Antibodies that bind GPRC5D
阅读说明：本技术 结合gprc5d的抗体 (Antibodies that bind GPRC5D ) 是由 G·费尔蒂希 C·克莱因 S·洛伦兹 W·许 M-L·贝尔纳斯科尼 A·布约齐克 于 2019-02-07 设计创作，主要内容包括：本发明一般涉及结合GPRC5D的抗体,包括双特异性抗原结合分子,例如用于活化T细胞。另外,本发明涉及编码此类抗体的多核苷酸,以及包含此类多核苷酸的载体和宿主细胞。本发明进一步涉及用于生成该抗体的方法,和在疾病的治疗中使用它们的方法。(The present invention relates generally to antibodies, including bispecific antigen binding molecules, that bind GPRC5D, e.g., for use in activating T cells. In addition, the invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and methods of using them in the treatment of disease.)
1. An antibody that binds GPRC5D, wherein the antibody comprises
(i) A heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO: 89;
(ii) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO: 89;
(iii) A heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO: 97;
(iv) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO: 97; or
(v) A heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:92, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO: 97.
2. The antibody according to claim 1, wherein said antibody is selected from the group consisting of,
(i) wherein the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO 13 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO 14; or
(ii) Wherein the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 15 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 16; or
(iii) Wherein the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO 48 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO 53; or
(iv) Wherein the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 49 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 52; or
(v) Wherein the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 57 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 64; or
(vi) Wherein the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO 58 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO 63.
3. The antibody of claim 1 or 2, wherein the antibody is an IgG antibody.
4. The antibody of claim 3, wherein the antibody is an IgG antibody.
5. The antibody of any one of claims 1 to 4, wherein the antibody is a full length antibody.
6. The antibody of any one of claims 1 to 4, wherein the antibody is selected from the group consisting of Fv molecules, scFv molecules, Fab molecules, and F (ab')2Antibody fragments of a panel of molecules.
7. The antibody of any one of claims 1 to 6, wherein the antibody is a multispecific antibody.
8. A bispecific antigen binding molecule comprising
(a) A first antigen-binding moiety that binds a first antigen,
wherein the first antigen is GPRC5D and the first antigen binding module comprises
(i) A heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO: 89;
(ii) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO: 89;
(iii) A heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO: 97;
(iv) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO: 97; or
(v) A heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:92, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO: 97; and
(b) a second antigen binding moiety that specifically binds a second antigen.
9. The bispecific antigen binding molecule of claim 8,
(i) wherein the VH of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 13, and wherein the VL of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 14; or
(ii) Wherein the VH of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 15, and wherein the VL of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 16; or
(iii) Wherein the VH of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 48, and wherein the VL of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 53; or
(iv) Wherein the VH of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 49, and wherein the VL of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 52; or
(v) Wherein the VH of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 57, and wherein the VL of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 64; or
(vi) Wherein the VH of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 58, and wherein the VL of the first antigen-binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 63.
10. The bispecific antigen binding molecule of claim 8 or 9, wherein the second antigen is CD 3.
11. The bispecific antigen binding molecule of claim 10, wherein the second antigen is CD 3.
12. The bispecific antigen binding molecule of claim 10 or 11, wherein the second antigen binding module comprises a VH comprising HCDR 1 of SEQ ID No. 29, HCDR 2 of SEQ ID No. 30, and HCDR 3 of SEQ ID No. 31 and a VL comprising LCDR 1 of SEQ ID No. 32, LCDR 2 of SEQ ID No. 33, and LCDR 3 of SEQ ID No. 34.
13. The bispecific antigen binding molecule of claim 12, wherein the VH of the second antigen binding moiety comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 35 and the VL of the second antigen binding moiety comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 36.
14. The bispecific antigen binding molecule of any one of claims 8 to 13, wherein the first and/or the second antigen binding moiety is a Fab molecule.
15. The bispecific antigen binding molecule of any one of claims 8 to 14, wherein the second antigen binding moiety is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains, in particular the variable domains VL and VH, are replaced with each other.
16. The bispecific antigen binding molecule of any one of claims 8 to 15, wherein the first antigen binding moiety is a Fab molecule wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) in the constant domain, and the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat index) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the EU index) in the constant domain CH 1.
17. The bispecific antigen binding molecule of any one of claims 8 to 16, wherein the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker.
18. The bispecific antigen binding molecule of any one of claims 8 to 17, wherein the first and the second antigen binding moiety are each a Fab molecule and wherein either (i) 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, or (ii) 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.
19. The bispecific antigen binding molecule of any one of claims 8 to 18, comprising a third antigen binding moiety.
20. The bispecific antigen binding molecule of claim 19, wherein the third antigen moiety is identical to the first antigen binding moiety.
21. The bispecific antigen binding molecule of any one of claims 8 to 20, comprising an Fc domain comprised of a first and a second subunit.
22. The bispecific antigen binding molecule of claim 21, wherein the first, the second and, when present, the third antigen binding moiety are each a Fab molecule;
and wherein either (i) 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 subunit of the Fc domain, or (ii) 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 subunit of the Fc domain;
And wherein the third antigen binding moiety, when present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
23. The bispecific antigen binding molecule of claim 21 or 22, wherein the Fc domain is an IgG Fc domain.
24. The bispecific antigen binding molecule of claim 23, wherein the Fc domain is IgG1An Fc domain.
25. The bispecific antigen binding molecule of any one of claims 21 to 24, wherein the Fc domain is a human Fc domain.
26. The bispecific antigen binding molecule of any one of claims 21 to 25, wherein 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 generating a protuberance within the CH3 domain of the first subunit that is placeable in a 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 generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is placeable.
27. The bispecific antigen binding molecule of any one of claims 21 to 26, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function.
28. One or more isolated polynucleotides encoding the antibody or bispecific antigen binding molecule of any one of claims 1 to 27.
29. One or more vectors, in particular expression vectors, comprising one or more polynucleotides according to claim 28.
30. A host cell comprising one or more polynucleotides of claim 28 or one or more vectors of claim 29.
31. A method of producing an antibody that binds GPRC5D comprising the steps of a) culturing the host cell of claim 30 under conditions suitable for expression of the antibody and b) optionally recovering the antibody.
32. An antibody that binds GPRC5D, produced by the method of claim 31.
33. A pharmaceutical composition comprising the antibody or bispecific antigen binding molecule of any one of claims 1 to 27 or 32 and a pharmaceutically acceptable carrier.
34. The antibody or bispecific antigen binding molecule of any one of claims 1 to 27 or 32 or the pharmaceutical composition of claim 33 for use as a medicament.
35. The antibody or bispecific antigen binding molecule of any one of claims 1 to 27 or 32 or the pharmaceutical composition of claim 33 for use in the treatment of a disease.
36. The antibody, bispecific antigen binding molecule or pharmaceutical composition of claim 35, wherein the disease is cancer or an autoimmune disease.
37. The antibody, bispecific antigen-binding molecule or pharmaceutical composition of claim 35, wherein the disease is multiple myeloma.
38. Use of the antibody or bispecific antigen binding molecule of any one of claims 1 to 27 or 32 in the manufacture of a medicament for the treatment of a disease.
39. A method of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of a composition comprising the antibody or bispecific antigen binding molecule of any one of claims 1 to 27 or 32 in a pharmaceutically acceptable form.
40. The use of claim 38 or the method of claim 39, wherein the disease is cancer or an autoimmune disease.
41. The use of claim 38 or method of claim 39, wherein the disease is multiple myeloma.
42. The invention as described in the specification.
Technical Field
The present invention relates generally to antibodies, including bispecific antigen binding molecules, that bind GPRC5D, e.g., for use in activating T cells. In addition, the invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and methods of using them in the treatment of disease.
Background
Multiple Myeloma (MM) is one of the most common hematological malignancies affecting 75,000 new patients annually in the european union and united states, and still has a high unmet medical need. Multiple myeloma is characterized by terminally differentiated plasma cells that secrete non-functional monoclonal immunoglobulins. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide, and proteasome inhibitors such as carfilzomib or bortezomib may remain the backbone of first-line therapy for multiple myeloma (Moreau, p.and s.v. rajkumar, multiple myelo-transformation of tertiary disorders in lancet,2016.388(10040): p.111-3). However, these drugs do not specifically target diseased tumor cells, such as diseased Plasma Cells (PCs). Efforts have been made to selectively deplete plasma cells in multiple myeloma. The lack of surface proteins that specifically label plasma cells has hindered the development of antibodies or cell therapies for multiple myeloma. To date, there are few cases of successful biologies, as represented, for example, by daratuzumab (anti-CD 38) and erlotinib (anti-CD 319), noting that these two molecules are not uniquely expressed by plasma cells. Thus, RNA sequencing was used to identify novel targets from plasma cells in multiple myeloma, such as the G-protein coupled receptor class C group 5 member D (GPRC 5D). GPRC5D is a specific surface protein expressed by plasma cells in multiple myeloma. GPRC5D has been reported to be associated with prognosis and tumor burden in patients with multiple myeloma (Atamaniuk, J., et al, Overexpression of G protein-coalplex hormone 5D in the bone marrow hormone with bone hormone secretion in multiple myeloma. Eur J Clin Invest,2012.42(9): p.953-60; and Cohen, Y., et al, GPRC5D a stimulating marker for monitoring the tumor load and target tumor hormone cells.hematology,2013.18(6): p.348-51).
GPRC5D is an orphan receptor with no known ligand or function in human and human cancers. The GPRC5D encoding gene located on chromosome 12p13.3 contains three exons and spans about 9.6kb (Brauner-Osborne, H., et al., Cloning and characterization of a human orphan family C G-protein linker GPRC 5. Biochim Biophys Acta,2001.1518(3): p.237-48). The larger first exon encodes the seven transmembrane domain. However, the biology of GPRC5D is largely unknown. GPRC5D has been shown to be involved in keratin formation in Hair follicles in animals (Gao, Y., et al., Comparative transaction Analysis of Fetal Skin vectors Key Genes Related to Hair follicule Morphogenesis in Cashmere Goates One,2016.11(3): p.e0151118; and Inoue, S., T.Nambu, andT.Shimomura, The RAIG family member, GPRC5D, associated with hard-keyed construction. J Invest Dermatol,2004.122(3): p.565-73).
WO 2018/017786a2 discloses GPRC 5D-specific antibodies or antigen-binding fragments.
There is a need for additional drugs to treat cancer, particularly multiple myeloma. Particularly useful drugs for this purpose include antibodies that bind GPRC5D, particularly bispecific antibodies that bind GPRC5D on target cells and activating T cell antigens such as CD3 on T cells. Simultaneous binding of such an antibody to its two targets will force a transient interaction between the target cell and the T cell, causing any cytotoxic T cell activation and subsequent lysis of the target cell.
The present invention provides novel antibodies, including bispecific antibodies, that specifically bind to human GPRC 5D. In particular, T cell bispecific antibodies targeting GPRC5D according to the present invention have efficacy in treating multiple myeloma.
Summary of The Invention
The inventors have developed novel antibodies that bind GPRC5D with unexpected, improved properties. Moreover, the inventors developed bispecific antigen binding molecules that bind GPRC5D and activating T cell antigens that incorporate the novel GPRC5D antibody.
In a first aspect, the invention provides an antibody that binds GPRC5D, wherein the antibody comprises (i) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83,
In yet another aspect, the invention provides a bispecific antigen binding molecule comprising (a) a first antigen binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen binding moiety comprises (i) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, HCDR2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:87,
In one embodiment, the first and/or the second antigen binding moiety is a Fab molecule. This means that either the first antigen binding moiety may be a Fab molecule, or the second antigen binding moiety may be a Fab molecule, or the first and second antigen binding moieties may be Fab molecules. In another embodiment, the second antigen binding moiety is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains, in particular the variable domains VL and VH, are replaced with each other. In another embodiment, the first antigen binding moiety is a Fab molecule, wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) in the constant domain, and the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat index) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) in
In another aspect, the invention provides one or more isolated polynucleotides encoding an antibody or bispecific antigen binding molecule as described herein. In a further aspect, the present invention provides one or more vectors, in particular expression vectors, comprising one or more polynucleotides as described herein. In another aspect, the invention provides a host cell comprising one or more polynucleotides as described herein or one or more vectors as described herein. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell.
In another aspect of the invention, a method of producing an antibody that binds GPRC5D is provided comprising the steps of a) culturing a host cell as described herein under conditions suitable for expression of the antibody and b) optionally recovering the antibody. In another aspect, the invention provides antibodies that bind GPRC5D produced by a method as described herein. Another aspect of the invention relates to a pharmaceutical composition comprising an antibody or bispecific antigen binding molecule as described herein and a pharmaceutically acceptable carrier. Another aspect of the invention relates to an antibody or bispecific antigen binding molecule as described herein or a pharmaceutical composition as described herein for use as a medicament. Another aspect of the invention relates to an antibody or bispecific antigen binding molecule as described herein or a pharmaceutical composition as described herein for use in the treatment of a disease. In one embodiment, the disease is cancer, in particular multiple myeloma. Alternatively, the disease is an autoimmune disease, such as systemic lupus erythematosus and/or rheumatoid arthritis.
The invention further provides the use of an antibody or bispecific antigen binding molecule as described herein in the manufacture of a medicament for the treatment of a disease, in particular cancer, more in particular multiple myeloma. Alternatively, the disease is an autoimmune disease, such as systemic lupus erythematosus and/or rheumatoid arthritis.
In another aspect, the invention relates to a method of treating a disease, in particular cancer, more in particular multiple myeloma, in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising a pharmaceutically acceptable form of an antibody or a bispecific antigen binding molecule as described herein. Alternatively, the disease is an autoimmune disease, such as systemic lupus erythematosus and/or rheumatoid arthritis. In any of the above embodiments, the individual is preferably a mammal, particularly a human.
Brief Description of Drawings
FIGS. 1A-Z. Exemplary configurations of bispecific antigen binding molecules of the invention. (FIG. 1A, FIG. 2D) "1 +1 CrossMab" molecule. (FIG. 1B, FIG. 1E) "2 +1IgG Crossfab" molecule diagram with alternative (alternative) order Crossfab and Fab components ("inverted"). (FIG. 1C, FIG. 1F) "2 +1IgG Crossfab" molecule. (FIG. 1G, FIG. 1K) Illustration of the "1 +1IgG Crossfab" molecule with alternative (alternative) order Crossfab and Fab components ("inverted"). (FIG. 1H, FIG. 1L) "1 +1IgG Crossfab" molecule. (FIG. 1I, FIG. 1M) "2 +1IgG Crossfab" molecule, which has two CrossFab. (FIG. 1J, FIG. 1N) "2 +1 IgGCrossfab" molecule diagram, with two CrossFab and alternative (alternative) order Crossfab and Fab components ("inverted"). (FIG. 1O, FIG. 1S) "Fab-Crossfab" molecule. (FIG. 1P, FIG. 1T) "Crossfab" molecule. (FIG. 1Q, FIG. 1U) "(Fab)2-representation of Crossfab "molecule. (FIG. 1R, FIG. 1V) "Crossfab- (Fab)2"molecular diagram. (FIG. 1W, FIG. 1Y) "Fab- (Crossfab)2"molecular diagram. (FIG. 1X, FIG. 1Z) "(Crossfab)2Representation of the Fab "molecule. Black dots modification in the optional Fc domain to promote heterodimerization. + +, -, CH1 and the CL domain optionally with oppositely charged amino acids. The Crossfab molecule is depicted as comprising a VH and VL region exchange, but may alternatively comprise an exchange of CH1 and CL domains in embodiments in which no charge modification is introduced in the CH1 and CL domains.
Fig. 2. Analysis of gene expression of tumor targets on plasma and B cells by RNAseq.
Fig. 3. Exemplary configurations of the 5E11 bispecific antigen binding molecules of the invention. Black dots, modification in the optional Fc domain to promote heterodimerization. + + + - -, optionally amino acids of opposite charge introduced into the CH1 and CL domains.
FIGS. 4A-C. Binding assays of bispecific antigen-binding molecules 5F11-TCB (FIG. 4A) and 5E11-TCB (FIG. 4B) and control antibody ET150-5-TCB (FIG. 4C) to multiple myeloma cell lines AMO-1, L636, NCI-H929, RPMI-8226, OPM-2 and WSU-
FIGS. 5A-E. GPRC5D-TCB mediated analysis of T cell cytotoxicity against multiple myeloma cell lines AMO-1 (FIG. 5A), NCI-H929 (FIG. 5B), RPMI-8226 (FIG. 5C) and L363 (FIG. 5D). The control cell line was WSU-DL CL2 (FIG. 5E). Test molecules 5E11-TCB,5F 11-TCB. Control molecules DP47-TCB (non-targeting) and ET 150-5-TCB.
Fig. 6. Analysis of GPRC5D-TCB activated T cell engagement multiple myeloma cell line NCI-H929 and negative control cell line WSU-DLCL2, which upregulated CD25 and CD 69.
FIGS. 7A-J. Analysis of GPRC 5-TCB activated T cell engagement multiple myeloma of CD69 in up-regulated cell lines AMO-1 (FIG. 7A), NCI-H929 (FIG. 7B), RPMI-8226 (FIG. 7C), L363 (FIG. 7D) and WSU-DLCL2 (FIG. 7E) and up-regulated cell lines AMO-1 (FIG. 7F), NCI-H929 (FIG. 7G), RPMI-8226 (FIG. 7H), L363 (FIG. 7I) and WSU-DLCL2 (FIG. 7J).
FIGS. 8A-B. Visualization of antibody localization and internalization by fluorescence confocal microscopy (fig. 8A) and analysis of membrane-to-cytosolic signal intensity (fig. 8B).
Fig. 9. The binding of the different anti-GPRC 5D antibodies to human, cynomolgus and murine GPRC5D was assessed by ELISA using stably transfected CHO clones expressing human GPRC5D (clone 12) or cynomolgus GPRC5D (clone 13), murine GPRC5D (clone 4) or human GPRC5A (clone 30).
FIGS. 10A-G. T cell-mediated lysis of various Multiple Myeloma (MM) cell lines induced by different GPRC5D or BCMA-targeted T cell bispecific molecules during 20 hours of co-incubation (E: T ═ 10:1, human pan T cells). Depicted are duplicates and SD.
FIGS. 11A-F. Allogeneic pan-human T cells and unprocessed bone marrow cells from healthy donors (E: T ═ 10:1, human pan T cells) were co-incubated for about 20 hours for T cell activation induced by different GPRC5D or BCMA-targeting T cell bispecific molecules (5E 11-TCB in fig. 11A; 5F11-TCB in fig. 11B; 10B10-TCB in fig. 11C; BCMA-TCB in fig. 11D; BCMA-TCB in fig. 11E; DP47-TCB in fig. 11F). Depicted are FACS dot plots from one representative donor showing upregulation of the activation marker CD69 on CD4 (upper row) or CD 8T cells (lower row) as a percentage of positive cells in all CD4 or CD 8T cells.
FIGS. 12A-B. Allogeneic pan-human T cells and unprocessed bone marrow cells from healthy donors (E: T ═ 10:1, human pan-T cells) were co-incubated for about 20 hours with T cell activation induced by different GPRC5D or BCMA-targeted T cell bispecific molecules. Depicted is a summary of all four donors evaluated, showing the upregulation of the activation marker CD69 on CD 8T cells at the selected fixed dose of 50nM (fig. 12A) or 5nM (fig. 12B) TCB.
FIGS. 13A-D. In vivo efficacy induced by different GPRC5D targeting T cell bispecific molecules (5F 11-TCB in FIG. 13A; BCMA-TCB in FIG. 13B; B72-TCB in FIG. 13C; vehicle in FIG. 13D) as depicted by tumor growth kinetics over time in a humanized NSG mouse model implanted with NCI-H929 tumor cells. Spiders were drawn, and each line indicated one mouse.
FIGS. 14A-D. In vivo efficacy induced by different GPRC5D targeting T cell bispecific molecules (5F 11-TCB in fig. 14A; 5E11-TCB in fig. 14B; B72-TCB in fig. 14C; vehicle in fig. 14D) as depicted by tumor growth kinetics over time in a humanized NSG mouse model implanted with OPM-2 tumor cells. Spiders were drawn, and each line indicated one mouse.
FIGS. 15A-B. PGLALA-CAR-J activation after approximately 16 hours of incubation, as determined by luminescence. The latter was induced following simultaneous binding of GPRC5D IgG (5F 11-IgG in FIG. 15A; 5E11-IgG in FIG. 15B) to GPRC5D expressing multiple myeloma cell line L-363 and PGLALA modified Fc domain to Jurkat-NFAT reporter cells genetically engineered to express TCR against PGLALA mutations in the Fc portion of these IgG molecules. Depicted are duplicates and SD.
Detailed Description
Definition of
Unless otherwise defined below, terms are used herein as generally used in the art.
As used herein, the term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives, e.g. fragments, thereof.
The term "bispecific" means that the antigen binding molecule is capable of specifically binding at least two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antigen binding molecule is capable of binding two antigenic determinants simultaneously, in particular two antigenic determinants expressed on two different cells.
As used herein, the term "valency" refers to the presence of a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen binding site in an antigen binding molecule that is specific for the antigen.
An "antigen binding site" refers to a site, i.e., one or more amino acid residues, on 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 a Complementarity Determining Region (CDR). Natural immunoglobulin molecules typically have two antigen binding sites, and Fab molecules typically have a single antigen binding site.
As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding moiety is capable of directing an entity attached thereto (e.g., a second antigen binding moiety) to a target site, e.g., to a particular type of antigenic determinant-bearing tumor cell. In another embodiment, the antigen binding moiety is capable of activating signaling via its target antigen, e.g., a T cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include the antigen binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moiety may comprise an antibody constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of the 5 isoforms α, γ or μ. Useful light chain constant regions include any of the following 2 isoforms κ and λ.
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 moiety binds, thereby forming an antigen-binding moiety-antigen complex (e.g., a contiguous stretch of amino acids or a conformational construct composed of different regions of non-contiguous 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 blood serum and/or in the extracellular matrix (ECM). Unless otherwise indicated, a protein referred to herein as an antigen (e.g., GPRC5D, CD3) can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human protein. Where reference is made to a particular protein herein, the term encompasses "full-length," unprocessed protein as well as any form of protein resulting from processing in the cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants. An exemplary human protein that can be used as an antigen is CD3, in particular the subunit of CD3 (for human sequences see UniProt No. P07766 (version 185), NCBI Refseq No. NP-000724.1, SEQ ID NO: 40; or for cynomolgus [ Macaca fascicularis ] sequences see UniProt No. Q95LI5 (version 69), NCBI GenBank No. BAB71849.1, SEQ ID NO:41), or GPRC5D (for human sequences see UniProt No. Q9NZDD 1 (version 115); NCBI Refq No. NP-061124.1, SEQ ID NO: 45). In certain embodiments, the antibodies or bispecific antigen binding molecules of the invention bind to an epitope of CD3 or GPRC5D that is conserved among CD3 or GPRC5D antigens from different species. In particular embodiments, the antibodies or bispecific antigen binding molecules of the invention bind to human GPRC 5D.
By "specific binding" is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding module to bind a particular antigenic determinant can be measured via enzyme-linked immunosorbent assays (ELISAs) or other techniques well known to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (e.g., analysis on a BIAcore instrument) (Liljeblad et al, Glyco J17, 323-. In one embodiment, the extent of binding of the antigen binding moiety to an unrelated protein is less than about 10% of the antigen binding moiety to the antigen, as measured, for example, by SPR. In certain embodiments, an antigen-binding moiety that binds an antigen, or an antigen-binding molecule comprising the antigen-binding moiety, has a molecular weight of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g.Such as 10-8M or less, e.g. 10-8M to 10-13M, e.g. 10-9M to 10-13M) dissociation constant (K)D)。
"affinity" refers to the strength of the sum 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 an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be in terms of the dissociation constant (K) D) Expressed as dissociation and association rate constants (K, respectively)DissociationAnd KBonding of) The ratio of (a) to (b). As such, equal 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. One particular method for measuring affinity is Surface Plasmon Resonance (SPR).
"reduced binding", e.g. reduced binding to Fc receptors, refers to a reduction in affinity of the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes a decrease in affinity to 0 (or below the detection limit of the analytical method), i.e. complete elimination of the interaction. Conversely, "increased binding" refers to an increase in the binding affinity of the corresponding interaction.
As used herein, an "activating T cell antigen" refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which upon interaction with an antigen binding molecule is capable of inducing T cell activation. Specifically, the interaction of the antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In a specific embodiment, the activating T cell antigen is a subunit of CD3, in particular CD3 (see UniProt No. P07766 (version 144), NCBI RefSeq No. NP-000724.1, SEQ ID NO:40 for human sequences, or UniProt No. Q95LI5 (version 49), NCBI GenBank No. BAB71849.1, SEQ ID NO:41 for cynomolgus [ Macaca fascicularis ] sequences).
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, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and are 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 cancer cell or a cell of a tumor stroma. In a particular embodiment, the target cell antigen is GPRC5D, in particular human GPRC5D according to SEQ ID NO: 45.
As used herein, the terms "first", "second" or "third" are used with respect to Fab molecules and the like to facilitate differentiation when there is more than one module of each class. Unless explicitly stated as such, the use of these terms is not intended to confer a particular order or orientation to the bispecific antigen binding molecule.
By "fusion" is meant that the components (e.g., Fab molecule and Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.
"Fab molecule" refers to a protein consisting of the VH and CH1 domains of the heavy chain of an immunoglobulin ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain").
By "exchanged" Fab molecule (also called "Crossfab") is meant a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e. replaced with each other), i.e. an exchanged Fab molecule comprising a peptide chain consisting of the light chain variable domain VL and the heavy chain constant domain 1CH1 (VL-CH1, N to C orientation), and a peptide chain consisting of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, N to C orientation). For clarity, in an exchanged Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (exchanged) Fab molecule. In contrast, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.
In contrast, a "conventional" Fab molecule is intended to mean a Fab molecule in its native form, i.e., comprising a heavy chain consisting of heavy chain variable and constant domains (VH-CH1, N-to-C orientation), and a light chain consisting of light chain variable and constant domains (VL-CL, N-to-C orientation).
Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL), also known as a variable light domain or light chain variable region, followed by a Constant Light (CL) domain, also known as a light chain constant region 1(IgG1),γ2(IgG2),γ3(IgG3),γ4(IgG4),α1(IgA1) And α2(IgA2). Based on the amino acid sequence of their constant domains, the light chains of immunoglobulins can fall into one of two classes called kappa (κ) and lambda (λ). An immunoglobulin essentially consists of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.
The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.
An "isolated" antibody is one that has been separated from components of its natural environment, i.e., is not in its natural environment. No specific level of purification is required. For example, an isolated antibody can be removed from its natural or native environment. For the purposes of the present invention, a recombinantly produced antibody expressed in a host cell is considered to be isolated, as are natural or recombinant antibodies that have been separated, fractionated, or partially or substantially purified by any suitable technique. Thus, the antibodies and bispecific antigen binding molecules of the invention are isolated. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromager.b 848:79-87 (2007).
The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure.
An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen that is bound to the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ') 2For reviews of certain antibody fragments, see Hudson et al, Nat Med 9,129-The Pharmacology of Monoclonal Antibodies, vol.113, eds Rosenburg and Moore, Springer-Verlag, New York, pp.269-315 (1994); see also WO 93/16185; and U.S. Pat. nos. 5,571,894 and 5,587,458. With respect to Fab and F (ab') comprising salvage receptor binding epitope residues and having extended half-life in vivo2See 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, Nat Med 9,129-134 (2003); and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Tri-and tetrabodies are also described in Hudson et al, Nat Med 9,129-134 (2003). Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain, or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1). Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., e.coli or phage), as described herein.
The term "antigen binding domain" refers to the portion of an antibody that comprises a region that specifically binds to part or the entire antigen and is complementary thereto. 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 domain (VL) and an antibody heavy chain variable domain (VH).
The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding 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 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freemanand co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. As used herein in connection with variable region Sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al, Sequences of Proteins of immunological Interest,5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).
As used herein, the amino acid positions of the constant regions and domains of all heavy and light chains are numbered according to the Kabat numbering system described in Kabat, et al, Sequences of Proteins of Immunological Interest,5th ed., Public Health service, National Institutes of Health, Bethesda, MD (1991), referred to herein as "numbering according to Kabat" or "Kabat numbering". In particular, the Kabat numbering system (see Kabat et al, Sequences of Proteins of Immunological Interest,5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) at page 647-.
As used herein, the term "hypervariable region" or "HVR" refers to regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs"; CDRs of the heavy chain variable region/domain are abbreviated such as HCDR1, HCDR2 and HCDR 3; CDRs of the light chain variable region/domain are abbreviated such as LCDR1, LCDR2 and LCDR3) and/or form structurally defined loops ("hypervariable loops") and/or each region which contains antigen-contacting residues ("antigen-contacting"). Typically, antibodies comprise 6 HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:
(a) hypervariable loops present at amino acid residues 26-32(L1),50-52(L2),91-96(L3),26-32(H1),53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));
(b) CDRs present at amino acid residues 24-34(L1),50-56(L2),89-97(L3),31-35b (H1),50-65(H2), and 95-102(H3) (Kabat et al, Sequences of Proteins of immunological interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigen contacts, present at amino acid residues 27c-36(L1),46-55(L2),89-96(L3),30-35b (H1),47-58(H2), and 93-101(H3) (MacCallum et al.J.mol.biol.262:732-745 (1996)); and
(d) A combination of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2),47-56(L2),48-56(L2),49-56(L2),26-35(H1),26-35b (H1),49-65(H2),93-102(H3), and 94-102 (H3).
Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.
"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of the variable domains typically consist of 4 FR domains FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in VH (or VL) in the order FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.
A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as "humanized variable regions". Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have undergone humanization. Other forms of "humanized antibodies" encompassed by the invention are those in which the constant regions have been additionally modified or altered from the constant regions of the original antibody to generate properties in accordance with the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.
"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. In certain embodiments, the human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, the human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from a human antibody library are also considered herein to be human antibodies or human antibody fragments.
The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region that a heavy chain possesses. There are 5 major classes of antibodies, IgA, IgD, IgE, IgG and IgM, and these several species can be further divided into subclasses (isotypes), e.g., IgG1,IgG2,IgG3,IgG4,IgA1And IgA2The constant domains of the heavy chains corresponding to the different immunoglobulin classes are called α, 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. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries 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 Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by the host cell may undergo post-translational cleavage, cleaving one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise the full-length heavy chain, or it may comprise a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain comprising the Fc domain (or a subunit of the Fc domain as defined herein) represents herein a C-terminal glycine-lysine dipeptide without, if not otherwise indicated. In one embodiment of the invention, the heavy chain of one subunit comprising an Fc domain as defined herein comprised in an antibody or bispecific antigen binding molecule according to the invention comprises a further C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). In one embodiment of the invention, the heavy chain of one subunit comprised in the antibody or bispecific antigen binding molecule according to the invention, comprising an Fc domain as defined herein, comprises a further C-terminal glycine residue (G446, numbering according to the EU index of Kabat). The compositions of the invention, such as the pharmaceutical compositions described herein, comprise a population of antibodies or bispecific antigen binding molecules of the invention. A population of antibodies or bispecific antigen-binding molecules can comprise molecules having full-length heavy chains and molecules having cleaved variant heavy chains. The population of antibodies or bispecific antigen-binding molecules can consist of a mixture of molecules having full-length heavy chains and molecules having cleavage variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or bispecific antigen-binding molecules have cleavage variant heavy chains. In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antigen binding molecules of the invention comprises an antibody or bispecific antigen binding molecule comprising one subunit comprising an Fc domain as defined herein and additionally a heavy chain of a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antigen binding molecules of the invention comprises an antibody or bispecific antigen binding molecule comprising a heavy chain comprising one subunit of an Fc domain as defined herein and additionally a C-terminal glycine residue (G446, numbering according to the EU index of Kabat). In one embodiment of the invention, such compositions comprise a population of antibodies or bispecific antigen binding molecules consisting of a molecule comprising a heavy chain comprising one subunit of an Fc domain as defined herein; a molecule comprising a heavy chain comprising one subunit of an Fc domain as defined herein and an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat), and a molecule comprising a heavy chain comprising one subunit of an Fc domain as defined herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD,1991 (see also above). 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 comprising a C-terminal constant region in an immunoglobulin heavy chain that is capable of stably associating with itself. For example, the subunits of the IgG Fc domain comprise IgG CH2 and IgG CH3 constant domains.
A "modification that facilitates association of a first subunit and a second subunit of an Fc domain" is a peptide backbone manipulation or post-translational modification of an Fc domain subunit that reduces or prevents association of a polypeptide comprising the Fc domain subunit with the same polypeptide to form a homodimer. As used herein, in particular, a modification that facilitates association includes a separate modification of each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to be associated, wherein the modifications are complementary to each other, thereby facilitating association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both Fc domain subunits, thereby promoting their association, sterically or electrostatically, respectively. As such, (hetero) dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be identical in the sense that the other components (e.g., antigen binding modules) fused to each subunit are different. In some embodiments, the modification facilitating the association comprises an amino acid mutation, in particular an amino acid substitution, in the Fc domain. In a specific embodiment, the modifications facilitating association comprise separate amino acid mutations, in particular amino acid substitutions, in each of the two subunits of the Fc domain.
The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include C1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, cell surface receptor (e.g., B cell receptor) downregulation and B cell activation.
As used herein, the term "engineered" is considered to include 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 approaches.
As used herein, the term "amino acid mutation" is meant to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications may be made to achieve the final construct, so long as the final construct possesses the desired properties, such as reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino and/or carboxy-terminal deletions and amino acid insertions. Particular amino acid mutations are amino acid substitutions. In order to alter the binding characteristics of, for example, the Fc region, non-conservative amino acid substitutions, i.e., the substitution of one amino acid with another having 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 considered that a method of changing the side chain group of an amino acid by a method other than genetic engineering such as chemical modification may also be usable. 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 may be indicated as 329G, G329, G 329P329G or Pro329 Gly.
"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA package. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are generated using the ggsearch program of the version FASTA package 36.3.8c or later and the BLOSUM50 comparison matrix. The FASTA package is described by W.R.Pearson and D.J.Lipman (1988) "Improved Tools for Biological sequential analysis", PNAS 85: 2444-2448; W.R. Pearson (1996) "Effective protein sequence composition" meth.enzymol.266: 227-; and Pearson et al (1997) Genomics 46:24-36 and is publicly available from http:// fasta. bioch. virginia. edu/fasta _ www2/fasta _ down. Alternatively, sequences can be compared using a common server available at http:// fasta. bioch.virginia.edu/fasta _ www2/index. cgi, using the ggsearch (global protein: protein) program and default options (BLOSUM 50; open: -10; extension: -2; Ktup ═ 2) to ensure that global, rather than local, alignments are performed. Percent amino acid identity is given in the output alignment headings.
The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mrna), virus-derived RNA or plasmid dna (pdna). Polynucleotides may comprise conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in Peptide Nucleic Acids (PNAs)). 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. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or (partially or substantially) purified polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell that generally contains the polynucleotide molecule, but which is extrachromosomal or at a chromosomal location different from its natural 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. Isolated polynucleotides or nucleic acids according to the invention also include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may be or may include regulatory elements such as a promoter, ribosome binding site or transcription terminator.
An "isolated polynucleotide (or nucleic acid) encoding [ e.g., an antibody or bispecific antigen binding molecule of the invention ] refers to one or more polynucleotide molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such polynucleotide molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.
The term "expression cassette" refers to a polynucleotide, recombinantly or synthetically produced, having 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. Typically, the recombinant expression cassette portion of the expression vector contains the nucleic acid sequence to be transcribed and a promoter, among other things. In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding an antibody or bispecific antigen binding molecule of the invention, or a fragment thereof.
The term "vector" or "expression vector" refers to a DNA molecule used to introduce a specific gene in operable association with it and direct its expression in a cell. The term includes vectors which are autonomously replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the cell, 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 antibody or 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 originally transformed cell and progeny derived therefrom (regardless of the number of passages). Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell. The host cell is any type of cellular system that can be used to produce the antibody or bispecific antigen binding molecule of the invention. Host cells include cultured cells, for example, mammalian culture cells such as HEK cells, 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, plant cells and the like, and also include cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues.
An "activating Fc receptor" is an Fc receptor that, upon engagement of the Fc domain of an antibody, initiates a signaling event that stimulates cells bearing the receptor to perform effector function. 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 to which an antibody or derivative thereof comprising an Fc region specifically binds, typically via a protein moiety at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed in a given time by the ADCC mechanism as defined above at a given concentration of antibody in the medium surrounding the target cells, and/or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time by the ADCC mechanism. The reduction in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell but not yet engineered, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art). For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution in its Fc domain that reduces ADCC is relative to 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 publication No. wo 2012/130831).
An "effective amount" of an agent refers to an amount necessary to effect a physiological change in the cell or tissue to which it is administered.
A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount (in a necessary dose and for a necessary time) effective to achieve the desired therapeutic or prophylactic 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 formulation in a form that allows the biological activity of the active ingredients contained therein to be effective, and that is free of other ingredients that would have unacceptable toxicity to a subject to whom the composition would be administered.
"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
As used herein, "treatment" refers to an attempt to alter the natural course of disease in a treated individual (and grammatical variants thereof), and may be a clinical intervention performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis. In some embodiments, the antibodies or bispecific antigen binding molecules of the invention are used to delay the development of a disease or delay the progression of a disease.
The term "package insert" is used to refer to instructions typically contained in commercial packages of therapeutic products that contain information about the indications, uses, dosages, administrations, combination therapies, contraindications and/or warnings concerning the use of such therapeutic products.
Detailed description of the embodiments
The present invention provides antibodies and bispecific antigen binding molecules that bind GPRC5D, particularly human GPRC 5D. In addition, the molecules have other properties that are advantageous for therapeutic applications, for example in terms of efficacy and/or safety and productivity.
GPRC5D antibody
In a first aspect, the invention provides an antibody that binds GPRC5D, wherein the antibody comprises (i) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR3 of SEQ ID NO:86, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO: 89; (ii) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR3 of SEQ ID NO:86, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO: 89; (iii) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO: 97; (iv) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO: 97; or (v) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:92, and HCDR3 of SEQ ID NO:93, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO: 97.
In some embodiments, the antibody is a humanized antibody. In one embodiment, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment, the antibody comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
In a particular embodiment, (i) the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 13, and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 14; or (ii) the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO. 15 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 16; or (iii) the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:48, and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53; or (iv) the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:49 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52; or (v) the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:57, and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64; or (vi) the VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:58 and the VL comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63.
In a particular embodiment, the antibody comprises (i) a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:13 and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14; or (ii) a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 15, and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 16; or (iii) a VH at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:48, and a VL at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53; or (iv) a VH at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 49 and a VL at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 52; or (v) a VH at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:57, and a VL at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64; or (vi) a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:58 and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63.
In another embodiment, the antibody is an IgG, particularly an IgG1, antibody. In one embodiment, the antibody is a full length antibody. In another embodiment, the antibody is selected from the group consisting of Fv molecules, scFv molecules, Fab molecules, and F (ab')2Antibody fragments of a panel of molecules. In one embodiment, the antibody is a multispecific antibody.
In certain embodiments, a VH or VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind GPRC 5D. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO. 13 and/or substituted in SEQ ID NO. 14, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO. 15, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO. 16, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO. 48, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO. 53, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO. 49, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO. 52, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO:57, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO:64, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO:58, a total of 1 to 10 amino acids are inserted and/or deleted and/or substituted in SEQ ID NO:63, a total of 1 to 10 amino acids are inserted and/or deleted.
In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the antibody comprises the VH sequence of SEQ ID NO 13 and/or the VL sequence of SEQ ID NO 14, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO. 15 and/or the VL sequence of SEQ ID NO. 16, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO:48 and/or the VL sequence of SEQ ID NO:53, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO. 49 and/or the VL sequence of SEQ ID NO. 52, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO:57 and/or the VL sequence of SEQ ID NO:64, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO:58 and/or the VL sequence of SEQ ID NO:63, including post-translational modifications of the sequences.
In one embodiment, the antibody comprises a VH comprising an amino acid sequence selected from the group of SEQ ID NO 13 and SEQ ID NO 15, and a VL comprising an amino acid sequence of SEQ ID NO 14.
In one embodiment, the antibody comprises a VH sequence selected from the group of SEQ ID NO 13 and SEQ ID NO 12, and a VL sequence of SEQ ID NO 16.
In a particular embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO. 13 and a VL comprising the amino acid sequence of SEQ ID NO. 14. In a particular embodiment, the antibody comprises the VH sequence of SEQ ID NO. 13 and the VL sequence of SEQ ID NO. 14.
In a particular embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO. 15 and a VL comprising the amino acid sequence of SEQ ID NO. 16. In a particular embodiment, the antibody comprises the VH sequence of SEQ ID NO. 15 and the VL sequence of SEQ ID NO. 16.
In a particular embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO. 48 and a VL comprising the amino acid sequence of SEQ ID NO. 53. In a particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:48 and the VL sequence of SEQ ID NO: 53.
In a particular embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO. 49 and a VL comprising the amino acid sequence of SEQ ID NO. 52. In a particular embodiment, the antibody comprises the VH sequence of SEQ ID NO. 49 and the VL sequence of SEQ ID NO. 52.
In a particular embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO. 57 and a VL comprising the amino acid sequence of SEQ ID NO. 64. In a particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:57 and the VL sequence of SEQ ID NO: 64.
In a particular embodiment, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO. 58 and a VL comprising the amino acid sequence of SEQ ID NO. 63. In a particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:58 and the VL sequence of SEQ ID NO: 63.
In one embodiment, the antibody comprises a human constant region. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region, in particular an immunoglobulin molecule of the IgG class comprising human CH1, CH2, CH3 and/or CL domains. Exemplary sequences of human constant domains are set forth in SEQ ID NOs: 37 and 38 (human kappa and lambda CL domains, respectively) and SEQ ID NO:39 (human IgG1 heavy chain constant domain CH1-CH2-CH 3). In some embodiments, the antibody comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO 37 or 39, particularly the amino acid sequence of SEQ ID NO 38. In some embodiments, the antibody comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 39. In particular, the heavy chain constant region may comprise an amino acid mutation in the Fc domain as described herein.
In one embodiment, the antibody is a monoclonal antibody.
In one embodiment, the antibody isThe body is an IgG, in particular an IgG1An antibody. In one embodiment, the antibody is a full length antibody.
In one embodiment, the antibody comprises an Fc domain, particularly an IgG Fc domain, more particularly an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain. The Fc domain of the antibody may incorporate any of the features described herein with respect to the Fc domain of the bispecific antigen binding molecule of the invention, singly or in combination.
In another embodiment, the antibody is selected from the group consisting of Fv molecules, scFv molecules, Fab molecules, and F (ab')2Antibody fragments of a group of molecules; in particular Fab molecules. In another embodiment, the antibody fragment is a diabody, a triabody, or a tetrabody.
In yet another aspect, an antibody according to any of the above embodiments may incorporate any feature, singly or in combination, as described in the sections below.
Glycosylation variants
In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.
In the case of an antibody comprising an Fc region, the oligosaccharide attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.
In one embodiment, antibody variants are provided that have a nonfucosylated oligosaccharide attached (directly or indirectly) to the Fc region, i.e., an oligosaccharide structure that lacks fucose. Such non-fucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides lacking the fucose residue attached to the first GlcNAc in the backbone of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided having an increased proportion of nonfucosylated oligosaccharides in the Fc region as compared to the native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides (e.g. complexed, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2006/082515. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between
Examples of cell lines capable of producing antibodies with reduced fucosylation include protein fucosylation-deficient Lec13 CHO cells (Ripka et al, arch, biochem, biophysis, 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially in example 11), and knockout cell lines, such as α -1, 6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-ohniki et al, biotech, bioeng, 87:614-622 (2004); Kanda, y.et al, biotechnol, bioeng, 94(4): 688 (2006); and WO 2003/085107), or cells with reduced or eliminated GDP-fucose synthesis or transporter activity (see, e.g., US2004259150, US 2001615033, US2004132140, US 2000280284112).
In yet another embodiment, antibody variants are provided having bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, for example, in Umana et al, Nat Biotechnol17,176-180 (1999); ferrara et al, Biotechn Bioeng 93,851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.
Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.
Cysteine engineered antibody variants
In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. Cysteine-engineered antibodies can be produced as described, for example, in U.S. Pat. nos. 7,521,541,8,30,930,7,855,275,9,000,130, or WO 2016040856.
Antibody derivatives
In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a therapy under specified conditions, and the like.
In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.
Immunoconjugates
The present invention also provides immunoconjugates comprising an anti-GPRC 5D antibody as described herein conjugated (chemically bonded) to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope.
In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more of the above-mentioned therapeutic agents. The antibody is typically linked to one or more therapeutic agents using a linker. For a Review of ADC technology (including examples of therapeutic agents and drugs and linkers) see Pharmacol Review 68:3-19 (2016).
In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to enzymatically active toxins, or fragments thereof, including but not limited to diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin (ricin) a chain, abrin (abrin) a chain, modeccin (modeccin) a chain, α -sarcin, aleurites (aleurites fordii) toxic protein, dianthus caryophyllus (dianthin) toxic protein, phytolacca americana (phytolaccai americana) protein (PAPII, PAPII and PAP-S), Momordica charantia (momordia) inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (saparilia pi), trichothecin (trichothecin), and trichothecin (irin), trichothecin (enomycin), and neomycin).
In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At211,I131,I125,Y90,Re186,Re188,Sm153,Bi212,P32,Pb212And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
A variety of bifunctional protein coupling agents may be used to generate conjugates of the antibody and cytotoxic agent, such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as
Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).
Multispecific antibodies
In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain embodiments, the multispecific antibody has three or more binding specificities. In certain embodiments, one of the binding specificities is for GPRC5D, while the other (two or more) specificity is for any other antigen. In certain embodiments, a bispecific antibody may bind two (or more) different epitopes of GPRC 5D. Multispecific (e.g., bispecific) antibodies may also be used to localize cytotoxic agents or cells to cells expressing GPRC 5D. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.
Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy and light chains with different specificities (see Milstein and Cuello, Nature 305:537(1983)), and "node-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168 and Atwell et al, J.mol.biol.270:26 (1997)). Effects can also be manipulated electrostatically by engineering the molecules for the generation of antibody Fc-heterodimers (see e.g. WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al, Science,229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553(1992) and WO 2011/034605); the use of common light chain technology to circumvent the light chain mismatch problem (see e.g. WO 98/50431); the "diabody" technique used to generate bispecific antibody fragments was used (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (sfv) dimers (see, e.g., Gruber et al, j.immunol.,152:5368 (1994)); and making a trispecific antibody to generate a multispecific antibody as described, for example, in Tutt et al j.
Also included herein are engineered antibodies having three or more antigen binding sites, including, for example, "octopus antibodies" or DVD-Ig (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies having three or more antigen binding sites may be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010/145792, and WO 2013/026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual action fabs" or "DAFs" comprising an antigen-binding site that binds GPRC5D and another, different antigen, or two different epitopes of GPRC5D (see, e.g., US 2008/0069820 and WO 2015/095539).
Multispecific antibodies may also be provided in an asymmetric form, with domain exchanges in one or more binding arms having the same antigen specificity, i.e. by exchanging VH/VL domains (see e.g. WO 2009/080252 and WO 2015/150447), CH1/CL domains (see e.g. WO 2009/080253) or the entire Fab arm (see e.g. WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS,108(2011)1187-1191, and Klein et al, MAbs 8(2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interface to direct proper Fab pairing. See, for example, WO 2016/172485.
Various additional molecular versions of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al, Mol Immunol 67(2015) 95-106).
Also included herein is a specific type of multispecific antibody that is a bispecific antibody designed to simultaneously bind to a target cell, e.g., an activated, invariant component of the surface antigen and T Cell Receptor (TCR) complex on a tumor cell, such as CD3, thereby retargeting the T cell to kill the target cell. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one of the binding specificities is for GPRC5D and the other is for CD 3.
Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engagement) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO2008/119567, Nagorsen and
Bispecific antigen binding molecules that bind GPRC5D and a second antigen
The present invention also provides a bispecific antigen binding molecule, i.e. an antigen binding molecule comprising at least two antigen binding moieties capable of specifically binding two distinct antigenic determinants (a first and a second antigen).
According to a particular embodiment of the invention, the antigen binding moiety comprised in the bispecific antigen binding molecule is a Fab molecule (i.e. an antigen binding domain consisting of a heavy and a light chain comprising a variable and a constant domain, respectively). In one embodiment, the first and/or second antigen binding moiety is a Fab molecule. In one embodiment, the Fab molecule is human. In a particular embodiment, the Fab molecule is humanized. In yet another embodiment, the Fab molecule comprises human heavy and light chain constant domains.
Preferably, at least one of the antigen binding moieties is an exchange Fab molecule. Such modifications reduce the mismatching of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the bispecific antigen binding molecules of the invention in recombinant production. In a particular exchanged Fab molecule useful in the bispecific antigen binding molecules of the present invention, the variable domains of the Fab light chain and Fab heavy chain (VL and VH, respectively) are exchanged. However, even with such domain exchanges, preparations of bispecific antigen-binding molecules may contain certain side products due to the so-called Bence Jones type of interaction between mismatched heavy and light chains (see Schaefer et al, PNAS,108(2011) 11187-. To further reduce the mismatches of the heavy and light chains from the different Fab molecules and thereby improve the purity and yield of the desired bispecific antigen binding molecule, charged amino acids with opposite charges may be introduced at specific amino acid positions in the CH1 and CL domains of the Fab molecule that binds to the first antigen (i.e., GPRC5D) or the Fab molecule that binds to the second antigen (e.g., an activating T cell antigen such as CD3), as further described herein. Charge modification is performed in a conventional Fab molecule comprised in a bispecific antigen binding molecule (such as e.g. shown in figures 1A-C, G-J) or in a VH/VL exchange Fab molecule comprised in a bispecific antigen binding molecule (such as e.g. shown in figures 1D-F, K-N), but not in both. In particular embodiments, charge modification is performed in a conventional Fab molecule included in the bispecific antigen binding molecule, which in particular embodiments binds to the first antigen, i.e., GPRC 5D.
In a specific embodiment according to the present invention, the bispecific antigen binding molecule is capable of simultaneously binding a first antigen (i.e. GPRC5D) and a second antigen (e.g. an activating T cell antigen, in particular CD 3). In one embodiment, the bispecific antigen binding molecule is capable of cross-linking a T cell and a target cell by simultaneously binding GPRC5D and an activating T cell antigen. In an even more specific embodiment, such simultaneous binding results in lysis of target cells (particularly GPRC 5D-expressing tumor cells). In one embodiment, such simultaneous binding results in the activation of T cells. In other embodiments, such simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the bispecific antigen binding molecule to an activating T cell antigen, particularly CD3, does not result in T cell activation in the absence of simultaneous binding to GPRC 5D.
In one embodiment, the bispecific antigen binding molecule is capable of redirecting the cytotoxic activity of a T cell to a target cell. In a specific embodiment, the redirecting is independent of MHC-mediated peptide antigen presentation by the target cell and/or specificity of 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.
First antigen binding module
The bispecific antigen binding molecules of the present invention comprise at least one antigen binding moiety, in particular a Fab molecule, that binds GPRC5D (first antigen). In certain embodiments, the bispecific antigen binding molecule comprises two antigen binding moieties, particularly Fab molecules, that bind GPRC 5D. In one particular such embodiment, each of the antigen binding moieties binds the same antigenic determinant. In an even more particular embodiment, all of these antigen binding moieties are the same, i.e., they comprise the same amino acid sequence, including the same amino acid substitutions (if any) in the CH1 and CL domains as described herein. In one embodiment, the bispecific antigen binding molecule comprises no more than two antigen binding moieties, particularly Fab molecules, that bind GPRC 5D.
In particular embodiments, the antigen binding moiety that binds GPRC5D is a conventional Fab molecule. In such embodiments, the antigen binding moiety that binds the second antigen is an exchanged Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other.
In an alternative embodiment, the antigen binding moiety that binds GPRC5D is an exchanged Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In such embodiments, the antigen binding moiety that binds the second antigen is a conventional Fab molecule.
The GPRC5D binding module is capable of directing the bispecific antigen binding molecule to a target site, such as a specific type of GPRC 5D-expressing tumor cell.
The first antigen-binding moiety of the bispecific antigen-binding molecule may incorporate any of the features described herein with respect to antibodies that bind GPRC5D, singly or in combination, unless it is clearly scientifically unreasonable or impossible.
Thus, in one aspect, the invention provides a bispecific antigen binding molecule comprising (a) a first antigen binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83,
In some embodiments, the first antigen-binding moiety is (derived from) a humanized antibody. In one embodiment, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment, the first antigen binding moiety comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
In one embodiment, the VH of the first antigen binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58, and the VL of the first antigen binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In one embodiment, the first antigen binding moiety comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58, and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In one embodiment, the first antigen binding moiety comprises a VH comprising an amino acid sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58 and a VL comprising an amino acid sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In one embodiment, the first antigen binding moiety comprises a VH sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58 and a VL sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In a particular embodiment, the first antigen-binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 13 and a VL comprising the amino acid sequence of SEQ ID NO. 14. In a particular embodiment, the first antigen-binding moiety comprises the VH sequence of SEQ ID NO. 13 and the VL sequence of SEQ ID NO. 14.
In a particular embodiment, the first antigen-binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 15 and a VL comprising the amino acid sequence of SEQ ID NO. 16. In a particular embodiment, the first antigen-binding moiety comprises the VH sequence of SEQ ID NO. 15 and the VL sequence of SEQ ID NO. 16.
In a particular embodiment, the first antigen-binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 48 and a VL comprising the amino acid sequence of SEQ ID NO. 53. In a particular embodiment, the first antigen binding moiety comprises the VH sequence of SEQ ID NO. 48 and the VL sequence of SEQ ID NO. 53.
In a particular embodiment, the first antigen binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 49 and a VL comprising the amino acid sequence of SEQ ID NO. 52. In a particular embodiment, the first antigen binding moiety comprises the VH sequence of SEQ ID NO. 49 and the VL sequence of SEQ ID NO. 52.
In a particular embodiment, the first antigen-binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 57 and a VL comprising the amino acid sequence of SEQ ID NO. 64. In a particular embodiment, the first antigen-binding moiety comprises the VH sequence of SEQ ID NO. 57 and the VL sequence of SEQ ID NO. 64.
In a particular embodiment, the first antigen-binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 58 and a VL comprising the amino acid sequence of SEQ ID NO. 63. In a particular embodiment, the first antigen binding moiety comprises the VH sequence of SEQ ID NO. 58 and the VL sequence of SEQ ID NO. 63.
In one embodiment, the first antigen binding moiety comprises a human constant region. In one embodiment, the first antigen binding moiety is a Fab molecule comprising a human constant region, in particular a human CH1 and/or CL domain. Exemplary sequences of human constant domains are set forth in SEQ ID NOs: 37 and 38 (human kappa and lambda CL domains, respectively) and SEQ ID NOs: 39 (human IgG)1Heavy chain constant domain CH1-CH2-CH 3). In some embodiments, the first antigen-binding moiety comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 37 or SEQ ID No. 38, particularly the amino acid sequence of SEQ ID No. 37. In particular, the light chain constant region may comprise amino acid mutations as described herein under "charge modification" and/or may comprise a deletion or substitution of one or more (in particular two) N-terminal amino acids, if in an exchange Fab molecule. In some embodiments, the first antigen binding moiety comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence comprised in the amino acid sequence of SEQ id No. 39. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise amino acid mutations as described herein under "charge modification".
Second antigen binding Module
The bispecific antigen binding molecules of the invention comprise at least one antigen binding moiety, in particular a Fab molecule, that binds to a second antigen (different from GPRC 5D).
In a particular embodiment, the antigen binding moiety that binds the second antigen is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In such embodiments, the antigen binding moiety that binds the first antigen (i.e., GPRC5D) is preferably a conventional Fab molecule. In embodiments in which the bispecific antigen binding molecule comprises more than one antigen binding moiety, particularly a Fab molecule, that binds to GPRC5D, the antigen binding moiety that binds to the second antigen is preferably an exchange Fab molecule and the antigen binding moiety that binds to GPRC5D is a conventional Fab molecule.
In an alternative embodiment, the antigen binding moiety that binds the second antigen is a conventional Fab molecule. In such embodiments, the antigen binding moiety that binds the first antigen (i.e., GPRC5D) is an exchanged Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In embodiments in which the bispecific antigen binding molecule comprises more than one antigen binding moiety that binds a second antigen, particularly a Fab molecule, the antigen binding moiety that binds GPRC5D is preferably an exchange Fab molecule and the antigen binding moiety that binds the second antigen is a conventional Fab molecule.
In some embodiments, the second antigen is an activating T cell antigen (also referred to herein as an "activating T cell antigen binding moiety, or an activating T cell antigen binding Fab molecule"). In a particular embodiment, the bispecific antigen binding molecule comprises no more than one antigen binding moiety capable of specifically binding an activating T cell antigen. In one embodiment, the bispecific antigen binding molecule provides monovalent binding to an activating T cell antigen.
In a particular embodiment, the second antigen is CD3, particularly human CD3(SEQ ID NO:40) or cynomolgus monkey CD3(SEQ ID NO:41), most particularly human CD 3. In one embodiment, the second antigen binding moiety is cross-reactive (i.e., specifically binds) to human and cynomolgus monkey CD 3. In some embodiments, the second antigen is the epsilonclon subunit of CD3 (CD 3).
In one embodiment, the second antigen binding module comprises
In one embodiment, the second antigen binding module comprises a
In some embodiments, the second antigen binding moiety is (derived from) a humanized antibody. In one embodiment, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment, the second antigen binding moiety comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
In one embodiment, the second antigen binding moiety comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 35. In one embodiment, the second antigen binding moiety comprises a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 36.
In one embodiment, the second antigen binding module comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 35, and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 36.
In one embodiment, the VH of the second antigen binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 35, and the VL of the second antigen binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 36.
In one embodiment, the second antigen binding module comprises a VH comprising the amino acid sequence of SEQ ID NO. 35 and a VL comprising the amino acid sequence of SEQ ID NO. 36.
In one embodiment, the second antigen binding module comprises the VH sequence of SEQ ID NO. 35, and the VL sequence of SEQ ID NO. 36.
In one embodiment, the second antigen binding moiety comprises a human constant region. In one embodiment, the second antigen binding moiety is a Fab molecule comprising human constant regions, in particular human CH1 and/or CL domains. Exemplary sequences of human constant domains are set forth in SEQ ID NOs: 37 and 38 (human kappa and lambda CL domains, respectively) and SEQ ID NOs: 39 (human IgG)1Heavy chain constant domain CH1-CH2-CH 3). In some embodiments, the second antigen binding moiety comprises an amino group that binds to SEQ ID NO 37 or SEQ ID NO 38A light chain constant region of an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO 37. In particular, the light chain constant region may comprise amino acid mutations as described herein under "charge modification" and/or may comprise a deletion or substitution of one or more (in particular two) N-terminal amino acids, if in an exchange Fab molecule. In some embodiments, the second antigen binding module comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence comprised in the amino acid sequence of SEQ id No. 39. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise amino acid mutations as described herein under "charge modification".
In some embodiments, the second antigen binding module is a Fab molecule in which the variable domains VL and VH or constant domains CL and CH1 of the Fab light and Fab heavy chains, in particular the variable domains VL and VH, are replaced by each other (i.e. according to such embodiments the second antigen binding module is a crossover Fab molecule in which the variable or constant domains of the Fab light and Fab heavy chains are swapped). In one such embodiment, the first (and third, if any) antigen binding moiety is a conventional Fab molecule.
In one embodiment, no more than one antigen binding moiety that binds a second antigen (e.g., an activating T cell antigen such as CD3) is present in the bispecific antigen binding molecule (i.e., the bispecific antigen binding molecule provides monovalent binding to the second antigen).
Charge modification
The bispecific antigen binding molecules of the present invention may comprise in the Fab molecules comprised therein amino acid substitutions which are particularly effective in reducing mismatches of light and non-matching heavy chains (Bence-Jones type by-products), mismatches may occur in the production of Fab-based bi/multispecific antigen binding molecules with VH/VL exchanges in one of their binding arms (or more, in the case where the molecule comprises more than two antigen binding Fab molecules) (see also PCT publication No. wo 2015/150447, especially the examples therein, incorporated herein by reference in their entirety). The ratio of desired bispecific antigen binding molecules to undesired by-products, in particular the Bence Jones type by-products, occurring in bispecific antigen binding molecules with VH/VL domain exchange in one of their binding arms can be improved by introducing charged amino acids of opposite charge at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").
Thus, in some embodiments wherein the first and second antigen binding moiety of the bispecific antigen binding molecule are both Fab molecules and the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other in one of the antigen binding moieties (in particular the second antigen binding moiety),
i) the amino acid at position 124 in the constant domain CL of the first antigen binding module is substituted with a positively charged amino acid (numbering according to Kabat), and wherein the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the first antigen binding module is substituted with a negatively charged amino acid (numbering according to Kabat EU index); or
ii) the amino acid at position 124 is replaced with a positively charged amino acid in the constant domain CL of the second antigen binding module (numbering according to Kabat), and wherein the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the second antigen binding module is replaced with a negatively charged amino acid (numbering according to Kabat EU index).
Bispecific antigen-binding molecules do not comprise both the modifications mentioned under i) and ii). The constant domains CL and CH1 of the antigen binding module with VH/VL swapping do not replace each other (i.e. remain un-swapped).
In a more specific embodiment of the process of the present invention,
i) the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the first antigen binding module is independently substituted with glutamic acid (E), or aspartic acid (D) (numbering according to the Kabat EU index); or
ii) the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) in the constant domain CL of the second antigen binding module (numbering according to Kabat), and the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E), or aspartic acid (D) in the constant domain CH1 of the second antigen binding module (numbering according to the Kabat EU index).
In one such embodiment, the amino acid at position 124 in the constant domain CL of the first antigen binding module is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the first antigen binding module is independently substituted with glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).
In yet another embodiment, the amino acid at position 124 in the constant domain CL of the first antigen binding module is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and the amino acid at position 147 in the constant domain CH1 of the first antigen binding module is independently substituted with glutamic acid (E), or aspartic acid (D) (numbering according to the Kabat EU index).
In a particular embodiment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) in the constant domain CL of the first antigen binding module and the amino acid at position 147 is independently substituted with glutamic acid (E) in the constant domain CH1 of the first antigen binding module, or aspartic acid (D) (numbering according to the Kabat index) and the amino acid at position 213 is independently substituted with glutamic acid (E), or aspartic acid (D) (numbering according to the Kabat EU index).
In a more specific embodiment, the amino acid at position 124 is substituted with lysine (K) and the amino acid at position 123 is substituted with lysine (K) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 is substituted with glutamic acid (E) in the constant domain CH1 of the first antigen binding module (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index).
In an even more specific embodiment, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with arginine (R) (numbering according to Kabat) in the constant domain CL of the first antigen binding module, and the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index) in the constant domain CH1 of the first antigen binding module.
In a particular embodiment, the constant domain CL of the first antigen binding module is of kappa isotype if amino acid substitutions according to the above embodiments are made in the constant domain CL and the constant domain CH1 of the first antigen binding module.
Alternatively, amino acid substitutions according to the above embodiments may be made in the constant domain CL and constant domain CH1 of the second antigen binding module instead of in the constant domain CL and constant domain CH1 of the first antigen binding module. In particular such embodiments, the constant domain CL of the second antigen binding module is of the kappa isotype.
Thus, in one embodiment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) in the constant domain CL of the second antigen binding module (numbering according to Kabat), and the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the second antigen binding module is independently substituted with glutamic acid (E), or aspartic acid (D) (numbering according to the EU index of Kabat).
In yet another embodiment, the amino acid at position 124 in the constant domain CL of the second antigen binding module is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and the amino acid at position 147 in the constant domain CH1 of the second antigen binding module is independently substituted with glutamic acid (E), or aspartic acid (D) (numbering according to the Kabat EU index).
In yet another embodiment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) in the constant domain CL of the second antigen binding module and the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat index) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat index) in the constant domain CH1 of the second antigen binding module.
In one embodiment, the amino acid at position 124 is substituted with lysine (K) and the amino acid at position 123 is substituted with lysine (K) in the constant domain CL of the second antigen binding module (numbering according to Kabat) and the amino acid at position 147 is substituted with glutamic acid (E) in the constant domain CH1 of the second antigen binding module (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index).
In another embodiment, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with arginine (R) (numbering according to Kabat) in the constant domain CL of the second antigen binding module, and the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index) in the constant domain CH1 of the second antigen binding module.
In a particular embodiment, the bispecific antigen binding molecules of the invention comprise
(a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
(b) a second antigen binding moiety that binds a second antigen, wherein the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
Wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 in constant domain CH1 of the first antigen binding module is glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index).
In a particular embodiment, the bispecific antigen binding molecules of the invention comprise
(a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
(b) A second antigen binding moiety that binds a second antigen, wherein the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 in constant domain CH1 of the first antigen binding module is glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index).
In a particular embodiment, the bispecific antigen binding molecules of the invention comprise
(a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
(b) A second antigen binding moiety that binds a second antigen, wherein the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 in constant domain CH1 of the first antigen binding module is glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index).
In a particular embodiment, the bispecific antigen binding molecules of the invention comprise
(a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
(b) A second antigen binding moiety that binds a second antigen, wherein the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 in constant domain CH1 of the first antigen binding module is glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index).
In a particular embodiment, the bispecific antigen binding molecules of the invention comprise
(a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:90,
(b) A second antigen binding moiety that binds a second antigen, wherein the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 in constant domain CH1 of the first antigen binding module is glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index).
In a particular embodiment, the bispecific antigen binding molecules of the invention comprise
(a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:1,
(b) A second antigen binding moiety that binds a second antigen, wherein the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 in constant domain CH1 of the first antigen binding module is glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index).
In a particular embodiment, the bispecific antigen binding molecules of the invention comprise
(a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:7,
(b) A second antigen binding moiety that binds a second antigen, wherein the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
wherein the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (independently substituted with lysine (K) or arginine (R) in a particular embodiment) in the constant domain CL of the first antigen binding module (numbering according to Kabat), and the amino acid at position 147 in constant domain CH1 of the first antigen binding module is glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E), or aspartic acid (D) independently (numbering according to the Kabat EU index).
Bispecific antigen binding molecule formats
The components of the bispecific antigen binding molecules according to the invention may be fused to each other in a variety of configurations. Exemplary configurations are depicted in fig. 1A-Z.
In a specific embodiment, the antigen binding moiety comprised in the bispecific antigen binding molecule is a Fab molecule. In such embodiments, the first, second, third, etc. antigen binding moiety may be referred to herein as a first, second, third, etc. Fab molecule, respectively.
In one embodiment, the first and second antigen binding moieties of the bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In particular embodiments, the first and second antigen binding moieties are each Fab molecules. In one such embodiment, 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. In another such embodiment, 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. In embodiments wherein either (i) 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 or (ii) 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, additionally the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may be fused to each other, optionally via a peptide linker.
Bispecific antigen binding molecules (e.g., as shown in figures 1A,1D,1G,1H,1K, 1L) having a single antigen binding moiety (such as a Fab molecule) capable of specifically binding a target cell antigen, such as GPRC5D, are useful, particularly where the target cell antigen is expected to be internalized following binding of the high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety specific for a target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.
However, in other cases, bispecific antigen binding molecules comprising two or more antigen binding moieties specific for a target cell antigen, such as Fab molecules, may be advantageous (see the examples shown in fig. 1B,1C,1E,1F,1I,1J,1M or 1N), e.g. in order to optimize targeting to a target site or to allow cross-linking of a target cell antigen.
Thus, in a particular embodiment, the bispecific antigen binding molecule according to the invention comprises a third antigen binding moiety.
In one embodiment, the third antigen binding moiety binds to the first antigen, i.e., GPRC 5D. In one embodiment, the third antigen binding moiety is a Fab molecule.
In one embodiment, the third antigen moiety is identical to the first antigen binding moiety.
The third antigen-binding moiety of the bispecific antigen-binding molecule may incorporate any of the features described herein with respect to the first antigen-binding moiety and/or the antibody that binds GPRC5D, singly or in combination, unless it is clearly scientifically apparent that it is not reasonable or possible.
In one embodiment, the third antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83,
In one embodiment, the third antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83,
In one embodiment, the third antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
In one embodiment, the third antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
In one embodiment, the third antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
In one embodiment, the third antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:1,
In one embodiment, the third antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:7,
In some embodiments, the third antigen binding moiety is (derived from) a humanized antibody. In one embodiment, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment, the third antigen binding moiety comprises a CDR as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
In one embodiment, the VH of the third antigen binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58, and the VL of the third antigen binding module comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In one embodiment, the third antigen binding moiety comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58 and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In one embodiment, the third antigen binding moiety comprises a VH comprising an amino acid sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58 and a VL comprising an amino acid sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In one embodiment, the third antigen binding moiety comprises a VH sequence selected from the group of SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 57 and SEQ ID NO 58 and a VL sequence selected from the group of SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63 and SEQ ID NO 64.
In a particular embodiment, the third antigen binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 13 and a VL comprising the amino acid sequence of SEQ ID NO. 14. In a particular embodiment, the third antigen binding moiety comprises the VH sequence of SEQ ID NO. 13 and the VL sequence of SEQ ID NO. 14.
In a particular embodiment, the third antigen binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 15 and a VL comprising the amino acid sequence of SEQ ID NO. 16. In a particular embodiment, the third antigen binding moiety comprises the VH sequence of SEQ ID NO. 15 and the VL sequence of SEQ ID NO. 16.
In a particular embodiment, the third antigen binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 48 and a VL comprising the amino acid sequence of SEQ ID NO. 53. In a particular embodiment, the third antigen binding moiety comprises the VH sequence of SEQ ID NO. 48 and the VL sequence of SEQ ID NO. 53.
In a particular embodiment, the third antigen binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 49 and a VL comprising the amino acid sequence of SEQ ID NO. 52. In a particular embodiment, the third antigen binding moiety comprises the VH sequence of SEQ ID NO. 49 and the VL sequence of SEQ ID NO. 52.
In a particular embodiment, the third antigen binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 57 and a VL comprising the amino acid sequence of SEQ ID NO. 64. In a particular embodiment, the third antigen binding moiety comprises the VH sequence of SEQ ID NO. 57 and the VL sequence of SEQ ID NO. 64.
In a particular embodiment, the third antigen binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO. 58 and a VL comprising the amino acid sequence of SEQ ID NO. 63. In a particular embodiment, the third antigen binding moiety comprises the VH sequence of SEQ ID NO. 58 and the VL sequence of SEQ ID NO. 63.
At one endIn one embodiment, the third antigen binding moiety comprises a human constant region. In one embodiment, the third antigen binding moiety is a Fab molecule comprising a human constant region, in particular a human CH1 and/or CL domain. Exemplary sequences of human constant domains are set forth in SEQ ID NOs: 37 and 38 (human kappa and lambda CL domains, respectively) and SEQ ID NOs: 39 (human IgG)1Heavy chain constant domain CH1-CH2-CH 3). In some embodiments, the third antigen-binding moiety comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 37 or SEQ ID No. 38, particularly the amino acid sequence of SEQ ID No. 37. In particular, the light chain constant region may comprise amino acid mutations as described herein under "charge modification" and/or may comprise a deletion or substitution of one or more (in particular two) N-terminal amino acids, if in an exchange Fab molecule. In some embodiments, the third antigen binding moiety comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence comprised in the amino acid sequence of SEQ id No. 39. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise amino acid mutations as described herein under "charge modification".
In a particular embodiment, the third and first antigen binding moiety are each a Fab molecule and the third antigen binding moiety is identical to the first antigen binding moiety. Thus, in these embodiments, the first and third antigen-binding modules comprise identical heavy and light chain amino acid sequences and have the same domain arrangement (i.e., conventional or swapped). Also, in these embodiments, the third antigen binding moiety comprises the same amino acid substitutions, if any, as the first antigen binding moiety. For example, amino acid substitutions described herein as "charge modifications" will be made in the constant domain CL and constant domain CH1 of each of the first and third antigen binding modules. Alternatively, this may be done in the constant domain CL and constant domain CH1 of the second antigen binding module (which in a particular embodiment is also a Fab molecule), but not in the constant domain CL and constant domain CH1 of the first and third antigen binding modules.
Like the first antigen binding moiety, the third antigen binding moiety is in particular a conventional Fab molecule. However, embodiments are also contemplated wherein the first and third antigen binding moiety are exchange Fab molecules (and the second antigen binding moiety is a conventional Fab molecule). Thus, in a particular embodiment, the first and third antigen binding moiety are each conventional Fab molecules and the second antigen binding moiety is an exchanged Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other embodiments, the first and third antigen binding moieties are each exchange Fab molecules and the second antigen binding moiety is a conventional Fab molecule.
If a third antigen binding moiety is present, in a particular embodiment, the first and third antigen binding moieties bind GPRC5D and the second antigen binding moiety binds a second antigen, particularly an activating T cell antigen, more particularly CD3, most particularly CD 3.
In particular embodiments, the bispecific antigen binding molecule comprises an Fc domain comprised of a first and a second subunit. The first and second subunits of the Fc domain are capable of stable association.
The bispecific antigen binding molecules according to the invention may have different configurations, i.e. the first, second (and optionally third) antigen binding moieties may be fused to each other and to the Fc domain in different ways. The individual building blocks can be fused to one another directly or preferably via one or more suitable peptide linkers. In the case where the Fab molecule is fused to the N-terminus of a subunit of the Fc domain, it is typically via an immunoglobulin hinge region.
In some embodiments, the first and second antigen binding moieties are each a Fab molecule 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 such embodiments, the first antigen binding moiety may be 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 or to the N-terminus of another subunit of the Fc domain. In certain such embodiments, the first antigen binding moiety is a conventional Fab molecule and the second antigen binding moiety is an exchanged Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other such embodiments, the first Fab molecule is an exchange Fab molecule and the second Fab molecule is a conventional Fab molecule.
In one embodiment, the first and second antigen binding moieties are each a Fab molecule, 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, 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. In a specific embodiment, the bispecific antigen binding molecule essentially consists of a first and a second Fab molecule, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab 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. Such a construct is schematically depicted in fig. 1G and 1K (the second antigen-binding domain in these examples is a VH/VL exchange Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.
In another embodiment, the first and second antigen binding moieties are each a Fab molecule and the first and second antigen binding moieties 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 a specific embodiment, the bispecific antigen binding molecule essentially consists of a first and a second Fab molecule, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and second Fab 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. Such a construct is schematically depicted in fig. 1A and 1D (in these examples the second antigen binding domain is a VH/VL exchange Fab molecule and the first antigen binding moiety is a conventional Fab molecule). The first and second Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a particular embodiment, the first and second Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG 1The hinge region, particularly in the Fc domain, is IgG1In the case of an Fc domain.
In some embodiments, the first and second antigen binding moieties are each Fab molecules 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 such embodiments, the second antigen binding moiety may be 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 or to the N-terminus of the other of the subunits of the Fc domain (as described above). In certain such embodiments, the first antigen binding moiety is a conventional Fab molecule and the second antigen binding moiety is an exchanged Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other such embodiments, the first Fab molecule is an exchange Fab molecule and the second Fab molecule is a conventional Fab molecule.
In one embodiment, the first and second antigen binding moieties are each a Fab molecule, 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, 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. In a specific embodiment, the bispecific antigen binding molecule essentially consists of a first and a second Fab molecule, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab 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. Such a construct is schematically depicted in fig. 1H and 1L (in these examples the second antigen-binding domain is a VH/VL exchange Fab molecule and the first antigen-binding moiety is a conventional Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.
In some embodiments, the third antigen binding moiety, in particular the third Fab 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 certain such embodiments, the first and third Fab molecules are each conventional Fab molecules, and the second Fab molecule is an exchanged Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other such embodiments, the first and third Fab molecules are each exchange Fab molecules and the second Fab molecule is a conventional Fab molecule.
In one particular such embodiment, the second and third antigen binding moieties 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 Fab molecule. In a specific embodiment, the bispecific antigen binding molecule consists essentially of a first, a second and a third Fab molecule, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule 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 Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such configurations are schematically depicted in fig. 1B and 1E (in these examples the second antigen binding moiety is a VH/VL exchange Fab molecule, and the first and third antigen binding moieties are conventional Fab molecules), and fig. 1J and 1N (in these examples the second antigen binding moiety is a conventional Fab molecule, and the first and third antigen binding moieties are VH/VL exchange Fab molecules). The second and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a particular embodiment, the second and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG 1The hinge region, particularly in the Fc domain, is IgG1In the case of an Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.
In another such embodiment, the first and third antigen binding moieties 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. In a specific embodiment, the bispecific antigen binding molecule essentially consists of a first, a second and a third Fab molecule, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the first subunit of the Fc domainAnd wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such configurations are schematically depicted in fig. 1C and 1F (in these examples the second antigen binding moiety is a VH/VL exchange Fab molecule, and the first and third antigen binding moieties are conventional Fab molecules) and fig. 1I and 1M (in these examples the second antigen binding moiety is a conventional Fab molecule, and the first and third antigen binding moieties are VH/VL exchange Fab molecules). The first and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a particular embodiment, the first and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG 1The hinge region, particularly in the Fc domain, is IgG1In the case of an Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.
In the construction of the bispecific antigen binding molecule in which the Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each subunit of the Fc domain via an immunoglobulin hinge region, the two Fab molecules, the hinge region and the Fc domain essentially form an immunoglobulin molecule. In a particular embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more particular 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 immunoglobulin comprises a human constant region, in particular a human Fc region.
In some bispecific antigen binding molecules of the invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces mismatches of the mismatched Fab heavy and light chains, and also reduces the number of plasmids required to express some bispecific antigen binding molecules of the invention.
The antigen binding moieties may be fused to the Fc domain or to each other 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. Suitable, non-immunogenic peptide linkers include, for example, (G)4S)n,(SG4)n,(G4S)nOr G4(SG4)nA peptide linker. "n" is generally an integer from 1 to 10, usually from 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment 5 to 100 amino acids, in yet another embodiment 10 to 50 amino acids. In one embodiment, the peptide linker is (GxS)nOr (GxS)nGmWhere G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3,4,5, or 6, and m ═ 0,1,2, or 3) or (x ═ 4, n ═ 2,3,4, or 5, and m ═ 0,1,2, or 3), in one embodiment, x ═ 4 and n ═ 2 or 3, and in yet another embodiment, x ═ 4 and n ═ 2. In one embodiment, the peptide linker is (G)4S)2. One peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is (G)4S)2. An exemplary peptide linker suitable for linking the Fab heavy chains of the first and second Fab fragments comprises the sequences (D) - (G)4S)2(SEQ ID NOS: 43 and 44). Another suitable such linker comprises the sequence (G) 4S)4. Additionally, the linker may comprise (a part of) an immunoglobulin hinge region. In particular, when the Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or portion thereof, with or without additional peptide linkers.
In certain embodiments, the bispecific antigen binding molecule according to the invention comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain variable region is replaced with a light chain variable region) which is followed by a polypeptideAnd sharing a carboxy-terminal peptide bond (VL) with an Fc domain subunit(2)-CH1(2)-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH)(1)-CH1(1)-CH2-CH3(-CH 4)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH)(2)-CL(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1)). In certain embodiments, the polypeptides are covalently linked, for example by disulfide bonds.
In certain embodiments, the bispecific antigen binding molecule according to the invention comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain with a heavy chain constant region replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2)-CL(2)-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH)(1)-CH1(1)-CH2-CH3(-CH 4)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL)(2)-CH1(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1)). In certain embodiments, the polypeptides are covalently linked, for example by disulfide bonds.
In some embodiments, the bispecific antigen binding molecule comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VL)(2)-CH1(2)-VH(1)-CH1(1)-CH2-CH3(-CH 4)). In other implementationsIn a version, the bispecific antigen binding molecule comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (1)-CH1(1)-VL(2)-CH1(2)-CH2-CH3(-CH4))。
In some of these embodiments, the bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide of a second Fab molecule, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH)(2)-CL(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1)). In other such embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VH)(2)-CL(2)-VL(1)-CL(1)) Or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL)(1)-CL(1)-VH(2)-CL(2)) Where appropriate.
The bispecific antigen binding molecule according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH)(3)-CH1(3)-CH2-CH3(-CH4)) and a Fab light chain polypeptide (VL) of a third Fab molecule(3)-CL(3)). In certain embodiments, the polypeptides are covalently linked, for example by disulfide bonds.
In some embodiments, the bispecific antigen binding molecule comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule is linked to the Fab light chain of the second Fab moleculeThe constant region shares a carboxy-terminal peptide bond (i.e. the second Fab molecule comprises an exchanged Fab heavy chain with a heavy chain constant region replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH)(2)-CL(2)-VH(1)-CH1(1)-CH2-CH3(-CH 4)). In other embodiments, the bispecific antigen binding molecule comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchanged Fab heavy chain with a heavy chain constant region replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(1)-CH1(1)-VH(2)-CL(2)-CH2-CH3(-CH4))。
In some of these embodiments, the bispecific antigen binding molecule further comprises an exchange Fab light chain polypeptide of a second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL) with the Fab heavy chain constant region of the second Fab molecule(2)-CH1(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule (1)-CL(1)). In other such embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VL)(2)-CH1(2)-VL(1)-CL(1)) Or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL)(1)-CL(1)-VL(2)-CH1(2)) Where appropriate.
The bispecific antigen binding molecule according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH)(3)-CH1(3)-CH2-CH3(-CH4)) andfab light chain polypeptides (VL) of three Fab molecules(3)-CL(3)). In certain embodiments, the polypeptides are covalently linked, for example by disulfide bonds.
In certain embodiments, the bispecific antigen binding molecule does not comprise an Fc domain. In certain such embodiments, the first and, if present, third Fab molecules are each conventional Fab molecules, and the second Fab molecule is an exchanged Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other such embodiments, the first and, if present, third Fab molecules are each exchange Fab molecules and the second Fab molecule is a conventional Fab molecule.
In one such embodiment, the bispecific antigen binding molecule consists essentially of a first and a second antigen binding moiety, and optionally one or more peptide linkers, wherein both the first and the second antigen binding moiety are Fab molecules 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. Such configurations are schematically depicted in fig. 1O and 1S (in these examples the second antigen-binding domain is a VH/VL exchange Fab molecule and the first antigen-binding moiety is a conventional Fab molecule).
In another such embodiment, the bispecific antigen binding molecule consists essentially of a first and a second antigen binding moiety, and optionally one or more peptide linkers, wherein both the first and the second antigen binding moiety are Fab molecules 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. Such configurations are schematically depicted in fig. 1P and 1T (in these examples the second antigen-binding domain is a VH/VL exchange Fab molecule and the first antigen-binding moiety is a conventional Fab molecule).
In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the bispecific antigen binding molecule further comprises a third antigen binding moiety, in particular a third Fab molecule, wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the bispecific antigen binding molecule consists essentially of a first, a second and a third Fab molecule, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. Such configurations are schematically depicted in fig. 1Q and 1U (in these examples the second antigen-binding domain is a VH/VL crossover Fab molecule and the first and third antigen-binding moieties are each conventional Fab molecules), or fig. 1X and 1Z (in these examples the second antigen-binding domain is a conventional Fab molecule and the first and third antigen-binding moieties are each a VH/VL crossover Fab molecule).
In some embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the bispecific antigen binding molecule further comprises a third antigen binding moiety, in particular a third Fab molecule, wherein the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the bispecific antigen binding molecule consists essentially of a first, a second and a third Fab molecule, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the first Fab molecule. Such configurations are schematically depicted in fig. 1R and 1V (in these examples the second antigen-binding domain is a VH/VL crossover Fab molecule and the first and third antigen-binding moieties are each conventional Fab molecules), or fig. 1W and 1Y (in these examples the second antigen-binding domain is a conventional Fab molecule and the first and third antigen-binding moieties are each a VH/VL crossover Fab molecule).
In certain embodiments, the bispecific antigen binding molecules according to the invention comprise a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region) (VH (1)-CH1(1)-VL(2)-CH1(2)). In some embodiments, the bispecific antigen binding molecule further comprises a peptide such asA polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH)(2)-CL(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1))。
In certain embodiments, the bispecific antigen binding molecule according to the invention comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VL)(2)-CH1(2)-VH(1)-CH1(1)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH)(2)-CL(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1))。
In certain embodiments, the bispecific antigen binding molecule according to the invention comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain with a heavy chain constant region replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VH (2)-CL(2)-VH(1)-CH1(1)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL)(2)-CH1(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1))。
In certain embodiments, the bispecific antigen binding molecule according to the invention comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain variable region is replaced with a light chain variable regionChain) which in turn shares a carboxy-terminal peptide bond (VL) with the Fab heavy chain of the first Fab molecule(2)-CH1(2)-VH(1)-CH1(1)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH)(2)-CL(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1))。
In certain embodiments, the bispecific antigen binding molecules according to the invention comprise a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region) (VH (3)-CH1(3)-VH(1)-CH1(1)-VL(2)-CH1(2)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH)(2)-CL(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1)). In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule(3)-CL(3))。
In certain embodiments, the bispecific antigen binding molecule according to the invention comprises a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain constant region is replaced with a light chain constant region) (VH)(3)-CH1(3)-VH(1)-CH1(1)-VH(2)-CL(2)). In some embodiments, bispecific antigen binding moleculesFurther comprising a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL) with the Fab heavy chain constant region of the second Fab molecule(2)-CH1(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1)). In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule (3)-CL(3))。
In certain embodiments, the bispecific antigen binding molecules according to the invention comprise a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule (VL)(2)-CH1(2)-VH(1)-CH1(1)-VH(3)-CH1(3)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH)(2)-CL(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1)). In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule(3)-CL(3))。
In certain embodiments, the bispecific antigen binding molecule according to the invention comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule (VH (2)-CL(2)-VH(1)-CH1(1)-VH(3)-CH1(3)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein Fa of the second Fab moleculeb light chain variable region sharing a carboxy terminal peptide bond (VL) with the Fab heavy chain constant region of a second Fab molecule(2)-CH1(2)) And a Fab light chain polypeptide (VL) of a first Fab molecule(1)-CL(1)). In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule(3)-CL(3))。
In certain embodiments, the bispecific antigen binding molecules according to the invention comprise a polypeptide wherein the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the third Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e. the third Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region) (VH chain variable region(2)-CH1(2)-VL(1)-CH1(1)-VL(3)-CH1(3)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH (1)-CL(1)) And a Fab light chain polypeptide (VL) of a second Fab molecule(2)-CL(2)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH)(3)-CL(3))。
In certain embodiments, the bispecific antigen binding molecules according to the invention comprise a polypeptide wherein the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the third Fab moleculeAnd sharing a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain constant region is replaced with a light chain constant region) (VH(2)-CH1(2)-VH(1)-CL(1)-VH(3)-CL(3)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL)(1)-CH1(1)) And a Fab light chain polypeptide (VL) of a second Fab molecule (2)-CL(2)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL)(3)-CH1(3))。
In certain embodiments, the bispecific antigen binding molecules according to the invention comprise a polypeptide wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e. the third Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule (VL)(3)-CH1(3)-VL(1)-CH1(1)-VH(2)-CH1(2)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH(1)-CL(1)) And a Fab light chain polypeptide (VL) of a second Fab molecule(2)-CL(2)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH) (3)-CL(3))。
In certain embodiments, the bispecific antigen binding molecules according to the invention comprise a polypeptide wherein the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e. the third Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule (VH(3)-CL(3)-VH(1)-CL(1)-VH(2)-CH1(2)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL)(1)-CH1(1)) And a Fab light chain polypeptide (VL) of a second Fab molecule(2)-CL(2)). In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL)(3)-CH1(3))。
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) An Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) An Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:1,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID No. 7, the
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) An Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:1,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID No. 7, the
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein
(i) The first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In all the different configurations of the bispecific antigen binding molecules according to the invention, the amino acid substitutions described herein, if present, may be in the CH1 and CL domains of the first and (if present) third antigen binding moiety/Fab molecule, or in the CH1 and CL domains of the second antigen binding moiety/Fab molecule. Preferably, they are in the CH1 and CL domains of the first and (if present) third antigen binding moiety/Fab molecule. In accordance with the concepts of the present invention, if an amino acid substitution as described herein is made in the first (and, if present, the third) antigen binding moiety/Fab molecule, no such amino acid substitution is made in the second antigen binding moiety/Fab molecule. In contrast, if an amino acid substitution as described herein is made in the second antigen binding moiety/Fab molecule, no such amino acid substitution is made in the first (and, if present, the third) antigen binding moiety/Fab molecule. In particular amino acid substitutions are made in bispecific antigen binding molecules comprising Fab molecules in which the variable domains VL and VH1 of the Fab light and Fab heavy chains are replaced with each other.
In a particular embodiment of the bispecific antigen binding molecule according to the invention, in particular wherein an amino acid substitution as described herein is made in the first (and, if present, the third) antigen binding moiety/Fab molecule, the constant domain CL of the first (and, if present, the third) Fab molecule is of the kappa isotype. In other embodiments of the bispecific antigen binding molecule according to the invention, in particular wherein the amino acid substitutions as described herein are made in a second antigen binding moiety/Fab molecule, the constant domain CL of the second antigen binding moiety/Fab molecule is of the kappa isotype. In some embodiments, the constant domain CL of the first (and, if present, the third) antigen binding moiety/Fab molecule and the constant domain CL of the second antigen binding moiety/Fab molecule are of the kappa isotype.
In one embodiment, the present invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C).
In one embodiment, the present invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C).
In one embodiment, the present invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) An Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C).
In one embodiment, the present invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C).
In one embodiment, the present invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) An Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C), or (ii) the second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C).
In one embodiment, the present invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:1,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C).
In one embodiment, the present invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID No. 7, the
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) An Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under C).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) and the third antigen binding module under c) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) and the third antigen binding module under c) is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:83,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) A third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) and the third antigen binding module under c) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) and the third antigen binding module under c) is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) and the third antigen binding module under c) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) and the third antigen binding module under c) is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) A third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) and the third antigen binding module under c) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) and the third antigen binding module under c) is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) and the third antigen binding module under c) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) and the third antigen binding module under c) is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:1,
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) A third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) and the third antigen binding module under c) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) and the third antigen binding module under c) is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In a particular embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID No. 7, the
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) a third antigen-binding moiety that binds to the first antigen and is identical to the first antigen-binding moiety; and
d) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) and the third antigen binding module under c) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) and the third antigen binding module under c) is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index); and is
Wherein
(i) The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under C) 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 under d), or
(ii) The second antigen binding moiety under b) 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 under a), and the first antigen binding moiety under a) and the third antigen binding moiety under C) 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 under d).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising a) a first antigen binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83, the
b) a second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) An Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising a) a first antigen binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:83,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising a) a first antigen binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:90,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO:1,
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In another embodiment, the invention provides a bispecific antigen binding molecule comprising
a) A first antigen-binding moiety that binds a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR)1 of SEQ ID No. 7, the
b) A second antigen binding moiety that binds a second antigen, wherein the second antigen is an activating T cell antigen, particularly CD3, more particularly CD3, and the second antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other;
c) an Fc domain comprised of first and second subunits;
wherein the amino acid at position 124 in the constant domain CL of the first antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the Kabat index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the Kabat index); and is
Wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under C).
In accordance with any of the above embodiments, the components of the bispecific antigen binding molecule (e.g., Fab molecule, Fc domain) can be fused directly or via various linkers described herein or known in the art, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. Suitable, non-immunogenic peptide linkers include, for example, (G) 4S)n,(SG4)n,(G4S)nOr G4(SG4)nA peptide linker, wherein n is generally an integer from 1 to 10, typically from 2 to 4.
In a particular aspect, the invention provides a bispecific antigen binding molecule comprising
a) First and third antigen binding moieties that bind to a first antigen; wherein the first antigen is GPRC5D and wherein the first and second antigen binding moieties are each a (conventional) Fab molecule comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 13 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 14;
b) a second antigen binding moiety that binds a second antigen; wherein the second antigen is CD3 and wherein the second antigen binding module is a Fab molecule wherein the variable domains VL and VH of the Fab light and Fab heavy chains are replaced with each other comprising the heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 35 and the light chain variable region comprising the amino acid sequence of SEQ ID NO. 36;
c) an Fc domain comprised of first and second subunits;
wherein
The amino acid at position 124 in the constant domain CL of the first and third antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first and third antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the EU index of Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index of Kabat);
And wherein further
The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under a) 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 under C).
In a particular aspect, the invention provides a bispecific antigen binding molecule comprising
a) First and third antigen binding moieties that bind to a first antigen; wherein the first antigen is GPRC5D and wherein the first and second antigen binding moieties are each a (conventional) Fab molecule comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 16;
b) a second antigen binding moiety that binds a second antigen; wherein the second antigen is CD3 and wherein the second antigen binding module is a Fab molecule wherein the variable domains VL and VH of the Fab light and Fab heavy chains are replaced with each other comprising the heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 35 and the light chain variable region comprising the amino acid sequence of SEQ ID NO. 36;
c) an Fc domain comprised of first and second subunits;
wherein
The amino acid at position 124 in the constant domain CL of the first and third antigen binding module under a) is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein the amino acid at position 147 in the constant domain CH1 of the first and third antigen binding module under a) is substituted with glutamic acid (E) (numbering according to the EU index of Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index of Kabat);
And wherein further
The first antigen binding moiety under a) 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 under b), and the second antigen binding moiety under b) and the third antigen binding moiety under a) 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 under C).
In one embodiment, in accordance with this aspect of the invention, the threonine residue at position 366 is replaced with a tryptophan residue in the first subunit of the Fc domain (T366W), and the tyrosine residue at position 407 is replaced with a valine residue in the second subunit of the Fc domain (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering in accordance with the Kabat EU index).
In yet another embodiment, in accordance with this aspect of the invention, the serine residue at position 354 is additionally replaced with a cysteine residue in the first subunit of the Fc domain (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular the serine residue at position 354 is replaced with a cysteine residue) and the tyrosine residue at position 349 is additionally replaced with a cysteine residue in the second subunit of the Fc domain (Y349C) (numbering according to the Kabat EU index).
In yet another embodiment according to this aspect of the invention, the leucine residue at position 234 is replaced with an alanine residue in each of the first and second subunits of the Fc domain (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbering according to the Kabat EU index).
In yet another embodiment according to this aspect of the invention, the Fc domain is a human IgG1An Fc domain.
In particular embodiments, the bispecific antigen binding molecule comprises a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 17, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 18, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 19, and a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 20. In yet another specific embodiment, the bispecific antigen binding molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO 17, a polypeptide comprising the amino acid sequence of SEQ ID NO 18, a polypeptide comprising the amino acid sequence of SEQ ID NO 19 and a polypeptide comprising the amino acid sequence of SEQ ID NO 20.
In another specific embodiment, the bispecific antigen binding molecule comprises a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 21, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 22, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 23, and a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID No. 24. In yet another specific embodiment, the bispecific antigen binding molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO 21, a polypeptide comprising the amino acid sequence of SEQ ID NO 22, a polypeptide comprising the amino acid sequence of SEQ ID NO 23 and a polypeptide comprising the amino acid sequence of SEQ ID NO 24.
Fc domain
In a particular embodiment, the bispecific antigen binding molecule of the invention comprises an Fc domain composed of a first and a second subunit. It is understood that the features of the Fc domains described herein in relation to bispecific antigen binding molecules are equally applicable to the Fc domains comprised in the antibodies of the invention.
The Fc domain of the 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 of an immunoglobulin g (IgG) molecule is a dimer, each subunit of which comprises a CH2 and CH3 IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably associating with each other. In one embodiment, the bispecific antigen binding molecule of the invention comprises no more than one Fc domain.
In one embodiment, the Fc domain of the 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 EU index numbering), in particular 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 Disposition 38,84-91 (2010)). In yet another specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is human IgG1An Fc domain. Human IgG1An exemplary sequence of the Fc region is given in SEQ ID NO 42.
Fc domain modification to promote heterodimerization
The bispecific antigen binding molecules according to the invention comprise different antigen binding modules which can be fused to one or the other of the two subunits of the Fc domain, such that 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 bispecific antigen binding molecules in recombinant production, it would be advantageous to introduce modifications in the Fc domain of the bispecific antigen binding molecule that facilitate the association of the desired polypeptide.
Thus, in a specific embodiment, the Fc domain of the bispecific antigen binding molecule according to 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.
There are several approaches to modify the CH3 domain of an Fc domain to enhance heterodimerization, which are described in detail in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, both the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (such that the first and second CH3 domains heterodimerize and no homodimer is formed between the two first CH3 domains or the two second CH3 domains). These different approaches for improving heavy chain heterodimerization are contemplated as different alternatives, in combination with heavy-light chain modifications in bispecific antigen binding molecules that reduce heavy/light chain mismatches and Bence Jones type by-products (e.g. VH and VL exchange/substitutions in one binding arm and substitutions that introduce charged amino acids with opposite charges in the CH1/CL interface).
In a particular embodiment, the modification that facilitates association of the first and second subunits of the Fc domain is a so-called "knob-to-hole" modification comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.
Node-in-point techniques are described, for example, in US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9,617- > 621(1996) and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide such that the protuberance can be placed in the cavity to promote heterodimer formation and hinder homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the first polypeptide interface with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).
Thus, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecule, one amino acid residue 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 is locatable within the cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, one amino acid residue 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, wherein the protuberance within the CH3 domain of the first subunit is locatable.
Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
The protuberances and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis.
In a particular embodiment, in the (CH 3 domain of the) first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), while in the (CH 3 domain of the) second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, additionally, the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).
In yet another embodiment, in the first subunit of the Fc domain, additionally the serine residue at position 354 is substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular, the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is substituted with a cysteine residue (Y349C) (numbering according to the Kabat index). The introduction of these two cysteine residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, jimmnol Methods 248,7-15 (2001)).
In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).
In a specific embodiment, an antigen binding moiety that binds a second antigen (e.g., an activating T cell antigen) is fused (optionally via the first antigen binding moiety and/or peptide linker that binds GPRC 5D) to the first subunit of the Fc domain (which comprises a "knob" modification). Without wishing to be bound by theory, fusion of an antigen binding moiety that binds a second antigen, such as an activating T cell antigen, to a knob-containing subunit of an Fc domain (further) minimizes the production of an antigen binding molecule comprising two antigen binding moieties that bind an activating T cell antigen (spatial collision of two knob-containing polypeptides).
Other techniques for modifying CH3 to enhance heterodimerization are contemplated as alternatives according to the present invention and are described in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.
In one embodiment, the heterodimerization approach described in EP 1870459 is alternatively used. This approach is based on the introduction of charged amino acids of opposite charge at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment of the bispecific antigen binding molecule of the invention is the amino acid mutation R409D in one of the two CH3 domains (of the Fc domain); amino acid mutation D399K in the other of K370E and the CH3 domain of the Fc domain; E357K (numbering according to Kabat EU index).
In another embodiment, the bispecific antigen binding molecule of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutation T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and further the amino acid mutation R409D in the CH3 domain of the first subunit of the Fc domain; K370E and the amino acid mutation in the CH3 domain of the second subunit of the Fc domain D399K; E357K (numbering according to Kabat EU index).
In another embodiment, the bispecific antigen binding molecule of the invention comprises the amino acid mutation S354C in the CH3 domain of the first subunit of the Fc domain, T366W and the amino acid mutation Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the bispecific antigen binding molecule comprises the amino acid mutation Y349C in the CH3 domain of the first subunit of the Fc domain, T366W and the amino acid mutation S354C in the CH3 domain of the second subunit of the Fc domain, T366S, L368A, Y407V and further the amino acid mutation R409D in the CH3 domain of the first subunit of the Fc domain; K370E and the amino acid mutation in the CH3 domain of the second subunit of the Fc domain D399K; E357K (all numbering according to Kabat EU index).
In one embodiment, the heterodimerization approach described in WO 2013/157953 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the Kabat EU index). In yet another embodiment, the first CH3 domain further comprises the amino acid mutation L351K. In yet another embodiment, the second CH3 domain further comprises an amino acid mutation selected from the group consisting of Y349E, Y349D and L368E (preferably L368E) (numbering according to the Kabat EU index).
In one embodiment, the heterodimerization approach described in WO 2012/058768 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutation L351Y, Y407A and the second CH3 domain comprises the amino acid mutation T366A, K409F. In yet another embodiment, the second CH3 domain further comprises an amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W, b) D399R, D399W, D399Y, or D399K, c) S400E, S400D, S400R, or S400K, D) F405I, F405M, F405T, F405S, F405V, or F405W, e) N390R, N390K, or N390D, F) K392V, K392M, K392R, K38392 392L, K392F, or K392E (numbering according to Kabat 389). In yet another embodiment, the first CH3 domain comprises the amino acid mutation L351Y, Y407A and the second CH3 domain comprises the amino acid mutation T366V, K409F. In yet another embodiment, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In yet another embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).
In one embodiment, the heterodimerization approach described in WO 2011/143545 is alternatively used, e.g., amino acid modifications at positions selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index) are performed.
In one embodiment, the heterodimerization approach described in WO2011/090762 is alternatively used, which also uses the node-in-hole technique described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the Kabat EU index).
In one embodiment, the bispecific antigen binding molecule or its Fc domain is an IgG2Subclassed and alternatively using the heterodimerization approach described in WO 2010/129304.
In an alternative 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. Generally, this method involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues, thereby electrostatically favoring homodimer formation and electrostatically favoring heterodimerization. In one such embodiment, the first CH3 domain comprises an amino acid substitution of a negatively charged amino acid (e.g., glutamic acid (E), or aspartic acid (D)) to K392 or N392 (preferably K392D or N392D) and the second CH3 domain comprises an amino acid substitution of a positively charged amino acid (e.g., lysine (K) or arginine (R)) to D399, E356, D356, or E357 (preferably D399K, E356K, D K, or E357K, more preferably D399K and E356K). In yet another embodiment, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E), or aspartic acid (D)), preferably K409D or R409D. In yet another embodiment, the first CH3 domain further or alternatively comprises an amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g., glutamic acid (E), or aspartic acid (D)) (all numbering according to the Kabat EU index).
In yet another embodiment, the heterodimerization approach described in WO 2007/147901 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K, and K322D and the second CH3 domain comprises the amino acid mutations D239K, E240K, and K292D (numbering according to the Kabat EU index).
In yet another embodiment, the heterodimerization approach described in WO 2007/110205 may alternatively be used.
In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).
Fc domain modifications that reduce Fc receptor binding and/or effector function
The Fc domain confers the bispecific antigen binding molecule (or antibody) with advantageous pharmacokinetic properties, including a long serum half-life, which contributes to better accumulation in the target tissue and a favorable tissue-to-blood partition ratio. At the same time, however, it may lead to unwanted targeting of the bispecific antigen binding molecule (or antibody) to Fc receptor expressing cells rather than to preferred antigen bearing cells. Furthermore, co-activation of the Fc receptor signaling pathway may lead to cytokine release, which in combination with the T cell activation properties of the bispecific antigen binding molecule (e.g., in embodiments of the bispecific antigen binding molecule where a second antigen binding module binds an activating T cell antigen) and long half-life, causes over-activation of cytokine receptors and severe side effects following systemic administration. Activation of immune cells (bearing Fc receptors) rather than T cells may even reduce the efficacy of bispecific antigen binding molecules, particularly bispecific antigen binding molecules in which a second antigen binding module binds an activating T cell antigen, due to potential destruction of T cells, e.g. by NK cells.
Thus, in a specific embodiment, the Fc domain of the bispecific antigen binding molecule according to the invention exhibits affinity to native IgG1Reduced binding affinity to Fc receptors and/or reduced effector function compared to Fc domains. In one such embodiment, the Fc domain (or bispecific antigen binding molecule comprising the Fc domain) exhibits affinity for native IgG1Fc domain (or comprising native IgG)1Bispecific antigen binding molecules for Fc domains) less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to Fc receptors, and/or to native IgG1Fc domain (or comprising native IgG)1Bispecific antigen binding molecules for Fc domains) less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of effector function. In one embodiment, the Fc domain (or bispecific antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or 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 particular embodiment, the Fc receptor is an activating human 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 the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits IgG identity to native IgG 1The Fc domain compares to substantially similar binding affinity to neonatal Fc receptor (FcRn). When the Fc domain (or 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)1Bispecific antigen binding molecules for Fc domains) binding affinity to FcRn, substantially similar binding to FcRn is achieved.
In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of the 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 these 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 bispecific antigen binding molecule comprising an engineered Fc domain exhibits a binding affinity for an Fc receptor of less than 20%, particularly less than 10%, more particularly less than 5% compared to a 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 particular embodiment, the Fc receptor is an activating human Fc γ receptor, more particularly human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most particularly human Fc γ RIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components, particularly to 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 when the Fc domain (or bispecific antigen binding molecule comprising the Fc domain) exhibits more than about 70% of the binding affinity of the non-engineered form of the Fc domain (or bispecific antigen binding molecule comprising the non-engineered form of the Fc domain) for FcRn, i.e. retains the binding affinity of the Fc domain for the receptor. The Fc domain or bispecific antigen binding molecule of the invention comprising said Fc domain may exhibit such affinity of more than about 80% and even more than about 90%. In certain embodiments, the Fc domain of the bispecific antigen binding molecule is engineered to have reduced effector function as compared to a non-engineered Fc domain. Reduced effector function may include, but is not limited to, one or more of reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce 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% ADCC induced by the non-engineered Fc domain (or the bispecific antigen binding molecule comprising a non-engineered Fc domain).
In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function 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 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group consisting of L234, L235, and P329 (numbering according to the KabatEU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG1Fc domain, in particular human IgG1An Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more particular embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain comprises at position P329An amino acid substitution and a further amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "lalapc"). In particular, in a specific embodiment, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and second subunits of the Fc domain the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A) and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbering according to Kabat EU index).
In one such embodiment, the Fc domain is an IgG1Fc domain, in particular human IgG1An Fc domain. Amino acid substitution combination "P329G LALA" almost completely eliminated human IgG1Fc gamma receptor (and complement) binding of the Fc domain, as described in PCT publication No. wo 2012/130831, which is incorporated herein in its entirety by reference. WO 2012/130831 also describes methods of making such mutant Fc domains and methods for determining properties thereof, such as Fc receptor binding or effector function.
IgG4Antibodies exhibit IgG binding1Reduced binding affinity to Fc receptors and reduced effector function compared to antibodies. Thus, in some embodiments, the Fc domain of the 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 an amino acid substitution at position S228, in particular amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce its binding affinity to Fc receptors and/or its effector function, in one embodiment, IgG4The Fc domain comprises an amino acid substitution at position L235, in particular amino acid substitution L235E (numbering according to the Kabat EU index). In another embodimentIn embodiments, the IgG4The Fc domain comprises an amino acid substitution at position P329, in particular amino acid substitution P329G (numbering according to the Kabat EU index). In a specific embodiment, the IgG is 4The Fc domain comprises amino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G (numbering according to the Kabat EU index). Such IgG4Fc domain mutants and their Fc γ receptor binding properties are described in PCT publication No. wo 2012/130831, which is incorporated herein by reference in its entirety.
In a specific embodiment, natural IgG is displayed1Fc domain with reduced binding affinity to Fc receptor and/or reduced effector function compared to Fc domain 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 P329G4Fc domain (numbering according to Kabat EU index).
In certain embodiments, N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the Kabat EU index).
Fc domains with reduced Fc receptor binding and/or effector function in addition to those described above and in PCT publication No. wo 2012/130831 also include those with substitutions of one or more of Fc domain residues 238,265,269,270,297,327 and 329 (U.S. Pat. No.6,737,056) (numbering according to the Kabat EU index). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).
Mutant Fc domains may be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide change can be verified by, for example, sequencing.
Binding to Fc receptors can be readily determined, for example by ELISA or by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and Fc receptors such as can be obtained by recombinant expression. Alternatively, cell lines known to express specific Fc receptors, such as human NK cells expressing Fc γ IIIa receptors, can be used to estimate the binding affinity of the Fc domain or bispecific antigen binding molecule comprising the Fc domain to the Fc receptor.
Effector function of an Fc domain or bispecific antigen binding molecule comprising an Fc domain can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of molecules 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 ExpMed 166, 1351-. Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry) TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and Cytotox
In some embodiments, Fc domain binding to complement components (particularly to C1q) is reduced. Thus, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function comprises reduced CDC. A C1q binding assay can be performed to determine whether an Fc domain or bispecific antigen binding molecule comprising an Fc domain is capable of binding C1q and thus has CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J ImmunolMethods 202,163 (1996); Cragg et al, Blood 101, 1045-.
FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b.et al, Int' l.immunol.18(12):1759-1769 (2006); WO 2013/120929).
Polynucleotide
The invention also provides an isolated polynucleotide encoding an antibody or bispecific antigen binding molecule as described herein, or a fragment thereof. In some embodiments, the fragment is an antigen-binding fragment.
The polynucleotide encoding the antibody or bispecific antigen binding molecule of the invention can be expressed as a single polynucleotide encoding the entire antibody or bispecific antigen binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by the co-expressed polynucleotides may associate via, for example, disulfide bonds or other means to form a functional antibody or bispecific antigen binding molecule. For example, the light chain portion of the antibody or bispecific antigen-binding molecule can be encoded by a separate polynucleotide from the portion of the antibody or bispecific antigen-binding molecule comprising the heavy chain of the antibody or bispecific antigen-binding molecule. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antibody or bispecific antigen binding molecule. In another example, the portion of the antibody or bispecific antigen binding molecule comprising one of the two Fc domain subunits and optionally (part of) one or more Fab molecules may be encoded by a separate polynucleotide from the portion of the antibody or bispecific antigen binding molecule comprising the other of the two Fc domain subunits and optionally (part of) a Fab molecule. When co-expressed, the Fc domain subunits associate to form an Fc domain.
In some embodiments, the isolated polynucleotide encodes an antibody or bispecific antigen binding molecule, entirely in accordance with the invention described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in an antibody or bispecific antigen binding molecule according to the invention described herein.
In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mrna). The RNA of the present invention may be single-stranded or double-stranded.
Recombination method
Antibodies or bispecific antigen-binding molecules of the invention can be obtained, for example, by solid phase peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody or bispecific antigen binding molecule (fragment), e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector (preferably an expression vector) comprising one or more polynucleotides of the invention is provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for the antibody or bispecific antigen binding molecule (fragment) and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, e.g., the discussion in Maniatis et al, Molecula clone: ALABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al, C URRENTPROTOCOLS INMOLECULARBIOLOGYGreene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector may be a plasmid, part of a virus or may be a nucleic acid fragment. The expression vector comprises an expression cassette in which a polynucleotide encoding an antibody or bispecific antigen binding molecule (fragment) (i.e., the coding region) is cloned in operable association with a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of the coding region (if present), but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or may be present in separate vectorsIn the context of the present invention, the term "promoter" refers to a promoter that directs the transcription of a gene product, such as a promoter region of a bovine retrovirus (e.g., a promoter region of a bovine retrovirus), such as a promoter region of a retrovirus promoter region of a bovine retrovirus (e.g., a promoter region of a retrovirus promoter region of a promoter that directs the promoter, such as a promoter region of promoter, such as a promoter region of promoter, such as a promoter region of promoter, such as a promoter region of promoter, such as a promoter region of promoter Other sequences that control gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (in particular, internal ribosome entry sites or IRES, also known as CITE sequences). The expression cassette may also comprise other features, such as an origin of replication and/or chromosomal integration elements, such as retroviral Long Terminal Repeats (LTRs) or adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).
The polynucleotide and nucleic acid coding regions of the invention may be associated with additional coding regions that encode secretion or signal peptides that direct secretion of the polypeptide encoded by the polynucleotide of the invention. For example, if secretion of the antibody or bispecific antigen binding molecule is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the antibody or bispecific antigen binding molecule of the invention, or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein upon initiation of export of the growing protein chain across the rough endoplasmic reticulum. One of ordinary skill in the art knows that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of a sequence that retains the ability to direct secretion of the polypeptide with which it is operably associated, is used. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.
DNA encoding short protein sequences that can be used to facilitate later purification (e.g., histidine tag) or to aid in labeling the antibody or bispecific antigen binding molecule (fragment) can be incorporated into or at the end of the antibody or bispecific antigen binding molecule encoding polynucleotide.
In a particular embodiment, host cells are provided that comprise one or more polynucleotides of the invention. 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 with respect to the polynucleotide and vector, respectively, alone or in combination. In one such embodiment, the host cell comprises one or more vectors (e.g. transformed or transfected with a vector), the one or more vectors comprising one or more polynucleotides encoding (part of) the antibody or bispecific antigen binding molecule of the invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce an antibody or bispecific antigen binding molecule of the invention, or a fragment thereof. Host cells suitable for replicating and supporting the expression of antibodies or bispecific antigen binding molecules are well known in the art. Such cells may be transfected or transduced with a particular expression vector as appropriate, and a large number of vector-containing cells may be cultured for inoculation into a large-scale fermentor to obtain a sufficient amount of antibody or 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 cells, and the like. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-. Host cells suitable for the expression (glycosylation) of polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A large number of baculovirus strains have been identified for use with insect cells, particularly for use Cells of Spodoptera frugiperda (Spodoptera frugiperda) were transfected. 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 (PLANTIBODIIES described for the production of antibodies in transgenic plantsTMA technique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of mammalian host cell lines that may be used are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, such as, for example, those described in Graham et al, J Gen Virol 36,59(1977)), baby hamster kidney cells (BHK), mouse Sertoli (TM4 cells, such as, for example, those described in 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), bovine mouse (buffalo rat) liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep 2), mouse mammary tumor cells (MMT 060562), TRI cells (such as, for example, those described in Mather et al, Annals N.Y. Acad Sci 383,44-68 (1982)), MRC 5 cells and FS4 cells. Other mammalian host cell lines that may be used include Chinese Hamster Ovary (CHO) cells, including dhfr -CHO cells (Urlaub et al, Proc Natl
Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide comprising an antigen binding domain, such as the heavy or light chain of an antibody, can be engineered such that the other antibody chain is also expressed, such that the product expressed is an antibody having both a heavy chain and a light chain.
In one embodiment, a method of producing an antibody or bispecific antigen binding molecule according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the antibody or bispecific antigen binding molecule (as provided herein) under conditions suitable for expression of the antibody or bispecific antigen binding molecule, and optionally recovering the antibody or bispecific antigen binding molecule from the host cell (or host cell culture medium).
The individual components of the bispecific antigen binding molecules (or antibodies) of the invention can be genetically fused to one another. Bispecific antigen binding molecules can be designed such that their 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 the efficacy can be tested. Examples of linker sequences between different building blocks of bispecific antigen binding molecules are provided herein. Additional sequences are included to incorporate cleavage sites to separate the individual components of the fusion, if desired, such as endopeptidase recognition sequences.
The antibodies or bispecific antigen binding molecules of the invention generally comprise at least an antibody variable region capable of binding an antigenic determinant. The variable regions may form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods for generating 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).
Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used for the antibody or bispecific antigen binding molecule 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 antibody or bispecific antigen binding molecule is intended for human use, a chimeric form of the antibody may be used in which 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 important for retaining better 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 intact non-human variable domains, but "cloak" them with human-like moieties by replacing surface residues. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature332:323-329 (1988); queen et al, Proc.Nat' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; kashmiri et al, Methods 36:25-34(2005) (specificity determining region (SDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (describes "resurfacing"); dall' Acqua et al, Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimkaet al, Br.J. cancer 83:252-260(2000) (describing the "guided selection" method of FR shuffling). Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al, J.Immunol.151:2296 (1993); framework regions derived from consensus sequences of a specific subset of human antibodies of the light or heavy chain variable regions (see, e.g., Carter et al proc. Natl.Acad.Sci.USA,89:4285 (1992); and Presta et al, J.Immunol.,151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al, J.biol.chem.272: 10678-.
Human antibodies can be generated using a variety of techniques known in the art. In general, human antibodies are described in vanDijk and van de Winkel, Curr, Opin, Pharmacol.5:368-74(2001), and Lonberg, Curr, Opin, Immunol.20: 450-. Human antibodies can be made by administering an immunogen to transgenic animals that have been modified to produce fully human antibodies or fully antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSETMA technique; U.S. Pat. No.5,770,429, which describes
Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for generating human Monoclonal antibodies have been described (see, e.g., Kozbor, J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York,1987), and Boerner et al, J.Immunol.,147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, proc.natl.acad.sci.usa,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiandai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandrein, Histology and Histopathlogy, 20(3): 927-.
Human antibodies can also be generated by isolation from a library of human antibodies, as described herein.
Antibodies useful in the present invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al, Nature Reviews 16:498-508 (2016). For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such methods are reviewed, for example, in Frenzel et al, mAbs 8:1177-1194 (2016); bazan et al, Human Vaccines and immunothereutics 8: 1817-.
In some phage display methods, the repertoire of VH and VL genes, respectively, are cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Annual Review of Immunology 12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the non-immune repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al, EMBO Journal 12:725-734 (1993). Finally, synthetic generation of non-immune libraries can also be achieved by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, Journal of molecular biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 and U.S. patent publication nos. 2005/0079574,2007/0117126,2007/0237764 and 2007/0292936. Other examples of methods known in the art for screening combinatorial libraries for antibodies having one or more desired activities include ribosome and mRNA display, as well as methods for displaying and selecting antibodies on bacterial, mammalian, insect or yeast cells. For reviews of Methods for yeast surface display see, for example, Scholler et al, Methods in Molecular Biology 503:135-56(2012) and Cherf et al, Methods in Molecular Biology 1319:155-175(2015) and Zhoo et al, Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, for example, in He et al, Nucleic Acids Research 25: 5132-.
Antibodies or bispecific antigen binding molecules prepared as described herein can be purified by techniques known in the art, 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 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 to which the antibody or bispecific antigen binding molecule binds can be used. For example, for affinity chromatography purification of the antibodies or bispecific antigen binding molecules of the invention, a matrix with protein a or protein G may be used. Antibodies or bispecific antigen binding molecules can be isolated using sequential protein a or G affinity chromatography and size exclusion chromatography, substantially as described in the examples. The purity of the antibody or 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.
Assay method
The antibodies or bispecific antigen binding molecules provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.
Affinity assay
The affinity of an antibody or bispecific antigen binding molecule for an Fc receptor or target antigen can be determined by Surface Plasmon Resonance (SPR), for example using standard instruments such as a BIAcore instrument (GE Healthcare), and the receptor or target protein can be obtained, for example, by recombinant expression. Alternatively, the binding of an antibody or bispecific antigen binding molecule to different receptors or target antigens can be assessed, for example, by flow cytometry (FACS), using cell lines expressing a particular receptor or target antigen. One particular illustrative and exemplary embodiment for measuring binding affinity is described below.
According to one embodiment, the use is by surface plasmon resonance
To analyze the interaction between the Fc portion and the Fc receptor, His-tagged recombinant Fc receptor was captured by anti-pentahis antibody (Qiagen) immobilized on CM5 chip and bispecific constructs were used as analytes. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-pentaHis antibody was diluted to 40. mu.g/ml with 10mM sodium acetate pH 5.0, followed by injection at a flow rate of 5. mu.l/min to achieve approximately 6500 Response Units (RU) of conjugated protein. After injection of the ligand, 1M ethanolamine was injected to block unreacted groups. Subsequently, Fc receptors were captured at 4 or 10nM for 60 seconds. For kinetic measurements, 4-fold serial dilutions (ranging from 500nM to 4000nM) of antibody or bispecific antigen-binding molecule were injected at 25 ℃ for 120 seconds at a flow rate of 30 μ l/min in HBS-EP (GE Healthcare,10mM HEPES,150mM NaCl,3mM EDTA, 0.05% surfactant P20, pH 7.4).
To determine affinity for the target antigen, the antibody or bispecific antigen binding molecule was captured by anti-human Fab specific antibody (GE Healthcare) immobilized on the surface of an activated CM5 sensor chip, as described for anti-penta-His antibody. The final amount of conjugated protein was about 12000 RU. The antibody or bispecific antigen binding molecule was captured at 300nM for 90 seconds. The target antigen was passed through the flow cell at a flow rate of 30 μ l/min for 180 seconds at a concentration ranging from 250 to 1000 nM. Dissociation was monitored for 180 seconds.
Bulk refractive index (bulk refractive index) differences were corrected by subtracting the response obtained on the reference flow cell. Derivation of dissociation constant K by nonlinear curve fitting of Langmuir binding isotherms using steady state responseD. Using a simple 1-to-1 Langmuir binding model: (T100 evaluation software version 1.1.1) calculate the association rate (k) by simultaneous fitting of the association and dissociation sensorgramsBonding of) And dissociation rate (k)Dissociation). Equilibrium dissociation constant (K)D) Is calculated as the ratio kDissociation/kBonding of. See, e.g., Chen et al, J Mol Biol 293,865- & 881 (1999).
Activity assay
The biological activity of the bispecific antigen binding molecules (or antibodies) of the invention can be measured by various assays, as described in the examples. Biological activities may include, for example, inducing proliferation of T cells, inducing signaling in T cells, inducing expression of activation markers in T cells, inducing cytokine secretion by T cells, inducing lysis of target cells such as 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 one of the antibodies or bispecific antigen binding molecules provided herein, e.g., for use in any one of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any one of the antibodies or bispecific antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any one of the antibodies or bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.
Also provided are methods of producing an antibody or bispecific antigen-binding molecule of the invention in a form suitable for in vivo administration, the method comprising (a) obtaining an antibody or bispecific antigen-binding molecule according to the invention, and (b) formulating the antibody or bispecific antigen-binding molecule with at least one pharmaceutically acceptable carrier, thereby formulating an antibody or bispecific antigen-binding molecule preparation for in vivo administration.
The pharmaceutical compositions of the invention comprise a therapeutically effective amount of the antibody or bispecific antigen binding molecule solubilized 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, i.e., do not produce an adverse, allergic, or other untoward reaction when administered to an animal such as, for example, a human, when appropriate at the dosages and concentrations employed. In accordance with the present disclosure, the preparation of pharmaceutical compositions containing antibodies or bispecific antigen binding molecules and optionally additional active ingredients will be known to those skilled in the art, as exemplified by Remington's pharmaceutical sciences,18th ed. In addition, for animal (e.g., human) administration, it will be understood that the formulations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of biologica standards or corresponding agencies 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, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants (disintegration agents), lubricants, sweeteners, fragrances, dyes, such like materials and combinations thereof, as will be known to those of ordinary skill in the art (see, e.g., Remington's pharmaceutical Sciences,18th ed. machine Printing Company,1990, pp.1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
The antibody or bispecific antigen binding molecule (and any other therapeutic agent) of the invention may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic.
Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the antibodies or bispecific antigen-binding molecules of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks 'solution, Ringer' solution or physiological saline buffer. The solution may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibody or bispecific antigen binding molecule may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibody or bispecific antigen-binding molecule of the invention in the required amount in the appropriate solvent with various other ingredients as enumerated below, as required. Sterility can be readily achieved, for example, by filtration through a sterile filtration membrane. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle which contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the 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 thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first made isotonic before injection with sufficient saline or glucose. The compositions must be stable under the conditions of manufacture and storage and provide protection against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum at safe levels, for example below 0.5ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; 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 carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant 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, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, 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-lipids or triglycerides or liposomes.
The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly- (methacrylate) microcapsules, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions (macroemulsions). Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack printing company, 1990). Sustained release formulations can be prepared. Examples of suitable sustained-release preparations 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 injectable compositions can be brought about by the use in the compositions of absorption delaying agents, such as, for example, aluminum monostearate, gelatin or combinations thereof.
In addition to the previously described compositions, the antibodies or bispecific antigen binding molecules can also be formulated as depot (depot) formulations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, an antibody or bispecific antigen-binding molecule can 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.
Pharmaceutical compositions comprising the antibodies or bispecific antigen binding molecules of the invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. The 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 antibody or bispecific antigen binding molecule can be formulated into compositions as free acids or bases, neutral or salt forms. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts (acid addition salts), such as those formed with the free amino groups of the proteinaceous composition, or with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or with organic acids, such as acetic, oxalic, tartaric or mandelic acids. The salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium hydroxide, potassium, ammonium, calcium or iron; or an organic base such as isopropylamine, trimethylamine, histidine or procaine (procaine). Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.
Therapeutic methods and compositions
Any of the antibodies or bispecific antigen binding molecules provided herein can be used in a method of treatment. The antibodies or bispecific antigen binding molecules of the invention are useful as immunotherapeutics, for example in the treatment of cancer.
For use in a method of treatment, the antibodies or bispecific antigen binding molecules of the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors 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 delivery of the agent, the method of administration, the timing of administration and other factors known to medical practitioners.
In one aspect, an antibody or bispecific antigen binding molecule of the invention for use as a medicament is provided. In a further aspect, an antibody or bispecific antigen binding molecule of the invention is provided for use in the treatment of a disease. In certain embodiments, the antibodies or bispecific antigen binding molecules of the invention are provided for use in a method of treatment. In one embodiment, the invention provides an antibody or 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 invention provides an antibody or 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 the antibody or 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, such as an anti-cancer agent (if the disease to be treated is cancer). In other embodiments, the invention provides an antibody or 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 invention provides an antibody or bispecific antigen binding molecule for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual, the method comprising administering to the individual an effective amount of the antibody or bispecific antigen binding molecule to induce lysis of the target cell. An "individual" according to any of the embodiments above is a mammal, preferably a human. In certain embodiments, the disease to be treated is an autoimmune disease, in particular systemic lupus erythematosus and/or rheumatoid arthritis. The production of pathogenic autoantibodies by autoreactive plasma cells is a hallmark of autoimmune disease. Therefore, GPRC5D may be used to target autoreactive plasma cells in autoimmune diseases.
In a further aspect, the invention provides the use of an antibody or 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 a further 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, such as an anti-cancer agent (if the disease to be treated is cancer). In a particular embodiment, the medicament is for inducing lysis of a target cell, particularly a tumor cell. In still other embodiments, the medicament is for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual, the method comprising administering to the individual an effective amount of the medicament to induce lysis of the target cell. An "individual" according to any of the embodiments above may be a mammal, preferably a human.
In a further aspect, the invention provides methods for treating a disease. In one embodiment, the method comprises administering to an individual having such a disease a therapeutically effective amount of an antibody or bispecific antigen binding molecule of the invention. In one embodiment, the individual is administered a composition comprising the antibody or bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form. 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, such as an anti-cancer agent (if the disease to be treated is cancer). An "individual" according to any of the embodiments above may be a mammal, preferably a human.
In a further 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 the antibody or bispecific antigen binding molecule of the invention in the presence of a T cell, particularly a cytotoxic T cell. In a further aspect, a method for inducing lysis of a target cell, in particular a tumor cell, in an individual is provided. In one such embodiment, the method comprises administering to the individual an effective amount of the antibody or bispecific antigen binding molecule to induce lysis of the target cells. 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, hematological cancer, skin cancer, squamous cell carcinoma, bone cancer, and renal cancer. Other cell proliferation disorders that can be treated using the antibodies or bispecific antigen binding molecules of the invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are precancerous conditions or lesions and cancer metastases. In certain embodiments, the cancer is selected from the group consisting of renal cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, and prostate cancer. In one embodiment, the cancer is prostate cancer. The skilled artisan readily recognizes that in many cases, an antibody or bispecific antigen binding molecule may not provide a cure but may only provide partial benefit. In some embodiments, physiological changes with some benefit are also considered therapeutically beneficial. As such, in some embodiments, the amount of antibody or 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 typically a mammal, more particularly a human.
In some embodiments, an effective amount of an antibody or bispecific antigen binding molecule of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an antibody or bispecific antigen binding molecule of the invention is administered to an individual to treat a disease.
For the prevention or treatment of disease, the appropriate dosage of an antibody or bispecific antigen binding molecule of the invention (either 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 weight of the patient, the type of antibody or bispecific antigen binding molecule, the severity and course of the disease, whether the antibody or bispecific antigen binding molecule is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the clinical history and response of the patient to the antibody or bispecific antigen binding molecule, and the judgment of the attending physician. The practitioner responsible for administration will determine at any event the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.
The antibody or bispecific antigen binding molecule is suitably administered to the patient in one or 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 antibody or bispecific antigen binding molecule can be an initial candidate dose for administration to a patient, whether, for example, by one or more divided administrations, or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of antibody or bispecific antigen binding molecule will be in the range of about 0.005mg/kg to about 10 mg/kg. In other non-limiting examples, the dose can further include from 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 1000mg/kg body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the amounts listed herein, based on the numbers described above, a range of about 5mg/kg body weight to about 100mg/kg body weight, a range of about 5 micrograms/kg body weight to about 500 milligrams/kg body weight, and the like may be administered. Thus, one or more doses of about 0.5mg/kg,2.0mg/kg,5.0mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, such as weekly or every 3 weeks (e.g., such that the patient receives from about 2 to about 20, or, for example, about 6 doses of the antibody or bispecific antigen binding molecule). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be used. The progress of this therapy is readily monitored by conventional techniques and assays.
The antibodies or bispecific antigen binding molecules of the invention will generally be used in an amount effective for the purpose intended. For use in treating or preventing a disease condition, the antibody or bispecific antigen binding molecule of the invention, or a pharmaceutical composition thereof, is administered or applied in a therapeutically effective amount. Determination of 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. The dosage can then be formulated in animal models to achieve inclusion of the IC as determined in cell culture50The circulating concentration range of (c). Such information can be used to more accurately determine the dosage available in a human.
Initial doses can also be estimated from in vivo data, such as animal models, using techniques well known in the art. One of ordinary skill in the art can readily optimize administration to humans based on animal data.
The dosage amount and time interval, respectively, can be adjusted to provide plasma levels of the antibody or bispecific antigen binding molecule sufficient to maintain a therapeutic effect. The dose range of available patients administered by injection is from about 0.1 to 50 mg/kg/day, usually about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.
In the case of local administration or selective uptake, the effective local concentration of the antibody or bispecific antigen binding molecule may not be related to the plasma concentration. One skilled in the art would be able to optimize therapeutically effective local dosages without undue experimentation.
A therapeutically effective dose of an antibody or bispecific antigen binding molecule described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of the antibody or bispecific antigen binding molecule can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. LD can be determined using cell culture assays and animal studies50(lethal dose for 50% of the population) and ED50(a 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. Antibodies or bispecific antigen-binding molecules exhibiting a large therapeutic index are preferred. In one embodiment, the antibody or bispecific antigen binding molecule according to the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage suitable for use in humans. Preferably, the dose is at a circulating concentration (including ED) with little or no toxicity 50) Within the range of (1). The dosage within this range may vary depending on a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be selected by The individual physician in view of The patient's condition (see, e.g., Fingl et al, 1975, in: The Pharmacological Basis of Therapeutics, ch.1, p.1, herein incorporated by reference in its entirety).
The attending physician of a patient being treated with an antibody or bispecific antigen binding molecule of the invention will know how and when to terminate, discontinue or adjust administration (due to toxicity, organ dysfunction, etc.). Conversely, if the clinical response is not adequate (excluding toxicity), the attending physician will also know to adjust the treatment to higher levels. The magnitude of the administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, 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 possibly the frequency of administration will also vary with the age, weight and response of the individual patient.
Other Agents and treatments
The antibodies and bispecific antigen binding molecules of the invention can be administered in combination with one or more other agents in therapy. For example, an antibody or bispecific antigen binding molecule of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may comprise 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 immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, 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 disruptor, an anti-metabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.
Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of antibody or bispecific antigen binding molecule used, the type of disorder or treatment, and other factors discussed above. The antibody or bispecific antigen binding molecule is generally used in the same dosages and administration routes as described herein, or in about 1 to 99% of the dosages described herein, or by any dosage and any route empirically/clinically determined to be appropriate.
Such combination therapies recited above encompass both combined administration (where two or more therapeutic agents are included in the same composition or separate compositions) and separate administration, in which case administration of the antibody or bispecific antigen binding molecule of the invention can occur prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants. The antibodies or bispecific antigen binding molecules of the invention may also be used in combination with radiotherapy.
Article of manufacture
In another aspect of the invention, articles of manufacture containing materials useful in the treatment, prevention and/or diagnosis of the conditions described above are provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition, either by itself or in combination with another composition, effective for treating, preventing and/or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody or bispecific antigen binding molecule of the invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises the antibody or bispecific antigen binding molecule of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may also comprise a package insert indicating that the composition is useful for treating 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 further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
Methods and compositions for diagnosis and detection
In certain embodiments, any of the anti-GPRC 5D antibodies provided herein can be used to detect the presence of GPRC5D in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue, such as prostate tissue.
In one embodiment, an anti-GPRC 5D antibody for use in a diagnostic or detection method is provided. In yet another aspect, a method of detecting the presence of GPRC5D in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-GPRC 5D antibody under conditions that allow the anti-GPRC 5D antibody to bind GPRC5D, as described herein, and detecting whether a complex is formed between the anti-GPRC 5D antibody and GPRC 5D. Such methods may be in vitro or in vivo. In one embodiment, an anti-GPRC 5D antibody is used to select a subject suitable for treatment with an anti-GPRC 5D antibody, e.g., where GPRC5D is a biomarker for selecting patients.
Exemplary disorders that can be diagnosed using the antibodies of the invention include cancer, in particular multiple myeloma.
In certain embodiments, labeled anti-GPRC 5D antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes 32P,14C,125I,3H, and131fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine (rhodamine) and derivatives thereof, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenaseHeterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to an enzyme that oxidizes a dye precursor with hydrogen peroxide such as HRP), lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.
In yet another aspect the invention relates to an antibody (10B10) that binds to GPRC5D comprising a variable heavy chain region (VH), wherein the VH may comprise an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 81. The antibody may comprise a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 82. The antibody may comprise a VH and a VL, wherein the VH may comprise an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:81 and wherein the VL comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 82. Preferably, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:81 and a VL comprising the amino acid sequence of SEQ ID NO: 82.
Yet another aspect of the invention relates to an antibody (10B 10-TCB). The antibody may comprise a first light chain, wherein the first light chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 67. The antibody may comprise a second light chain, wherein the second light chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 68. The antibody may comprise a first heavy chain, wherein the first heavy chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 69. The antibody may comprise a second heavy chain, wherein the second heavy chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID No. 70. In a preferred embodiment, the antibody comprises a first light chain comprising the amino acid sequence of SEQ ID NO 67, a second light chain comprising the amino acid sequence of SEQ ID NO 68, a first heavy chain comprising the amino acid sequence of SEQ ID NO 69 and a second heavy chain comprising the amino acid sequence of SEQ ID NO 70.
Amino acid sequence
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