Multiple specific binding compounds that bind to PD-L1

Abstract

This invention provides a multispecific binding compound that binds to PD-L1. [Solution] A bispecific antibody is provided, comprising a first binding unit that binds to PD-L1, each containing a heavy chain variable region and a light chain variable region, each containing a specific amino acid sequence, and a second binding unit that binds to CD47, each containing a single chain Fv(scFv), the single chain Fv(scFv) each containing a heavy chain variable region and a light chain variable region, each containing a different specific amino acid sequence.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of 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 includes a sequence listing 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 present invention further relates to methods of making such binding compounds, compositions comprising such binding compounds in pharmaceutical compositions, and their use for treating disorders characterized by the expression of PD - L1.

Background Art

[0004] Cancer is a major cause of death worldwide. In advanced metastatic cancer, conventional treatment regimens such as radiotherapy and chemotherapy have only a marginal effect on extending survival. Targeted therapies such as small - molecule inhibitors and inhibitory monoclonal antibodies have brought significant improvements in the management of disease progression, but nevertheless are limited to subsets of cancers with specific mutations or cancers that overexpress targetable receptors. Furthermore, tumor cells may further mutate or switch to another signaling pathway to avoid the inhibitory effect of the drug, so resistance to these therapies is common. Immunotherapy holds new promise in the fight against cancer by harnessing the body's own immune system. For example, checkpoint inhibitors targeting PD - 1 and CTLA - 4 have led the research and development in this field. The development of multispecific antibodies has enabled new therapeutic approaches.

[0005] PD-L1 multispecific antibodies are intended 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 between PD-L1 and its receptor PD-1. Third, they crosslink costimulatory targets such as 4-1BB on T cells, which then stimulates T cell proliferation only in the presence of PD-L1 expressing tumor cells. Furthermore, the constant region of the antibody may contain mutations that eliminate Fc gamma receptor-mediated functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). Thus, multispecific antibodies against PD-L1 have a potent immune response limited to tumor tissue and protect normal tissue from undesirable toxicity often observed with standalone costimulatory antibodies. [Overview of the project]

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

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

[0008] In some embodiments, the two binding units that bind to 4-1BB each include: a heavy chain variable region comprising a CDR1 sequence containing sequence number 13; a CDR2 sequence containing sequence number 14; and a CDR3 sequence containing sequence number 15; and a light chain variable region comprising a CDR1 sequence containing sequence number 19; a CDR2 sequence containing sequence number 20; and a CDR3 sequence containing sequence number 21.

[0009] In some embodiments, the two binding units that bind to 4-1BB each include: a heavy chain variable region comprising a CDR1 sequence containing sequence number 16; a CDR2 sequence containing sequence number 17; and a CDR3 sequence containing sequence number 18; and a light chain variable region comprising a CDR1 sequence containing sequence number 22; a CDR2 sequence containing sequence number 23; and a CDR3 sequence containing sequence number 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, each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 25. In some embodiments, each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing sequence number 25. In some embodiments, each of the two binding units that bind to PD-L1 includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 27. In some embodiments, each of the two binding units that bind to PD-L1 includes a light chain variable region containing sequence number 27. In some embodiments, each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 26. In some embodiments, each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing sequence number 26. In some embodiments, each of the two binding units that bind to PD-L1 includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 28.

[0011] In some embodiments, each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 29. In some embodiments, each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing sequence number 29. In some embodiments, each of the two binding units that bind to 4-1BB includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 31. In some embodiments, each of the two binding units that bind to 4-1BB includes a light chain variable region containing sequence number 31. In some embodiments, each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 30. In some embodiments, each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing sequence number 30. In some embodiments, each of the two binding units that bind to 4-1BB includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 32. In some embodiments, each of the two binding units that bind to 4-1BB includes a light chain variable region containing sequence number 32.

[0012] In some embodiments, the antibody further comprises a heavy chain constant region sequence including a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region sequence comprises the wild-type human IgG1 constant region sequence (SEQ ID NO: 92). In some embodiments, the heavy chain constant region sequence comprises the L234A mutation, the L235A mutation, the 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 bonding 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 includes a G4S linker sequence (sequence number 36). In some embodiments, the G4S linker sequence (sequence number 36) includes sequence number 36, sequence number 37, or sequence number 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 includes a G4S linker sequence (SEQ ID NO: 36). In some embodiments, the G4S linker sequence (SEQ ID NO: 36) includes SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

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

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

[0018] Aspects of the present invention include a bispecific antibody that binds to PD-L1 and CD47, the antibody comprising a first binding unit that binds to PD-L1, which comprises: a heavy chain variable region comprising a CDR1 sequence including SEQ ID NO: 1 or 4; a CDR2 sequence including SEQ ID NO: 2 or 5; and a CDR3 sequence including SEQ ID NO: 3 or 6; and a light chain variable region comprising a CDR1 sequence including SEQ ID NO: 7 or 10; a CDR2 sequence including SEQ ID NO: 8 or 11; and a CDR3 sequence including SEQ ID NO: 9 or 12. The material comprises 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 containing sequence number 50 or 53; a CDR2 sequence containing sequence number 51 or 54; and a CDR3 sequence containing sequence number 52 or 55; and a light-chain variable region comprising a CDR1 sequence containing sequence number 56 or 59; a CDR2 sequence containing sequence number 57 or 60; and a CDR3 sequence containing sequence number 58 or 61.

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

[0020] In some embodiments, the second binding unit that binds to CD47 includes: a heavy chain variable region comprising a CDR1 sequence containing sequence number 50; a CDR2 sequence containing sequence number 51; and a CDR3 sequence containing sequence number 52; and a light chain variable region comprising a CDR1 sequence containing sequence number 56, a CDR2 sequence containing sequence number 57; and a CDR3 sequence containing sequence number 58. In some embodiments, the second binding unit that binds to CD47 includes: a heavy chain variable region comprising a CDR1 sequence containing sequence number 53; a CDR2 sequence containing sequence number 54; and a CDR3 sequence containing sequence number 55; and a light chain variable region comprising a CDR1 sequence containing sequence number 59, a CDR2 sequence containing sequence number 60; and a CDR3 sequence containing sequence number 61.

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

[0022] In some embodiments, the second binding unit that binds to CD47 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 62. In some embodiments, the second binding unit that binds to CD47 includes a heavy chain variable region containing sequence number 62. In some embodiments, the second binding unit that binds to CD47 includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 64. In some embodiments, the second binding unit that binds to CD47 includes a light chain variable region containing sequence number 64. In some embodiments, the second binding unit that binds to CD47 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 63. In some embodiments, the second binding unit that binds to CD47 includes a heavy chain variable region containing sequence number 63. In some embodiments, the second binding unit that binds to CD47 includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 65. In some embodiments, the second binding unit that binds to CD47 includes a light chain variable region containing sequence number 65.

[0023] In some embodiments, the bispecific antibody further comprises a heavy chain constant region sequence including a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region sequence comprises the wild-type human IgG1 constant region sequence (SEQ ID NO: 92). In some embodiments, the heavy chain constant region sequence comprises the L234A mutation, the L235A mutation, the 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 to CD47, the heavy chain variable region and the light chain variable region are linked by a linker sequence. In some embodiments, the linker sequence includes a G4S linker sequence (sequence number 36). In some embodiments, the G4S linker sequence (sequence number 36) includes sequence number 36, sequence number 37, or sequence number 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 includes a G4S linker sequence (SEQ ID NO: 36). In some embodiments, the G4S linker sequence (SEQ ID NO: 36) includes SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38. In some embodiments, the bispecific antibody further includes a heavy chain constant region containing one or more knob-in-hole mutations that facilitate heterodimerization of two different heavy chain polypeptides.

[0027] Aspects of the present invention include a bispecific antibody that binds to PD-L1 and CD47, which comprises (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] Aspects of the present invention include a bispecific antibody that binds to PD-L1 and CD47, which comprises (a) a first light chain polypeptide containing the sequence of SEQ ID NO: 69; (b) a first heavy chain polypeptide containing the sequence of SEQ ID NO: 70; and (c) a second heavy chain polypeptide containing the sequence of SEQ ID NO: 71.

[0029] An aspect of the present invention comprises an antibody comprising one or more IL15 polypeptides bound to PD-L1 and fused to the C-terminus of a heavy-chain polypeptide subunit of a bispecific antibody, the antibody comprising a first binding unit that binds to PD-L1, the first binding unit 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 with any one of SEQ ID NOs: 86 to 90.

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

[0031] In some embodiments, the IL15 polypeptide comprises one of the sequences from SEQ ID NOs: 86-90. In some embodiments, the IL15 polypeptide is linked to the heavy chain polypeptide subunit of the antibody by a linker sequence. In some embodiments, the linker sequence comprises one of the sequences from 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 within the human VH or human VL framework. In some embodiments, the first binding unit that binds to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 25. In some embodiments, the first binding unit that binds to PD-L1 includes a heavy chain variable region containing sequence number 25. In some embodiments, the first binding unit that binds to PD-L1 includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 27. In some embodiments, the first binding unit that binds to PD-L1 includes a light chain variable region containing sequence number 27. In some embodiments, the first binding unit that binds to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with sequence number 26. In some embodiments, the first binding unit that binds to PD-L1 includes a heavy chain variable region containing sequence number 26. In some embodiments, the first binding unit that binds to PD-L1 includes a light chain variable region containing a sequence having at least 95% sequence identity with sequence number 28.

[0033] In some embodiments, the antibody further comprises a heavy chain constant region sequence including a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region sequence comprises the wild-type human IgG1 constant region sequence (SEQ ID NO: 92). In some embodiments, the heavy chain constant region sequence comprises the L234A mutation, the L235A mutation, the 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 containing one or more knob-in-hole mutations that promote heterodimerization of two different heavy chain polypeptides.

[0036] Aspects of the present invention include a bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy-chain polypeptide subunit, which comprises: (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] Aspects of the present invention include a bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy-chain polypeptide subunit, which comprises: (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] Aspects of the present invention include a bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy-chain polypeptide subunit, which comprises: (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] Aspects of the present invention include a bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of one heavy-chain polypeptide subunit, which comprises: (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] Aspects of the present invention include a bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of one heavy-chain polypeptide subunit, which comprises: (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] Aspects of the present invention include a bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy-chain polypeptide subunit, which comprises: (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 present invention include pharmaceutical compositions comprising antibodies as described herein.

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

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

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

[0046] Aspects of the present invention include a polynucleotide encoding an antibody as described herein, a vector comprising a polynucleotide as described herein, and a cell comprising a vector as described in the claims herein.

[0047] Aspects of the present invention include a method for producing an antibody as described herein, comprising growing the cells described herein under conditions that allow for antibody expression, and isolating the antibody from these cells.

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

[0049] These and further embodiments, including examples, are described further in the remainder of this disclosure. [Brief explanation of the drawing]

[0050] [Figure 1] Panel A is a graph showing the binding of the indicated antibody constructs to HEK293 cells expressing human PD-L1. Panel B is a graph showing the binding of the indicated antibody constructs to HEK293 cells expressing human 4-1BB. Panel C is a table showing the 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 reaction kinetics of antibody constructs to HIS-tagged PD-L1 and 4-1BB, respectively. Panel C is a table showing the 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 demonstrated antibody constructs in HEK293 cells expressing PD-L1. Panel B is a graph showing the 4-1BBL blocking activity of the demonstrated antibody constructs in HEK293 cells expressing 4-1BB. Panel C is a table showing the IC50 values ​​for PD-1 and 4-1BBL for the demonstrated antibody constructs. [Figure 5] Panel A is a graph showing the bifunctional ELISA binding of the indicated antibody construct as a function of concentration. Panel B is a graph showing the NF-κB reporter activity of the indicated antibody construct 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 antibody constructs at the indicated concentrations. 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 antibody constructs at the indicated concentrations. [Figure 7] Panel A is a graph showing IL2 release in an SEB-stimulated assay using the antibody constructs indicated at the concentrations shown. Panel B is a graph showing CD8+ T cell proliferation in the presence of anti-CD3 antibody (OKT3) and PD-L1+ A431 cells, along with the antibody constructs indicated at the concentrations shown. [Figure 8] Panel A is a graph showing tumor volume as a function of days after initial administration for MC38 mouse tumor models treated with the prescribed antibody construct at the prescribed dose. Panel B is a graph showing tumor-infiltrating immune cells (CD8+ T cells) for each dose group collected at the end of the study. [Figure 9] Panel A is a graph showing tumor volume as a function of days after initial administration for A431 human tumor models treated with the prescribed antibody construct at the prescribed dose. Panel B is a graph showing tumor-infiltrating immune cells (CD8+ T cells) for each dose group collected at the end of the study. [Figure 10] This table shows the calculated percentages of monomers, aggregates, and fragments in the HPLC-SEC profile of QL301 obtained from accelerated temperature stress tests. [Figure 11] Panel A is a graph showing the bifunctional ELISA binding as a function of antibody concentration against QL301 after incubation in human serum for 7 days, and as a stock control. Panel B is a graph showing IL2 release from PBMCs in the SEB-stimulated assay as a function of antibody concentration using human serum-incubated and stock control QL301 antibodies at the indicated concentrations. [Figure 12] Panel A is a schematic diagram of a PD-L1-CD47 bispecific antibody. Panel B is a graph showing ELISA binding to PD-L1 and CD47 as a function of antibody concentration. [Figure 13] Panels A through E are a series of graphs showing the binding of the indicated antibody constructs to cells as a function of antibody concentration. [Figure 14] This graph shows the PD-1 blocking activity of the CD47-PD-L1 bispecific antibody construct against stimulated A431 cells as a function of antibody concentration. [Figure 15] This graph shows the PD-1 blocking activity of the CD47-PD-L1 bispecific antibody construct against huPD-L1+HEK293 cells as a function of antibody concentration. [Figure 16] Panel A is a graph showing the SIRPα blockade of A431 cells by the indicated CD47-PD-L1 bispecific antibody construct as a function of antibody concentration. Panel B is a graph showing the SIRPα blockade of huCD47+CHO cells by the indicated CD47-PD-L1 bispecific antibody construct as a function of antibody concentration. [Figure 17] Panel A is a graph showing the antibody-mediated phagocytosis of Raji cells using the indicated CD47-PD-L1 antibody construct at the indicated concentration. Panel B is a graph showing the antibody-mediated phagocytosis of MM.1S cells using the indicated CD47-PD-L1 antibody construct at the indicated concentration. [Figure 18] Panels A and B are graphs showing the binding of the indicated CD47-PD-L1 bispecific antibody to red blood cells at the indicated antibody concentrations for two different RBC donors. [Figure 19] This image shows hemagglutination of red blood cells induced by the indicated antibody construct at the indicated concentration. [Figure 20] Panels A-F are a series of graphs showing tumor volume as a function of days in the A431, hPBMC co-grafted tumor model in ICR-SCID mice. Treatment groups G1-G5 represent different antibody constructs or controls (PBS). [Figure 21] Panels A through F are graphs showing the efficacy evaluation items from the tumor model described in Figure 20. [Figure 22] Panels A-F are a series of graphs showing tumor volume as a function of days in the A431, hPBMC co-grafted tumor model in NOD-SCID mice. Treatment groups G1-G5 represent different antibody constructs or controls (PBS). [Figure 23] Panels A-F are schematic diagrams of various bispecific antibody constructs containing an IL15 fusion at the C-terminus. [Figure 24] Panels A-C are a series of graphs showing the binding of the indicated PD-L1-IL15 bispecific antibody construct to the indicated cells at the indicated antibody concentrations. [Figure 25] Panels A–C 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 antibody or monoclonal anti-PD-L1 or isotype-controlled IL15 antibody at the indicated concentrations. [Figure 27] Panels A-C are a series of graphs showing the proliferation of indicated cell types in response to exposure to PD-L1-IL15 bispecific antibodies at the indicated concentrations. [Figure 28] Panels A–D are a series of graphs showing the proliferation of the indicated cell types in response to exposure to PD-L1-IL15 bispecific antibodies at the indicated concentrations. [Figure 29] Panels A–D are a series of graphs showing the proliferation of the indicated cell types in response to exposure to PD-L1-IL15 bispecific antibodies at the indicated concentrations. Panel E is a table showing the antibodies used for staining (listed by BioLegend catalog number). [Figure 30] Panels A-E are a series of graphs showing the cell types, as well as the cell number as a function of time for the indicated PD-L1-IL15 bispecific antibody constructs and administration schedules. [Figure 31] Panels A-F are a series of graphs showing the number of cells as a function of time for the indicated cell types, as well as the indicated PD-L1-IL15 bispecific antibody constructs and administration schedules. [Figure 32] Panels A-D are a series of graphs showing the pharmacokinetic properties of PD-L1-IL15 bispecific antibody constructs demonstrated in C57BL / 6 and NSG mice. [Figure 33] Panels A-F are a series of graphs showing the inhibition of tumor growth in PD-L1-expressing MC38 mouse colon cancer cells by the indicated PD-L1-IL15 antibody construct at the indicated doses. Panel G is a graph showing tumor volume as a function of the number of days after tumor rechallenge. [Figure 34] Panels A–G are a series of graphs showing tumor growth inhibition of a human PBMC tumor model and a co-implanted A431 xenograft for the indicated PD-L1-IL15 bispecific antibody construct at the indicated doses. [Figure 35] Panels A–G are a series of graphs showing phenotypic analysis of cells isolated from tumors of A431 xenografts co-implanted with the PD-L1-IL15 bispecific antibody construct shown in Figure 34, in addition to human PBMC tumor models treated with the indicated PD-L1-IL15 bispecific antibody construct. [Figure 36]Panels A–G are a series of graphs showing phenotypic analysis of cells isolated from tumors of A431 xenografts co-implanted with the PD-L1-IL15 bispecific antibody construct shown in Figure 34, in addition to human PBMC tumor models treated with the indicated PD-L1-IL15 bispecific antibody construct. [Figure 37] Panels A-E are a series of graphs showing the inhibition of tumor growth in PD-L1-expressing MC38 mouse colon cancer cells by the indicated PD-L1-IL15 antibody construct at the indicated doses in C57BL / 6 mice. [Figure 38] Panels A-F are a series of graphs showing the phenotypic analysis of cells isolated from tumors of the MC38 mouse colon cancer tumor model, treated with the indicated PD-L1-IL15 bispecific antibody construct at the doses shown in Figure 37. [Figure 39] Panels A-G are a series of graphs showing the inhibition of tumor growth in NCI-H1650 cells co-implanted with human PBMCs in CD17-SCID mice by the indicated PD-L1-IL15 bispecific antibody at the indicated doses. [Figure 40] Panel A is a model of QL301, a bispecific PD-L1x4-1BB antibody having two identical binding regions that bind to PD-L1 and two identical scFvs that bind to 4-1BB. Panel B is a diagram of tumor cells and T cells crosslinked with the QL301 bispecific antibody. [Figure 41] Panels A and B are graphs showing the percentage of RBC phagocytosis mediated by bispecific PD-L1xCD47 antibody for two different donors. [Figure 42] This graph shows the binding activity of the PD-L1-IL15 antibody construct. [Figure 43] This graph shows the proliferation of NK92 cells in response to the PD-L1-IL15 antibody construct. [Figure 44] Panels A and B are graphs showing AST and ALT levels observed in repeated-dose toxicological studies in rhesus monkeys using PD-L1-4-1BB bispecific antibody constructs according to several embodiments of the present invention. [Figure 45] This graph shows the inhibition of A375 tumor growth in a tumor model of NOG mice using PD-L1-CD47 bispecific antibody constructs according to several embodiments of the present invention. [Figure 46] This graph shows the inhibition of Raji tumor growth in a NOG mouse tumor model using a PD-L1-CD47 bispecific antibody construct according to an embodiment of the present invention. [Figure 47] This graph shows the number of red blood cells observed in repeated-dose toxicological studies in cynomolgus monkeys using a PD-L1-CD47 bispecific antibody construct according to an embodiment of the present invention. [Figure 48] This graph shows the stimulation of cDC1 observed in an MC38 tumor model performed in C57BL / 6 mice using a mouse cross-reactivity substitute for the PD-L1-IL15-T2A construct according to an embodiment of the present invention. [Modes for carrying out the invention]

[0051] Unless otherwise specified, the implementation of this invention will utilize conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope 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 This is adequately explained in literature such as Laboratory (1998) and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

[0052] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, as well as any other indicated or intermediate values ​​within that range, are included in the present invention. These upper and lower limits of smaller ranges may be independently included within the smaller range and are also included herein, according to any specific exclusion limits within the range. Where a 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 included in the present invention.

[0053] Unless otherwise specified, antibody residues in this specification 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] The following description includes many specific details to provide a more complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without one or more of these specific details. In other examples, well-known features and procedures well-known to those skilled in the art are omitted to avoid obscuring the invention.

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

[0056] I. Definition "Includes" means that the elements described are necessary for the composition / method / kit, but other elements may be included within the claims to form the composition / method / kit, etc.

[0057] "Essentially consisting of..." means limiting the scope of the composition or method described to specific substances or steps that do not substantially affect the basic and novel properties of the present invention.

[0058] "Consists of" means that any element, step, or component not specified in the claims is excluded from the composition, method, or kit.

[0059] The antibody residues described 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 1-113 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 constant region of the immunoglobulin heavy chain (e.g., the EU index reported by Kabat et al. (above)). "The EU index described in 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 mean residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0060] Antibodies, also known as immunoglobulins, conventionally contain at least one heavy chain and one light chain. The amino-terminal domains of the heavy and light chains are sequence-variable and are therefore generally referred to as variable region domains, or variable heavy chain (VH) or variable light chain (VH) domains. Conventionally, the two domains associate to form a specific binding region. However, as discussed herein, specific binding can also be obtained with a variable sequence of the heavy chain alone, and various non-natural structures of antibodies are known and used in the art.

[0061] A “functional” or “biologically active” antibody or conjugate is one that can exert one or more activities in a structural, regulatory, biochemical, or biophysical event. For example, a functional antibody or other conjugate may have the ability to specifically bind to an antigen, and this binding can then induce or modify cellular or molecular events, such as signaling or enzymatic activity. A functional antibody or other conjugate may also be able to block ligand activation of a receptor, or it may function as an agonist or antagonist. The ability of an antibody or other conjugate to exert one or more activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0062] In this specification, the term “antibody” is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), triple-chain antibodies, single-chain Fv (scFv), nanobodies, and antibody fragments insofar as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be derived from mouse, human, humanized, chimeric, or other species.

[0063] The term antibody may also refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or a polypeptide containing an immunologically active portion of any of these polypeptides, i.e., an antigen-binding site that immune-specifically binds to an antigen of a target or part thereof, such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may 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 having modified Fc portions that provide reduced or enhanced effector cell activity. The immunoglobulins may originate from any species.

[0064] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies constituting that population are identical except for the possibility of naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are directed to a single antigen site and are highly specific. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an 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, for example, by recombinant protein production methods (see, for example, U.S. Patent No. 4,816,567).

[0065] The term "variable" as used in relation to antibodies refers to the fact that the sequences of specific parts of the antibody's variable domain differ significantly between antibodies, and that these sequences are used for the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody's variable domain. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The variable domains of the natural heavy and light chains each contain four FRs, which primarily employ a β-sheet structure and are linked by three hypervariable regions. These hypervariable regions form loops that connect to the β-sheet structure, and in some cases form loops that form part of it. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs, contributing to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domain is not directly involved in antibody binding to antigens, but it exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity (ADCC).

[0066] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region generally includes amino acid residues derived from the “complementarity-determining region” or “CDR” (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 derived from the “hypervariable loop” residues 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)). The “framework region” or “FR” residues are variable domain residues other than the hypervariable region residues as defined herein.

[0067] While exemplary CDR designations are given herein, those skilled in the art will understand that several definitions of CDR 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), based on sequence variability. The Chothia definition is based on the location of the structural loop region (Chothia et al. "Conformations of immunoglobulin hypervariableRegions." Nature. 1989;342:877-883).Alternative CDR definitions for the purpose are, but are not limited to, 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 that Recognize 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 This includes the definitions disclosed in J.1995;9:133-139, each of which is specifically incorporated herein by reference.

[0068] As used herein, the term “multispecific conjugate” means a conjugate compound comprising two or more antigen-binding sites. Multispecific conjugate compounds according to embodiments of the present invention may comprise, essentially consist of, or comprise two, three, or four polypeptide subunits, each of which may include one or more variable region domains having binding affinity to a target antigen (e.g., PD-L1). In some embodiments, the multispecific conjugate 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 conjugate compound comprises a pair of variable region domains in the form of a single-chain Fv(scFv), where the first and second variable region domains are linked by a linker to form a binding unit. The multispecific conjugate compounds of the present invention may have any suitable combination or configuration of binding units, including but not limited to the specific configurations described herein.

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

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

[0071] Depending on the amino acid sequence of the heavy chain's Fc (constant domain), antibodies and various antigen-binding proteins can be offered as different classes. There are five major classes of the heavy chain Fc region: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to different classes of antibodies may be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinged or unhinged 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 derived from any vertebrate species can be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

[0072] A "functional Fc region" possesses the "effector function" of a native sequence Fc region. Non-limited examples of effector function include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, and downregulation of cell surface receptors (e.g., B cell receptors). Such effector function generally requires the Fc region to interact with receptors, such as FcγRI, FCγRIIA, FcγRIIB1, FCγRIIB2, FCγRIIIA, FCγRIIIB receptors, and low-affinity FcRn receptors, and can be evaluated using various assays known in the art. A "dead" or "silenced" Fc is, for example, an Fc that has been mutated to retain activity with respect to serum half-life extension, but does not activate high-affinity Fc receptors or has reduced affinity for Fc receptors.

[0073] A "natural sequence Fc region" contains an amino acid sequence identical to that of a naturally occurring Fc region. Examples of natural sequence human Fc regions include, for example, the natural sequence human IgG1 Fc region (non-A and A allotypes), the natural sequence human IgG2 Fc region, the natural sequence human IgG3 Fc region, and the natural sequence human IgG4 Fc region, as well as their natural variants.

[0074] A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the natural sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the natural sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions, in the natural sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region as described herein preferably has at least about 80% homology to the natural sequence Fc region and / or Fc region of the parent polypeptide, most preferably at least about 90% homology to them, and more preferably at least about 95% homology to them.

[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 include 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 (these disclosures are incorporated herein by reference in their entirety)). In some embodiments, the variant Fc sequence may include the following amino acid substitutions: L234A; L235A; and G237A. When these three amino acid substitutions are present in the IgG1 Fc sequence, they may be referred to as G1AAA.

[0077] Two amino acid substitutions at 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 for human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 significantly reduce ADCC and CDC (see, for example, Armour KL. et al., 1999 Eur J Immunol.29(8):2613-24 and Shields RL. et al., 2001. J Biol Chem.276(9):6591-604).

[0078] Other Fc variants are also possible, including, but not limited to, variants in which a region capable of forming a disulfide bond is deleted, or in which a specific amino acid residue is removed from the N-terminus of the native Fc, or a methionine residue is added thereto. Thus, in some embodiments, one or more Fc moieties of the conjugated compound may contain one or more mutations in the hinge region to eliminate the disulfide bond. In yet another embodiment, the hinge region of Fc may be completely removed. In yet another embodiment, the conjugated compound may contain 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 result in complement binding or Fc receptor binding. For example, deletions may be made at complement binding sites, such as C1q binding sites, but are not limited to these. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO97 / 34631 and WO96 / 32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

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

[0081] The embodiments of the present invention are not limited to those including conjugated compounds having multispecific configurations such as bispecificity and tripspecificity. A wide variety of methods and protein configurations are known and used for bispecificity monoclonal antibodies (BsMABs), tripspecificity antibodies, and the like.

[0082] Various methods have been developed for the production of polyvalent artificial antibodies by recombinant fusion of the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are linked 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, and n is an integer in the range of 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., 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 in this specification and may be incorporated into the antibody of the present invention in any suitable configuration.

[0083] The antibodies and multispecificity conjugates described herein may be in the form of dimers in which two heavy chains are disulfide-bonded or otherwise covalently or noncovalently bonded to each other, and may optionally include an asymmetric interface between two or more CH domains to facilitate proper pairing between polypeptide chains (generally referred to as a "knob-into-hole" interface). Knob-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. Patent No. 8,216,805, the disclosures of which are incorporated herein by reference in their entirety. The Fc region including the asymmetric interface may be referred herein by the abbreviation "KiH," which means knob-into-hole. For example, aspects of the present invention include a variant Fc region sequence such as a G1AAA sequence that includes an asymmetric interface and is referred herein by the abbreviation "G1AAA KiH."

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

[0085] As used herein, the term “human PD-L1” includes any variant, isoform, and species homolog (UniProt Q9NZQ7) of human PD-L1, regardless of its source or preparation method. Therefore, “human PD-L1” includes human PD-L1 expressed naturally in 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 conjugate,” and “PD-L1 conjugate” are used interchangeably herein to refer to antibodies or conjugates defined herein that bind immunospecifically 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, specifically including human 4-1BB and 4-1BB of non-human mammals.

[0088] As used herein, the term “Human 4-1BB” includes any variant, isoform, and species homolog (UniProt Q07011) of Human 4-1BB, regardless of its source or preparation method. Therefore, “Human 4-1BB” includes cells expressed on cells transfected with the Human 4-1BB gene and Human 4-1BB spontaneously expressed by 4-1BB.

[0089] The terms “anti-4-1BB antibody,” “4-1BB antibody,” “anti-4-1BB conjugate,” and “4-1BB conjugate” are used interchangeably herein to refer to antibodies or conjugates defined herein that bind immunospecifically 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, specifically including human CD47 and CD47 of non-human mammals.

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

[0092] The terms “anti-CD47 antibody,” “CD47 antibody,” “anti-CD47 conjugate,” and “CD47 conjugate” are used interchangeably herein to refer to antibodies or conjugate compounds, as defined herein, that bind immunospecifically to CD47, including human CD47 as defined herein.

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

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

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

[0096] The “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 amino acid residues in the reference polypeptide sequence, after the sequences have been aligned and gaps introduced to achieve the maximum sequence identity percentage, if necessary, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved using 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 appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2.

[0097] An “isolated” antibody or conjugated compound is one identified, separated, and / or recovered from components of its natural environment. Contaminations from the natural environment are materials that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified to homogeneity by (1) more than 95% by weight, most preferably more 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 the N-terminal sequence or internal amino acid sequence using a spinning cup sequenator; or (3) by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver staining. Since isolated antibodies lack at least one component of the antibody’s natural environment, they include in situ antibodies in recombinant cells. However, typically, isolated antibodies will be prepared by at least one purification step.

[0098] The conjugation compounds of the present invention include multispecific conjugation compounds. Multispecific conjugation compounds have two or more binding specificities. The term "multispecificity" specifically includes "bispecificity" and "triple specificity," as well as higher-order independent specific binding affinities such as higher-order polyepitope specificity, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody" and "multispecific conjugation compound" are used most broadly herein and encompass all antibodies and antibody-like molecules having two or more binding specificities. The multispecific anti-PD-L1 conjugation compounds of the present invention specifically include conjugation compounds that immunospecifically bind to epitopes on PD-L1 proteins, such as human PD-L1 protein, and to epitopes on different proteins, such as 4-1BB protein or CD47 protein.

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

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

[0101] As used herein, the term "valence" refers to a specific number of binding sites in an antibody molecule or binding compound.

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

[0103] A "polyvalent" binding compound has two or more binding sites. Therefore, the terms "divalent," "trivalent," and "tetravalent" refer to the presence of two, three, and four binding sites, respectively. Thus, the bispecific binding compound according to the present invention is at least divalent and may be trivalent, tetravalent, or otherwise polyvalent. The divalent binding compound according to embodiments of the present invention may have two binding sites to the same epitope (i.e., divalent, monoparatopic) or two binding sites to two different epitopes (i.e., divalent, biparatopic).

[0104] A wide variety of methods and protein structures are publicly known, and they are used in the preparation of bispecific monoclonal antibodies (BsMABs) and their conjugates, tripspecific antibodies and their conjugates, and so on.

[0105] The term "human antibody" as used herein includes antibodies having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human antibodies as used herein may include amino acid residues not encoded by a human germline immunoglobulin sequence, for example, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. Specifically, the term "human antibody" includes antibodies and conjugating compounds having a human heavy chain variable region sequence.

[0106] As used herein, the term “chimeric” antibody refers to an antibody having a variable sequence 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 generating chimeric antibodies are known in the art. See, for example, 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. Patents No. 5,807,715, No. 4,816,567, and No. 4,816,397, all of which are incorporated herein by reference. Specifically, the term “chimeric antibody” includes antibodies and conjugating compounds having a variable region sequence derived from a non-human immunoglobulin and a human immunoglobulin constant region sequence.

[0107] As used herein, the term “humanized antibody” refers to an antibody or conjugated compound containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the framework (FR) region is from a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically the sequence of the human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. For example, Riechmann et al., 1988, Nature 332:323-7; US Patent No. 5,530,101; US ​​Patent No. 5,585,089; US Patent No. 5,693,761; US ​​Patent No. 5,693,762; and US Patent No. 6,180,370 by Queen et al.; European Patent No. 239400; PCT Publication WO91 / 09967; US 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. See al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332 (all of which are incorporated herein by reference in their entirety).

[0108] As used herein, the term “effector cell” refers to immune cells involved in the effector phase of the immune response, as opposed to the cognitive and activation phases of the immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, may induce antibody-dependent cell-mediated cytotoxicity (ADCC). For example, monocytes and macrophages expressing FcR are involved in the specific death of target cells and the presentation of antigens to other components of the immune system, or binding to antigen-presenting cells. In some embodiments, effector cells may phagocytose target antigens or target cells.

[0109] "Human effector cells" are leukocytes that express receptors such as T cell receptors or FcR and perform effector functions. Preferably, these cells express at least FcγRIII and perform ADCC effector functions. 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 natural sources, for example, from blood or PBMCs, as described herein.

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

[0111] The "effector function" of an antibody refers to the biological activity attributed to the antibody's Fc region (either the Fc region of the natural sequence or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors, BCRs).

[0112] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, subsequently causing lysis of the target cells. NK cells, the main cells mediating ADCC, express only FcγRIII, while 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 evaluate the ADCC activity of a target molecule, an in vitro ADCC assay, such as those described in U.S. Patent No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer cells. Alternatively, the ADCC activity of the target molecule may be evaluated in vivo, for example, in an animal model disclosed in Clynes et al. PNAS(USA)95:652-656(1998).

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

[0114] As used interchangeably herein, “directed T cell-mediated cytotoxicity” and “redirected T cell-mediated cytotoxicity” refer to cell-mediated reactions in which a crosslinking 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 between T cells and target cells promotes the killing of target cells by T cells via the cytotoxic activity of the T cells. Redirected T cell-mediated cytotoxicity is described, for example, in Velasquez et al., Blood 2018 131:30-38.

[0115] "Binding affinity" refers to the sum of the 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 the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art. Low-affinity antibodies typically tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies typically tend to bind more quickly to antigens and remain bound.

[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 a reaction kinetic manner. For example, an anti-mouse Fc sensor is loaded with a mouse-Fc fusion antigen and then immersed in an antibody-containing well to measure the concentration-dependent association rate (kon). The antibody dissociation rate (koff) is measured in the final step by immersing the sensor in a well containing only buffer. KD is the koff / kon ratio. (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0117] The terms “treatment,” “procedure,” and “to treat” are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents the disease or symptoms, and / or therapeutic in that it partially or completely cures the disease and / or any side effects resulting from it. As used herein, “treatment,” “procedure” encompasses any treatment of a disease in a mammal, including (a) preventing its onset in a subject susceptible to the disease but not yet diagnosed with it, (b) inhibiting the disease, i.e., preventing its manifestation, or (c) reducing the disease, i.e., causing its regression. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of a progressing disease that stabilizes or reduces undesirable clinical symptoms in the patient is of particular interest. Such treatments are preferably performed before the complete loss of function of the affected tissue. The therapeutic agents of interest may be administered during the symptomatic phase of the disease, and in some cases, after the symptomatic phase.

[0118] The term "therapeutic dose" refers to the amount of active drug necessary to produce a therapeutic effect on a target. For example, the therapeutic dose is the amount that induces, induces remission of, or causes improvement in disease-related pathological symptoms, disease progression, or physiological condition, or improves tolerance to the disorder.

[0119] The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth. A “tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer "NSCLC"), adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (e.g., 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, hepatocellular carcinoma, 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. Examples of cancers include (e.g., renal cell carcinoma, nephroblastoma, or Wilms' tumor), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. Additional examples of cancers, but not limited to, include retinoblastoma, tecoma, allenoblastoma, hepatocellular carcinoma, hematological malignancies, such as non-Hodgkin lymphoma (NHL), multiple myeloma, and acute hematological 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 cancers.

[0120] The term "metastatic cancer" refers to a type of cancer in which cancer cells originating from tissue spread from their original site to one or more other sites in the body via blood vessels or lymphatic vessels, forming one or more secondary tumors in one or more organs other than the tissue of origin. A prominent example is metastatic breast cancer.

[0121] The term "characterized by PD-L1 expression" broadly refers to any disease or disorder in which PD-L1 expression is associated with or involved in one or more pathological processes characteristic of the disease or disorder. Specifically, but not limited to, diseases or disorders characterized by PD-L1 expression include, for example, cancers in which tumor cells express PD-L1, and / or cancers in which tumor-associated stroma expresses PD-L1, and / or cancers in which PD-L1 is expressed in immune cells. Examples of such disorders, though not limited to them, include invasive breast cancer, colonic 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 cervical adenocarcinoma, bile duct cancer, 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 "proliferative disorder" and "proliferative disorder" refer to disorders associated with a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0123] When used herein, "tumor" refers to any neoplastic cell growth and proliferation, whether malignant or benign, as well as any precancerous and cancerous cells and tissues.

[0124] The terms “to treat, to treat,” “treatment, treatment,” and “treating, treating” as used herein refer to both therapeutic treatments and preventive measures, the purpose of which is to prevent or slow (mitigate) a targeted physiological condition or disorder. Subjects requiring treatment include those who already have a particular condition or disorder, as well as those who are prone to developing the disorder or whose disorder should be prevented.

[0125] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to mammals being evaluated for and / or treated for treatment. 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 pathogenic infections, etc. The subject may be a human, but may also include other mammals, particularly mammals useful as laboratory models for human diseases, such as mice and rats.

[0126] The term "pharmaceutical preparation" 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 preparation is administered. Such preparations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is an excipient that, when administered in appropriate amounts to the target mammal, can provide an effective dose of the active ingredient to be used.

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

[0128] A “stable” formulation is one in which the internal proteins inherently retain their physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation inherently retains its physical and chemical stability, as well as its biological activity, during 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 outlined, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (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 assessed qualitatively and / or quantitatively by a variety of different methods, including assessment of aggregate formation (e.g., by measuring turbidity using size exclusion chromatography and / or by visual inspection), assessment of charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis, amino-terminal or carboxy-terminal sequence analysis, mass spectrometry, SDS-PAGE analysis comparing reduced intact antibodies, peptide mapping (e.g., trypsin or LYS-C) analysis, and assessment of the biological activity or antigen-binding function of the antibody. Instability may include one or more of the following: 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 elongation, C-terminal processing, and glycosylation differences.

[0129] II. Detailed explanation PD-L1x 4-1BB bispecific antibody Aspects of the present invention include multispecific conjugate compounds that bind to PD-L1 and 4-1BB, such as bispecific antibodies. Multispecific conjugate compounds may have various configurations, and each binding unit may include a set of CDR sequences. The PD-L1 heavy chain CDR sequences include SEQ ID NOs: 1 to 6. The PD-L1 light chain CDR sequences include SEQ ID NOs: 7 to 12. The anti-4-1BB heavy chain CDR sequences include SEQ ID NOs: 13 to 18, and the anti-4-1BB light chain CDR sequences include SEQ ID NOs: 19 to 24. In some embodiments, the multispecific conjugate compound includes a CDR sequence having two or fewer amino acid substitutions in any one of SEQ ID NOs: 1 to 24.

[0130] The multispecific conjugated compounds according to embodiments of the present invention may include any suitable combination of heavy chain 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 conjugated compounds include variable region sequences having at least about 80% identity, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity, with respect to any one of the variable region sequences of SEQ ID NOs: 25-32 and 45-48.

[0131] The multispecific conjugated compounds according to embodiments of the present invention may comprise one or more anti-4-1BB scFv sequences, as enumerated herein. The anti-4-1BB scFv sequences include SEQ ID NOs. 129-132. In some embodiments, the multispecific conjugated compound comprises an scFv sequence having at least about 80% identity to any one of the scFv sequences of SEQ ID NOs. 129-132, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity. In some embodiments, the scFv sequence is linked to a polypeptide subunit (e.g., a heavy-chain 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 conjugate compounds described herein offer numerous advantages that contribute to their usefulness as clinical therapeutic agents. These multispecific conjugate compounds include members with various conjugate unit configurations, enabling the selection of specific molecules that exhibit therapeutic benefits.

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

[0134] In a preferred embodiment, the bispecificity conjugate compound is conjugated to PD-L1 and 4-1BB and comprises a first light chain polypeptide containing the sequence of SEQ ID NO: 43, a first heavy chain polypeptide containing the sequence of SEQ ID NO: 41, a second heavy chain polypeptide containing the sequence of SEQ ID NO: 41, and a second light chain polypeptide containing the sequence of SEQ ID NO: 43. This bispecificity antibody is called 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 called QL301 (without signal sequence).

[0136] Determination of the affinity for a candidate protein can be carried out using methods known in the art such as Biacore measurements. The multispecific binding compounds described herein have a Kd of about 10 -10 , -9 ~ about 10 -11 , by way of example and not 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 may have an affinity for PD-L1 or 4-1BB with a Kd comprising any value within these ranges. The selection of the affinity can be confirmed by biological evaluation for modulating 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] The present invention encompasses various forms of multispecific conjugation compounds, including but not limited to the quadruple-chain polypeptides described herein. Specifically, multispecific conjugation compounds in this specification include bispecific conjugation compounds having binding affinity to PD-L1 and 4-1BB (e.g., anti-PD-L1 × anti-4-1BB conjugation compounds). Such bispecific conjugation compounds induce potent T-cell-mediated death of tumor cells, as shown in panel B of Figure 40.

[0138] PD-L1xCD47 bispecific antibody Aspects of the present invention include multispecific binding compounds that bind to PD-L1 and CD47, such as bispecific antibodies. Multispecific binding compounds may have various configurations, and each binding unit may include 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, the multispecific binding compound includes a CDR sequence having two or fewer amino acid substitutions in either SEQ ID NOs: 1-12 or 50-61.

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

[0140] The multispecific conjugated compounds according to embodiments of the present invention may comprise one or more anti-CD47 scFv sequences, as enumerated herein. The anti-CD47 scFv sequences include SEQ ID NOs. 72-75. In some embodiments, the multispecific conjugated compound comprises an scFv sequence having at least about 80% identity to any one of the scFv sequences of SEQ ID NOs. 72-75, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity. In some embodiments, the scFv sequence is linked to a polypeptide subunit (e.g., a heavy-chain 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 conjugate compounds described herein offer numerous advantages that contribute to their usefulness as clinical therapeutic agents. These multispecific conjugate compounds include members with diverse conjugate unit configurations, enabling the selection of specific molecules that exhibit therapeutic benefits.

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

[0143] Aspects of the present invention include a multispecificity binding compound comprising a knob-into-hole (KiH) interface between its heavy chain subunits to promote heterodimerization of a desired component of a multispecificity 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., anti-CD47 scFv)).

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

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

[0146] The affinity for candidate proteins can be determined using methods known in the art, such as Biacore measurements. The multispecificity conjugate compounds described herein are approximately 10 -6 ~about 10 -11 For example, though not limited to: approximately 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 Kd value within these ranges may have affinity for PD-L1 or CD47. Affinity selection can be confirmed by biological evaluations to modulate the biological activity of PD-L1 or CD47, including in vitro assays, preclinical models, and clinical trials, as well as evaluations of potential toxicity.

[0147] The present invention encompasses various forms of multispecific conjugation compounds, including but not limited to the triple-chain or quadruple-chain polypeptides described herein. Specifically, multispecific conjugation compounds as described herein include bispecific conjugation compounds having binding affinity to PD-L1 and CD47 (e.g., anti-PD-L1 × anti-CD47 conjugation compounds). Such bispecific conjugation compounds induce potent T-cell-mediated death of tumor cells.

[0148] PD-L1xIL15 binding compound Aspects of the present invention include multispecific binding compounds, such as bispecific antibodies, that contain an IL15 region that binds to PD-L1 and facilitates interaction with the IL15 receptor. Multispecific binding compounds may have various configurations, and each PD-L1 binding unit may contain a set of CDR sequences. The PD-L1 heavy chain CDR sequences include SEQ ID NOs: 1 to 6. The PD-L1 light chain CDR sequences include SEQ ID NOs: 7 to 12. In some embodiments, the multispecific binding compound includes a CDR sequence having two or fewer amino acid substitutions in any one of SEQ ID NOs: 1 to 12.

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

[0150] The multispecific conjugated compounds according to embodiments of the present invention may comprise one or more anti-IL15 sequences, as enumerated herein. The IL15 sequences include SEQ ID NOs. 86-90. In some embodiments, the multispecific conjugated compound comprises an IL15 sequence having at least about 80% identity to any one of the IL15 sequences of SEQ ID NOs. 86-90, e.g., about 85%, about 90%, about 95%, about 99%, or about 99.9% identity. In some embodiments, the IL15 sequence is linked to a polypeptide subunit (e.g., a heavy-chain 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 conjugate compounds described herein offer numerous advantages that contribute to their usefulness as clinical therapeutic agents. These multispecific conjugate compounds include members with diverse conjugate unit configurations, enabling the selection of specific molecules that exhibit therapeutic benefits.

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

[0153] Aspects of the present invention include multispecificity binding compounds comprising a knob-into-hole (KiH) interface between their heavy chain subunits to promote heterodimerization of desired components of a multispecificity 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 a preferred embodiment, the bispecificity binding compound binds to PD-L1 and comprises two IL15 proteins (one on each heavy chain polypeptide subunit), a first light chain polypeptide containing the sequence of SEQ ID NO: 104, a first heavy chain polypeptide containing the sequence of SEQ ID NO: 105, a second heavy chain polypeptide containing the sequence of SEQ ID NO: 105, and a second light chain polypeptide containing the sequence of SEQ ID NO: 104. This molecule is called D39.5.2.3-G1AAA-IL15RaSu-IL15-T2A and is schematically shown in panel A of Figure 23.

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

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

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

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

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

[0160] The affinity for candidate proteins can be determined using methods known in the art, such as Biacore measurements. The multispecificity conjugate compounds described herein are approximately 10 -6 ~about 10 -11 For example, though not limited to: approximately 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 Kd value within these ranges may have affinity for PD-L1. Affinity selection can be confirmed by biological evaluations to modulate the biological activity of PD-L1 or IL15, including in vitro assays, preclinical models, and clinical trials, as well as evaluations of potential toxicity.

[0161] Various forms of multispecific conjugation compounds, including but not limited to the triple-chain or quadruple-chain polypeptides described herein, are within the scope of the present invention. Multispecific conjugation compounds as described herein include, in particular, bispecific conjugation compounds (e.g., anti-PD-L1 × IL15 conjugation compounds) that have binding affinity to PD-L1 and contain one or more IL15 proteins that facilitate interaction with the IL15 receptor. Such bispecific conjugation compounds induce potent T cell-mediated death of tumor cells.

[0162] The following table provides various sequences used when assembling the conjugated 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 the conjugated compound The multispecific binding compounds of the present invention can be prepared by methods known in the art. For example, the binding compounds and their antigen-binding fragments can also be produced by recombinant DNA technology by expression of encoding nucleic acids in a suitable eukaryotic or prokaryotic host, such as mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0164] Pharmaceutical composition, method of use and treatment Another aspect of the present invention is to provide a pharmaceutical composition comprising one or more multispecific conjugate compounds of the present invention mixed with a suitable pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers used herein are, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

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

[0166] Pharmaceutical compositions of the conjugated compounds used in accordance with the present invention are prepared for storage, for example, in the form of lyophilized formulations or aqueous solutions, by mixing a protein of 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 nontoxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides, serum albumin, This includes 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 dextrin; 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 nonionic 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 may be provided in unit dosage forms (i.e., single-dose doses). The formulation depends on the chosen route of administration. The conjugated compounds described herein may be administered by intravenous injection or infusion, or subcutaneously. With respect to injection administration, the conjugated compounds described herein may be formulated in aqueous solution, preferably in a physiologically compatible buffer, to reduce injection site discomfort. The solution may contain carriers, excipients, or stabilizers as described above. Alternatively, the conjugated compounds may be lyophilized before use for formulation in a suitable vehicle, e.g., sterile, pyrogen-free water.

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

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

[0170] In one embodiment, the multispecific conjugates and pharmaceutical compositions of this specification may be used to treat cancers characterized by PD-L1 expression. As used herein, cancers "characterized by PD-L1 expression" include, but are not limited to, cancers in which one or more tumor cells express PD-L1, and / or cancers in which tumor-associated stroma expresses PD-L1, and / or cancers in which immune cells express PD-L1. Examples of such disorders, though not limited to them, include invasive breast cancer, colonic 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 cervical adenocarcinoma, bile duct cancer, 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 varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, such as companion animals like dogs, cats, and horses, and laboratory mammals like rabbits, mice, and rats, can also be treated. The treatment dose can be gradually increased to optimize safety and efficacy.

[0172] The dose level can be easily determined by those skilled in the art, but may be varied as needed, for example, to alter the subject's response to treatment. The amount of active ingredient that can be combined with a carrier substance to produce a single dosage form varies depending on the host being treated and the specific mode of administration. Generally, a unit dosage form contains about 1 mg to about 500 mg of the active ingredient.

[0173] In some embodiments, the therapeutic dose of the drug may be in the range of approximately 0.0001 to 100 mg / kg of the host's body weight, more generally, 0.01 to 5 mg / kg. For example, the dose may be in the range of 1 mg / kg body weight, 10 mg / kg body weight, or 1 to 10 mg / kg. An exemplary treatment plan involves administration every two weeks, once a month, or once every three to six months. The therapeutic substance of the present invention is usually administered multiple times. The interval between single doses may be weekly, monthly, or yearly. The interval may also be irregular, as specified by measuring the blood levels of the therapeutic substance in the patient. Alternatively, the therapeutic substance of the present invention may be administered as a sustained-release formulation, in which case less frequent administration is required. The dose and frequency vary considerably depending on the half-life of the polypeptide in the patient.

[0174] Generally, the compositions are prepared as injectable liquid solutions or suspensions, and solid forms suitable for dissolving or suspending in a liquid vehicle before injection can also be prepared. The pharmaceutical compositions herein are suitable for direct or intravenous or subcutaneous administration after reconstitution of solid (e.g., lyophilized) compositions. Preparations may also be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides, or copolymers to enhance the 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 be administered in the form of depot injections or implantable preparations that can be formulated in a manner that allows for sustained or pulsed release of the active ingredient. Pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with all Good Manufacturing Practices (GMP) regulations of the U.S. Food and Drug Administration.

[0175] The toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by measuring the LD50 (lethal dose for 50% of the population) or LD100 (lethal dose for 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. Data obtained from these cell culture assays and animal experiments can be used in formulating dose ranges that are non-toxic for human use. The doses of the antibodies described herein are preferably within the range of circulating concentrations that include effective doses with little to no toxicity. Doses may vary considerably within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage may be selected by the individual physician in consideration of the patient's condition.

[0176] The administration composition will generally contain an antibody or other drug (e.g., another abrasive) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances (pH adjusters and buffers, toxicity modifiers, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate) as needed to approximate physiological conditions. The concentration of the active agent in these formulations can vary considerably and is selected mainly based on volume, viscosity, body weight, etc., according to the chosen specific mode of administration and patient needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0177] The present invention also includes the activator and formulation thereof, as well as a kit comprising instructions for use. The kit may further include at least one additional reagent, such as a chemotherapeutic agent. The kit typically includes a label indicating the intended use of the kit's contents. As used herein, the term “label” includes any written or recorded material that is on or supplied with the kit, or otherwise accompanies the kit.

[0178] It will be apparent to those skilled in the art that the present invention, as fully described herein, can be modified in various ways without departing from the spirit or scope of the invention. [Examples]

[0179] Example 1: Binding of QL301 to HEK293 cells expressing PD-L1 or 4-1BB. HEK293 cells expressing PD-L1 or 4-1BB were placed in 1 × 10⁶ wells of a 96-well V-bottom plate. 5 Cells were seeded at the specified density. Serially diluted antibodies were added to the cells and incubated on ice for 30 minutes. After washing twice with FACS buffer, AF647-labeled 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 survival dye and analyzed by flow cytometry. The results are shown in panels A-C of Figure 1.

[0180] Example 2: Reaction Kinetics of Bonds The kinetics of the binding reaction were measured using the OctetRED96 system. After loading the antibody onto a ForteBio anti-human Fc capture (AHC) sensor, his-tagged recombinant PD-L1 or 4-1BB protein was conjugated. The results are shown in panels A-C of Figure 2.

[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 placed in 1 × 10⁶ wells of a 96-well V-bottom plate. 5Plated at the density of. Serial diluted antibodies were added to the cells and incubated on ice for 30 minutes. After washing twice with FACS buffer, AF647-labeled 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 cytometry. The results are shown in panels A - C of Figure 3.

[0182] Example 4: QL301 Competition Assay HEK293 cells expressing PD-L1 or 4-1BB were plated in 96-well V-bottom plates at a density of 1×10 5 . Serial 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-tag 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 cytometry. The results are shown in panels A - C of Figure 4.

[0183] Example 5: QL301 Bifunctional ELISA and NF-kB Reporter Assay The recombinant His-tagged 4-1BB protein was coated on a 96-well plate at room temperature overnight with shaking. After washing the plate with PBS containing 0.05% Tween-20, the plate was blocked with 2% BSA for 60 minutes, and the antibody was 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 streptavidin conjugated with HRP (horseradish peroxidase) 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, and then 0.16 M sulfuric acid was added to stop the reaction. The absorbance was read with a plate reader. For the reporter assay, HEK293 cells expressing 4-1BB, which also contains a luciferase reporter element under the control of NF-kB transcriptional regulation, were seeded in a 96-well plate at 5×10 4 cells per well. Parental HEK293 cells or HEK293 cells expressing PD-L1 were added at the same cell number per well. Next, serially diluted antibodies were added and incubated at 37 °C with 5% CO2 for 24 hours. Then, the supernatant was collected and transferred to a 96-well white-wall plate, and QuantiLuc reagent (Invivogen) was added. Luminescence was read immediately with a plate reader. The results are shown in Panels A - B of Figure 5.

[0184] Example 6: Cytokine Release Human PBMCs were stimulated with anti-CD3 (OKT3) and incubated with QL301, PD-L1 or 4-1BB monoclonal antibodies, or combinations thereof, together with PD-L1+A431 cells. QL301 induced the release of IL2 and IFNγ, but was not induced by anti-PD-L1 or 4-1BB alone, or the combination of the two. When A431 cells were absent, the induction of IL2 was significantly less. The results are shown in Panels A - C of Figure 6. Each dot represents an individual donor, and the values are multiples of the control antibody.

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

[0186] Example 8: MC38 tumor model MC38 mouse cancer cells expressing human PD-L1 were transplanted into the flanks of C57BL / 6 mice with double knock-in of human PD-L1 and 4-1BB. The average tumor volume was approximately 100 mm². 3 After reaching a certain stage, QL301, PD-L1 monoclonal, or saline were administered intraperitoneally twice a week. 10 mg / kg of QL301 was significantly more effective than equimolar doses of 8 mg / kg of PD-L1 monoclonal antibody (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 had a higher concentration of CD8+ T cells compared to those treated with saline or PD-L1 monoclonal (p<0.01). The results are shown in panels A and B of Figure 8.

[0187] Example 9: A431 Tumor Model In the second model, A431 human cancer cells were co-transplanted with human PBMCs into the flanks of CB17-SCID mice. Consistent with the results in the MC38-hPD-L1 model, QL301 showed a greater tumor growth inhibitory effect and a higher proportion of CD8+ T cells in the tumors than the PD-L1 monoclonal antibody (n=8). The results are shown in panels A and B of Figure 9.

[0188] Example 10: Accelerated Temperature Stress Test In an accelerated temperature stress test at 42°C for 28 days, the HPLC-SEC profile of QL301 showed minimal changes (overlay of chromatograms at five time points). The calculated percentages of monomers, aggregates, and fragments remained within 1% of the initial production 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 a SEB stimulation assay. No significant changes were observed between molecules incubated in serum and stock controls at 4°C. The results are shown in panels A and B of Figure 11.

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

[0191] Example 13: Binding of PD-L1 × CD47 bispecific antibody to HEK293 cells Cells were harvested and washed once with FACS buffer. 1 × 10⁶ cells per well. 5The cells were distributed into a 96-well V-bottom plate. Serial dilutions of the test antibody were added, and the cells were incubated on ice for 20 minutes. The cells were then washed twice with 200 μL of FACS buffer. Next, the cells were treated with 50 μL of secondary antibody, AF647 F(ab'). 2 The cells were resuspended in goat anti-human IgG, Fc-specific, 1:500 dilution (Jackson ImmunoResearch, catalog no. 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 × 10⁶ cells per well. 5 The cells were distributed into a 96-well V-bottom plate. 0.5 μg / mL of PD-1-biotin (final concentration) was added to the cells and incubated on ice for 5 minutes. Next, serial dilutions of the test antibody were added and incubated on ice for 20 minutes. The cells were washed twice with 200 μL of FACS buffer. Then, the cells were treated with 50 μL of secondary antibody, streptavidin-APC (R&D, catalog number F0050), at a rate of 10 μL / 10 6 The cells were resuspended 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 × 10⁶ cells per well. 5The cells were distributed into a 96-well V-bottom plate. 1.25 μg / mL of SIRPα-biotin (final concentration) was added to the cells and incubated on ice for 5 minutes. Next, serial dilutions of the test antibody were added and incubated on ice for 20 minutes. The cells were washed twice with 200 μL of FACS buffer. Then, the cells were treated with 50 μL of secondary antibody, streptavidin-APC (R&D, catalog number F0050), at a rate of 10 μL / 10 6 The cells were resuspended 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 Macrophages derived from recombinant human M-CSF (Miltenyi Biotec, catalog number 130-096-492) and recombinant human IL-10 (Miltenyi Biotec, catalog number 130-098-448) were generated from newly isolated human peripheral blood mononuclear cells (PBMCs). After removing non-adherent cells on day 0, Rh M-CSF (20 ng / ml) was added to adherent cells in a tissue culture flask. Fresh medium was replenished on days 3 and 7, and Rh IL-10 (10 ng / ml) was added on day 7. The cells were then incubated for a further 2 days in RPMI-1640 containing 10% thermally inactivated FBS. Subsequently, CFSE-labeled target cells (1 × 10⁶) were subjected to further incubation. 5 Cells / well) and effector cells (2.5 × 10⁻¹⁰ 4Cells (per well) were incubated at 37°C for 2 hours in a 5% CO2 incubator with serial dilutions of the test antibody in a 96-well ultra-low attachment U-bottom plate (Costar, catalog no. 7007). Next, the cells were transferred to a 96-well v-bottom PP plate, spun down to form a 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 BD LSR Fortessa, and further analyzed using Live CFSE / APC double positivity and FlowJo gating to indicate phagocytosis of target cells by macrophages induced by CD47-PDL1 antibody. The results are shown in panels A and B of Figure 17.

[0195] Example 17: Binding of PD-L1 / CD47 to red blood cells Human erythrocytes (RBCs) obtained from the buffy coat after separation of mononuclear cells by density gradient centrifugation were carefully transferred to 50 mL conical tubes. The RBCs were washed three times with DPBS, and the supernatant was carefully removed after centrifugation. Next, DPBS was added to prepare a 10% solution of erythrocytes. 1 × 10⁶ cells per well 6The cells were distributed into a 96-well V-bottom plate. Serial dilutions of the test antibody were added and incubated at 4°C for 30 minutes. The RBCs were then washed twice with 200 μL of DPBS (FACS buffer) containing 2% FBS and 0.05% sodium azide. Next, the RBCs were resuspended in 100 μL of secondary antibody, AF647 F(ab')2 goat anti-human IgG, Fc-specific, 1:500 dilution (Jackson ImmunoResearch, catalog no. 109-606-098) and incubated at 4°C for 15 minutes. 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: Hemagglutination of red blood cells induced by CD47 antibody Fresh whole blood obtained from the Stanford Blood Center was diluted in DPBS at a 1:1 ratio. Then, 2 μL of the diluted blood was distributed into 96-well U-bottom plates. 50 μL of serially diluted test antibody was added, and the plates were 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 simultaneous transplantation tumor model in ICR-SCID mice Each mouse is 5x10 6 A431 cells and 1.5 × 10⁶ 7 To enable the acceptance of individual human PBMCs, a mixture of A431 cells, human PBMCs, and Matrigel was subcutaneously transplanted into ICR-SCID mice. The tumors averaged 140 mm. 3 When tumor growth reached a certain level, mice were administered 10 mg / kg IP test antibody or an equivalent amount of PBS on days 0, 4, 7, 11, and 15 (n=8). Tumor growth and mouse body weight were monitored twice weekly. Tumors were collected on day 18, and lymphocyte and monocyte content was analyzed. The results are shown in panels A-F of Figure 20 and panels A-F of Figure 21.

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

[0199] Example 21: The PDL1 - IL15 antibody binds 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 dispensed into a 96 - well V - bottom plate. Serial dilutions of the test antibody were added and incubated for 20 minutes on ice. 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 number 109 - 606 - 098) and incubated for 25 minutes on ice. 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 - C of Figure 24. 5

[0200] Example 22: Proliferation of NK92 or M07e cells in response to the PD - L1 - IL15 antibody ​​NK92 cells were cultured in MEM-alpha medium (Gibo, 12561056) supplemented with 12.5% ​​equine 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 growth assays, cells were harvested and washed twice with appropriate medium free of IL-2 and GM-CSF. Cells were distributed at 20,000 cells / well into white 96-well plates and starved at 37°C in 5% CO2 for 4 hours. Serial dilutions of the test antibody were then added, and the plates were incubated for a further 3 days. Cell growth was measured using CellTiter-Glo reagent (Promega) according to the manufacturer's instructions. Luminescence was recorded using FlexStation3. The results are shown in panels A-C of Figure 25.

[0201] Example 23: Induction of pSTAT5 in M07e cells by PD-L1-IL15 antibody M07e cells were harvested and washed twice in IMDM medium supplemented with 20% FBS without GM-CSF. The cells were depleted of GM-CSF by 24 hours at 37°C in 5% CO2, and then transferred to a 96-well V-bottom plate at a rate of 1.5 × 10⁶ cells per well. 5Cells were partitioned. Serial dilutions of the test antibody were added, and the cells were incubated in 5% CO2 at 37°C for 20 minutes. For the wells stimulated with GM-CSF as a positive control, GM-CSF was added after 10 minutes, for a total stimulation time of 10 minutes. Upon completion of incubation, 4% paraformaldehyde was added directly to the culture medium to fix the cells, resulting in a final concentration of 1.5% paraformaldehyde, and the cells were incubated at room temperature for 10 minutes. Next, 100 μL of ice-cold methanol was added to permeabilize the cells, and the mixture was vigorously pipettered. After incubation at 4°C for 10 minutes, the cells were washed twice with staining buffer (PBS containing 1% BSA), and then resuspended in 50 μL of staining buffer containing human Fc blocks. Next, an anti-pSTAT5 antibody (AF647 mouse anti-STAT5 pY694, BD catalog number 612599) or isotype control (mouse IgG1 isotype control, BD, catalog number 557714) was added, and the cells were incubated at room temperature for 15–30 minutes. Then, the cells were washed twice with staining buffer and resuspended in 120 μL of staining buffer for analysis in LSF Fortessa. The results are shown in panels A–B of Figure 26.

[0202] Example 24: PD-L1-IL15 antibody increases the 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 in RBC lysis buffer (eBiosciences) according to the manufacturer's protocol. After isolation, PBMCs were washed once with PBS supplemented with 2% FBS and 2 × 10⁻⁶ ions. 7 The cells were resuspended at a concentration of 6 μM. CellTrace Violet was prepared at 6 μM and added to the PBMCs to a final concentration of 3 μM. After incubation at room temperature in the dark for 10 minutes, the reaction was stopped by adding an equal volume of FBS. Next, the PBMCs were washed twice with PBS containing 2% FBS and 2 × 10⁶ units were added to RPMI containing 10% heat-inactivated FBS. 6 The cells were resuspended at a concentration of cells / mL. Then, the PBMCs were placed in a 96-well plate at a rate of 2 × 10⁶ per well. 5The cells were then partitioned. Serial dilutions of the test antibody were added, and the plates were incubated at 37°C and 5% CO2 for 5 days. Next, the proliferation of PBMCs was analyzed by staining with antibodies against the following human proteins possessing 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 antibody against mouse lymphocyte count C57BL / 6 mice were administered via IP with either a test antibody or an equal volume of PBS (n=3). Whole blood was collected on days 1, 4, 6, 8, and 11. Mouse Fc block CD16 / CD32 clone 2.4G2 (BD catalog number 553142) was added to 50 μL of anticoagulated mouse whole blood at a rate of 1.2–1.5 μL per 50 μL of blood, and incubated at 4°C for 5 minutes. Fluorescent dye-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, erythrocytes were lysed in BD Lysing Buffer (BD, catalog number 555899) by adding 800 μL to each sample and vigorously vortexing. After incubation at room temperature in the dark for 15 minutes, the samples were centrifuged at 350 × g for 5 minutes, and the supernatant was discarded. The cells were washed once with 2 mL of BD staining buffer (BD catalog number 554657), and then resuspended in 350 μL of BD staining buffer containing 7-AAD and 50 μL of counting beads (Biolegen catalog number 424902) per sample. The results are shown in panels A to E of Figure 30.

[0204] Example 26: Pharmacodynamics of PD-L1-IL15 antibody against mouse lymphocyte count C57BL / 6 mice were administered via IP (n=3) with either a test antibody (0.5 mg / kg) or an equal volume of PBS. Whole blood was collected at 4 hours and on days 1, 2, 3, 6, and 8. Mouse Fc block CD16 / CD32 clone 2.4G2 (BD catalog number 553142) was added to 50 μL of anti-agglutinating mouse whole blood at a rate of 1.2–1.5 μL per 50 μL of blood, and incubated at 4°C for 5 minutes. Fluorescent dye-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, erythrocytes were lysed in BD Lysing Buffer (BD, catalog number 555899) by adding 800 μL to each sample and vigorously vortexing. After incubation at room temperature in the dark for 15 minutes, the samples were centrifuged at 350 × g for 5 minutes, and the supernatant was discarded. The cells were washed once with 2 mL of BD staining buffer (BD catalog number 554657) and resuspended in 350 μL of BD staining buffer containing 7-AAD and 50 μL of counting beads (Biolegend catalog number 424902) per sample. The results are shown in panels A to F of Figure 31.

[0205] Example 27: Pharmacokinetic measurements of D39.5-G1AAA-IL15 type T2A, T2B, T3, and T2A mono in C57BI / 6 and NSG mice Panels A-C: Study 27: C57BL / 6 mice (n=6 and n=3 at each time point) received one IV dose on day 0. Whole blood and plasma were collected at 4 hours and on days 1, 2, 3, 6, and 8. Study 28: U118 cells (5×10) were administered to NSG mice along with Matrigel. 6 Cells (in mice) were transplanted. The tumor was 250 mm. 3 When the stage was reached, each mouse with a tumor was divided into two groups: one with a tumor and one with a non-tumor-bearing NSG mouse. The PBS group contained two non-tumor-bearing mice. On day 0, the mice were intravenously injected with human PBMC (5 × 10⁻¹⁰). 6Cells / mouse). On day 1, mice were administered either the test antibody or the same amount of PBS. Whole blood and plasma were collected on days 2, 4, 7, and 11. Panel D: C57Bl / 6 mice (n=6 at each time point, n=3 at each time point) received one 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 panels A-D of Figure 32.

[0206] Example 28: Inhibition of tumor growth in MC38 mouse colon cancer cells expressing human PD-L1 by PDL1-G1AAA-IL15-T2A MC38-hPDL1 cells were introduced into C57BL / 6 mice at a rate of 5 × 10⁶ cells per mouse. 6 The cells were transplanted subcutaneously along with Matrigel. The tumors averaged 165 mm in size. 3 When this stage was reached, mice were randomized (n=10) and administered IP with the test molecule or an equivalent amount of PBS on days 0, 7, and 14. Tumor growth and body weight were monitored twice weekly. The results are shown in panels A-F of Figure 33. Tumor-free mice treated with PDL1-G1AAA-IL15-T2A were rechallenged with either MC38-hPD-L1 or B16F10 cancer cells. The data are shown in panel G of Figure 33. MC38-hPD-L1 did not proliferate, suggesting persistent protective immune memory.

[0207] Example 29: Inhibition of tumor growth in A431 xenografts co-implanted with human PBMCs. A431 cells (5 x 10 6 Cells / mouse) to human PBMC (15×10 6 The cells (mouse) and Matrigel (1:1) were mixed and then subcutaneously transplanted into CB17-SCID mice. The tumors averaged 100 mm. 3When the tumor reached [a certain size], the mice were randomized (n = 8), and the test molecule or an equal volume of PBS was administered IP on days 0, 6, and 13. Tumor growth and body weight were monitored twice a week. The mice were euthanized, and tumors were harvested on day 27. The 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. The results are shown in panels A - G of Figure 34. Data from tumor phenotype analysis are shown in panels A - G of Figure 35 and panels A - G of Figure 36. In groups 1, 2, 3, and 4, human T cells, NK cells, and NKT cells were significantly increased.

[0208] Example 30: Tumor growth inhibition of MC38 mouse colon cancer cells expressing human PD - L1 by PDL1 - G1AAA - IL15 - T2A in C57BL / 6 mice MC38 cells were implanted subcutaneously into C57BL / 6 mice at [a certain cell number] per mouse 6 The cells were transplanted subcutaneously together with Matrigel. When the tumor reached an average of 145 mm 3 When the tumor reached [a certain size], the mice were randomized (n = 5 for PBS, n = 8), and the test molecule or an equal volume of PBS was administered IP on days 0, 7, and 14. Tumor growth and body weight were monitored twice a week. The tumors were homogenized and stained for mouse 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 panels A - E of Figure 37. Data from tumor phenotype analysis are shown in panels A - F of Figure 38.

[0209] Example 31: Tumor growth inhibition of NCI - H1650 cells co - transplanted with human PBMC in CB17 - SCID mice NCI - H1650 cells ([a certain cell number] cells / mouse) were co - transplanted with human PBMC ([a certain cell number] cells / mouse) 6 cells / mouse) with human PBMC ([a certain cell number] cells / mouse) 6The cells (or mouse cells) were mixed with Matrigel (1:1 ratio) and then subcutaneously transplanted into CB17-SCID mice. The tumors averaged 95 mM. 3 When this was reached, mice were randomized (n=8) and administered IP with the test molecule or an equivalent amount of PBS on days 0, 7, and 14. Tumor growth and body weight were monitored twice weekly. The results are shown in panels A-G of Figure 39.

[0210] Example 32: The phagocytosis of red blood cells (RBCs) induced by the CD47-PDL1 bispecific antibody is less than that of the monoclonal anti-CD47 antibody. 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, catalog number C34554). CFSE-labeled RBCs (1 × 10⁻¹⁶) 5 Cells / well and macrophages (2.5 × 10⁻⁶) 4 Cells (per well) were incubated at 37°C for 2 hours in 5% CO2 in a 96-well ultra-low attachment U-bottom plate (Costar, catalog no. REF7007) with serial dilutions of the test antibody. The cells were then transferred to a 96-well v-bottom PP plate, spun down to form a pellet, and washed once with DPBS containing 20% ​​heat-inactivated FBS. The cells were resuspended in DPBS containing 20% ​​HI FBS, and APC-conjugated anti-human CD36 antibody (ThermoFisher Scientific, catalog no. MA1-10210) was added to the wells containing the test antibody. The cells were incubated on ice for 20 minutes. The cells were washed twice with 200 μL DPBS containing 20% ​​HI FBS. Finally, the cells were resuspended in a buffer containing 7-AAD. Samples were analyzed by flow cytometry using BD LSR Fortessa and further analyzed using FlowJo gating with Live CFSE / APC double positivity, indicating RBC phagocytosis by macrophages induced by anti-CD47 antibody. The results are shown in panels A and B of Figure 41.

[0211] Example 33: Binding of Type 2A mask antibodies to CHOK1-IL2Rb / g cells before and after cleavage with MMP14 and uPA. Each antibody (16 μg) was digested overnight at 37°C with 0.4 μg of furin-activated MMP14 and 0.4 μg of uPA supplemented with zinc chloride. After digestion, CHOK1-IL2Rb / g cells were collected and washed twice with FACS buffer. 1 × 10⁶ cells per well. 5 The cells were distributed into a 96-well v-bottom plate. Serial dilutions of the test antibody (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, showing that the antibody using only IL15Rb D1 did not reduce binding; however, masking with IL15Rb reduced binding by more than 10 times.

[0212] Example 34: Proliferation of NK92 cells responsive to Type 2A mask antibodies before and after cleavage with MMP14 and uPA NK92 cells were cultured in MEM-alpha medium (Gibo, 12561056) supplemented with 12.5% ​​equine 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-activated MMP14 and 0.4 μg of uPA supplemented with zinc chloride. After digestion, NK92 cells were harvested and washed twice with IL-2-free culture medium. The cells were distributed at 20,000 cells / well into white 96-well plates and starved at 37°C in 5% CO2 for 4 hours. Serial dilutions of the test antibodies were then added, and the plates were incubated for a further 3 days. Growth was measured using CellTiter-Glo reagent (Promega) according to the manufacturer's instructions. Luminescence was recorded with FlexStation3. The results are shown in Figure 43, demonstrating that masking with IL15Rb reduces growth to 1 / 5 to 1 / 15.

[0213] Example 35: AST and ALT levels in rhesus monkeys in a 4-week repeated-dose toxicological study of PD-L1×4-1BB bispecific antibody. A 4-week repeated-dose toxicity study of the bispecific antibody PD-L1x4-1BB was conducted in rhesus monkeys, and aspartate transaminase (AST) and alanine transaminase (ALT) levels were measured. The results are shown in panels A and B of Figure 44. There were no chronic elevations of AST or ALT levels after repeated administration, suggesting that PD-L1x4-1BB at doses of 3, 10, and 30 mg / kg minimized hepatic toxicity.

[0214] Example 36: Inhibition of A375 tumor growth in NOG mice by PD-L1 × CD47 (QL401) bispecific antibody Human PBMCs were transplanted into NOG mice before inoculation with A375 cells. The results from this tumor model are shown in Figure 45. The antitumor effect of PD-L1xCD47 (QL401) at 10 mg / kg was equivalent to or greater than that of maglorimab, durvalumab, or a combination thereof.

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

[0216] Example 38: Red blood cell count in cynomolgus monkeys in a 4-week repeated-dose toxicological study of PD-L1 × CD47 bispecific antibody. A 4-week repeated-dose toxicity study was conducted in cynomolgus monkeys using a PD-L1x CD47 bispecific antibody. The results from this model are shown in Figure 47. After repeated administration of PD-L1x CD47 at doses of 10, 30, and 100 mg / kg, the red blood cell count did not fall significantly below the normal range.

[0217] Example 39: Stimulation of cDC1 with mouse cross-response substitute for PDL1-G1AAA-IL15-T2A MC38 tumor cells were transplanted into C57BL / 6 mice, and approximately 100 mM 3 The mice were allowed to proliferate until [a certain stage]. Mice were treated with saline, a non-targeted IL-15 fusion protein, and a mouse cross-reactive substitute for PD-PDL1-G1AAA-IL15-T2A. Tumor drainage lymph nodes were collected, and antigen-presenting cells were analyzed by FACS. The results are shown in Figure 48. The PD-L1x IL-15 substitute molecule induced a higher percentage of antigen-presenting cells than conventional dendritic cells 1 (cDC1), suggesting a secondary mechanism of antitumor effect through antigen-presenting cell stimulation.

[0218] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. Those skilled in the art will find numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. The following claims define the scope of the present invention, and the methods and structures within these claims, as well as their equivalents, are intended to be encompassed thereby.

Claims

1. A bispecific antibody that binds to PD-L1 and 4-1BB, and is as follows: Two binding units that bind to PD-L1, each of which is as follows: Heavy chain variable region, A CDR1 sequence containing sequence number 1 or 4; A CDR2 sequence containing sequence number 2 or 5; The heavy chain variable region comprising a CDR3 sequence including sequence number 3 or 6; and Light chain variable region, A CDR1 sequence containing sequence number 7 or 10; A CDR2 sequence containing sequence number 8 or 11; The CDR3 sequence including sequence number 9 or 12; the light chain variable region including; the two binding units including; and 4-1 Two bonding units that bond to BB, each containing a single chain Fv(scFv), wherein the single chain Fv(scFv) is: In the heavy chain variable region: A CDR1 sequence containing sequence number 13 or 16; A CDR2 sequence containing sequence number 14 or 17; The heavy chain variable region includes a CDR3 sequence containing sequence number 15 or 18; and In the light chain variable region: A CDR1 sequence containing sequence number 19 or 22; A CDR2 sequence containing sequence number 20 or 23; The two binding units include the CDR3 sequence containing sequence number 21 or 24, and the light chain variable region containing, The bispecific antibody comprising the above.

2. The bispecific antibody according to claim 1, wherein the two binding units that bind to PD-L1 are as follows: In the heavy chain variable region: The CDR1 sequence containing sequence number 1; The CDR2 sequence containing sequence number 2; The heavy chain variable region includes the CDR3 sequence containing Sequence ID 3; and In the light chain variable region: The CDR1 sequence containing sequence number 7; The CDR2 sequence containing sequence number 8; The light chain variable region includes the CDR3 sequence containing sequence number 9, The bispecific antibody comprising the above.

3. The bispecific antibody according to claim 1, wherein the two binding units that bind to PD-L1 are as follows: In the heavy chain variable region: The CDR1 sequence containing sequence number 4; The CDR2 sequence containing sequence number 5; The heavy chain variable region includes the CDR3 sequence containing sequence number 6, and In the light chain variable region: The CDR1 sequence containing sequence number 10; The CDR2 sequence containing sequence number 11; A CDR3 sequence containing sequence number 12, and a light chain variable region containing The bispecific antibody comprising the above.

4. The bispecific antibody according to claim 1, wherein the two binding units that bind to 4-1BB are each: In the heavy chain variable region: The CDR1 sequence containing sequence number 13; The CDR2 sequence containing sequence number 14; The heavy chain variable region includes the CDR3 sequence containing sequence number 15; and In the light chain variable region: The CDR1 sequence containing sequence number 19; The CDR2 sequence containing sequence number 20; The light chain variable region includes the CDR3 sequence containing sequence number 21, The bispecific antibody comprising the above.

5. The bispecific antibody according to claim 1, wherein the two binding units that bind to 4-1BB are each: In the heavy chain variable region: The CDR1 sequence containing sequence number 16; The CDR2 sequence containing sequence number 17; The heavy chain variable region includes the CDR3 sequence containing sequence number 18; and In the light chain variable region: The CDR1 sequence containing sequence number 22; The CDR2 sequence containing sequence number 23; The light chain variable region includes the CDR3 sequence containing sequence number 24, The bispecific antibody comprising the above.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the CDR1, CDR2, and CDR3 sequences in each binding unit are present in a human VH or human VL framework.

7. The bispecific antibody according to claim 2, wherein each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

25.

8. The bispecific antibody according to claim 7, wherein each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing SEQ ID NO:

25.

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

27.

10. The bispecific antibody according to claim 9, wherein each of the two binding units that bind to PD-L1 includes a light chain variable region containing SEQ ID NO:

27.

11. The bispecific antibody according to claim 2, wherein each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

26.

12. The bispecific antibody according to claim 11, wherein each of the two binding units that bind to PD-L1 includes a heavy chain variable region containing SEQ ID NO:

26.

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

28.

14. The bispecific antibody according to claim 13, wherein each of the two binding units that bind to PD-L1 includes a light chain variable region containing SEQ ID NO:

28.

15. The bispecific antibody according to claim 2, wherein each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

29.

16. The bispecific antibody according to claim 15, wherein each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing Sequence ID No.

29.

17. The bispecific antibody according to claim 2, wherein each of the two binding units that bind to 4-1BB includes a light chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

31.

18. The bispecific antibody according to claim 17, wherein each of the two binding units that bind to 4-1BB includes a light chain variable region containing SEQ ID NO:

31.

19. The bispecific antibody according to claim 2, wherein each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

30.

20. The bispecific antibody according to claim 19, wherein each of the two binding units that bind to 4-1BB includes a heavy chain variable region containing SEQ ID NO:

30.

21. The bispecific antibody according to claim 2, wherein each of the two binding units that bind to 4-1BB includes a light chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

32.

22. The bispecific antibody according to claim 21, wherein each of the two binding units that bind to 4-1BB includes a light chain variable region containing SEQ ID NO:

32.

23. A bispecific antibody according to any one of claims 1 to 22, further comprising a heavy chain constant region sequence including a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain.

24. The bispecific antibody according to claim 23, wherein the heavy chain constant region sequence includes a wild-type human IgG1 constant region sequence (SEQ ID NO: 92).

25. The bispecific antibody according to claim 24, wherein the heavy chain constant region sequence includes the L234A mutation, the L235A mutation, the G237A mutation, or any combination thereof.

26. The bispecific antibody according to claim 25, wherein the heavy chain constant region sequence includes sequence number 93.

27. A bispecific antibody according to any one of claims 1 to 26, further comprising a light chain constant region sequence.

28. The bispecific antibody according to claim 27, wherein the light chain constant region sequence includes a human κ light chain constant region sequence (SEQ ID NO: 91).

29. The bispecific antibody according to claim 27, wherein the light chain constant region sequence includes a human λ light chain constant region sequence.

30. The bispecific antibody according to any one of claims 1 to 29, wherein in each of the binding units bound to 4-1BB, the heavy chain variable region and the light chain variable region are linked by a linker sequence.

31. The linker array is G ４ A bispecific antibody according to claim 30, comprising an S-linker sequence (SEQ ID NO: 36).

32. The aforementioned G ４ The bispecific antibody according to claim 31, wherein the S linker sequence (sequence number 36) comprises sequence number 36, sequence number 37, or sequence number 38.

33. The bispecific antibody according to any one of claims 23 to 32, wherein each of the second binding units is linked to the C-terminus of the heavy chain constant region sequence by a linker sequence.

34. The linker array is G ４ A bispecific antibody according to claim 33, comprising an S-linker sequence (SEQ ID NO: 36).

35. The aforementioned G ４ The bispecific antibody according to claim 34, wherein the S linker sequence (sequence number 36) comprises sequence number 36, sequence number 37, or sequence number 38.

36. A bispecific antibody that binds to PD-L1 and 4-1BB: (a) a first light chain polypeptide containing the sequence of Sequence ID No. 43; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 41; (c) a second light chain polypeptide containing the sequence of Sequence ID No. 43; (d) The bispecific antibody comprising a second heavy chain polypeptide containing the sequence of SEQ ID NO:

41.

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

42.

38. A bispecific antibody that binds to PD-L1 and CD47: A first binding unit that binds to PD-L1, In the heavy chain variable region: A CDR1 sequence containing sequence number 1 or 4; A CDR2 sequence containing sequence number 2 or 5; The heavy chain variable region comprising a CDR3 sequence including sequence number 3 or 6; and In the light chain variable region: A CDR1 sequence containing sequence number 7 or 10; A CDR2 sequence containing sequence number 8 or 11; The first binding unit comprising the CDR3 sequence including sequence number 9 or 12, the light chain variable region including; and A second bonding unit that binds to CD47, comprising a single chain Fv(scFv), wherein the single chain Fv(scFv) is: In the heavy chain variable region: A CDR1 sequence containing sequence number 50 or 53; A CDR2 sequence containing sequence number 51 or 54; The heavy chain variable region includes a CDR3 sequence containing sequence number 52 or 55; and In the light chain variable region: A CDR1 sequence containing sequence number 56 or 59; A CDR2 sequence containing sequence number 57 or 60; The second binding unit includes the CDR3 sequence containing sequence number 58 or 61, the light chain variable region containing, The bispecific antibody.

39. The bispecific antibody according to claim 38, wherein the first binding unit that binds to PD-L1 is: In the heavy chain variable region: The CDR1 sequence containing sequence number 1; The CDR2 sequence containing sequence number 2; The heavy chain variable region includes the CDR3 sequence containing Sequence ID 3; and In the light chain variable region: The CDR1 sequence containing sequence number 7; The CDR2 sequence containing sequence number 8; The light chain variable region includes the CDR3 sequence containing sequence number 9, The bispecific antibody comprising the above.

40. The bispecific antibody according to claim 38, wherein the first binding unit that binds to PD-L1 is: In the heavy chain variable region: The CDR1 sequence containing sequence number 4; The CDR2 sequence containing sequence number 5; The heavy chain variable region includes the CDR3 sequence containing sequence number 6, and In the light chain variable region: The CDR1 sequence containing sequence number 10; The CDR2 sequence containing sequence number 11; The light chain variable region includes the CDR3 sequence containing sequence number 12, The bispecific antibody comprising the above.

41. The bispecific antibody according to claim 38, wherein the second binding unit that binds to CD47 is: In the heavy chain variable region: The CDR1 sequence containing sequence number 50; The CDR2 sequence containing sequence number 51; The heavy chain variable region includes the CDR3 sequence containing sequence number 52; and In the light chain variable region: The CDR1 sequence containing sequence number 56; The CDR2 sequence containing sequence number 57; The light chain variable region includes the CDR3 sequence containing sequence number 58, The bispecific antibody comprising the above.

42. The bispecific antibody according to claim 38, wherein the second binding unit that binds to CD47 is: In the heavy chain variable region: The CDR1 sequence containing sequence number 53; The CDR2 sequence containing sequence number 54; The heavy chain variable region includes the CDR3 sequence containing sequence number 55; and In the light chain variable region: The CDR1 sequence containing sequence number 59; The CDR2 sequence containing sequence number 60; The light chain variable region includes the CDR3 sequence containing sequence number 61, The bispecific antibody comprising the above.

43. A bispecific antibody according to any one of claims 38 to 42, wherein the CDR1, CDR2, and CDR3 sequences in each binding unit are present in a human VH or human VL framework.

44. The bispecific antibody according to claim 39, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

25.

45. The bispecific antibody according to claim 44, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing SEQ ID NO:

25.

46. The bispecific antibody according to claim 39, wherein the first binding unit that binds to PD-L1 includes a light chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

27.

47. The bispecific antibody according to claim 46, wherein the first binding unit that binds to PD-L1 includes a light chain variable region containing SEQ ID NO:

27.

48. The bispecific antibody according to claim 39, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

26.

49. The bispecific antibody according to claim 48, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing SEQ ID NO:

26.

50. The bispecific antibody according to claim 39, wherein the first binding unit that binds to PD-L1 includes a light chain variable region having at least 95% sequence identity with SEQ ID NO:

28.

51. The bispecific antibody according to claim 50, wherein the first binding unit that binds to PD-L1 includes a light chain variable region containing SEQ ID NO:

28.

52. The bispecific antibody according to claim 39, wherein the second binding unit that binds to CD47 includes a heavy chain variable region having at least 95% sequence identity with sequence number 62.

53. The bispecific antibody according to claim 52, wherein the second binding unit that binds to CD47 includes a heavy chain variable region containing SEQ ID NO:

62.

54. The bispecific antibody according to claim 39, wherein the second binding unit that binds to CD47 includes a light chain variable region having at least 95% sequence identity with sequence number 64.

55. The bispecific antibody according to claim 54, wherein the second binding unit that binds to CD47 includes a light chain variable region containing SEQ ID NO:

64.

56. The bispecific antibody according to claim 39, wherein the second binding unit that binds to CD47 includes a heavy chain variable region having at least 95% sequence identity with sequence number 63.

57. The bispecific antibody according to claim 56, wherein the second binding unit that binds to CD47 includes a heavy chain variable region containing SEQ ID NO:

63.

58. The bispecific antibody according to claim 39, wherein the second binding unit that binds to CD47 includes a light chain variable region having at least 95% sequence identity with sequence number 65.

59. The bispecific antibody according to claim 58, wherein the second binding unit that binds to CD47 includes a light chain variable region containing SEQ ID NO:

65.

60. A bispecific antibody according to any one of claims 38 to 59, further comprising a heavy chain constant region sequence including a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain.

61. The bispecific antibody according to claim 60, wherein the heavy chain constant region sequence includes a wild-type human IgG1 constant region sequence (SEQ ID NO: 92).

62. The bispecific antibody according to claim 61, wherein the heavy chain constant region sequence includes the L234A mutation, the L235A mutation, the G237A mutation, or any combination thereof.

63. The bispecific antibody according to claim 62, wherein the heavy chain constant region sequence includes sequence number 93.

64. A bispecific antibody according to any one of claims 38 to 63, further comprising a light chain constant region sequence.

65. The bispecific antibody according to claim 64, wherein the light chain constant region sequence includes a human κ light chain constant region sequence (SEQ ID NO: 91).

66. The bispecific antibody according to claim 64, wherein the light chain constant region sequence includes a human λ light chain constant region sequence.

67. The bispecific antibody according to any one of claims 38 to 66, wherein in the second binding unit bound to CD47, the heavy chain variable region and the light chain variable region are linked by a linker sequence.

68. The linker array is G ４ A bispecific antibody according to claim 67, comprising an S-linker sequence (SEQ ID NO: 36).

69. The aforementioned G ４ The bispecific antibody according to claim 68, wherein the S linker sequence (sequence number 36) comprises sequence number 36, sequence number 37, or sequence number 38.

70. The bispecific antibody according to any one of claims 60 to 69, wherein each of the second binding units is linked to the C-terminus of the heavy chain constant region sequence by a linker sequence.

71. The linker array is G ４ A bispecific antibody according to claim 70, comprising an S-linker sequence (SEQ ID NO: 36).

72. The aforementioned G ４ The bispecific antibody according to claim 71, wherein the S linker sequence (sequence number 36) comprises sequence number 36, sequence number 37, or sequence number 38.

73. A bispecific antibody according to any one of claims 38 to 72, further comprising a heavy chain constant region containing one or more knob-in-hole mutations that promote heterodimerization of two different heavy chain polypeptides.

74. A bispecific antibody that binds to PD-L1 and DCD47: (a) a first light chain polypeptide comprising the sequence of Sequence ID No. 66; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 67; (c) The bispecific antibody comprising a second heavy chain polypeptide containing the sequence of SEQ ID NO:

68.

75. A bispecific antibody that binds to PD-L1 and DCD47: (a) a first light chain polypeptide containing the sequence of Sequence ID No. 69; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 70; (c) The bispecific antibody comprising a second heavy chain polypeptide containing the sequence of SEQ ID NO:

71.

76. A bispecific antibody comprising one or more IL15 polypeptides bound to PD-L1 and fused to the C-terminus of the heavy chain polypeptide subunit of the bispecific antibody, A first binding unit that binds to PD-L1, In the heavy chain variable region: A CDR1 sequence containing sequence number 1 or 4; A CDR2 sequence containing sequence number 2 or 5; The heavy chain variable region comprising a CDR3 sequence including sequence number 3 or 6; and In the light chain variable region: A CDR1 sequence containing sequence number 7 or 10; A CDR2 sequence containing sequence number 8 or 11; The first binding unit comprising the CDR3 sequence including sequence number 9 or 12, the light chain variable region including; and IL15 polypeptide containing a sequence having at least 95% identity with any of sequence numbers 86-90, The bispecific antibody comprising the above.

77. The bispecific antibody according to claim 76, wherein the first binding unit that binds to PD-L1 is: In the heavy chain variable region: The CDR1 sequence containing sequence number 1; The CDR2 sequence containing sequence number 2; The heavy chain variable region includes the CDR3 sequence containing Sequence ID 3; and In the light chain variable region: The CDR1 sequence containing sequence number 7; The CDR2 sequence containing sequence number 8; The light chain variable region includes the CDR3 sequence containing sequence number 9, The bispecific antibody comprising the above.

78. The bispecific antibody according to claim 76, wherein the first binding unit that binds to PD-L1 is: In the heavy chain variable region: The CDR1 sequence containing sequence number 4; The CDR2 sequence containing sequence number 5; The heavy chain variable region includes the CDR3 sequence containing sequence number 6, and In the light chain variable region: The CDR1 sequence containing sequence number 10; The CDR2 sequence containing sequence number 11; The light chain variable region includes the CDR3 sequence containing sequence number 12, The bispecific antibody comprising the above.

79. The bispecific antibody according to claim 76, wherein the IL15 polypeptide comprises one of the sequences of SEQ ID NOs: 86 to 90.

80. The bispecific antibody according to any one of claims 76 to 79, wherein the IL15 polypeptide is linked to the heavy chain polypeptide subunit of the antibody by a linker sequence.

81. The bispecific antibody according to claim 80, wherein the linker sequence includes one of sequence numbers 36, 37, 38, 49, 120, 121, 122, 123, 124, 125, 126, 127, or 128.

82. The bispecific antibody according to any one of claims 76 to 81, wherein the CDR1, CDR2, and CDR3 sequences are present in a human VH or human VL framework.

83. The bispecific antibody according to claim 77, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

25.

84. The bispecific antibody according to claim 83, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing SEQ ID NO:

25.

85. The bispecific antibody according to claim 77, wherein the first binding unit that binds to PD-L1 includes a light chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

27.

86. The bispecific antibody according to claim 85, wherein the first binding unit that binds to PD-L1 includes a light chain variable region containing SEQ ID NO:

27.

87. The bispecific antibody according to claim 77, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing a sequence having at least 95% sequence identity with SEQ ID NO:

26.

88. The bispecific antibody according to claim 87, wherein the first binding unit that binds to PD-L1 includes a heavy chain variable region containing SEQ ID NO:

26.

89. The bispecific antibody according to claim 77, wherein the first binding unit that binds to PD-L1 includes a light chain variable region having at least 95% sequence identity with SEQ ID NO:

28.

90. The bispecific antibody according to claim 89, wherein the first binding unit that binds to PD-L1 includes a light chain variable region containing SEQ ID NO:

28.

91. A bispecific antibody according to any one of claims 76 to 90, further comprising a heavy chain constant region sequence including a CH1 domain, a hinge region sequence, a CH2 domain, and a CH3 domain.

92. The bispecific antibody according to claim 91, wherein the heavy chain constant region sequence includes a wild-type human IgG1 constant region sequence (SEQ ID NO: 92).

93. The bispecific antibody according to claim 92, wherein the heavy chain constant region sequence includes the L234A mutation, the L235A mutation, the G237A mutation, or any combination thereof.

94. The bispecific antibody according to claim 93, wherein the heavy chain constant region sequence includes sequence number 93.

95. A bispecific antibody according to any one of claims 76 to 94, further comprising a light chain constant region sequence.

96. The bispecific antibody according to claim 95, wherein the light chain constant region sequence includes a human κ light chain constant region sequence (SEQ ID NO: 91).

97. The bispecific antibody according to claim 95, wherein the light chain constant region sequence includes a human λ light chain constant region sequence.

98. A bispecific antibody according to any one of claims 76 to 97, further comprising a heavy chain constant region containing one or more knob-in-hole mutations that promote heterodimerization of two different heavy chain polypeptides.

99. A bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy chain polypeptide subunit, (a) a first light chain polypeptide containing the sequence of Sequence ID No. 104; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 105; (c) a second heavy chain polypeptide containing the sequence of Sequence ID No. 105; (d) A second light chain polypeptide containing the sequence of Sequence ID No. 104, The bispecific antibody comprising the above.

100. A bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy chain polypeptide subunit, (a) a first light chain polypeptide comprising the sequence of Sequence ID No. 106; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 107; (c) A second heavy chain polypeptide containing the sequence of Sequence ID No. 107; (d) A second light chain polypeptide containing the sequence of Sequence ID No. 106, The bispecific antibody comprising the above.

101. A bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy chain polypeptide subunit, (a) a first light chain polypeptide comprising the sequence of Sequence ID No. 108; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 109; (c) A second heavy chain polypeptide containing the sequence of SEQ ID NO: 110; (d) A second light chain polypeptide containing the sequence of Sequence ID No. 108, The bispecific antibody comprising the above.

102. A bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of one heavy-chain polypeptide subunit, (a) a first light chain polypeptide containing the sequence of SEQ ID NO: 111; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 112; (c) a second heavy chain polypeptide containing the sequence of SEQ ID NO: 113; (d) A second light chain polypeptide containing the sequence of sequence number 111, The bispecific antibody comprising the above.

103. A bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of one heavy-chain polypeptide subunit, (a) a first light chain polypeptide containing the sequence of SEQ ID NO: 114; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 115; (c) A second heavy chain polypeptide containing the sequence of Sequence ID No. 116; (d) A second light chain polypeptide containing the sequence of SEQ ID NO: 114, The bispecific antibody comprising the above.

104. A bispecific antibody comprising an IL15 polypeptide bound to PD-L1 and fused to the C-terminus of each heavy chain polypeptide subunit, (a) a first light chain polypeptide containing the sequence of Sequence ID No. 117; (b) A first heavy chain polypeptide containing the sequence of Sequence ID No. 118; (c) A second heavy chain polypeptide containing the sequence of Sequence ID No. 119; (d) A second light chain polypeptide containing the sequence of Sequence ID No. 117, The bispecific antibody comprising the above.

105. A pharmaceutical composition comprising the antibody described in any one of claims 1 to 104.

106. A method for treating a disorder characterized by the expression of PD-L1, comprising administering to a subject having the disorder an antibody according to any one of claims 1 to 104, or a pharmaceutical composition according to claim 105.

107. Use of an antibody according to any one of claims 1 to 104 in the preparation of a pharmaceutical product for treating a disorder characterized by PD-L1 expression.

108. An antibody according to any one of claims 1 to 104, for use in treating disorders characterized by PD-L1 expression.

109. The method, use, or antibody according to any one of claims 106 to 108, wherein the disorder is cancer.

110. A polynucleotide encoding an antibody according to any one of claims 1 to 104.

111. A vector comprising the polynucleotide described in claim 110.

112. A cell comprising the vector according to claim 111.

113. A method for producing an antibody according to any one of claims 1 to 104, comprising growing the cells according to claim 112 under conditions that allow the expression of the antibody, and isolating the antibody from the cells.

114. A treatment method comprising administering an effective dose of the antibody according to any one of claims 1 to 104, or the pharmaceutical composition according to claim 105, to an individual in need.

Images