaffold, such as a binding protein comprising at least one ankyrin repeat motif (e.g., Darpin). In one embodiment, the Fab molecule or Single Domain Antigen Binding (SDAB) molecule is human. In another embodiment, the Fab molecule or Single Domain Antigen Binding (SDAB) molecule is humanized. In yet another embodiment, the Fab molecule comprises a human heavy chain constant region and a light chain constant region.
At least one antigen binding portion is a single chain Fab molecule or an exchange Fab molecule. Such modifications prevent heavy and light chain mis-pairing from different Fab molecules, thereby improving the yield and purity of the T cell activating bispecific antigen binding molecules of the invention when recombinantly produced. In a particular single chain Fab molecule useful in the T cell activating bispecific antigen binding molecules of the invention, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain by a peptide linker. The peptide linker allows the Fab heavy and light chains to be aligned to form a functional antigen binding portion. Suitable peptide linkers for linking the Fab heavy and light chains include, for example, (G)4S)6-GG (SEQ ID NO:152) or (SG)3)2-(SEG3)4-(SG3) SG (SEQ ID NO: 153). In a specific exchanged Fab molecule useful in the T cell activating bispecific antigen binding molecules of the invention, the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In another exchanged Fab molecule useful in the T cell activating bispecific antigen binding molecules of the invention, the Fab light chain variable region and the Fab heavy chain variable region are exchanged.
In a particular embodiment of the invention, the T cell activating bispecific antigen binding molecule is capable of binding to both a target cell antigen (in particular a tumor cell antigen) and a T cell activating antigen. In one embodiment, the T cell activating bispecific antigen binding molecule is capable of cross-linking a T cell and a target cell by simultaneous binding to the target cell antigen and a T cell activating antigen. In an even more specific embodiment, such simultaneous binding results in lysis of target cells, in particular tumor cells. In one embodiment, such simultaneous binding results in T cell activation. In other embodiments, such simultaneous binding results in a cellular response of T lymphocytes, in particular cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity and activation marker expression. In one embodiment, binding of the T cell activating bispecific antigen binding molecule to a T cell activating antigen without simultaneous binding to the target cell antigen does not result in T cell activation.
In one embodiment, the T cell activating bispecific antigen binding molecule is capable of redirecting the cytotoxic activity of the T cell to the target cell. In a specific embodiment, the redirection is independent of MHC-mediated specificity of presentation of the peptide antigen by the target cell and/or the T cell.
In particular, the T cell according to any embodiment of the invention is a cytotoxic T cell. In some embodiments, the T cell is CD4+Or CD8+T cells, in particular CD8+T cells.
T cell activating antigen binding moieties
The T cell activating bispecific antigen binding molecules of the invention comprise at least one antigen binding moiety (also referred to herein as "T cell activating antigen binding moiety") capable of binding to a T cell activating antigen. In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises no more than one antigen binding moiety capable of specifically binding to a T cell activating antigen. In one embodiment, the T cell activating bispecific antigen binding molecule provides a monovalent binding effect to a T cell activating antigen. The T cell activating antigen binding moiety may be a conventional Fab molecule or a modified Fab molecule, i.e. a single chain or exchanged Fab molecule, or a Single Domain Antigen Binding (SDAB) molecule, or a binding protein comprising at least one ankyrin repeat motif. In embodiments wherein there is more than one antigen binding moiety comprised in the T cell activating bispecific antigen binding molecule capable of specifically binding to a target cell antigen, the antigen binding moiety capable of specifically binding to a T cell activating antigen is preferably a modified Fab molecule.
In a particular embodiment, the T cell activating antigen is CD3, particularly human CD3(SEQ ID NO:265) or cynomolgus monkey CD3(SEQ ID NO:266), most particularly
In one embodiment, the T cell activating antigen binding moiety may compete with monoclonal antibody H2C (described in PCT publication No. WO 2008/119567) for binding to an epitope of
In one embodiment, the T cell activating antigen binding portion comprises heavy chain CDR1 of SEQ ID NO. 249, heavy chain CDR2 of SEQ ID NO. 251, heavy chain CDR3 of SEQ ID NO. 253, light chain CDR1 of SEQ ID NO. 257, light chain CDR2 of SEQ ID NO. 259 and light chain CDR3 of SEQ ID NO. 261. In one embodiment, the T cell activating antigen binding portion may compete for binding to an epitope of CD3 with an antigen binding portion comprising heavy chain CDR1 of SEQ ID NO:249, heavy chain CDR2 of SEQ ID NO:251, heavy chain CDR3 of SEQ ID NO:253, light chain CDR1 of SEQ ID NO:257, light chain CDR2 of SEQ ID NO:259, and light chain CDR3 of SEQ ID NO: 261. In one embodiment, the T cell activating antigen binding portion binds to the same epitope of CD3 as the antigen binding portion comprising heavy chain CDR1 of SEQ ID NO:249, heavy chain CDR2 of SEQ ID NO:251, heavy chain CDR3 of SEQ ID NO:253, light chain CDR1 of SEQ ID NO:257, light chain CDR2 of SEQ ID NO:259 and light chain CDR3 of SEQ ID NO: 261. In yet another embodiment, the T cell activating antigen binding portion comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 255 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 263, or a functionally preserved variant thereof. In one embodiment, the T cell activation antigen binding portion can compete for binding to an epitope of CD3 with an antigen binding portion comprising the heavy chain variable region sequence of SEQ ID NO:255 and the light chain variable region sequence of SEQ ID NO: 263. In one embodiment, the T cell activating antigen binding portion binds to the same epitope of CD3 as the antigen binding portion comprising the heavy chain variable region sequence of SEQ ID NO. 255 and the light chain variable region sequence of SEQ ID NO. 263. In another embodiment, the T cell activating antigen binding portion comprises a humanized form of the heavy chain variable region sequence of SEQ ID NO:255 and a humanized form of the light chain variable region sequence of SEQ ID NO: 263. In one embodiment, the T cell activating antigen binding portion comprises heavy chain CDR1 of SEQ ID NO:249, heavy chain CDR2 of SEQ ID NO:251, heavy chain CDR3 of SEQ ID NO:253, light chain CDR1 of SEQ ID NO:257, light chain CDR2 of SEQ ID NO:259 and light chain CDR3 of SEQ ID NO:261, and the variable region framework sequences of human heavy and light chains.
In one embodiment, the T cell activating antigen binding portion comprises at least one heavy chain Complementarity Determining Region (CDR) selected from SEQ ID NO 270, 271 and 272 and at least one light chain CDR selected from SEQ ID NO 274, 275 and 276.
In one embodiment, the T cell activating antigen binding portion comprises a variable heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:269, SEQ ID NO:298 and SEQ ID NO:299 and a variable light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:273 and SEQ ID NO: 297.
In one embodiment, the T cell activating antigen binding portion comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 269 and a variable light chain comprising the amino acid sequence of SEQ ID NO 273.
Target cell antigen binding moieties
The T cell activating bispecific antigen binding molecules of the invention comprise at least one antigen binding moiety capable of binding to a target cell antigen (also referred to herein as "target cell antigen binding moiety"). In certain embodiments, the T cell activating bispecific antigen binding molecule comprises two antigen binding moieties capable of binding to a target cell antigen. In one such specific embodiment, each of these antigen binding portions specifically binds to the same antigenic determinant. In one embodiment, the T cell activating bispecific antigen binding molecule comprises no more than two antigen binding moieties capable of binding to a target cell antigen.
The target cell antigen binding portion may be a conventional Fab molecule or a modified Fab molecule, i.e., a single chain or exchanged Fab molecule, or a Single Domain Antigen Binding (SDAB) molecule, or a binding protein comprising at least one ankyrin repeat motif. The target cell antigen binding portion binds to a specific antigenic determinant and is capable of directing the T cell activating bispecific antigen binding molecule to a target site, e.g. to a specific type of tumor cell carrying the antigenic determinant.
In certain embodiments, the target cell antigen-binding moiety is directed to an antigen associated with a pathological condition, such as an antigen presented on a tumor cell or on a virus-infected cell. Suitable antigens are cell surface antigens, such as, but not limited to, cell surface receptors. In a specific embodiment, the antigen is a human antigen. In a specific embodiment, the target cell antigen is selected from Fibroblast Activation Protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, and
In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises at least one antigen binding moiety specific for melanoma associated chondroitin sulfate proteoglycan (MCSP). In one embodiment, the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody LC007 (see SEQ ID NOS: 75 and 83, and European patent application No. EP11178393.2, which are incorporated herein by reference in their entirety) for binding to an epitope of MCSP. In one embodiment, the antigen-binding portion specific for MCSP comprises the heavy chain CDR1 of SEQ ID NO. 69, the heavy chain CDR2 of SEQ ID NO. 71, the heavy chain CDR3 of SEQ ID NO. 73, the light chain CDR1 of SEQ ID NO. 77, the light chain CDR2 of SEQ ID NO. 79, and the light chain CDR3 of SEQ ID NO. 81. In yet another embodiment, the antigen-binding portion specific for MCSP comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO 75 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO 83, or a functionally-retaining variant thereof. In one embodiment, the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with the monoclonal antibody M4-3 ML2 (see SEQ ID NOs: 239 and 247, and European patent application No. EP11178393.2, which is incorporated herein by reference in its entirety) for binding to a epitope of MCSP. In one embodiment, the antigen binding portion specific for MCSP binds to the same epitope of MCSP as monoclonal antibody M4-3
In one embodiment, the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO. 1, the polypeptide sequence of SEQ ID NO. 3 and the polypeptide sequence of SEQ ID NO. 5, or a functionally retained variant thereof. In yet another embodiment, the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of
In one embodiment, the antigen-binding portion specific for MCSP comprises at least one heavy chain Complementarity Determining Region (CDR) selected from SEQ ID NO 280, 281, 282, 301, 303, 304 and 306 and at least one light chain CDR selected from SEQ ID NO 284, 285, 286, 310, 311, 314, 315 and 316.
In one embodiment, the antigen-binding portion specific for MCSP comprises at least one heavy chain Complementarity Determining Region (CDR) selected from SEQ ID NO:280, SEQ ID NO:281 and SEQ ID NO:282 and at least one light chain CDR selected from SEQ ID NO:284, SEQ ID NO:285 and SEQ ID NO: 286.
In one embodiment, the antigen-binding portion specific for MCSP comprises the heavy chain CDR1 of SEQ ID NO. 280, the heavy chain CDR2 of SEQ ID NO. 281, the heavy chain CDR3 of SEQ ID NO. 282, the light chain CDR1 of SEQ ID NO. 284, the light chain CDR2 of SEQ ID NO. 285, and the light chain CDR3 of SEQ ID NO. 286.
In yet another embodiment, the antigen-binding portion specific for MCSP comprises a variable heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:279, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:305, and SEQ ID NO:307 and a variable light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:283, SEQ ID NO:309, SEQ ID NO:312, SEQ ID NO:313, and SEQ ID NO: 317.
In one embodiment, the antigen-binding portion specific for MCSP comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 279 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 283.
In yet another embodiment, the antigen-binding portion specific for MCSP comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 279 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 283, or a functionally-retaining variant thereof.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 278, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 319, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 320, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 321.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 369, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 370, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 371.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 372, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 373, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 374, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 375.
In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence encoding a polypeptide sequence selected from the group consisting of SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 78, SEQ ID NO 80, SEQ ID NO 82, SEQ ID NO 84, SEQ ID NO 234, SEQ ID NO 236, SEQ ID NO 238, SEQ ID NO 240, SEQ ID NO 242, SEQ ID NO 244, SEQ ID NO 246, SEQ ID NO 248, SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 180, SEQ ID NO 182, SEQ ID NO 184, SEQ ID NO 186, SEQ ID NO 188, SEQ ID NO 190, SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 196, SEQ ID NO 200, SEQ ID NO 202, SEQ ID NO 216, SEQ ID NO 218, SEQ ID NO 220 and SEQ ID NO 329 to 388 are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical in sequence.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises at least one antigen binding moiety specific for Epidermal Growth Factor Receptor (EGFR). In another embodiment, the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody GA201 for binding to an EGFR epitope. See PCT publication WO 2006/082515, which is incorporated by reference herein in its entirety. In one embodiment, the antigen-binding portion specific for EGFR comprises the heavy chain CDR1 of SEQ ID NO. 85, the heavy chain CDR2 of SEQ ID NO. 87, the heavy chain CDR3 of SEQ ID NO. 89, the light chain CDR1 of SEQ ID NO. 93, the light chain CDR2 of SEQ ID NO. 95, and the light chain CDR3 of SEQ ID NO. 97. In yet another embodiment, the antigen-binding portion specific for EGFR comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 91 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to
In yet another embodiment, the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of
In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of
In one embodiment, the T cell activating bispecific antigen binding molecule comprises at least one antigen binding moiety specific for Fibroblast Activation Protein (FAP). In another embodiment, the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody 3F2 for binding to an epitope of FAP. See PCT publication WO 2012/020006, which is incorporated by reference herein in its entirety. In one embodiment, the antigen-binding portion specific for FAP comprises the heavy chain CDR1 of
In yet another embodiment, the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO:55, the polypeptide sequence of SEQ ID NO:51 and the polypeptide sequence of SEQ ID NO:11, or a functionally retained variant thereof. In yet another embodiment, the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of
In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of
In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises at least one antigen binding moiety specific for carcinoembryonic antigen (CEA). In one embodiment, the T cell activating bispecific antigen binding molecule comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody BW431/26 (described in european patent No. EP160,897 and Bosslet et al,
In one embodiment, the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of
In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of
In one embodiment, the antigen-binding portion specific for CEA comprises at least one heavy chain Complementarity Determining Region (CDR) selected from SEQ ID NO:290, SEQ ID NO:291, and SEQ ID NO:292 and at least one light chain CDR selected from SEQ ID NO: 294, SEQ ID NO:295, and SEQ ID NO: 296.
In one embodiment, the antigen-binding portion specific for CEA comprises the heavy chain CDR1 of SEQ ID NO. 290, the heavy chain CDR2 of SEQ ID NO. 291, the heavy chain CDR3 of SEQ ID NO. 292, the light chain CDR1 of SEQ ID NO. 294, the light chain CDR2 of SEQ ID NO. 295, and the light chain CDR3 of SEQ ID NO. 296.
In one embodiment, the antigen-binding portion specific for CEA comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 289 and a variable light chain comprising the amino acid sequence of SEQ ID NO 293.
In yet another embodiment, the antigen-binding portion specific for CEA comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 289 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 293, or a functionally-retaining variant thereof.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 288, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 322, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 323, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 324.
In one embodiment, the T cell activating bispecific antigen binding molecule comprises at least one antigen binding moiety specific for
In one embodiment, the T cell activating bispecific antigen binding molecule comprises the polypeptide sequence of
In a specific embodiment, the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO 134, SEQ ID NO 136, SEQ ID NO 138,
Polynucleotide
The invention also provides an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule or fragment thereof as described herein.
The polynucleotides of the invention include those polynucleotides that hybridize to SEQ ID NOs 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 216, 212, 218, 226, 220, 232, 220, 230, 234, 224, 228, 234, 224, 228, 234, 230, 234, 224, 220, 230, 236. 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, and 388 are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, including functional fragments or variants thereof.
The polynucleotide encoding the T cell activating bispecific antigen binding molecule of the invention may be expressed as a single polynucleotide encoding the entire T cell activating bispecific antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides may be combined to form a functional T cell activating bispecific antigen binding molecule, e.g., via disulfide bonds or other means. For example, the light chain portion of the antigen binding portion may be encoded by separate polynucleotides from a portion of a T cell activating bispecific antigen binding molecule comprising the heavy chain portion of the antigen binding portion, the Fc domain subunit and optionally (a part of) another antigen binding portion. When co-expressed, the heavy chain polypeptide will bind to the light chain polypeptide to form the antigen-binding portion. In another example, the T cell activating bispecific antigen binding molecule part comprising one of the two Fc domain subunits and optionally (part of) the one or more antigen binding moieties may be encoded by an isolated polynucleotide from a T cell activating bispecific antigen binding molecule part comprising the other of the two Fc domain subunits and optionally (part of) the antigen binding moiety. When co-expressed, the Fc domain subunits associate to form an Fc domain.
In certain embodiments, the isolated polynucleotides of the present invention encode a fragment of a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding portion and an Fc domain consisting of two subunits, wherein the first antigen binding portion is a single chain Fab molecule. In one embodiment, the isolated polynucleotide of the invention encodes the first antigen binding portion and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the single-chain Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. In another embodiment, the isolated polynucleotide of the invention encodes the heavy chain of the second antigen-binding portion and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the Fab heavy chain and the Fc domain subunit share a carboxy-terminal peptide bond. In yet another embodiment, the isolated polynucleotide of the invention encodes the heavy chain of the first antigen-binding portion, the second antigen-binding portion, and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the single chain Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit.
In certain embodiments, the isolated polynucleotide of the invention encodes a fragment of a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding portion and an Fc domain consisting of two subunits, wherein the first antigen binding portion is an crossover Fab molecule. In one embodiment, the isolated polynucleotide of the invention encodes the heavy chain of the first antigen-binding portion and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the Fab light chain variable region shares a carboxy-terminal peptide bond with the Fab heavy chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the Fab heavy chain variable region shares a carboxy-terminal peptide bond with the Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit. In another embodiment, the isolated polynucleotide of the invention encodes the heavy chain of the second antigen-binding portion and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the Fab heavy chain and the Fc domain subunit share a carboxy-terminal peptide bond. In yet another embodiment, the isolated polynucleotide of the invention encodes the heavy chain of the first antigen-binding portion, the heavy chain of the second antigen-binding portion, and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab light chain variable region shares a carboxy-terminal peptide bond with a Fab heavy chain constant region, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit. In another specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab heavy chain variable region shares a carboxy-terminal peptide bond with a Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit. In yet another specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab heavy chain shares a carboxy-terminal peptide bond with the Fab light chain variable region, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit. In yet another specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab heavy chain shares a carboxy-terminal peptide bond with the Fab heavy chain variable region, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit.
In other embodiments, the isolated polynucleotide of the invention encodes a heavy chain of the third antigen binding portion and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the Fab heavy chain and the Fc domain subunit share a carboxy-terminal peptide bond.
In other embodiments, the isolated polynucleotides of the invention encode a light chain of an antigen-binding portion. In some embodiments, the isolated polynucleotide encodes a polypeptide in which the Fab light chain variable region shares a carboxy-terminal peptide bond with the Fab heavy chain constant region. In other embodiments, the isolated polynucleotide encodes a polypeptide wherein the Fab heavy chain variable region shares a carboxy-terminal peptide bond with the Fab light chain constant region. In additional embodiments, the isolated polynucleotide of the invention encodes a light chain of the first antigen-binding portion and a light chain of the second antigen-binding portion. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the Fab heavy chain variable region shares a carboxy-terminal peptide bond with the Fab light chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fab light chain. In another specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab light chain shares a carboxy-terminal peptide bond with the Fab heavy chain variable region, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region. In yet another specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab light chain variable region shares a carboxy-terminal peptide bond with the Fab heavy chain constant region, which in turn shares a carboxy-terminal peptide bond with the Fab light chain. In yet another specific embodiment, the isolated polynucleotide encodes a polypeptide wherein the Fab light chain shares a carboxy-terminal peptide bond with the Fab light chain variable region, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region.
In another embodiment, the present invention relates to an isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of the present invention or a fragment thereof, wherein said polynucleotide comprises a sequence encoding a variable region sequence as shown in
In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, e.g., in the form of messenger RNA (mrna). The RNA of the present invention may be single-stranded or double-stranded.
Recombination method
The expression vector may be constructed by any of the polynucleotide coding region of the invention, such as a retroviral promoter, such as a retroviral enhancer, or a promoter sequence, such as a retroviral promoter, and a promoter sequence, such as a promoter sequence, or a promoter sequence, which is operably linked to a heterologous promoter sequence, such as a promoter sequence, or a promoter sequence, which is operably linked to a promoter sequence encoding a polypeptide, such as a promoter sequence, or a promoter sequence, which is operably linked to a heterologous promoter sequence, such as a promoter sequence encoding a polypeptide, such as a promoter sequence, or a promoter sequence, such as a promoter sequence, or a promoter sequence, which is not a promoter sequence, and a promoter sequence which is operably linked to a promoter sequence of a heterologous promoter, such as a ribosome, or a promoter sequence, such as a promoter sequence, and a promoter sequence, such as a promoter sequence, which is not a promoter sequence, such as a promoter sequence, and a promoter sequence, such as a promoter sequence, which is operably linked to a promoter sequence which is a promoter, such as a promoter sequence which is not a promoter, such as a promoter, which is a promoter sequence which is a promoter, and which is a promoter sequence which is capable of a promoter, such as a promoter, or a promoter sequence which is a promoter, or a promoter, and which is a promoter, which is operably linked to a promoter sequence which is a promoter, which is a promoter sequence which is not capable of a promoter, or a promoter, which is operably linked to a promoter, such as a promoter, or a promoter, which is operably linked to direct, or a promoter, which is not to a promoter, which is a promoter, or a promoter, which is operably linked to a promoter, which is a promoter, or which is a promoter, or which is operably linked to a promoter, which is a promoter, or which is a promoter, which is not a promoter, which is operably linked to a promoter, which is a promoter, or which is operably linked to a promoter, or which is a promoter, which is not a promoter, or which is not a promoter, which is a promoter, or which is a promoter, which is not a promoter, which is operably linked to a promoter, which is a promoter, or which is a promoter, which is a promoter, or which is a promoter, which is a promoter, or which is a promoter, which is not capable of a promoter, which is a promoter, or which is a promoter, which is not a promoter, which is a promoter.
The polynucleotides and nucleic acid coding regions of the present invention may be linked to additional coding regions encoding a secretion peptide or signal peptide that directs secretion of the polypeptide encoded by the polynucleotides of the present invention.A DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the T cell activating bispecific antigen binding molecule or fragment thereof of the present invention, for example, if secretion of the T cell activating bispecific antigen binding molecule is desired.A protein secreted by a mammalian cell has a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
The polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment) may comprise DNA encoding a short protein sequence either internally or at the end, which may be used to facilitate later purification (e.g. histidine tag) or to aid in labeling the T cell activating bispecific antigen binding molecule.
In yet another embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments, host cells comprising one or more vectors of the invention are provided. The polynucleotide and vector may incorporate any of the features described herein, individually or in combination, with respect to the polynucleotide and vector, respectively. In one such embodiment, the host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide encoding a T cell activating bispecific antigen binding molecule (portion) of the invention. The term "host cell" as used herein refers to any kind of cell system that can be engineered to produce the T cell activating bispecific antigen binding molecules of the invention or fragments thereof. Host cells suitable for replicating and supporting expression of T cell activating bispecific antigen binding molecules are well known in the art. Such cells can be transfected or transduced with a particular expression vector, as desired, and a large number of the vector-containing cells can be grown for inoculation into a large-scale fermentor to obtain sufficient quantities of the T cell activating bispecific antigen binding molecule for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, or various eukaryotic cells such as Chinese Hamster Ovary (CHO), insect cellsAnd the like. For example, the polypeptide may be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and the polypeptide may be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning hosts or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in production of polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-. Suitable host cells for the expression (glycosylation) of polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfecting spodoptera frugiperda (spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES described for generating antibodies in transgenic plantsTMA technique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension culture may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described in Graham et al, J Gen Virol36,59 (1977)), baby hamster kidney cells (BHK), mouse support cells (TM4 cells as described in e.g.Mather, Biol Reprod 23,243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Bufaro rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (as described in e.g.Mather et al, Annals N.Y. Acad Sci 383,44-68 (1982)), C5 cells and 4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77,4216(1980))(ii) a And myeloma cell lines such as YO, NS0, P3X63, and
Standard techniques for expressing foreign genes in these systems are known in the art. A cell expressing a polypeptide (e.g., an antibody) comprising a heavy or light chain of an antigen binding domain can be engineered so as to also express the other chain of the antibody, such that the expression product is an antibody having a heavy chain and a light chain.
In one embodiment, there is provided a method of producing a T cell activating bispecific antigen binding molecule of the invention, wherein the method comprises culturing a host cell as provided herein comprising a polynucleotide encoding the T cell activating bispecific antigen binding molecule under conditions suitable for expression of the T cell activating bispecific antigen binding molecule, and recovering the T cell activating bispecific antigen binding molecule from the host cell (or host cell culture medium).
The components of the T cell activating bispecific antigen binding molecule are genetically fused to each other. The T cell activating bispecific antigen binding molecule may be designed such that its components are fused to each other directly or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and can be tested for efficacy. Examples of linker sequences between the different components of the T cell activating bispecific antigen binding molecule are present in the sequences provided herein. Additional sequences may also be included to incorporate cleavage sites to separate the various components of the fusion, such as endopeptidase recognition sequences, if desired.
In certain embodiments, the one or more antigen binding portions of the T cell activating bispecific antigen binding molecule comprise at least an antibody variable region capable of binding an antigenic determinant. The variable region may form part of, and may be derived from, naturally or non-naturally occurring antibodies and fragments thereof. Methods for producing polyclonal and monoclonal Antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, antigen manual," Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. patent No. 4,186,567), or can be obtained, for example, by screening combinatorial libraries comprising variable heavy and variable light chains (see, e.g., U.S. patent No. 5,969,108 to McCafferty).
Antibodies, antibody fragments, antigen binding domains or variable regions of any animal species may be used in the T cell activating bispecific antigen binding molecules of the invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the invention may be of murine, primate or human origin. If the T cell activating bispecific antigen binding molecule is intended for use in humans, a chimeric form of an antibody may be used, wherein the constant region of the antibody is from a human. Antibodies in humanized or fully human form can also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods including, but not limited to, (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions with or without retention of critical framework residues (e.g., those residues important for retaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) onto human frameworks and constant regions, or (c) grafting entire non-human variable domains, but "masking" them with human-like moieties by replacing surface residues. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson,
In certain embodiments, the antigen binding moieties used in the present invention are engineered to have enhanced binding affinity according to methods disclosed, for example, in U.S. patent application publication No. 2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of the T cell activating bispecific antigen binding molecules of the invention to bind to a specific epitope can be measured by enzyme linked immunosorbent assay (ELISA) or other techniques familiar to the person skilled in the art, such as surface plasmon resonance techniques (analysis on the BIACORE T100 system) (Liljeblad et al, Glyco J17, 323-. Competition assays can be used to identify antibodies, antibody fragments, antigen binding domains or variable domains that compete with a reference antibody for binding to a particular antigen, e.g., antibodies that compete with the V9 antibody for binding to
The T cell activating bispecific antigen binding molecule prepared as described herein can be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on a number of factors, such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens that bind to the T cell activating bispecific antigen binding molecule can be used. For example, for affinity chromatography purification of the T cell activating bispecific antigen binding molecule, a matrix with protein a or protein G may be used. Protein a or G affinity chromatography and size exclusion chromatography in sequence can be used to isolate the T cell activating bispecific antigen binding molecule substantially as described in the examples above. The purity of the T cell activating bispecific antigen binding molecule can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, heavy chain fusion proteins expressed as described in were shown to be intact and correctly assembled as displayed by reducing SDS-PAGE (see, e.g., fig. 2). Three bands were isolated at approximately relative molecular weights of 25,000, 50,000 and 75,000, which correspond to the predicted molecular weights of the light chain, heavy chain and heavy/light chain fusion proteins of the T cell activating bispecific antigen binding molecule.
Assay method
The physical/chemical properties and/or biological activities of the T cell activating bispecific antigen binding molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.
Affinity assay
The affinity of the T cell activating bispecific antigen binding molecule for the Fc receptor or target antigen can be determined by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) according to the methods described in the examples and the receptor or target protein can be obtained, for example, by recombinant expression. Alternatively, the binding effect of a T cell activating bispecific antigen binding molecule to a different receptor or target antigen can be assessed using a cell line expressing the specific receptor or target antigen, e.g. by flow cytometry (FACS). Specific illustrative and exemplary embodiments for measuring binding affinity are described below and in the examples below.
According to one embodiment, use is made ofK measurement by surface plasmon resonance at 25 ℃ with T100 instrument (GE Healthcare)D。
To analyze the interaction between the Fc portion and Fc receptor, His-tagged recombinant Fc receptor was captured by anti-pentahis antibody (Qiagen) immobilized on a CM5 chip and a specific construct was used as the analyte. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide hydrochloride (NHS) according to the supplier's instructions. The anti-pentaHis antibody was diluted to 40. mu.g/ml with 10mM sodium acetate, pH5.0, and then loaded at a flow rate of 5. mu.l/min to achieve approximately 6500 Response Units (RU) of the coupled protein. After loading of the ligand, 1M ethanolamine was injected to block unreacted groups. Subsequently, the Fc receptor was captured at 4 or 10nM for 60 seconds. For kinetic measurements, 4-fold serial dilutions of the bispecific construct (ranging between 500nM and 4000 nM) were loaded in HBS-EP (GE Healthcare, 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at 25 ℃ for 120 seconds at a flow rate of 30. mu.l/min.
To determine affinity for the target antigen, bispecific constructs were captured by anti-human Fab specific antibodies (GE Healthcare) immobilized on the surface of an activated CM5 sensor chip, as described for anti-pentahis antibody. The final amount of coupled protein was about 12000 RU. The bispecific construct was captured at 300nM for 90 sec. The target antigen was passed through the flow cell at a concentration range of 250nM to 1000nM for 180 seconds at a flow rate of 30. mu.l/min. The dissociation process was monitored for 180 seconds.
Bulk refractive index differences were corrected by subtracting the responses obtained on the reference flow cell. Steady state response was used to derive the dissociation constant K by nonlinear curve fitting of Langmuir binding isothermsD. Using a simple one-to-one Langmuir binding model: (
Activity assay
The biological activity of the T cell activating bispecific antigen binding molecules of the invention can be measured by a variety of assays as described in the examples. Biological activities may, for example, include inducing T cell proliferation, inducing signaling of T cells, inducing expression of activation markers in T cells, inducing secretion of cytokines by T cells, inducing lysis of target cells (e.g., tumor cells), and inducing tumor regression and/or improving survival.
Compositions, formulations and routes of administration
In a further aspect, the invention provides a pharmaceutical composition comprising any of the T cell activating bispecific antigen binding molecules provided herein, e.g., for use in any of the treatment methods described below. In one embodiment, the pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.
Further provided is a method of producing a T cell activating bispecific antigen binding molecule of the invention in a form suitable for in vivo administration, the method comprising (a) obtaining a T cell activating bispecific antigen binding molecule of the invention, and (b) formulating the T cell activating bispecific antigen binding molecule with at least one pharmaceutically acceptable carrier, thereby formulating a preparation of the T cell activating bispecific antigen binding molecule for in vivo administration.
The pharmaceutical compositions of the invention comprise a therapeutically effective amount of one or more T cell activating bispecific antigen binding molecules dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce an adverse, strained, or other untoward reaction when administered to an animal (e.g., a human, if appropriate). In accordance with the present disclosure, it will be known to those skilled in the art to prepare Pharmaceutical compositions containing at least one T cell activating bispecific antigen binding molecule, and optionally additional active ingredients, as exemplified by Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it is understood that the article should meet sterility, pyrogenicity, overall safety and purity standards as required by the FDA office of biological standards or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such like materials, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, 1289-1329, which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, use of the carrier in therapeutic or pharmaceutical compositions is contemplated.
The composition may contain different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form and whether it needs to be sterile for such routes of administration as an injection. The T cell activating bispecific antigen binding molecules of the invention (and any other therapeutic agent) may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularly, mucosally, intrapericardially, intraumbilically, intraventricularly, orally, topically, by inhalation (e.g., aerosol inhalation), by injection, infusion, by continuous infusion, by direct localized perfusion bathing of target cells, by catheter, by lavage, in a paste, in a lipid composition (e.g., liposomes), or by other methods or any combination of the foregoing methods, as known to one of ordinary skill in the art (see, e.g., Remington's pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, incorporated by reference). Parenteral administration, especially intravenous injection, is most commonly used for administration of polypeptide molecules, such as the T cell activating bispecific antigen binding molecules of the invention.
Parenteral compositions include those designed to be administered by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the T cell activating bispecific antigen binding molecule of the invention may be formulated in aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution or physiological saline. The solution may contain formulating agents such as suspending, solubilizing, stabilizing and/or dispersing agents. Alternatively, the T cell activating bispecific antigen binding molecule may be in a form that is constituted prior to use with a suitable vehicle (e.g., sterile pyrogen-free water) powder. Sterile injectable solutions are prepared by: the T cell activating bispecific antigen binding molecule of the present invention is incorporated in a suitable solvent in the required amount, as required, together with various other ingredients enumerated below. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the in situ preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium. If desired, the liquid medium should be suitably buffered and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage; and are therefore protected from the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum, at a safe level, for example, less than 0.5ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citric acid and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (e.g. octadecyl benzyl dimethyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzalkonium bromide; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannose, trehalose or sorbose; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or materials that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.
The active ingredient may be embedded in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly (methylmethacylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or emulsions, for example, prepared by coacervation techniques or interfacial polymerization, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18 th edition, Mack Printing Company, 1990). Sustained release articles can be prepared. Suitable examples of sustained-release articles include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In particular embodiments, prolonged absorption of the composition can be brought about by the use in the injectable compositions of agents delaying absorption, for example, aluminum monostearate, gelatin, or combinations thereof.
In addition to the compositions described previously, the T cell activating bispecific antigen binding molecule may also be formulated as a depot formulation. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the T cell activating bispecific antigen binding molecule may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., a sparingly soluble salt.
The pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of the invention may be manufactured in any manner known in the art, e.g. by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen.
The T cell activating bispecific antigen binding molecule may be formulated into the composition as a free acid or base, a neutral salt or a salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These salts include acid addition salts, for example, those formed with the free amino groups of the protein composition or with inorganic acids such as hydrochloric acid or phosphoric acid or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous solvents and other protic solvents that are not the corresponding free base forms.
Therapeutic methods and compositions
Any of the T cell activating bispecific antigen binding molecules provided herein can be used in a method of treatment. The T cell activating bispecific antigen binding molecules of the invention may be used as immunotherapeutic agents, e.g. for the treatment of cancer.
For use in a method of treatment, the T cell activating bispecific antigen binding molecules of the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of drug delivery, the method of administration, the administration schedule and other factors known to the medical practitioner.
In one aspect, there is provided a T cell activating bispecific antigen binding molecule of the invention for use as a medicament. In other aspects, the T cell activating bispecific antigen binding molecules of the invention are provided for use in the treatment of a disease. In certain embodiments, there is provided a T cell activating bispecific antigen binding molecule of the invention for use in a method of treatment. In one embodiment, the present invention provides a T cell activating bispecific antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the present invention provides a T cell activating bispecific antigen binding molecule for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of a T cell activating bispecific antigen binding molecule. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In other embodiments, the invention provides a T cell activating bispecific antigen binding molecule as described herein for use in inducing lysis of a target cell, in particular a tumor cell. In certain embodiments, the present invention provides a method of using a T cell activating bispecific antigen binding molecule for inducing lysis of a target cell, particularly a tumor cell, in an individual, the method comprising administering to the individual an effective amount of a T cell activating bispecific antigen binding molecule to induce lysis of the target cell. An "individual" according to any of the above embodiments is a mammal, preferably a human.
In a further aspect, the invention provides the use of a T cell activating bispecific antigen binding molecule of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating a disease in an individual in need thereof. In yet another embodiment, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In yet another embodiment, the medicament is for inducing lysis of target cells, in particular tumor cells. In yet another embodiment, the medicament is for use in a method of inducing lysis of target cells, particularly tumor cells, in an individual, said method comprising administering to the individual an effective amount of the medicament to induce lysis of the target cells. An "individual" according to any of the above embodiments may be a mammal, preferably a human.
In yet another aspect, the invention provides a method for treating a disease. In one embodiment, the method comprises administering to an individual having such a disease a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention. In one embodiment, a composition comprising a T cell activating bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form is administered to the individual. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. An "individual" according to any of the above embodiments may be a mammal, preferably a human.
In yet another aspect, the invention provides a method for inducing lysis of a target cell, in particular a tumor cell. In one embodiment, the method comprises contacting the target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, in particular a cytotoxic T cell. In yet another aspect, a method for inducing lysis of a target cell, in particular a tumor cell, in an individual is provided. In such an embodiment, the method comprises administering to the individual an effective amount of a T cell activating bispecific antigen binding molecule to induce target cell lysis. In one embodiment, the "individual" is a human.
In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell cancer, bone cancer, and renal cancer. Other cell proliferation disorders that can be treated using the T cell activating bispecific antigen binding molecules of the invention include, but are not limited to, tumors located at: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, (central and peripheral) nervous system, lymphatic system, pelvic cavity, skin, soft tissue, spleen, chest and urogenital system. Also included are precancerous conditions or lesions and cancer metastases. In certain embodiments, the cancer is selected from renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. The skilled person readily recognizes that in many cases the T cell activating bispecific antigen binding molecule may not provide a cure, but may only provide partial benefits. In some embodiments, physiological changes with certain benefits are also considered therapeutically beneficial. Thus, in some embodiments, the amount of T cell activating bispecific antigen binding molecule that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient or individual in need of treatment is generally a mammal, more particularly a human.
In some embodiments, an effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to an individual to treat a disease.
For the prevention or treatment of a disease, the appropriate dosage of a T cell activating bispecific antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the type of T cell activating bispecific antigen binding molecule, the severity and course of the disease, whether the T cell activating bispecific antigen binding molecule is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic intervention, the patient's clinical history and response to the T cell activating bispecific antigen binding molecule, and the discretion of the attending physician. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
The T cell activating bispecific antigen binding molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of the T cell activating bispecific antigen binding molecule may be an initial candidate dose for administration to a patient, whether administered, for example, by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a common daily dose may be from about 1. mu.g/kg to 100mg/kg or more. For repeated administration over a range of days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the T cell activating bispecific antigen binding molecule is in the range of about 0.005mg/kg to about 10 mg/kg. In other non-limiting examples, the dose may further comprise about 1 microgram/kg body weight, about 5 microgram/kg body weight, about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligrams/kg body weight, about 10 milligrams/kg body weight, about 50 milligrams/kg body weight, about 100 milligrams/kg body weight, about 200 milligrams/kg body weight, about 350 milligrams/kg body weight, about 500 milligrams/kg body weight to about 1000 milligrams/kg body weight or more per administration, and any range useful therein. In non-limiting examples of ranges available from the values listed herein, based on the values described above, ranges of about 5mg/kg body weight to about 100mg/kg body weight, about 5 micrograms/kg body weight to about 500 milligrams/kg body weight, and the like, can be administered. Thus, one or more doses (or any combination thereof) of about 0.5mg/kg, 2.0 mg/kg, 5.0mg/kg, or 10mg/kg may be administered to the patient. Such doses may be administered intermittently, e.g., weekly or every 3 weeks (e.g., whereby the patient receives from about 2 to about 20 or, e.g., about 6 doses of the T cell activating bispecific antigen binding molecule). A higher initial loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be used. The progress of such therapy is readily monitored by conventional techniques and assays.
The T cell activating bispecific antigen binding molecules of the invention will generally be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease state, the T cell activating bispecific antigen binding molecule of the invention or a pharmaceutical composition thereof is administered or applied in a therapeutically effective amount. Determining a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can be formulated in animal models to achieve inclusion of IC50As determined in cell culture. This information can be used to more accurately determine the dosage for use in humans.
Initial doses can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data.
The number of doses and the interval can be adjusted individually to provide plasma levels of the T cell activating bispecific antigen binding molecule sufficient to maintain the therapeutic effect. A typical dose for patients administered by injection is about 0.1 to 50 mg/kg/day, typically about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma may be measured, for example, by HPLC.
In the case of topical administration or selective uptake, the effective local concentration of the T cell activating bispecific antigen binding molecule may not be related to the plasma concentration. One skilled in the art will be able to optimize therapeutically effective topical dosages without undue experimentation.
A therapeutically effective dose of the T cell activating bispecific antigen binding molecules described herein will generally provide therapeutic benefit without causing significant toxicity. Toxicity and therapeutic efficacy of T cell activating bispecific antigen binding molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine LD50(dose lethal to 50% of the population) and ED50(therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD50/ED50And (4) the ratio. Preferably, the T cell activating bispecific antigen binding molecule exhibits a large therapeutic index. In one embodiment, the T cell activating bispecific antigen binding molecule of the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dose is preferably at a low or no toxicity level including ED50A series of circulating concentration ranges. The dosage may vary within this range depending on a variety of factors, e.g., the dosage form used, the route of administration used, the condition of the subject, etc. The exact formulation, route of administration and dosage can be selected by The individual physician according to The disease of The patient (see, e.g., Fingl et al, 1975, cited in The Pharmacological basis of Therapeutics,
The attending physician of a patient treated with a T cell activating bispecific antigen binding molecule of the invention will know how and when to terminate, suspend or adjust administration due to resulting toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (not including toxicity), the attending physician will also know to adjust the treatment to higher levels. The size of the dose administered to treat a condition of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition can be assessed, for example, in part, by standard prognostic assessment methods. In addition, the dosage and perhaps the frequency of administration will also vary according to the age, weight and response of the individual patient.
Other Agents and treatments
The T cell activating bispecific antigen binding molecules of the invention may be administered in combination with one or more other agents in therapy. For example, the T cell activating bispecific antigen binding molecule of the invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered for the treatment of a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of apoptosis, or an agent that increases the sensitivity of a cell to an inducer of apoptosis. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalating agent, an alkylating agent, a hormonal therapy agent, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.
Such other agents are suitably present in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of T cell activating bispecific antigen binding molecule used, the type of disease or therapy, and other factors discussed above. The T cell activating bispecific antigen binding molecule is typically used at the same dosage and using the same route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and any route empirically/clinically determined to be appropriate.
Such combination therapies indicated above encompass combined administration (wherein two or more therapeutic agents are comprised in the same or separate compositions), and separate administration, in which case the administration of the T cell activating bispecific antigen binding molecules of the invention may be performed before, simultaneously with and/or after the administration of additional therapeutic agents and/or adjuvants. The T cell activating bispecific antigen binding molecules of the present invention may also be used in combination with radiotherapy.
Article of manufacture
In another aspect of the invention, an article of manufacture is provided, which comprises a substance as described above, which is useful in the treatment, prevention and/or diagnosis of a condition. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition that is effective, by itself or in combination with another composition, in the treatment, prevention and/or diagnosis of a condition and may have a sterile access port (e.g., the container may be an intravenous bag or a vial having a stopper penetrable by a hypodermic injection needle). At least one active agent in the composition is a T cell activating bispecific antigen binding molecule of the invention. The label or package insert indicates that the composition is for use in treating a selected condition. Moreover, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises a T cell activating bispecific antigen binding molecule of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic or therapeutic agent. In this embodiment of the invention, the article of manufacture may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials welcomed from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.