Multispecific binding compounds that bind to PD-L1

Abstract

Multispecific binding compounds that bind to PD-L1 are disclosed, along with methods of making such binding compounds, compositions, including pharmaceutical compositions, comprising such binding compounds, and their uses for treating disorders characterized by expression of PD-L1.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 093,109, filed October 16, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on December 30, 2021, is named QLS-0002-WO_SL.txt and is 228,930 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to multispecific binding compounds that bind to PD-L1. The invention further relates to methods of making such binding compounds, compositions, including pharmaceutical compositions, comprising such binding compounds, and their use for treating disorders characterized by PD-L1 expression. [Background technology]

[0004] Cancer is a leading cause of death worldwide. In advanced metastatic cancer, traditional treatment regimens, such as radiation therapy and chemotherapy, have only modest benefits in extending survival. Targeted therapies, such as small-molecule inhibitors and inhibitory monoclonal antibodies, have brought about significant improvements in managing disease progression; however, they are still limited to subsets of cancers that harbor specific mutations or overexpress targetable receptors. Furthermore, resistance to these therapies is common, as tumor cells can undergo further mutations or switch to alternative signaling pathways that evade the inhibitory effects of drugs. Immunotherapy offers new possibilities for combating cancer by harnessing the body's own immune system. For example, checkpoint inhibitors targeting PD-1 and CTLA-4 have led the way in research and development in this field. The development of multispecific antibodies has enabled new therapeutic approaches.

[0005] PD-L1 multispecific antibodies are designed to target PD-L1 and one or more costimulatory receptors, such as 4-1BB. Such antibodies function in several ways. First, they bind to tumor cells expressing PD-L1, which is immunosuppressive. Second, they act as standard checkpoint inhibitors by blocking the interaction of PD-L1 with its receptor PD-1. Third, they crosslink costimulatory targets, such as 4-1BB, on T cells, which then stimulate T cell proliferation only in the presence of tumor cells expressing PD-L1. Furthermore, the antibody constant regions may contain mutations that eliminate Fc gamma receptor-mediated functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, multispecific antibodies against PD-L1 can induce potent immune responses that are restricted to tumor tissues and protect normal tissues from the unwanted toxicity often observed with standalone costimulatory antibodies. Summary of the Invention

[0006] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and 4-1BB, wherein the antibody comprises two binding units that bind to PD-L1, each of which comprises: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 1 or 4; a CDR2 sequence comprising SEQ ID NO: 2 or 5; and a CDR3 sequence comprising SEQ ID NO: 3 or 6; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 7 or 10; a CDR2 sequence comprising SEQ ID NO: 8 or 11; and a CDR3 sequence comprising SEQ ID NO: 9 or 12. and two binding units that bind to 4-1BB, each of the two binding units comprising a single-chain Fv (scFv), which comprises: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 13 or 16; a CDR2 sequence comprising SEQ ID NO: 14 or 17; and a CDR3 sequence comprising SEQ ID NO: 15 or 18; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 19 or 22; a CDR2 sequence comprising SEQ ID NO: 20 or 23; and a CDR3 sequence comprising SEQ ID NO: 21 or 24.

[0007] In some embodiments, the two binding units that bind to PD-L1 each comprise a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 1, a CDR2 sequence comprising SEQ ID NO: 2, and a CDR3 sequence comprising SEQ ID NO: 3; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 7, a CDR2 sequence comprising SEQ ID NO: 8, and a CDR3 sequence comprising SEQ ID NO: 9. In some embodiments, the two binding units that bind to PD-L1 each comprise a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 4, a CDR2 sequence comprising SEQ ID NO: 5, and a CDR3 sequence comprising SEQ ID NO: 6; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 10, a CDR2 sequence comprising SEQ ID NO: 11, and a CDR3 sequence comprising SEQ ID NO: 12.

[0008] In some embodiments, the two binding units that bind to 4-1BB each comprise: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 13; a CDR2 sequence comprising SEQ ID NO: 14; and a CDR3 sequence comprising SEQ ID NO: 15; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 19, a CDR2 sequence comprising SEQ ID NO: 20; and a CDR3 sequence comprising SEQ ID NO: 21.

[0009] In some embodiments, the two binding units that bind to 4-1BB each comprise: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 16; a CDR2 sequence comprising SEQ ID NO: 17; and a CDR3 sequence comprising SEQ ID NO: 18; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 22, a CDR2 sequence comprising SEQ ID NO: 23; and a CDR3 sequence comprising SEQ ID NO: 24.

[0010] In some embodiments, the CDR1, CDR2, and CDR3 sequences of each binding unit are present in a human VH or human VL framework. In some embodiments, the two binding units that bind PD-L1 each comprise a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 25. In some embodiments, the two binding units that bind PD-L1 each comprise a heavy chain variable region comprising SEQ ID NO: 25. In some embodiments, the two binding units that bind PD-L1 each comprise a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the two binding units that bind PD-L1 each comprise a light chain variable region comprising SEQ ID NO: 27. In some embodiments, the two binding units that bind PD-L1 each comprise a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, the two binding units that bind PD-L1 each comprise a heavy chain variable region comprising SEQ ID NO: 26. In some embodiments, the two binding units that bind to PD-L1 each comprise a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, the two binding units that bind to PD-L1 each comprise a light chain variable region comprising SEQ ID NO: 28.

[0011] In some embodiments, two binding units that bind to 4-1BB each comprise a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 29. In some embodiments, two binding units that bind to 4-1BB each comprise a heavy chain variable region comprising SEQ ID NO: 29. In some embodiments, two binding units that bind to 4-1BB each comprise a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 31. In some embodiments, two binding units that bind to 4-1BB each comprise a light chain variable region comprising SEQ ID NO: 31. In some embodiments, two binding units that bind to 4-1BB each comprise a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 30. In some embodiments, two binding units that bind to 4-1BB each comprise a heavy chain variable region comprising SEQ ID NO: 30. In some embodiments, two binding units that bind to 4-1BB each comprise a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 32. In some embodiments, two binding units that bind to 4-1BB each comprise a light chain variable region comprising SEQ ID NO: 32.

[0012] In some embodiments, the antibody further comprises a heavy chain constant region sequence comprising a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region sequence comprises a wild-type human IgG1 constant region sequence (SEQ ID NO: 92). In some embodiments, the heavy chain constant region sequence comprises an L234A mutation, an L235A mutation, a G237A mutation, or any combination thereof. In some embodiments, the heavy chain constant region sequence comprises SEQ ID NO: 93.

[0013] In some embodiments, the antibody further comprises a light chain constant region sequence. In some embodiments, the light chain constant region sequence comprises a human kappa light chain constant region sequence (SEQ ID NO: 91). In some embodiments, the light chain constant region sequence comprises a human lambda light chain constant region sequence.

[0014] In some embodiments, in each of the binding units that bind to 4-1BB, the heavy chain variable region and the light chain variable region are linked by a linker sequence. In some embodiments, the linker sequence comprises a G4S linker sequence (SEQ ID NO: 36). In some embodiments, the G4S linker sequence (SEQ ID NO: 36) comprises SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0015] In some embodiments, each of the second binding units is connected to the C-terminus of the heavy chain constant region sequence by a linker sequence. In some embodiments, the linker sequence comprises a G4S linker sequence (SEQ ID NO: 36). In some embodiments, the G4S linker sequence (SEQ ID NO: 36) comprises SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0016] Embodiments of the invention include a bispecific antibody that binds to PD-L1 and 4-1BB, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO:43; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO:41; (c) a second light chain polypeptide comprising the sequence of SEQ ID NO:43; and (d) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:41.

[0017] Embodiments of the invention include a bispecific antibody that binds to PD-L1 and 4-1BB, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO:44; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO:42; (c) a second light chain polypeptide comprising the sequence of SEQ ID NO:44; and (d) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:42.

[0018] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and CD47, wherein the antibody comprises a first binding unit that binds to PD-L1, comprising: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 1 or 4; a CDR2 sequence comprising SEQ ID NO: 2 or 5; and a CDR3 sequence comprising SEQ ID NO: 3 or 6; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 7 or 10; a CDR2 sequence comprising SEQ ID NO: 8 or 11; and a CDR3 sequence comprising SEQ ID NO: 9 or 12. and a second binding unit that binds to CD47, the second binding unit comprising a single-chain Fv (scFv), which comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 50 or 53; a CDR2 sequence comprising SEQ ID NO: 51 or 54; and a CDR3 sequence comprising SEQ ID NO: 52 or 55; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 56 or 59; a CDR2 sequence comprising SEQ ID NO: 57 or 60; and a CDR3 sequence comprising SEQ ID NO: 58 or 61.

[0019] In some embodiments, the first binding unit that binds to PD-L1 comprises the following: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 1, a CDR2 sequence comprising SEQ ID NO: 2, and a CDR3 sequence comprising SEQ ID NO: 3; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 7, a CDR2 sequence comprising SEQ ID NO: 8, and a CDR3 sequence comprising SEQ ID NO: 9. In some embodiments, the first binding unit that binds to PD-L1 comprises the following: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 4, a CDR2 sequence comprising SEQ ID NO: 5, and a CDR3 sequence comprising SEQ ID NO: 6; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 10, a CDR2 sequence comprising SEQ ID NO: 11, and a CDR3 sequence comprising SEQ ID NO: 12.

[0020] In some embodiments, the second binding unit that binds CD47 comprises the following: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 50, a CDR2 sequence comprising SEQ ID NO: 51, and a CDR3 sequence comprising SEQ ID NO: 52; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 56, a CDR2 sequence comprising SEQ ID NO: 57, and a CDR3 sequence comprising SEQ ID NO: 58. In some embodiments, the second binding unit that binds CD47 comprises the following: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 53, a CDR2 sequence comprising SEQ ID NO: 54, and a CDR3 sequence comprising SEQ ID NO: 55; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 59, a CDR2 sequence comprising SEQ ID NO: 60, and a CDR3 sequence comprising SEQ ID NO: 61.

[0021] In some embodiments, the CDR1, CDR2, and CDR3 sequences of each binding unit are present in a human VH or human VL framework. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising a sequence having at least 95% identity to SEQ ID NO: 25. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising SEQ ID NO: 25. In some embodiments, the first binding unit that binds PD-L1 comprises a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the first binding unit that binds PD-L1 comprises a light chain variable region comprising SEQ ID NO: 27. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising SEQ ID NO: 26. In some embodiments, the first binding unit that binds to PD-L1 comprises a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, the first binding unit that binds to PD-L1 comprises a light chain variable region comprising SEQ ID NO: 28.

[0022] In some embodiments, the second binding unit that binds CD47 comprises a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 62. In some embodiments, the second binding unit that binds CD47 comprises a heavy chain variable region comprising SEQ ID NO: 62. In some embodiments, the second binding unit that binds CD47 comprises a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 64. In some embodiments, the second binding unit that binds CD47 comprises a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 63. In some embodiments, the second binding unit that binds CD47 comprises a heavy chain variable region comprising SEQ ID NO: 63. In some embodiments, the second binding unit that binds CD47 comprises a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 65. In some embodiments, the second binding unit that binds CD47 comprises a light chain variable region comprising SEQ ID NO: 65.

[0023] In some embodiments, the bispecific antibody further comprises a heavy chain constant region sequence comprising a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region sequence comprises a wild-type human IgG1 constant region sequence (SEQ ID NO: 92). In some embodiments, the heavy chain constant region sequence comprises an L234A mutation, an L235A mutation, a G237A mutation, or any combination thereof. In some embodiments, the heavy chain constant region sequence comprises SEQ ID NO: 93.

[0024] In some embodiments, the bispecific antibody further comprises a light chain constant region sequence. In some embodiments, the light chain constant region sequence comprises a human kappa light chain constant region sequence (SEQ ID NO: 91). In some embodiments, the light chain constant region sequence comprises a human lambda light chain constant region sequence.

[0025] In some embodiments, in the second binding unit that binds CD47, the heavy chain variable region and the light chain variable region are linked by a linker sequence. In some embodiments, the linker sequence comprises a G4S linker sequence (SEQ ID NO: 36). In some embodiments, the G4S linker sequence (SEQ ID NO: 36) comprises SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0026] In some embodiments, each of the second binding units is connected to the C-terminus of the heavy chain constant region sequence by a linker sequence. In some embodiments, the linker sequence comprises a G4S linker sequence (SEQ ID NO: 36). In some embodiments, the G4S linker sequence (SEQ ID NO: 36) comprises SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38. In some embodiments, the bispecific antibody further comprises a heavy chain constant region comprising one or more knobs-into-holes mutations that promote heterodimerization of two different heavy chain polypeptides.

[0027] Embodiments of the invention include a bispecific antibody that binds to PD-L1 and CD47, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 66; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 67; and (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 68.

[0028] Embodiments of the invention include a bispecific antibody that binds to PD-L1 and CD47, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 69; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 70; and (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 71.

[0029] Embodiments of the invention include antibodies that bind to PD-L1 and comprise one or more IL15 polypeptides fused to the C-terminus of a heavy chain polypeptide subunit of a bispecific antibody, comprising a first binding unit that binds to PD-L1, comprising: a heavy chain variable region comprising: a CDR1 sequence comprising SEQ ID NO: 1 or 4; a CDR2 sequence comprising SEQ ID NO: 2 or 5; and a CDR3 sequence comprising SEQ ID NO: 3 or 6; a light chain variable region comprising: a CDR1 sequence comprising SEQ ID NO: 7 or 10, a CDR2 sequence comprising SEQ ID NO: 8 or 11; and a CDR3 sequence comprising SEQ ID NO: 9 or 12; and an IL15 polypeptide comprising a sequence having at least 95% identity to any one of SEQ ID NOs: 86-90.

[0030] In some embodiments, the first binding unit that binds to PD-L1 comprises: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 1, a CDR2 sequence comprising SEQ ID NO: 2, and a CDR3 sequence comprising SEQ ID NO: 3; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 7, a CDR2 sequence comprising SEQ ID NO: 8, and a CDR3 sequence comprising SEQ ID NO: 9. In some embodiments, the first binding unit that binds to PD-L1 comprises: a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 4, a CDR2 sequence comprising SEQ ID NO: 5, and a CDR3 sequence comprising SEQ ID NO: 6; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 10, a CDR2 sequence comprising SEQ ID NO: 11, and a CDR3 sequence comprising SEQ ID NO: 12.

[0031] In some embodiments, the IL15 polypeptide comprises the sequence of any one of SEQ ID NOs: 86-90. In some embodiments, the IL15 polypeptide is linked to the antibody heavy chain polypeptide subunit by a linker sequence. In some embodiments, the linker sequence comprises the sequence of any one of SEQ ID NOs: 36, 37, 38, 49, 120, 121, 122, 123, 124, 125, 126, 127, or 128.

[0032] In some embodiments, the CDR1, CDR2, and CDR3 sequences are present in a human VH or human VL framework. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 25. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising SEQ ID NO: 25. In some embodiments, the first binding unit that binds PD-L1 comprises a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the first binding unit that binds PD-L1 comprises a light chain variable region comprising SEQ ID NO: 27. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, the first binding unit that binds PD-L1 comprises a heavy chain variable region comprising SEQ ID NO: 26. In some embodiments, the first binding unit that binds to PD-L1 comprises a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, the first binding unit that binds to PD-L1 comprises a light chain variable region comprising SEQ ID NO: 28.

[0033] In some embodiments, the antibody further comprises a heavy chain constant region sequence comprising a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region sequence comprises a wild-type human IgG1 constant region sequence (SEQ ID NO: 92). In some embodiments, the heavy chain constant region sequence comprises an L234A mutation, an L235A mutation, a G237A mutation, or any combination thereof. In some embodiments, the heavy chain constant region sequence comprises SEQ ID NO: 93.

[0034] In some embodiments, the antibody further comprises a light chain constant region sequence. In some embodiments, the light chain constant region sequence comprises a human kappa light chain constant region sequence (SEQ ID NO: 91). In some embodiments, the light chain constant region sequence comprises a human lambda light chain constant region sequence.

[0035] In some embodiments, the antibody further comprises a heavy chain constant region comprising one or more knobs-into-holes mutations that promote heterodimerization of two different heavy chain polypeptides.

[0036] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and comprise an IL15 polypeptide fused to the C-terminus of each heavy chain polypeptide subunit, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 104; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 105; (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 105; and (d) a second light chain polypeptide comprising the sequence of SEQ ID NO: 104.

[0037] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and comprise an IL15 polypeptide fused to the C-terminus of each heavy chain polypeptide subunit, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 106; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 107; (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 107; and (d) a second light chain polypeptide comprising the sequence of SEQ ID NO: 106.

[0038] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and comprise an IL15 polypeptide fused to the C-terminus of each heavy chain polypeptide subunit, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 108; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 109; (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 110; and (d) a second light chain polypeptide comprising the sequence of SEQ ID NO: 108.

[0039] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and comprise an IL15 polypeptide fused to the C-terminus of one heavy chain polypeptide subunit, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO:111; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO:112; (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:113; and (d) a second light chain polypeptide comprising the sequence of SEQ ID NO:111.

[0040] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and comprise an IL15 polypeptide fused to the C-terminus of one heavy chain polypeptide subunit, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 114; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 115; (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 116; and (d) a second light chain polypeptide comprising the sequence of SEQ ID NO: 114.

[0041] Embodiments of the invention include bispecific antibodies that bind to PD-L1 and comprise an IL15 polypeptide fused to the C-terminus of each heavy chain polypeptide subunit, comprising: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 117; (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 118; (c) a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 119; and (d) a second light chain polypeptide comprising the sequence of SEQ ID NO: 117.

[0042] Aspects of the invention include pharmaceutical compositions comprising antibodies as described herein.

[0043] Aspects of the invention relate to methods for the treatment of a disorder characterized by expression of PD-L1, comprising administering to a subject having such a disorder an antibody described herein or a pharmaceutical composition described herein.

[0044] Aspects of the invention include the use of an antibody described herein in the preparation of a medicament for the treatment of a disorder characterized by expression of PD-L1.

[0045] Aspects of the invention include an antibody described herein for use in treating a disorder characterized by expression of PD-L1. In some embodiments, the disorder is cancer.

[0046] Aspects of the invention include polynucleotides encoding the antibodies described herein, vectors comprising the polynucleotides described herein, and cells comprising the vectors claimed herein.

[0047] Aspects of the invention include methods for producing the antibodies described herein, comprising growing a cell described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell.

[0048] Aspects of the invention include methods of treatment comprising administering to an individual an effective amount of an antibody described herein, or a pharmaceutical composition described herein.

[0049] These and additional aspects are further described in the remainder of the disclosure, including the examples. [Brief explanation of the drawings]

[0050] [Figure 1] Panel A is a graph showing binding of the indicated antibody constructs to HEK293 cells expressing human PD-L1. Panel B is a graph showing binding of the indicated antibody constructs to HEK293 cells expressing human 4-1BB. Panel C is a table showing EC50 values ​​of human PD-L1 and human 4-1BB for the indicated antibody constructs. [Figure 2] Panels A and B are graphs showing the binding kinetics of antibody constructs to HIS-tagged PD-L1 and 4-1BB, respectively. Panel C is a table showing KD values ​​for binding to PD-L1 and 4-1BB. [Figure 3] Panel A is a graph showing the binding of the indicated antibody constructs to HEK293 cells expressing cynoPD-L1. Panel B is a graph showing the binding of the indicated antibody constructs to HEK293 cells expressing cyno 4-1BB. Panel C is a table showing the EC50 values ​​of cynoPD-L1 and cyno 4-1BB for the indicated antibody constructs. [Figure 4]Panel A is a graph showing the PD-1 blocking activity of the indicated antibody constructs in PD-L1-expressing HEK293 cells. Panel B is a graph showing the 4-1BBL blocking activity of the indicated antibody constructs in 4-1BB-expressing HEK293 cells. Panel C is a table showing the PD-1 and 4-1BBL IC50 values ​​for the indicated antibody constructs. [Figure 5] Panel A is a graph showing bifunctional ELISA binding of the indicated antibody constructs as a function of concentration. Panel B is a graph showing NF-kB reporter activity for the indicated antibody constructs as a function of antibody concentration. [Figure 6] Panels A and B are graphs showing IL2 release and IFNγ release, respectively, from human PBMCs stimulated with anti-CD3 antibody (OKT3) and then co-cultured with PD-L1+A431 cells, along with the indicated antibody constructs at the concentrations indicated. Panel C is a graph showing IL2 release from human PBMCs stimulated with anti-CD3 antibody (OKT3) and then cultured with or without PD-L1+A431 cells, along with the indicated antibody constructs at the concentrations indicated. [Figure 7] Panel A is a graph showing IL2 release in an SEB stimulation assay using the indicated antibody constructs at the indicated concentrations. Panel B is a graph showing CD8+ T cell proliferation in the presence of anti-CD3 antibody (OKT3) and PD-L1+ A431 cells with the indicated antibody constructs at the indicated concentrations. [Figure 8] Panel A is a graph showing tumor volume as a function of days after first dose for the MC38 mouse tumor model using treatment with the indicated antibody constructs at the indicated doses. Panel B is a graph showing tumor-infiltrating immune cells (CD8+ T cells) for each dose group harvested at the end of the study. [Figure 9] Panel A is a graph showing tumor volume as a function of days after first dose for the A431 human tumor model using treatment with the indicated antibody constructs at the indicated doses. Panel B is a graph showing tumor-infiltrating immune cells (CD8+ T cells) for each dose group harvested at the end of the study. [Figure 10] 1 is a table showing the calculated percentages of monomers, aggregates, and fragments in the HPLC-SEC profile of QL301 obtained from accelerated temperature stress testing. [Figure 11] Panel A is a graph showing bifunctional ELISA binding as a function of antibody concentration for QL301 and stock control after 7 days of incubation in human serum. Panel B is a graph showing IL2 release from PBMCs in an SEB stimulation assay as a function of antibody concentration using human serum-incubated and stock control QL301 antibody at the indicated concentrations. [Figure 12] Panel A is a schematic diagram of PD-L1-CD47 bispecific antibodies. Panel B is a graph showing ELISA binding to PD-L1 and CD47 as a function of antibody concentration. [Figure 13] Panels A-E are a series of graphs showing binding of the indicated antibody constructs to the indicated cells as a function of antibody concentration. [Figure 14] Figure 10 is a graph showing PD-1 blocking activity on stimulated A431 cells by the indicated CD47-PD-L1 bispecific antibody constructs as a function of antibody concentration. [Figure 15] Figure 10 is a graph showing the PD-1 blocking activity of the indicated CD47-PD-L1 bispecific antibody constructs on huPD-L1+ HEK293 cells as a function of antibody concentration. [Figure 16] Panel A is a graph showing SIRPα blockade on A431 cells by the indicated CD47-PD-L1 bispecific antibody constructs as a function of antibody concentration.Panel B is a graph showing SIRPα blockade on huCD47+ CHO cells by the indicated CD47-PD-L1 bispecific antibody constructs as a function of antibody concentration. [Figure 17] Panel A is a graph showing antibody-mediated phagocytosis of Raji cells using the indicated CD47-PD-L1 antibody constructs at the indicated concentrations. Panel B is a graph showing antibody-mediated phagocytosis of MM.1S cells using the indicated CD47-PD-L1 antibody constructs at the indicated concentrations. [Figure 18] Panels A and B are graphs showing the binding of the indicated CD47-PD-L1 bispecific antibodies to red blood cells at the indicated antibody concentrations for two different RBC donors. [Figure 19] 1 is an image showing hemagglutination of red blood cells induced by the indicated antibody constructs at the indicated concentrations. [Figure 20] Panels A-F are a series of graphs showing tumor volume as a function of days in an A431, hPBMC co-implanted tumor model in ICR-SCID mice. Treatment groups G1-G5 represent different antibody constructs or control (PBS). [Figure 21] Panels A-F are graphs showing efficacy endpoints from the tumor models described in FIG. [Figure 22] Panels A-F are a series of graphs showing tumor volume as a function of days in an A431, hPBMC co-implanted tumor model in NOD-SCID mice. Treatment groups G1-G5 represent different antibody constructs or control (PBS). [Figure 23] Panels A-F are schematic representations of various bispecific antibody constructs containing C-terminal IL15 fusions. [Figure 24] Panels AC are a series of graphs showing binding of the indicated PD-L1-IL15 bispecific antibody constructs to the indicated cells at the indicated antibody concentrations. [Figure 25] Panels AC are a series of graphs showing representative examples of NK92 or M07e cell proliferation in response to the indicated PD-L1-IL15 bispecific antibody constructs at the indicated concentrations. [Figure 26] Panels A and B are graphs showing the induction of pSTAT5 on MO7e cells using the indicated PD-L1-IL15 bispecific antibodies, or monoclonal anti-PD-L1 or isotype control IL15 antibodies, at the concentrations indicated. [Figure 27] Panels AC are a series of graphs showing proliferation of the indicated cell types in response to PD-L1-IL15 bispecific antibody exposure at the indicated concentrations. [Figure 28] Panels AD are a series of graphs showing proliferation of the indicated cell types in response to PD-L1-IL15 bispecific antibody exposure at the indicated concentrations. [Figure 29] Panels A-D are a series of graphs showing proliferation of the indicated cell types in response to PD-L1-IL15 bispecific antibody exposure at the indicated concentrations. Panel E is a table showing the antibodies used for staining (BioLegend catalog numbers are listed). [Figure 30] Panels A-E are a series of graphs showing cell numbers as a function of time for the indicated cell types and the indicated PD-L1-IL15 bispecific antibody constructs and dosing schedules. [Figure 31] Panels A-F are a series of graphs showing cell numbers as a function of time for the indicated cell types and the indicated PD-L1-IL15 bispecific antibody constructs and dosing schedules. [Figure 32] Panels AD are a series of graphs showing the pharmacokinetic properties of the indicated PD-L1-IL15 bispecific antibody constructs in C57BL / 6 and NSG mice. [Figure 33] Panels A-F are a series of graphs showing tumor growth inhibition of PD-L1-expressing MC38 murine colon carcinoma cells by the indicated PD-L1-IL15 antibody constructs at the indicated doses. Panel G is a graph showing tumor volume as a function of days after tumor re-challenge. [Figure 34] Panels A-G are a series of graphs showing tumor growth inhibition of A431 xenografts co-implanted with human PBMC tumor models for the indicated PD-L1-IL15 bispecific antibody constructs at the indicated doses. [Figure 35] Panels A-G are a series of graphs showing phenotypic analysis of cells harvested from tumors isolated from A431 xenografts co-implanted with human PBMC tumor models treated with the indicated PD-L1-IL15 bispecific antibody constructs at the doses indicated in Figure 34. [Figure 36]Panels A-G are a series of graphs showing phenotypic analysis of cells harvested from tumors isolated from A431 xenografts co-implanted with human PBMC tumor models treated with the indicated PD-L1-IL15 bispecific antibody constructs at the doses indicated in Figure 34. [Figure 37] Panels A-E are a series of graphs showing tumor growth inhibition of PD-L1-expressing MC38 murine colon carcinoma cells in C57BL / 6 mice by the indicated PD-L1-IL15 antibody constructs at the indicated doses. [Figure 38] Panels A-F are a series of graphs showing phenotypic analysis of cells harvested from tumors isolated from the MC38 mouse colon cancer tumor model treated with the indicated PD-L1-IL15 bispecific antibody constructs at the doses indicated in Figure 37. [Figure 39] Panels A-G are a series of graphs showing tumor growth inhibition of NCI-H1650 cells co-implanted with human PBMCs in CD17-SCID mice for the indicated PD-L1-IL15 bispecific antibodies at the indicated doses. [Figure 40] Panel A is a model of QL301, a bispecific PD-L1x4-1BB antibody with two identical binding domains that bind to PD-L1 and two identical scFvs that bind to 4-1BB. Panel B is an image of tumor cells and T cells cross-linked by the QL301 bispecific antibody. [Figure 41] Panels A and B are graphs showing the % of RBC phagocytosis mediated by bispecific PD-L1xCD47 antibodies for two different donors. [Figure 42] Figure 10 is a graph showing the binding activity of the indicated PD-L1-IL15 antibody constructs. [Figure 43] Figure 10 is a graph showing the proliferation of NK92 cells in response to the indicated PD-L1-IL15 antibody constructs. [Figure 44] Panels A and B are graphs showing AST and ALT levels observed in a repeat-dose toxicology study in rhesus monkeys using a PD-L1-4-1BB bispecific antibody construct according to some embodiments of the invention. [Figure 45] Figure 10 is a graph showing inhibition of A375 tumor growth in a NOG mouse tumor model using PD-L1-CD47 bispecific antibody constructs according to some embodiments of the invention. [Figure 46] Figure 10 is a graph showing Raji tumor growth inhibition in a NOG mouse tumor model using PD-L1-CD47 bispecific antibody constructs according to embodiments of the invention. [Figure 47] Figure 10 is a graph showing the red blood cell counts observed in a repeat dose toxicology study in cynomolgus monkeys, using a PD-L1-CD47 bispecific antibody construct according to embodiments of the invention. [Figure 48] Figure 10 is a graph showing the stimulation of cDC1 observed in an MC38 tumor model performed in C57BL / 6 mice using a mouse cross-reactive surrogate of the PD-L1-IL15-T2A construct, in accordance with embodiments of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0051] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); Biology” (FMAusubel et al., eds., 1987, and periodic updates), “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994), “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988), “Phage Display: A Laboratory Manual” (Barbas et al., 2001), Harlow, Lane and Harlow, Using Antibodies:A Laboratory Manual:Portable Protocol No.I,Cold Spring Harbor Laboratory (1998), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0052] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0053] Unless otherwise specified, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0054] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0055] All references cited throughout this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0056] I. Definition "Comprising" means that the recited element is necessary for the composition / method / kit, but other elements may be included to form the composition / method / kit, etc., within the scope of the claim.

[0057] "Consisting essentially of" means limiting the scope of the described composition or method to certain substances or steps that do not materially affect the basic and novel characteristic(s) of the invention.

[0058] "Consisting of" means that any element, step, or ingredient not specified in the claim is excluded from the composition, method, or kit.

[0059] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1 to 113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index according to Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system.

[0060] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, with the amino-terminal domains of the heavy and light chains being variable in sequence and therefore commonly referred to as variable region domains, or variable heavy (VH) or variable light (VH) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be achieved with variable sequence in the heavy chain alone, and various non-native antibody structures are known and used in the art.

[0061] A "functional" or "biologically active" antibody or binding compound is one that can exert one or more activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding compound may have the ability to specifically bind to an antigen, and the binding may then trigger or alter a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule may also be capable of blocking ligand activation of a receptor, or may function as an agonist or antagonist. The ability of an antibody or other binding compound to exert one or more activities depends on several factors, including proper folding and assembly of the polypeptide chain.

[0062] The term "antibody" as used herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), triple-chain antibodies, single-chain Fvs (scFvs), nanobodies, and the like, as well as antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0063] The term antibody may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen of a desired target or portion thereof, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass, including engineered subclasses with modified Fc portions that provide reduced or enhanced effector cell activity. The immunoglobulins may be derived from any species.

[0064] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies according to the present invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can also be produced, for example, via recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0065] The term "variable" in reference to antibodies refers to the fact that the sequences of certain portions of antibody variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy-chain and light-chain variable domains each contain four FRs, which adopt a primarily β-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0066] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity-determining regions" or "CDRs" (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy-chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the "hypervariable loops" 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy-chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework Region" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein.

[0067] While exemplary CDR designations are provided herein, those skilled in the art will appreciate that several definitions of CDRs are commonly used, including the most commonly used Kabat definition (see "Zhao et al. A germline knowledge-based computational approach for determining antibody complementarity determining regions." Mol. Immunol. 2010;47:694-700), which is based on sequence variability. The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable Regions." Nature. 1989;342:877-883).Alternative CDR definitions of interest include, but are not limited to, those described in Honegger, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol. 2001;309:657-670; Ofran et al. "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes," J Immunol. 2008;181:6230-6235; Almagro "Identification of differences in the specificity-determining residues of antibodies thatRecognize antigens of different size: implications for the rational design of antibody repertoires," J Mol Recognit. 2004;17:132-143; and Padlan et al. "Identification of specificity-determining residues in antibodies." Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0068] As used herein, the term "multispecific binding compound" refers to a binding compound comprising two or more antigen-binding sites. Multispecific binding compounds according to embodiments of the invention may be antibody-like molecules comprising, consisting essentially of, or consisting of two, three, or four polypeptide subunits, any of which may comprise one or more variable region domains having binding affinity for a target antigen (e.g., PD-L1). In some embodiments, the multispecific binding compound comprises a pair of variable region domains (e.g., a heavy chain variable region domain and a light chain variable region domain) that together form a binding unit. In some embodiments, the multispecific binding compound comprises a pair of variable region domains in a single-chain Fv (scFv) format, in which a first variable region domain and a second variable region domain are linked by a linker and together form a binding unit. The multispecific binding compounds of the invention may have any suitable combination or configuration of binding units, including, but not limited to, the specific configurations described herein.

[0069] The multispecific binding compounds described herein can belong to any immunoglobulin subclass, including IgG, IgM, IgA, IgD, and IgE subclasses. In certain embodiments, the multispecific binding compounds are of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. Modifications of the CH domain to alter effector function are further described herein.

[0070] As used herein, an "intact antibody chain" refers to an antibody chain comprising a full-length variable region and a full-length constant region (Fc). An intact, "traditional" antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, hinge, CH2, and CH3, for secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (a native-sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors. Constant region variants include variants that alter effector properties, binding to Fc receptors, and the like.

[0071] Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be provided as different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the different classes of antibodies can be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Forms of Ig include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090, Lund et al (2000) Eur. J. Biochem. 267:7246-7256, US2005 / 0048572, US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0072] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with a receptor, e.g., FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, and can be assessed using various assays known in the art. A "dead" or "silenced" Fc is an Fc that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0073] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0074] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide. A variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0075] The human IgG1 amino acid sequence is provided by UniProtKB No. P01857, which is incorporated herein by reference in its entirety. The human IgG2 amino acid sequence is provided by UniProtKB No. P01859, which is incorporated herein by reference in its entirety. The human IgG3 amino acid sequence is provided by UniProtKB No. P01860, which is incorporated herein by reference in its entirety. The human IgG4 amino acid sequence is provided by UniProtKB No. P01861, which is incorporated herein by reference in its entirety.

[0076] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563; Hezareh et al., (2001) J. Virology 75:12161; U.S. Patent No. 5,624,821, the disclosures of which are incorporated herein by reference in their entireties). In some embodiments, the variant Fc sequence may contain the following amino acid substitutions: L234A; L235A; and G237A. When these three amino acid substitutions are present in an IgG1 Fc sequence, they may be referred to as G1AAA.

[0077] Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions with human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 significantly reduce ADCC and CDC (see, e.g., Armour KL et al., 1999 Eur J Immunol. 29(8):2613-24, and Shields R L et al., 2001 J Biol Chem. 276(9):6591-604).

[0078] Other Fc variants are possible, including, but not limited to, variants in which regions capable of forming disulfide bonds are deleted or in which specific amino acid residues are removed or a methionine residue is added at the N-terminus of a native Fc. Thus, in some embodiments, one or more Fc portions of a binding compound may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, a binding compound may comprise an Fc variant.

[0079] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to confer complement binding or Fc receptor binding. For example, but not limited to, deletions may be made in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0080] The term "antibody comprising an Fc region" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, antibodies having an Fc region according to the present invention may or may not include antibodies with K447.

[0081] Aspects of the present invention include binding compounds with multispecific configurations, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein configurations for bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc. are known and used.

[0082] Various methods have been developed for producing multivalent artificial antibodies by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are connected by a polypeptide linker. One non-limiting example of such a polypeptide linker is the GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, where this sequence is repeated n times, where n is an integer ranging from 1 to about 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9 (SEQ ID NO: 133). Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 36) (n=1) and GGGGSGGGGS (SEQ ID NO: 37) (n=2). Other suitable linkers may also be used, e.g., as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety. Additional linker sequences are described elsewhere herein and may be incorporated into the antibodies of the invention in any suitable configuration.

[0083] The antibodies and multispecific binding compounds described herein may be in the form of dimers in which two heavy chains are disulfide-bonded or otherwise covalently or noncovalently bound to one another, and may optionally contain an asymmetric interface between two or more CH domains (commonly referred to as "knobs-into-holes") to facilitate proper pairing between the polypeptide chains. Knobs-into-hole antibody engineering techniques for heavy chain heterodimerization are discussed, for example, in Ridgway et al., Protein Eng. 1996 Jul;9(7):17-21, and U.S. Pat. No. 8,216,805, the disclosures of which are incorporated herein by reference in their entireties. Fc regions containing an asymmetric interface may be referred to herein by the abbreviation "KiH," which stands for knobs-into-holes. For example, embodiments of the present invention include variant Fc region sequences, such as the G1AAA sequence, which contain an asymmetric interface and are referred to herein as "G1AAA KiH."

[0084] The terms "PD-L1" and "programmed death-ligand 1" include PD-L1 proteins of any human and non-human animal species, and specifically include human PD-L1 and PD-L1 of non-human mammals.

[0085] As used herein, the term "human PD-L1" includes any variant, isoform, and species homologue of human PD-L1 (UniProt Q9NZQ7), regardless of its source or mode of preparation. Thus, "human PD-L1" includes human PD-L1 naturally expressed by cells, and PD-L1 expressed on cells transfected with the human PD-L1 gene.

[0086] The terms "anti-PD-L1 antibody", "PD-L1 antibody", "anti-PD-L1 binding compound" and "PD-L1 binding compound" are used interchangeably herein to refer to an antibody or binding compound, as defined herein, that immunospecifically binds to PD-L1, including human PD-L1, as defined herein.

[0087] The term "4-1BB" refers to the 4-1BB protein of any human and non-human animal species, and specifically includes human 4-1BB and non-human mammalian 4-1BB.

[0088] As used herein, the term "human 4-1BB" includes any variant, isoform, and species homologue of human 4-1BB (UniProt Q07011), regardless of its source or mode of preparation. Thus, "human 4-1BB" includes human 4-1BB naturally expressed by cells and 4-1BB expressed on cells transfected with the human 4-1BB gene.

[0089] The terms "anti-4-1BB antibody," "4-1BB antibody," "anti-4-1BB binding compound," and "4-1BB binding compound" are used interchangeably herein to refer to an antibody or binding compound as defined herein that immunospecifically binds to 4-1BB, including human 4-1BB as defined herein.

[0090] The terms "CD47" and "leukocyte surface antigen CD47" refer to the CD47 protein of any human and non-human animal species, and specifically include human CD47 and non-human mammalian CD47.

[0091] The term "human CD47," as used herein, includes any variant, isoform, and species homologue of human CD47 (UniProt Q08722), regardless of its source or mode of preparation. Thus, "human CD47" includes human CD47 naturally expressed by cells and CD47 expressed on cells transfected with the human CD47 gene.

[0092] The terms "anti-CD47 antibody," "CD47 antibody," "anti-CD47 binding compound," and "CD47 binding compound" are used interchangeably herein to refer to an antibody or binding compound as defined herein that immunospecifically binds to CD47, including human CD47 as defined herein.

[0093] The terms "IL15" and "interleukin-15" refer to the IL15 protein of any human and non-human animal species, and specifically include human IL15 and IL15 of non-human mammals.

[0094] The term "human IL15," as used herein, includes any variant, isoform, and species homologue of human IL15 (UniProt P40933), regardless of its source or mode of preparation. Thus, "human IL15" includes human IL15 naturally expressed by cells and IL15 expressed on cells transfected with the human IL15 gene.

[0095] As used herein to describe a multispecific antibody or multispecific binding compound, the term "IL15" refers to an antibody or binding compound that comprises a polypeptide subunit (e.g., an antibody heavy chain or an antibody light chain) to which an IL15 protein sequence is fused, as structurally shown in Figure 23, panels A-F, thereby facilitating the interaction between the fused IL15 protein and the IL15 receptor.

[0096] "Amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps to achieve the maximum sequence identity percentage, if necessary, without considering any conservative substitutions as part of the sequence identity.Alignment for determining amino acid sequence identity percentage can be achieved by various methods within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.However, for the purposes of this specification, amino acid sequence identity percentage values ​​are generated using the sequence comparison computer program ALIGN-2.

[0097] An "isolated" antibody or binding compound is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue, or preferably silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0098] Binding compounds of the invention include multispecific binding compounds. Multispecific binding compounds have two or more binding specificities. The term "multispecific" specifically includes "bispecific" and "trispecific," as well as higher orders of independent specific binding affinities, such as higher orders of polyepitopic specificity, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody" and "multispecific binding compound" are used in the broadest sense herein to encompass all antibodies and antibody-like molecules with two or more binding specificities. Multispecific anti-PD-L1 binding compounds of the invention specifically include binding compounds that immunospecifically bind to an epitope on a PD-L1 protein, such as the human PD-L1 protein, and to an epitope on a different protein, such as the 4-1BB protein or the CD47 protein.

[0099] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and will react with many different antibodies. The term specifically includes linear and conformational epitopes.

[0100] Antibody epitopes can be linear or conformational. Linear epitopes are formed by a contiguous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0101] The term "valency," as used herein, refers to a particular number of binding sites in an antibody molecule or binding compound.

[0102] A "monovalent" binding compound has one binding site. Thus, a monovalent binding compound is also monospecific.

[0103] A "multivalent" binding compound has two or more binding sites. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, bispecific binding compounds according to the invention are at least bivalent and may be trivalent, tetravalent, or otherwise multivalent. Bivalent binding compounds according to embodiments of the invention may have two binding sites for the same epitope (i.e., bivalent, monoparatopic) or may have two binding sites for two different epitopes (i.e., bivalent, biparatopic).

[0104] A wide variety of methods and protein structures are known and used to prepare bispecific monoclonal antibodies (BsMABs) and binding compounds, trispecific antibodies and binding compounds, and the like.

[0105] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term "human antibody" specifically includes antibodies and binding compounds having human heavy chain variable region sequences.

[0106] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entireties. The term "chimeric antibody" specifically includes antibodies and binding compounds having variable region sequences derived from a non-human immunoglobulin and human immunoglobulin constant region sequences.

[0107] The term "humanized antibody" as used herein refers to an antibody or binding compound that contains minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically the sequence of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and Queen et al., U.S. Patent No. 6,180,370; European Patent No. 239400; PCT Publication WO 91 / 09967; U.S. Patent No. 5,225,539; European Patent No. 592106; European Patent No. 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al. al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and US Pat. No. 5,565,332, all of which are incorporated herein by reference in their entireties.

[0108] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector stage of an immune response, as opposed to the recognition and activation stages of an immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce antibody-dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages, which express FcRs, are involved in the specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0109] "Human effector cells" are leukocytes that express a receptor, such as a T cell receptor or FcR, and perform effector function. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells can be isolated from their native source, e.g., from blood or PBMCs, as described herein.

[0110] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0111] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like.

[0112] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0113] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0114] As used interchangeably herein, "directed T cell-mediated cytotoxicity" and "redirected T cell-mediated cytotoxicity" refer to a cell-mediated reaction in which a bridging molecule (e.g., a bispecific antibody) crosslinks a surface antigen on a T cell (e.g., CD3) with an antigen on a target cell (e.g., a surface antigen on a cancer cell). Crosslinking of the T cell and the target cell promotes the killing of the target cell by the T cell through the cytotoxic activity of the T cell. Redirected T cell-mediated cytotoxicity is described, for example, in Velasquez et al., Blood 2018 131:30-38.

[0115] "Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art. Low-affinity antibodies usually bind antigens slowly and tend to dissociate easily, while high-affinity antibodies usually bind antigens more quickly and tend to remain bound to them.

[0116] As used herein, "KD" or "KD value" refers to the dissociation constant determined by BioLayer interferometry using an Octet Red96 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, the anti-mouse Fc sensor is loaded with mouse-Fc fusion antigen, and then immersed in an antibody-containing well to measure the concentration-dependent association rate (k). The antibody dissociation rate (koff) is measured in the final step, and the sensor is immersed in a well containing only buffer. KD is the ratio of koff / koff. (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009.)

[0117] The terms "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the onset of the disease in a subject who may be susceptible to, but has not yet been diagnosed with, the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of disease or injury. Treatment of ongoing diseases, where the treatment stabilizes or alleviates undesirable clinical symptoms in the patient, is of particular interest. Such treatment is desirably administered before complete loss of function in affected tissues. The subject therapeutic agent may also be administered during, and in some cases after, the symptomatic period of the disease.

[0118] By "therapeutically effective amount" is intended the amount of active agent required to provide a therapeutic effect to a subject, e.g., an amount that induces, ameliorates, or causes an improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or improves resistance to a disorder.

[0119] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer, including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung, and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or cancer of the stomach, including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer. Cancers include, but are not limited to, retinoblastoma, thecoma, allenoblastoma, hepatoma, hematologic malignancies, such as non-Hodgkin's lymphoma (NHL), multiple myeloma, and acute hematologic malignancies, endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, liver cancer, anal cancer, penile cancer, nasopharyngeal cancer, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, schwannoma, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, and urinary tract cancer.

[0120] The term "metastatic cancer" refers to a cancerous condition in which cancer cells of tissue origin spread from the original site by blood vessels or lymph nodes to one or more other sites in the body, forming one or more secondary tumors in one or more organs other than the tissue of origin. A notable example is metastatic breast cancer.

[0121] The term "characterized by PD-L1 expression" refers broadly to any disease or disorder in which PD-L1 expression is associated with or contributes to one or more pathological processes characteristic of the disease or disorder. Specifically, diseases or disorders characterized by PD-L1 expression include, but are not limited to, cancers in which tumor cells express PD-L1 and / or tumor-associated stroma exhibits PD-L1 expression, and / or cancers in which PD-L1 is expressed on immune cells. Such disorders include, but are not limited to, invasive breast cancer, colon adenocarcinoma, lymphoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, rectal adenocarcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma and adenocarcinoma, bile duct carcinoma, glioblastoma multiforme, hepatocellular carcinoma, mesothelioma, Merkel cell carcinoma, renal cell carcinoma, sarcoma (e.g., undifferentiated sarcoma), cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, uterine carcinosarcoma, osteosarcoma, glioblastoma, melanoma, ovarian cancer, gastric cancer, and colorectal cancer.

[0122] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0123] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0124] The terms "treat," "treatment," and "treating," as used herein, refer to both therapeutic treatment and prophylactic, preventative measures, the purpose of which is to prevent or slow down (alleviate) the targeted physiological condition or disorder. Subjects in need of treatment include those already with the particular condition or disorder as well as those prone to having the disorder or those in whom the disorder is to be prevented.

[0125] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, and the like. The subject may be a human, but also includes other mammals, particularly mammals useful as laboratory models of human disease, e.g., mice, rats, and the like.

[0126] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be competently administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0127] A "sterile" formulation is one that is sterile or free or essentially free of all viable microorganisms and their spores. A "frozen" formulation is one that has a temperature below 0°C.

[0128] A "stable" formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones. A. Adv. Drug Delivery Rev. 10:29-90) (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection), cation exchange chromatography, by assessing charge heterogeneity using image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis, amino- or carboxy-terminal sequence analysis, mass spectrometry, SDS-PAGE analysis comparing reduced and intact antibody, peptide mapping (e.g., trypsin or LYS-C) analysis, assessment of antibody biological activity or antigen-binding function, etc. Instability can include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, differential glycosylation, etc.

[0129] II. Detailed Description PD-L1x 4-1BB bispecific antibody Aspects of the invention include multispecific binding compounds, e.g., bispecific antibodies, that bind to PD-L1 and 4-1BB. Multispecific binding compounds may comprise a variety of configurations, and each binding unit may comprise a set of CDR sequences. PD-L1 heavy chain CDR sequences include SEQ ID NOs: 1-6. PD-L1 light chain CDR sequences include SEQ ID NOs: 7-12. Anti-4-1BB heavy chain CDR sequences include SEQ ID NOs: 13-18, and anti-4-1BB light chain CDR sequences include SEQ ID NOs: 19-24. In some embodiments, the multispecific binding compound comprises a CDR sequence with no more than two amino acid substitutions in any one of SEQ ID NOs: 1-24.

[0130] Multispecific binding compounds according to embodiments of the invention may comprise any suitable combination of heavy and light chain variable region sequences, as provided herein. Anti-PD-L1 heavy chain variable region sequences include SEQ ID NOs: 25 and 26. Anti-PD-L1 light chain variable region sequences include SEQ ID NOs: 27-28. Anti-4-1BB heavy chain variable region sequences include SEQ ID NOs: 29, 30, 45, and 46. Anti-4-1BB light chain variable region sequences include SEQ ID NOs: 31, 32, 47, and 48. In some embodiments, the multispecific binding compound comprises a variable region sequence having at least about 80% identity, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity, to the variable region sequence of any one of SEQ ID NOs: 25-32 and 45-48.

[0131] Multispecific binding compounds according to embodiments of the present invention may comprise one or more anti-4-1BB scFv sequences, as enumerated herein. Anti-4-1BB scFv sequences include SEQ ID NOs: 129-132. In some embodiments, a multispecific binding compound comprises an scFv sequence having at least about 80% identity, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity, to any one of the scFv sequences of SEQ ID NOs: 129-132. In some embodiments, the scFv sequence is linked to a polypeptide subunit (e.g., a heavy or light chain polypeptide subunit) by a linker sequence. Non-limiting examples of linker sequences include SEQ ID NOs: 36, 37, 38, 49, and 120-128.

[0132] The multispecific binding compounds described herein offer many advantages that contribute to their usefulness as clinical therapeutic agent(s): they contain members with a variety of binding unit configurations that allow for the selection of specific molecules that exhibit therapeutic benefit.

[0133] Suitable binding compounds can be selected from the binding compounds provided herein for development and therapeutic or other uses, including, but not limited to, use as bispecific binding compounds, for example, as shown in panel A of Figure 40.

[0134] In a preferred embodiment, the bispecific binding compound binds to PD-L1 and 4-1BB, and comprises a first light chain polypeptide comprising the sequence of SEQ ID NO: 43, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 41, a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 41, and a second light chain polypeptide comprising the sequence of SEQ ID NO: 43. This bispecific antibody is designated QL301 (with signal sequence).

[0135] In a preferred embodiment, the bispecific binding compound binds to PD-L1 and 4-1BB, and comprises a first light chain polypeptide comprising the sequence of SEQ ID NO: 44, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 42, a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 42, and a second light chain polypeptide comprising the sequence of SEQ ID NO: 44. This bispecific antibody is designated QL301 (without signal sequence).

[0136] Determining affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. The multispecific binding compounds described herein have affinity of about 10 -6 ~about 10 -11 , for example and without limitation, about 10 -6 ~about 10 -10 ;about 10 -6 ~about 10 -9 ;about 10 -6 ~about 10 -8 ;about 10 -8 ~about 10 -11 ;about 10 -8 ~about 10 -10 ;about 10 -8 ~about 10 -9 ;about 10 -9 ~about 10 -11 ;~about 10 -9 ~about 10 -10 or any value within these ranges. The affinity selection may be confirmed by biological evaluation to modulate the biological activity of PD-L1 or 4-1BB, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0137] Various formats of multispecific binding compounds are within the scope of the present invention, including, but not limited to, the four-chain polypeptides described herein. Multispecific binding compounds herein specifically include bispecific binding compounds that have binding affinity for PD-L1 and 4-1BB (e.g., anti-PD-L1 x anti-4-1BB binding compounds). Such bispecific binding compounds induce potent T cell-mediated killing of tumor cells, as shown in panel B of Figure 40.

[0138] PD-L1xCD47 bispecific antibody Aspects of the invention include multispecific binding compounds, e.g., bispecific antibodies, that bind to PD-L1 and CD47. Multispecific binding compounds may comprise a variety of configurations, and each binding unit may comprise a set of CDR sequences. PD-L1 heavy chain CDR sequences include SEQ ID NOs: 1-6. PD-L1 light chain CDR sequences include SEQ ID NOs: 7-12. Anti-CD47 heavy chain CDR sequences include SEQ ID NOs: 50-55, and anti-CD47 light chain CDR sequences include SEQ ID NOs: 56-61. In some embodiments, multispecific binding compounds comprise CDR sequences with no more than two amino acid substitutions in any one of SEQ ID NOs: 1-12 or 50-61.

[0139] Multispecific binding compounds according to embodiments of the invention may comprise any suitable combination of heavy and light chain variable region sequences, as provided herein. Anti-PD-L1 heavy chain variable region sequences include SEQ ID NOs: 25 and 26. Anti-PD-L1 light chain variable region sequences include SEQ ID NOs: 27-28. Anti-CD47 heavy chain variable region sequences include SEQ ID NOs: 62-63. Anti-CD47 light chain variable region sequences include SEQ ID NOs: 64-65. In some embodiments, the multispecific binding compounds comprise a variable region sequence having at least about 80% identity, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity, to the variable region sequence of any one of SEQ ID NOs: 25-28 and 62-65.

[0140] Multispecific binding compounds according to embodiments of the invention may comprise one or more anti-CD47 scFv sequences, as enumerated herein. Anti-CD47 scFv sequences include SEQ ID NOs: 72-75. In some embodiments, a multispecific binding compound comprises an scFv sequence having at least about 80% identity, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity, to any one of the scFv sequences of SEQ ID NOs: 72-75. In some embodiments, the scFv sequence is linked to a polypeptide subunit (e.g., a heavy or light chain polypeptide subunit) by a linker sequence. Non-limiting examples of linker sequences include SEQ ID NOs: 36, 37, 38, 49, and 120-128.

[0141] The multispecific binding compounds described herein offer many advantages that contribute to their usefulness as clinical therapeutic agent(s): Multispecific binding compounds include members with different binding unit configurations that allow for the selection of specific molecules that exhibit therapeutic benefit.

[0142] Suitable binding compounds can be selected from the binding compounds provided herein for development and therapeutic or other uses, including, but not limited to, use as bispecific binding compounds, for example, as shown in panel A of Figure 12.

[0143] Embodiments of the present invention include multispecific binding compounds that comprise a knobs-into-hole (KiH) interface between their heavy chain subunits to promote heterodimerization of the desired components of the multispecific compound (e.g., a first heavy chain polypeptide subunit comprising an anti-PD-L1 binding domain, and a second heavy chain polypeptide subunit comprising an anti-CD47 binding domain (e.g., an anti-CD47 scFv)).

[0144] In a preferred embodiment, the bispecific binding compound binds to PD-L1 and CD47 and comprises a first light chain polypeptide comprising the sequence of SEQ ID NO: 66, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 67, and a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 68. This molecule is referred to as huD39.5.2.3-huG4a_hole_RF_huE15.1_scFvds-huG4a_hingeFc_knob_KiHss.

[0145] In a preferred embodiment, the bispecific binding compound binds to PD-L1 and CD47 and comprises a first light chain polypeptide comprising the sequence of SEQ ID NO: 69, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 70, and a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 71. This molecule is referred to as huD39.5.2.3-huG4a_hole_RF_huE24.6_scFvds-huG4a_hingeFc_knob_KiHss.

[0146] Determining affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. The multispecific binding compounds described herein have affinity of about 10 -6 ~about 10 -11 For example, but not limited to, about 10 -6 ~about 10 -10 ;about 10 -6 ~about 10 -9 ;about 10 -6 ~about 10 -8 ;about 10 -8 ~about 10 -11 ;about 10 -8 ~about 10 -10 ;about 10 -8 ~about 10 -9 ;about 10 -9 ~about 10 -11 ;about 10 -9 ~about 10 -10or may have affinity for PD-L1 or CD47 with a Kd of any value within these ranges. Affinity selection may be confirmed by biological evaluation to modulate the biological activity of PD-L1 or CD47, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0147] Various formats of multispecific binding compounds are within the scope of the present invention, including, but not limited to, the three-chain or four-chain polypeptides described herein. Multispecific binding compounds herein specifically include bispecific binding compounds that have binding affinity for PD-L1 and CD47 (e.g., anti-PD-L1 x anti-CD47 binding compounds). Such bispecific binding compounds induce potent T cell-mediated killing of tumor cells.

[0148] PD-L1xIL15 binding compound Aspects of the invention include multispecific binding compounds, e.g., bispecific antibodies, that contain an IL15 region that binds to PD-L1 and promotes interaction with the IL15 receptor. Multispecific binding compounds may comprise a variety of configurations, and each PD-L1 binding unit may comprise a set of CDR sequences. PD-L1 heavy chain CDR sequences include SEQ ID NOs: 1-6. PD-L1 light chain CDR sequences include SEQ ID NOs: 7-12. In some embodiments, multispecific binding compounds comprise CDR sequences with no more than two amino acid substitutions in any one of SEQ ID NOs: 1-12.

[0149] Multispecific binding compounds according to embodiments of the invention may comprise any suitable combination of heavy and light chain variable region sequences, as provided herein. Anti-PD-L1 heavy chain variable region sequences include SEQ ID NOs: 25 and 26. Anti-PD-L1 light chain variable region sequences include SEQ ID NOs: 27-28. In some embodiments, the multispecific binding compounds comprise a variable region sequence having at least about 80% identity, for example, about 85%, about 90%, about 95%, about 99%, or about 99.9% identity, to the variable region sequence of any one of SEQ ID NOs: 25-28.

[0150] Multispecific binding compounds according to embodiments of the invention may comprise one or more anti-IL15 sequences, as enumerated herein. IL15 sequences include SEQ ID NOs: 86-90. In some embodiments, the multispecific binding compound comprises an IL15 sequence having at least about 80% identity, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity, to any one of the IL15 sequences of SEQ ID NOs: 86-90. In some embodiments, the IL15 sequence is linked to a polypeptide subunit (e.g., a heavy or light chain polypeptide subunit) by a linker sequence. Non-limiting examples of linker sequences include SEQ ID NOs: 36, 37, 38, 49, and 120-128.

[0151] The multispecific binding compounds described herein offer many advantages that contribute to their usefulness as clinical therapeutic agent(s): Multispecific binding compounds contain members with a variety of binding unit configurations, allowing for the selection of specific molecules that exhibit therapeutic benefit.

[0152] Suitable binding compounds can be selected from the binding compounds provided herein for development and therapeutic or other uses, including, but not limited to, use as bispecific binding compounds, for example, as shown in panels A-F of Figure 23.

[0153] Embodiments of the present invention include multispecific binding compounds that comprise a knobs-into-hole (KiH) interface between their heavy chain subunits to promote heterodimerization of the desired components of the multispecific compound (e.g., a first heavy chain polypeptide subunit comprising an anti-PD-L1 binding domain and a first IL15 protein, and a second heavy chain polypeptide subunit comprising an anti-PD-L1 binding domain and a second IL15 protein).

[0154] In one preferred embodiment, the bispecific binding compound binds to PD-L1 and comprises two IL15 proteins (one on each heavy chain polypeptide subunit), comprising a first light chain polypeptide comprising the sequence of SEQ ID NO: 104, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 105, a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 105, and a second light chain polypeptide comprising the sequence of SEQ ID NO: 104. This molecule is designated D39.5.2.3-G1AAA-IL15RaSu-IL15-T2A and is shown schematically in Panel A of Figure 23.

[0155] In one preferred embodiment, the bispecific binding compound binds to PD-L1 and comprises two IL15 proteins (one on each heavy chain polypeptide subunit), comprising a first light chain polypeptide comprising the sequence of SEQ ID NO: 106, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 107, a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 107, and a second light chain polypeptide comprising the sequence of SEQ ID NO: 106. This molecule is designated D39.5.2.3-G1AAA-IL15-IL15RaSu-T2B and is shown schematically in Figure 23 panel D.

[0156] In one preferred embodiment, the bispecific binding compound binds to PD-L1 and comprises two IL15 proteins (one on each heavy chain polypeptide subunit), comprising first and second light chain polypeptides comprising the sequence of SEQ ID NO: 108, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 109, and a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 110. The molecule is a four-chain molecule and contains a KiH interface between the heavy chain polypeptides to promote heterodimerization. This molecule is designated D39.5.2.3-G1AAA.KiH-IL15+IL15RaSu-T3 and is shown schematically in Figure 23, panel F.

[0157] In one preferred embodiment, the bispecific binding compound binds to PD-L1 and comprises one IL15 protein (on one heavy chain polypeptide subunit) comprising first and second light chain polypeptides comprising the sequence of SEQ ID NO: 111, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 112, and a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 113. The molecule is a four-chain molecule and contains a KiH interface between the heavy chain polypeptides to promote heterodimerization. This molecule is referred to as D39.5.2.3-G1AAA-IL15RaSu-IL15-T2A-mono and is shown schematically in Figure 23, panel B.

[0158] In one preferred embodiment, the bispecific binding compound binds to PD-L1 and comprises one IL15 protein (on one heavy chain polypeptide subunit) comprising first and second light chain polypeptides comprising the sequence of SEQ ID NO: 114, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 115, and a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 116. The molecule is a four-chain molecule and contains a KiH interface between the heavy chain polypeptides to promote heterodimerization. This molecule is referred to as D39.5.2.3-G1AAA-IL15-IL15RaSu-T2B-mono and is shown schematically in Figure 23, panel E.

[0159] In one preferred embodiment, the bispecific binding compound binds to PD-L1 and comprises two IL15 proteins (one on each heavy chain polypeptide subunit), comprising a first light chain polypeptide comprising the sequence of SEQ ID NO: 117, a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 118, and a second heavy chain polypeptide comprising the sequence of SEQ ID NO: 119, and a second light chain polypeptide comprising the sequence of SEQ ID NO: 117. The molecule is a four-chain molecule and contains a KiH interface between the heavy chain polypeptides to promote heterodimerization. This molecule is referred to as D39.5.2.3-G1AAA-IL15RaSu-IL15-T2A-Mask and is shown schematically in Figure 23, panel C.

[0160] Determining affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. The multispecific binding compounds described herein have affinity of about 10 -6 ~about 10 -11 For example, but not limited to, about 10 -6 ~about 10 -10 ;about 10 -6 ~about 10 -9 ;about 10 -6 ~about 10 -8 ;about 10 -8 ~about 10 -11 ;about 10 -8 ~about 10 -10 ;about 10 -8 ~about 10 -9 ;about 10 -9 ~about 10 -11 ;about 10 -9 ~about 10 -10 or may have affinity for PD-L1 with a Kd of any value within these ranges. Affinity selection may be confirmed by biological evaluation to modulate the biological activity of PD-L1 or IL15, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0161] Various formats of multispecific binding compounds are within the scope of the present invention, including, but not limited to, the three-chain or four-chain polypeptides described herein. Multispecific binding compounds herein particularly include bispecific binding compounds (e.g., anti-PD-L1 x IL15 binding compounds) that contain one or more IL15 proteins that have binding affinity for PD-L1 and promote interaction with the IL15 receptor. Such bispecific binding compounds induce potent T cell-mediated killing of tumor cells.

[0162] The following table provides various sequences used in assembling the binding compounds described herein. [Table 1] [Table 2] Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16-1 Table 16-2 Table 17 Table 18 Table 19-1 [Table 19-2] [Table 20] [Table 21] [Table 22-1] [Table 22-2] [Table 22-3] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6] [Table 23-7] [Table 23-8]

[0163] Preparation of binding compounds The multispecific binding compounds of the present invention can be prepared by methods known in the art. For example, the binding compounds and antigen-binding fragments thereof can also be produced by recombinant DNA technology by expression of encoding nucleic acids in suitable eukaryotic or prokaryotic hosts, including, for example, mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0164] Pharmaceutical Compositions, Methods of Use and Treatment Another aspect of the present invention is to provide pharmaceutical compositions comprising one or more multispecific binding compounds of the present invention in admixture with a suitable pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier is exemplified by, but not limited to, an adjuvant, a solid carrier, water, a buffer, or other carrier used in the art to carry therapeutic ingredients, or a combination thereof.

[0165] In one embodiment, the pharmaceutical composition comprises a multispecific binding compound that binds to PD-L1 and 4-1BB. In one embodiment, the pharmaceutical composition comprises a multispecific binding compound that binds to PD-L1 and CD47. In one embodiment, the pharmaceutical composition comprises a multispecific binding compound that binds to PD-L1 and comprises one or more IL15 proteins.

[0166] Pharmaceutical compositions of the binding compounds used in accordance with the present invention are prepared for storage, for example, in the form of a lyophilized formulation or aqueous solution, by mixing the protein having the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, 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, serum albumin, These include proteins such as 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 non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0167] Pharmaceutical compositions for oral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., single-administered doses). Formulations depend on the selected route of administration. The binding compounds herein can be administered by intravenous injection or infusion, or subcutaneously. For injection administration, the binding compounds herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution can contain carriers, excipients, or stabilizers as described above. Alternatively, the binding compounds can be in lyophilized form for constitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

[0168] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations may be used for the binding compounds of the present invention. Subcutaneous antibody formulations are described, for example, in U.S. Patent Application Publication Nos. 20160355591 and 20160166689.

[0169] How to use The multispecific binding compounds and pharmaceutical compositions described herein can be used to treat diseases and conditions characterized by expression of PD-L1, including, but not limited to, the conditions and diseases described herein above.

[0170] In one aspect, the multispecific binding compounds and pharmaceutical compositions herein may be used to treat cancers characterized by expression of PD-L1. As used herein, a cancer "characterized by expression of PD-L1" includes, but is not limited to, a cancer in which one or more tumor cells express PD-L1, and / or in which tumor-associated stroma exhibits PD-L1 expression, and / or in which immune cells exhibit PD-L1 expression. Such disorders include, but are not limited to, invasive breast cancer, colon adenocarcinoma, lymphoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, rectal adenocarcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma and adenocarcinoma, bile duct carcinoma, glioblastoma multiforme, hepatocellular carcinoma, mesothelioma, Merkel cell carcinoma, renal cell carcinoma, sarcoma (e.g., undifferentiated sarcoma), cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumor, uterine carcinosarcoma, osteosarcoma, glioblastoma, melanoma, ovarian cancer, gastric cancer, and colorectal cancer.

[0171] The effective amount of the composition of the present invention for treating a disease will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, such as companion animals such as dogs, cats, and horses, and laboratory mammals such as rabbits, mice, and rats, can also be treated. Treatment doses can be titrated to optimize safety and efficacy.

[0172] Dosage levels can be easily determined by those skilled in the art and may be varied as needed, for example, to accommodate changes in the subject's response to treatment. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. Generally, a unit dosage form contains about 1 mg to about 500 mg of active ingredient.

[0173] In some embodiments, the therapeutic dose of the agent can range from about 0.0001 to 100 mg / kg of host body weight, more typically 0.01 to 5 mg / kg. For example, the dose can be 1 mg / kg or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. Therapeutic agents of the present invention are typically administered multiple times. The interval between single doses can be weekly, monthly, or yearly. Intervals can also be irregular, as determined by measuring blood levels of the therapeutic agent in the patient. Alternatively, therapeutic agents of the present invention can be administered as sustained-release formulations, requiring less frequent administration. The dose and frequency will vary depending on the half-life of the polypeptide in the patient.

[0174] Generally, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolving or suspending in liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, either directly or after reconstitution of a solid (e.g., lyophilized) composition. The preparations may also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides, or copolymers, to enhance adjuvant effect, as discussed above. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention may also be administered in the form of depot injections or implant preparations, which can be formulated in a manner that allows sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0175] The toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0176] Compositions for administration will generally contain an antibody or other agent (e.g., another abrasive) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions (pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.). The concentration of the active agent in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0177] Also within the scope of the present invention are kits containing the active agents of the present invention and their formulations, as well as instructions for use. The kits may further include at least one additional reagent, such as a chemotherapeutic agent. The kits typically include a label indicating the intended use of the contents of the kit. As used herein, the term "label" includes any writing or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0178] Now that the present invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit or scope of the invention. [Example]

[0179] Example 1: Binding of QL301 to HEK293 cells expressing PD-L1 or 4-1BB HEK293 cells expressing PD-L1 or 4-1BB were plated in a 96-well V-bottom plate at 1 × 10 5 Cells were seeded at a density of 1000 μg / ml. Serially diluted antibodies were added to the cells and incubated on ice for 30 minutes. After washing twice with FACS buffer, AF647-conjugated anti-human Fc secondary antibody was added and incubated on ice for 20 minutes. After washing twice with FACS buffer, cells were resuspended in FACS buffer containing 7AAD viability dye and analyzed by flow cytometer. The results are shown in Figure 1, panels A-C.

[0180] Example 2: Binding kinetics Binding kinetics were measured using an OctetRED96 system. Antibodies were loaded onto a ForteBio anti-human Fc capture (AHC) sensor, followed by binding of his-tagged recombinant PD-L1 or 4-1BB proteins. The results are shown in Figure 2, panels A-C.

[0181] Example 3: Binding of QL301 to HEK293 cells expressing PD-L1 or cyno 4-1BB HEK293 cells expressing cynomolgus monkey PD-L1 or 4-1BB were plated in a 96-well V-bottom plate at 1 × 10 5The cells were plated at a density of 1000 μg / ml. Serially diluted antibodies were added to the cells and incubated on ice for 30 minutes. After washing twice with FACS buffer, AF647-conjugated anti-human Fc secondary antibody was added and incubated on ice for 20 minutes. After washing twice with FACS buffer, the cells were resuspended in FACS buffer containing 7AAD viability dye and analyzed by flow cytometer. The results are shown in Figure 3, panels A-C.

[0182] Example 4: QL301 competition assay 1 x 10 HEK293 cells expressing PD-L1 or 4-1BB were plated in a 96-well V-bottom plate. 5 The cells were plated at a density of 1000 x g. Serially diluted antibodies were added to the cells and incubated on ice for 15 minutes. His-tagged recombinant PD-L1 or 4-1BB protein was added to each plate and incubated on ice for an additional 15 minutes. After washing twice with FACS buffer, APC-labeled anti-His-tagged secondary antibody was added and incubated on ice for 20 minutes. After washing twice with FACS buffer, the cells were resuspended in FACS buffer containing 7AAD viability dye and analyzed on a flow cytometer. The results are shown in Figure 4, panels A-C.

[0183] Example 5: QL301 bifunctional ELISA and NF-kB reporter assay Recombinant His-tagged 4-1BB protein was coated onto a 96-well plate overnight at room temperature with shaking. After washing the plate with PBS containing 0.05% Tween-20, the plate was blocked with 2% BSA for 60 minutes. Antibodies were added and incubated at room temperature for 60 minutes with shaking. After washing, biotinylated recombinant PD-L1 protein was added to the plate and incubated at room temperature for 60 minutes. The plate was washed, and HRP (horseradish peroxidase)-conjugated streptavidin was added and incubated at room temperature for 30 minutes. After washing, TMB (3,3',5,5'-tetramethylbenzidine) substrate was added and incubated for 5-10 minutes to develop color. The reaction was then stopped by adding 0.16M sulfuric acid. The absorbance was read using a plate reader. For reporter assays, HEK293 cells expressing 4-1BB, which also contains a Renilla luciferase reporter element under NF-kB transcriptional control, were plated at 5 x 10 cells per well in a 96-well plate. 4 The cells were seeded with PD-L1-expressing HEK293 cells. Parental HEK293 cells or PD-L1-expressing HEK293 cells were added at equal cell numbers per well. Serially diluted antibodies were then added and incubated at 37°C with 5% CO2 for 24 hours. The supernatants were then collected and transferred to a 96-well white-walled plate, and QuantiLuc reagent (Invivogen) was added. Luminescence was immediately read using a plate reader. The results are shown in Figure 5, panels A-B.

[0184] Example 6: Cytokine Release Human PBMCs were stimulated with anti-CD3 (OKT3) and then incubated with QL301, PD-L1, or 4-1BB monoclonal antibodies, or their combinations, along with PD-L1+A431 cells. QL301 induced the release of IL2 and IFNγ, whereas anti-PD-L1 or 4-1BB alone, or the combination of the two, did not. In the absence of A431 cells, IL2 induction was significantly less. The results are shown in Figure 6, panels A–C. Each dot represents an individual donor, and values ​​are fold increases relative to the control antibody.

[0185] Example 7: Cytokine release in SEB stimulation assay IL2 release was also observed in SEB stimulation assays in the presence of QL301, but not with PD-L1 or 4-1BB monoclonals, or the combination of the two. QL301 induced CD8+ T cell proliferation in the presence of anti-CD3 (OKT3) and PD-L1+ A431 cells, but not with PD-L1 or 4-1BB monoclonals, or the combination of the two. The results are shown in Figure 7, panels A-B.

[0186] Example 8: MC38 tumor model MC38 mouse cancer cells expressing human PD-L1 were implanted into the flanks of human PD-L1 and 4-1BB double knock-in C57BL / 6 mice. The average tumor volume was approximately 100 mm. 3 After reaching a median age of 10 years, QL301, PD-L1 monoclonal, or saline was administered intraperitoneally twice weekly. QL301 at 10 mg / kg was significantly more effective than the equimolar dose of PD-L1 monoclonal antibody at 8 mg / kg (p<0.0001, n=6). Analysis of tumor-infiltrating immune cells at the end of the study showed that tumors in animals treated with QL301 contained more CD8+ T cells than those treated with saline or PD-L1 monoclonal (p<0.01). The results are shown in panels A and B of FIG.

[0187] Example 9: A431 tumor model In the second model, A431 human cancer cells were co-injected with human PBMCs into the flanks of CB17-SCID mice. Consistent with the results from the MC38-hPD-L1 model, QL301 was more effective in inhibiting tumor growth and induced a higher percentage of intratumoral CD8+ T cells than the PD-L1 monoclonal antibody (n=8). The results are shown in Figure 9, Panels A-B.

[0188] Example 10: Accelerated Temperature Stress Test An accelerated temperature stress test at 42°C over 28 days resulted in minimal changes in the HPLC-SEC profile of QL301 (overlaid chromatograms from five time points). The calculated percentages of monomer, aggregates, and fragments remained within 1% of the initial product composition. The results are shown in Figure 10.

[0189] Example 11: Incubation of QL301 in human serum QL301 was incubated in human serum for 7 days and tested for its ability to bind and stimulate IL2 release from PBMCs in an SEB stimulation assay. No significant changes were observed between the 4°C stock control and the serum-incubated molecule. The results are shown in Figure 11, panels A-B.

[0190] Example 12: ELISA binding to PD-L1 and CD47 ELISA binding to PD-L1 and CD47 was assessed. Immulon HBX plates were coated with 2 μg / mL hPDL1-FC (R&D Systems) overnight at 4°C. Plates were then washed three times with PBST and blocked with 4% NFDM / PBS for 1 hour at room temperature. The block was removed, and antibodies diluted in 4% NFDM / PBS were added and incubated for 1 hour at room temperature. Plates were then washed three times with PBST. 1 μg / mL huCD47-C33S_his was added to each well and incubated for 1 hour at room temperature, followed by three washes with PBST. Anti-His-HRP (AbCam, 1:20,000) was added to each well and incubated for 45 minutes at room temperature. After six washes with PBST, the assay was developed with TBM followed by 2N sulfuric acid. The results are shown in Figure 12, panel B. Figure 12, panel A, is a schematic diagram of the PD-L1 x CD47 bispecific antibody.

[0191] Example 13: Binding of PD-L1 x CD47 bispecific antibodies to HEK293 cells Cells were harvested and washed once with FACS buffer. 1 x 10 cells per well were collected. 5Cells were distributed into a 96-well V-bottom plate. Serial dilutions of test antibodies were added and incubated on ice for 20 minutes. The cells were washed twice with 200 μL of FACS buffer. The cells were then incubated with 50 μL of the secondary antibody, AF647 F(ab'). 2 The cells were resuspended in goat anti-human IgG, Fc specific, 1:500 dilution (Jackson ImmunoResearch, catalog number 109-606-098) and incubated on ice for 15 minutes. Finally, the cells were washed twice with 200 μL of FACS buffer and resuspended in 100 μL of FACS buffer containing 7-AAD. The results are shown in panels A-E of Figure 13.

[0192] Example 14: PD-1 Fc-biotin blocking Cells were harvested and washed once with FACS buffer. 1.2 × 10 cells per well were collected. 5 Cells were distributed into a 96-well V-bottom plate. 0.5 μg / mL PD-1-biotin (final concentration) was added to the cells and incubated on ice for 5 minutes. Serial dilutions of test antibodies were then added and incubated on ice for 20 minutes. The cells were washed twice with 200 μL of FACS buffer. The cells were then diluted with 50 μL of secondary antibody, streptavidin-APC (R&D, Cat. No. F0050), at 10 μL / 10 6 The cells were resuspended in 7-AAD and incubated on ice for 15 minutes. Finally, the cells were washed twice with 200 μL of FACS buffer and resuspended in 120 μL of FACS buffer containing 7-AAD. The results are shown in Figures 14 and 15.

[0193] Example 15: SIRPα Fc-biotin blocking Cells were harvested and washed once with FACS buffer. 1.2 × 10 cells per well were cultured. 5Cells were distributed into a 96-well V-bottom plate. 1.25 μg / mL SIRPα-biotin (final concentration) was added to the cells and incubated on ice for 5 minutes. Serial dilutions of test antibodies were then added and incubated on ice for 20 minutes. The cells were washed twice with 200 μL of FACS buffer. The cells were then soaked in 50 μL of secondary antibody, streptavidin-APC (R&D, Cat. No. F0050), at 10 μL / 10 6 The cells were resuspended in 7-AAD and incubated on ice for 15 minutes. Finally, the cells were washed twice with 200 μL of FACS buffer and resuspended in 120 μL of FACS buffer containing 7-AAD. The results are shown in panels A and B of Figure 16.

[0194] Example 16: PD-L1 / CD47-mediated phagocytosis of Raji and MM.1S cells Recombinant human M-CSF (Miltenyi Biotec, catalog no. 130-096-492) and recombinant human IL-10 (Miltenyi Biotec, catalog no. 130-098-448)-derived macrophages were generated from freshly isolated human peripheral blood mononuclear cells (PBMCs). After removal of nonadherent cells on day 0, Rh M-CSF (20 ng / ml) was added to adherent cells in tissue culture flasks, replenished with fresh medium on days 3 and 7, Rh IL-10 (10 ng / ml) was added on day 7, and the cells were incubated for an additional 2 days in RPMI-1640 containing 10% heat-inactivated FBS. CFSE-labeled target cells (1 × 10 5 cells / well) and effector cells (2.5 × 10 4Cells (0.16 cells / well) were incubated with serial dilutions of test antibodies in a 96-well ultra-low attachment U-bottom plate (Costar, catalog no. 7007) for 2 hours at 37°C in a 5% CO2 incubator. Next, the cells were transferred to a 96-well v-bottom PP plate, spun down to pellet, and washed once with DPBS containing 20% ​​heat-inactivated FBS. The cells were then resuspended in DPBS containing 20% ​​HI FBS. APC-conjugated anti-human CD36 antibody (ThermoFisher Scientific, catalog no. MA1-10210) was added to the wells containing the test antibody and incubated on ice for 30 minutes. The cells were washed twice with 200 μL of DPBS containing 20% ​​HI FBS. Finally, the cells were resuspended in buffer containing 7-AAD. Samples were analyzed by flow cytometry using a BD LSR Fortessa and further analyzed using FlowJo gating for Live CFSE / APC double positivity, demonstrating phagocytosis of target cells by macrophages induced by the CD47-PDL1 antibody. The results are shown in Figure 17, panels A-B.

[0195] Example 17: PD-L1 / CD47 binding to erythrocytes Human red blood cells (RBCs) obtained from buffy coats after separation of mononuclear cells by density gradient centrifugation were carefully transferred to a 50 mL conical tube. The RBCs were washed three times with DPBS, and the supernatant was carefully removed after centrifugation. DPBS was then added to create a 10% solution of red blood cells. 1 × 10 cells per well were added. 6Cells were distributed into a 96-well V-bottom plate. Serial dilutions of test antibodies were added and incubated for 30 minutes at 4°C. The RBCs were then washed twice with 200 μL of DPBS (FACS buffer) containing 2% FBS and 0.05% sodium azide. The RBCs were then resuspended in 100 μL of secondary antibody, AF647 F(ab')2 goat anti-human IgG, Fc specific, at a 1:500 dilution (Jackson ImmunoResearch, catalog number 109-606-098), and incubated for 15 minutes at 4°C. Finally, the RBCs were washed twice with 200 μL of FACS buffer and resuspended in 200 μL of FACS buffer for flow cytometry analysis. The results are shown in panels A-B of Figure 18.

[0196] Example 18: CD47 antibody-induced hemagglutination of red blood cells Fresh whole blood obtained from the Stanford Blood Center was diluted 1:1 with DPBS. 2 μL of diluted blood was then dispensed into a 96-well U-bottom plate. 50 μL of serially diluted test antibody was added and incubated at room temperature for 2 hours. Photographs of the results were taken. The results are shown in Figure 19.

[0197] Example 19: A431 / hPBMC co-implanted tumor model in ICR-SCID mice Each mouse is 5x10 6 A431 cells and 1.5 x 10 7 A431 cells and a mixture of human PBMCs and Matrigel were subcutaneously implanted into ICR-SCID mice so that they received 100 human PBMCs. Tumors grew to an average size of 140 mm. 3 When tumor size reached 10 mg / kg IP, mice were administered 10 mg / kg IP of the test antibody or an equivalent volume of PBS on days 0, 4, 7, 11, and 15 (n=8). Tumor growth and mouse weights were monitored twice weekly. Tumors were harvested on day 18 and analyzed for lymphocyte and monocyte content. The results are shown in Figure 20, panels A-F and Figure 21, panels A-F.

[0198] Example 20: A431 tumor model in NOD-SCID mice NOD-SCID mice were treated with 5 × 10 6 A431 cells were subcutaneously implanted. The tumors grew to an average of 110 mm 3 When tumor size reached 100 mg / kg IP, mice were administered 20 mg / kg IP of the test antibody or an equal volume of PBS on days 0, 4, 8, 11, 14, and 18 (n=6). Tumor growth and mouse weights were monitored twice weekly. The results are shown in Figure 22, panels A-F.

[0199] Example 21: PDL1-IL15 antibodies bind to cells expressing human or cynomolgus monkey PD-L1 or human IL2Rβ and human IL2Rγ Cells were harvested and washed twice with FACS buffer. 2 × 10 cells per well were cultured. 5 Cells were dispensed into a 96-well V-bottom plate. Serial dilutions of test antibodies were added and incubated on ice for 20 minutes. Cells were washed twice with 200 μL of FACS buffer. Next, cells were resuspended in 50 μL of secondary antibody, AF647 F(ab')2 goat anti-hu IgG, Fc specific, 1:500 dilution (Jackson ImmunoResearch, catalog number 109-606-098), and incubated on ice for 25 minutes. Finally, cells were washed twice with 200 μL of FACS buffer and resuspended in 100 μL of FACS buffer containing 7-AAD. The results are shown in panels A-C of Figure 24.

[0200] Example 22: Proliferation of NK92 or M07e cells in response to PD-L1-IL15 antibodies NK92 cells were cultured in MEM-alpha medium (Gibco, 12561056) supplemented with 12.5% ​​horse serum, 12.5% ​​fetal bovine serum, 0.2 mM inositol, 0.02 mM folic acid, 0.1 mM 2-mercaptoethanol, and 100-200 U / mL IL-2 (Pepro Tech). M07e cells were cultured in IMDM (Gibco, 12440046) supplemented with 20% fetal bovine serum and 10 ng / mL GM-CSF. For these proliferation assays, cells were harvested and washed twice with the appropriate medium without IL-2 or GM-CSF. Cells were dispensed at 20,000 cells / well into white 96-well plates and starved for 4 hours at 37°C in 5% CO2. Serial dilutions of test antibodies were then added, and the plates were incubated for an additional 3 days. Proliferation was measured using CellTiter-Glo reagent (Promega) according to the manufacturer's instructions. Luminescence was recorded on a FlexStation 3. The results are shown in Figure 25, panels A-C.

[0201] Example 23: Induction of pSTAT5 in M07e cells by PD-L1-IL15 antibodies M07e cells were harvested and washed twice with IMDM medium supplemented with 20% FBS without GM-CSF. Cells were starved of GM-CSF for 24 hours at 37°C in 5% CO2, and then plated at 1.5 x 10 cells per well in a 96-well V-bottom plate. 5The cells were then distributed to the wells. Serial dilutions of the test antibody were added and incubated for 20 minutes at 37°C in 5% CO2. For wells stimulated with GM-CSF as a positive control, GM-CSF was added after 10 minutes for a total of 10 minutes of stimulation. At the end of the incubation, cells were fixed by adding 4% paraformaldehyde directly to the culture medium to a final concentration of 1.5% paraformaldehyde and incubated at room temperature for 10 minutes. Next, cells were permeabilized by adding 100 μL of ice-cold methanol and mixed vigorously by pipetting. After a 10-minute incubation at 4°C, cells were washed twice with staining buffer (PBS containing 1% BSA) and then resuspended in 50 μL of staining buffer containing human Fc block. Next, anti-pSTAT5 antibody (AF647 mouse anti-STAT5 pY694, BD catalog no. 612599) or isotype control (mouse IgG1 isotype control, BD, catalog no. 557714) was added and incubated for 15-30 minutes at room temperature. Cells were then washed twice with staining buffer and resuspended in 120 μL of staining buffer for analysis on the LSF Fortessa. The results are shown in panels A-B of Figure 26.

[0202] Example 24: PD-L1-IL15 antibodies increase proliferation of CD4+ and CD8+ T cells, NKT cells, and NK cells PBMCs were isolated according to the Miltenyi Biotech Density Centrifugation protocol. Red blood cells were lysed with RBC lysis buffer (eBiosciences) according to the manufacturer's protocol. After isolation, PBMCs were washed once with PBS supplemented with 2% FBS and collected at a concentration of 2 × 10 7 CellTrace Violet was prepared at 6 μM and added to PBMCs to a final concentration of 3 μM. After 10 minutes of incubation at room temperature in the dark, the reaction was stopped by adding an equal volume of FBS. PBMCs were then washed twice with PBS containing 2% FBS and resuspended at 2 × 10 cells / mL in RPMI containing 10% heat-inactivated FBS. 6 PBMCs were then resuspended at 2 x 10 cells / mL per well in a 96-well plate. 5The cells were distributed over 1000 cells. Serial dilutions of the test antibodies were then added, and the plates were incubated at 37°C, 5% CO for 5 days. PBMC proliferation was then analyzed by staining with antibodies against the following human proteins with the corresponding fluorophores: CD3-BB515, CD4-APC-H7, CD8-APC, CD56-BV786, and CD25 BUV395. The results are shown in panels A-C of Figure 27, panels A-D of Figure 28, and panels A-E of Figure 29.

[0203] Example 25: Pharmacodynamics of PD-L1-IL15 antibodies on mouse lymphocyte counts C57BL / 6 mice were administered the test antibody or an equal volume of PBS IP (n=3). Whole blood was collected on days 1, 4, 6, 8, and 11. Mouse Fc-block CD16 / CD32 clone 2.4G2 (BD catalog no. 553142) was added to 50 μL of anticoagulated mouse whole blood at 1.2–1.5 μL per 50 μL of blood and incubated at 4°C for 5 minutes. Fluorochrome-conjugated antibodies against mouse lymphocyte markers were mixed and added to the blood samples, which were then incubated in the dark at 4°C for 15–20 minutes. After incubation, red blood cells were lysed with BD Lysing Buffer (BD, catalog no. 555899) by adding 800 μL to each sample and vortexing vigorously. After 15 minutes of incubation at room temperature in the dark, the samples were centrifuged at 350 × g for 5 minutes and the supernatant was discarded. Cells were washed once with 2 mL of BD staining buffer (BD catalog no. 554657) and resuspended in 350 μL of BD staining buffer containing 7-AAD and 50 μL of counting beads (Biolegen catalog no. 424902) per sample. The results are shown in panels A-E of Figure 30.

[0204] Example 26: Pharmacodynamics of PD-L1-IL15 antibodies on mouse lymphocyte counts C57BL / 6 mice were administered test antibodies (0.5 mg / kg) or an equal volume of PBS IP (n=3). Whole blood samples were collected 4 hours later and on days 1, 2, 3, 6, and 8. Mouse Fc-block CD16 / CD32 clone 2.4G2 (BD catalog no. 553142) was added to 50 μL of anti-agglutinating mouse whole blood at 1.2–1.5 μL per 50 μL of blood and incubated at 4°C for 5 minutes. Fluorochrome-conjugated antibodies against mouse lymphocyte markers were mixed and added to the blood samples, which were then incubated in the dark at 4°C for 15–20 minutes. After incubation, red blood cells were lysed with BD Lysing Buffer (BD, catalog no. 555899) by adding 800 μL to each sample and vortexing vigorously. After 15 minutes of incubation at room temperature in the dark, the samples were centrifuged at 350 × g for 5 minutes and the supernatant was discarded. Cells were washed once with 2 mL of BD stain buffer (BD catalog no. 554657) and resuspended in 350 μL of BD stain buffer containing 7-AAD and 50 μL of counting beads (Biolegend catalog no. 424902) per sample. The results are shown in panels A-F of Figure 31.

[0205] Example 27: Pharmacokinetic measurements of D39.5-G1AAA-IL15 T2A, T2B, T3, and T2A monoclonal antibodies in C57BI / 6 and NSG mice Panels A-C: Study 27: C57BL / 6 mice (n=6, n=3 per time point) were dosed IV once on day 0. Whole blood and plasma were collected at 4 hours and on days 1, 2, 3, 6, and 8. Study 28: NSG mice were dosed with U118 cells (5x10) with Matrigel. 6 The tumors were transplanted into the mice. 3 When tumor-bearing mice reached 100 mg / kg / day, each tumor-bearing mouse was grouped with a tumor-free NSG mouse. The PBS group had two non-tumor-bearing mice. On day 0, mice were injected IV with human PBMCs (5 × 10 6Cells / mouse). On day 1, mice were administered the test antibody or an equal volume of PBS. Whole blood and plasma were collected on days 2, 4, 7, and 11. Panel D: C57B1 / 6 mice (n=6, n=3 per time point) were administered a single IP dose on day 0. Whole blood and plasma were collected at 4 hours and on days 1, 2, 3, 7, and 9. The results are shown in Figure 32, panels A-D.

[0206] Example 28: Tumor growth inhibition of MC38 murine colon cancer cells expressing human PD-L1 by PDL1-G1AAA-IL15-T2A MC38-hPDL1 cells were injected into C57BL / 6 mice at 5 × 10 6 The cells were implanted subcutaneously with Matrigel. The tumors averaged 165 mm 3 When tumor growth reached 100%, mice were randomized (n=10) and administered the test molecule or an equal volume of PBS IP on days 0, 7, and 14. Tumor growth and body weight were monitored twice weekly. The results are shown in Figure 33, Panels A-F. Tumor-free mice treated with PDL1-G1AAA-IL15-T2A were re-challenged with either MC38-hPD-L1 or B16F10 cancer cells. The data are shown in Figure 33, Panel G. MC38-hPD-L1 did not grow, suggesting durable protective immune memory.

[0207] Example 29: Tumor growth inhibition of A431 xenografts co-transplanted with human PBMCs A431 cells (5 x 10 6 cells / mouse) to human PBMCs (15 × 10 6 The cells were mixed with Matrigel (1:1) and then subcutaneously implanted into CB17-SCID mice. The tumors grew to an average size of 100 mm. 3Upon reaching a tumor size of 1000 mg / kg, mice were randomized (n=8) and administered the test molecule or an equal volume of PBS IP on days 0, 6, and 13. Tumor growth and body weight were monitored twice weekly. Mice were euthanized, and tumors were harvested on day 27. Tumors were homogenized and stained for human T cell and NK cell markers CD45, CD3, CD8, CD4, and CD56. Samples were analyzed by flow cytometry using a BD LSR Fortessa and further analyzed using FlowJo. Results are shown in panels A-G of Figure 34. Data from tumor phenotyping are shown in panels A-G of Figure 35 and panels A-G of Figure 36. Groups 1, 2, 3, and 4 showed significant expansion of human T cells, NK cells, and NKT cells.

[0208] Example 30: PDL1-G1AAA-IL15-T2A inhibits tumor growth of MC38 murine colon carcinoma cells expressing human PD-L1 in C57BL / 6 mice MC38 cells were injected into C57BL / 6 mice at a dose of 0.6 × 10 per mouse. 6 The cells were implanted subcutaneously with Matrigel. The tumors averaged 145 mm 3 Upon reaching a tumor size of 1000 mg / kg, mice were randomized (n=5 for PBS, n=8 for PBS) and administered the test molecule or an equivalent volume of PBS IP on days 0, 7, and 14. Tumor growth and body weight were monitored twice weekly. Tumors were homogenized and stained for murine T cell and NK cell markers CD45, CD90.2, CD8, CD4, and NK1.1. Samples were analyzed by flow cytometry using a BD LSR Fortessa and further analyzed using FlowJo. The results are shown in Figure 37, panels A-E. Data from tumor phenotyping are shown in Figure 38, panels A-F.

[0209] Example 31: Tumor growth inhibition of NCI-H1650 cells co-implanted with human PBMCs in CB17-SCID mice NCI-H1650 cells (10 x 10 6 cells / mouse) to human PBMCs (10 × 10 6The tumors were mixed with 1:1 HCl (cells / mouse) and Matrigel and then subcutaneously implanted into CB17-SCID mice. 3 Upon reaching 14 days, mice were randomized (n=8) and administered IP with the test molecule or an equal volume of PBS on days 0, 7, and 14. Tumor growth and body weight were monitored twice weekly. The results are shown in Figure 39, panels A-G.

[0210] Example 32: Phagocytosis of red blood cells (RBCs) induced by CD47-PDL1 bispecific antibodies is less than that of monoclonal anti-CD47 antibodies Recombinant human M-CSF and recombinant human IL-10 derived macrophages were generated as described in Example 16 and Figure 17. Carefully isolated RBCs were labeled with 1 μM CellTrace CFSE (ThermoFisher Scientific, Cat. No. C34554). CFSE-labeled RBCs (1 × 10 5 cells / well) and macrophages (2.5 × 10 4 Cells (0.175 cells / well) were incubated with serial dilutions of test antibodies in a 96-well ultra-low attachment U-bottom plate (Costar, catalog no. REF7007) for 2 hours at 37°C in 5% CO2. Cells were then transferred to a 96-well v-bottom PP plate, spun down to pellet, and washed once with DPBS containing 20% ​​heat-inactivated FBS. Cells were resuspended in DPBS containing 20% ​​HI FBS, and an APC-conjugated anti-human CD36 antibody (ThermoFisher Scientific, catalog no. MA1-10210) was added to the wells containing the test antibody and incubated on ice for 20 minutes. Cells were washed twice with 200 μL DPBS containing 20% ​​HI FBS. Finally, cells were resuspended in buffer containing 7-AAD. Samples were analyzed by flow cytometry using a BD LSR Fortessa and further analyzed using FlowJo gating on Live CFSE / APC double positivity, which indicates phagocytosis of RBCs by macrophages induced by anti-CD47 antibodies. The results are shown in panels A and B of Figure 41.

[0211] Example 33: Binding of Type2A masked antibody to CHOK1-IL2Rb / g cells before and after cleavage with MMP14 and uPA. 16 μg of each antibody was digested overnight at 37° with 0.4 μg of furin-activating MMP14 and 0.4 μg of uPA supplemented with zinc chloride. After digestion, CHOK1-IL2Rb / g cells were harvested and washed twice with FACS buffer. 1 × 10 cells per well were added. 5 Cells were dispensed into a 96-well v-bottom plate. Serial dilutions of test antibodies (cleaved and uncleaved) were added and incubated on ice for 30 minutes. The cells were washed twice with 200 μL of FACS buffer. Next, the cells were resuspended in 50 μL of secondary antibody, AF647 F(ab')2 goat anti-hu IgG, Fc specific, 1:500 dilution (Jackson ImmunoResearch, catalog no. 109-606-098), and incubated on ice for 20 minutes. Finally, the cells were washed twice with 200 μL of FACS buffer and resuspended in 120 μL of FACS buffer containing 7-AAD. The results are shown in Figure 42, demonstrating that using only the D1 antibody of IL15Rb does not reduce binding; however, masking with IL15Rb reduces binding by more than 10-fold.

[0212] Example 34: Proliferation of NK92 cells in response to Type 2A-masked antibodies before and after cleavage by MMP14 and uPA NK92 cells were cultured in MEM-alpha medium (Gibo, 12561056) supplemented with 12.5% ​​horse serum, 12.5% ​​fetal bovine serum, 0.2 mM inositol, 0.02 mM folic acid, 0.1 mM 2-mercaptoethanol, and 100-200 U / ml IL-2 (Pepro Tech). 16 μg of each antibody was digested overnight at 37°C with 0.4 μg of furin-activating MMP14 and 0.4 μg of uPA supplemented with zinc chloride. After digestion, NK92 cells were harvested and washed twice with culture medium without IL2. The cells were distributed at 20,000 cells / well into white 96-well plates and starved for 4 hours at 37°C in 5% CO2. Serial dilutions of the test antibodies were then added, and the plates were incubated for an additional 3 days. Proliferation was measured using CellTiter-Glo reagent (Promega) according to the manufacturer's instructions. Luminescence was recorded on a FlexStation 3. The results are shown in Figure 43 and demonstrate that masking with IL15Rb reduces proliferation 5-15 fold.

[0213] Example 35: AST and ALT levels in rhesus monkeys in a 4-week repeat-dose toxicology study of PD-L1x4-1BB bispecific antibody A 4-week repeat-dose toxicity study of the PD-L1x4-1BB bispecific antibody was conducted in rhesus monkeys, and aspartate transaminase (AST) and alanine transaminase (ALT) levels were measured. The results are shown in Figure 44, panels A and B. There were no chronic elevations in AST or ALT levels after repeat dosing, suggesting that 3, 10, and 30 mg / kg of PD-L1x 4-1BB had minimal toxic effects on the liver.

[0214] Example 36: Inhibition of A375 tumor growth by PD-L1 x CD47 (QL401) bispecific antibody in NOG mice Human PBMCs were transferred into NOG mice prior to inoculation with A375 cells. Results from this tumor model are shown in Figure 45. The antitumor effect of PD-L1xCD47 (QL401) at 10 mg / kg was as potent or more potent than that of magrolimab, durvalumab, or their combination.

[0215] Example 37: Inhibition of Raji tumor growth by PD-L1 x CD47 (QL401) bispecific antibody in NOG mice Human PBMCs were transferred into NOG mice prior to inoculation with Raji cells. Results from this tumor model are shown in Figure 46. The antitumor effect of PD-L1xCD47(QL401) at 10 mg / kg was comparable to that of magrolimab.

[0216] Example 38: Red Blood Cell Counts in Cynomolgus Monkeys in a 4-Week Repeat-Dose Toxicology Study of a PD-L1 x CD47 Bispecific Antibody A 4-week repeat-dose toxicity study was conducted with the PD-L1xCD47 bispecific antibody in cynomolgus monkeys. Results from this model are shown in Figure 47. After repeated administration of PD-L1xCD47 at doses of 10, 30, and 100 mg / kg, red blood cell counts did not fall significantly below the normal range.

[0217] Example 39: Stimulation of cDC1 with a mouse cross-reactive surrogate of PDL1-G1AAA-IL15-T2A MC38 tumor cells were implanted into C57BL / 6 mice, and approximately 100 mM 3 The tumors were grown to 100% IL-15 expression levels. Mice were treated with saline, a non-targeted IL-15 fusion protein, and a murine cross-reactive surrogate of PD-PDL1-G1AAA-IL15-T2A. Tumor-draining lymph nodes were collected and antigen-presenting cells were analyzed by FACS. The results are shown in Figure 48. The PD-L1x IL-15 surrogate molecule induced a higher percentage of conventional dendritic cells 1 (cDC1), suggesting a secondary mechanism of anti-tumor effect through stimulation of antigen-presenting cells.

[0218] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will recognize numerous variations, changes, and substitutions that do not depart from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A bispecific antibody that binds to PD-L1 and 4-1BB, comprising: Two binding units that bind to PD-L1, each of which is: a CDR1 sequence comprising SEQ ID NO:1; a CDR2 sequence comprising SEQ ID NO:2; a heavy chain variable region comprising: a CDR3 sequence comprising SEQ ID NO:3; and a CDR1 sequence comprising SEQ ID NO:7; a CDR2 sequence comprising SEQ ID NO:8; a light chain variable region comprising: a CDR3 sequence comprising SEQ ID NO: 9; and Two binding units that bind to 4-1BB, each comprising a single chain Fv (scFv), wherein the single chain Fv (scFv) comprises: a CDR1 sequence comprising SEQ ID NO: 13; a CDR2 sequence comprising SEQ ID NO: 14; a heavy chain variable region comprising: a CDR3 sequence comprising SEQ ID NO: 15; and a CDR1 sequence comprising SEQ ID NO: 19; a CDR2 sequence comprising SEQ ID NO: 20; and a light chain variable region comprising: a CDR3 sequence comprising SEQ ID NO: 21; The bispecific antibody comprising:

2. 2. The bispecific antibody of claim 1, wherein the CDR1, CDR2, and CDR3 sequences in each binding unit are present in a human VH or human VL framework.

3. The bispecific antibody of claim 1, wherein the two binding units that bind to PD-L1 each comprise a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO:

25.

4. The bispecific antibody of claim 3, wherein the two binding units that bind to PD-L1 each comprise a heavy chain variable region comprising SEQ ID NO:

25.

5. 2. The bispecific antibody of claim 1, wherein the two binding units that bind to PD-L1 each comprise a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO:

27.

6. The bispecific antibody of claim 5, wherein the two binding units that bind to PD-L1 each comprise a light chain variable region comprising SEQ ID NO:

27.

7. The bispecific antibody of claim 1, wherein the two binding units that bind to 4-1BB each comprise a heavy chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO:

45.

8. The bispecific antibody of claim 7, wherein the two binding units that bind to 4-1BB each comprise a heavy chain variable region comprising SEQ ID NO:

45.

9. The bispecific antibody of claim 1, wherein the two binding units that bind to 4-1BB each comprise a light chain variable region comprising a sequence having at least 95% sequence identity to SEQ ID NO:

47.

10. 10. The bispecific antibody of claim 9, wherein the two binding units that bind to 4-1BB each comprise a light chain variable region comprising SEQ ID NO:

47.

11. 2. The bispecific antibody of claim 1, further comprising a heavy chain constant region sequence comprising a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain, wherein the heavy chain constant region sequence comprises a L234A mutation, a L235A mutation, a G237A mutation, or any combination thereof.

12. 2. The bispecific antibody of claim 1 , further comprising a light chain constant region sequence, wherein the light chain constant region sequence comprises a human λ light chain constant region sequence.

13. In each of the binding units that bind to 4-1BB, the heavy chain variable region and the light chain variable region are G comprising SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO:

38. ４ The bispecific antibody of claim 1 , which is linked by an S linker sequence.

14. A G1000 antibody comprising a 4-1BB-binding domain, wherein each of the two binding units binds to 4-1BB and comprises SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO:

38. ４ The bispecific antibody of claim 1 , wherein the heavy chain constant region sequence is linked to the C-terminus of the heavy chain constant region sequence by an S linker sequence.

15. 1. A bispecific antibody that binds to PD-L1 and 4-1BB: (a) a first light chain polypeptide comprising the sequence of SEQ ID NO: 44; and (b) a first heavy chain polypeptide comprising the sequence of SEQ ID NO: 42; and (c) a second light chain polypeptide comprising the sequence of SEQ ID NO: 44; and (d) a second heavy chain polypeptide comprising the sequence of SEQ ID NO:

42.

16. A pharmaceutical composition comprising the antibody of claim 1.

17. A pharmaceutical for treating a subject having a disorder characterized by expression of PD-L1, the pharmaceutical comprising the antibody of claim 1 or the pharmaceutical composition of claim 16.

18. The method of claim 17, wherein the disorder is cancer.

19. The bispecific antibody of claim 1, further comprising a heavy chain constant region comprising SEQ ID NO:

93.

20. A polynucleotide encoding the antibody of claim 1.

21. A vector comprising the polynucleotide of claim 20.

22. A cell comprising the vector of claim 21.

23. 23. A method for producing the antibody of claim 1, comprising growing the cells of claim 22 under conditions that allow expression of the antibody, and isolating the antibody from the cells.

24. A medicament comprising an effective dose of the antibody of claim 1 or the pharmaceutical composition of claim 16.

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