Single-domain PD-L1 antibody

Abstract

Provision of a single-domain PD-L1 antibody. [Solution] Various single-domain anti-PD-L1 antibodies and polypeptides containing these single-domain antibodies (e.g., bispecific antibodies and chimeric antigen receptors) are provided. These antibodies, including their humanized counterparts, have shown excellent activity and are therefore suitable for use in various bispecific antibody forms. Methods of using the antibodies or polypeptides of the present invention to treat and diagnose various diseases, such as cancer and infectious diseases, are also provided.

Description

[Technical Field]

[0001] This invention claims priority to international patent application number PCT / CN2021 / 090058 (filed April 26, 2021), the contents of which are incorporated herein by reference in their entirety.

[0002] This invention claims priority to international patent application number PCT / CN2021 / 090046 (filed April 26, 2021), the contents of which are incorporated herein by reference in their entirety.

[0003] This invention claims priority to International Patent Application No. PCT / CN2021 / 090049 (filed April 26, 2021), the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0004] background Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. With a molecular weight of only 12–15 kDa, single-domain antibodies are significantly smaller than typical antibodies (150–160 kDa). Given their small size and single-chain nature, single-domain antibodies may be particularly suitable for inclusion as fragments within other proteins (e.g., bispecific antibodies).

[0005] Various antibodies specific to programmed death ligand 1 (PD-L1), also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1), are used for cancer treatment and other clinical applications. PD-L1 is a 40 kDa type 1 transmembrane protein thought to play a major role in suppressing the immune system during certain events, such as pregnancy, allogeneic tissue transplantation, autoimmune diseases, and other disease states (e.g., hepatitis). When PD-L1 binds to PD-1 or B7.1, it transmits an inhibitory signal that reduces the proliferation of CD8+ T cells in lymph nodes. Complementarily, PD-1 can also regulate the accumulation of exogenous antigen-specific T cells in lymph nodes via apoptosis, which is further mediated by reduced regulation of the gene Bcl-2.

[0006] In addition to its use in treating cancer, PD-L1 inhibition has also shown promise in treating infectious diseases. In a mouse model of intracellular infection, L. monocytogenes induced PD-L1 protein expression in T cells, NK cells, and macrophages. PD-L1 blockade (e.g., using blocking antibodies) resulted in increased mortality in infected mice. Blockade reduced TNFα and nitric oxide production by macrophages, reduced granzyme B production by NK cells, and decreased proliferation of L. monocytogenes antigen-specific CD8 T cells (not CD4 T cells). This evidence suggests that PD-L1 acts as an active co-stimulatory molecule in intracellular infection. [Overview of the project] [Means for solving the problem]

[0007] Summary of the Invention This disclosure provides a variety of novel single-domain antibodies targeting the human PD-L1 protein. These single-domain antibodies exhibited excellent binding affinity and biological function despite their small size. When incorporated into various different forms of bispecific antibodies, some of the resulting bispecific antibodies demonstrated superior properties.

[0008] In one aspect, a single-domain antibody is provided that has binding specificity to the human PD-L1 protein and comprises one complementarity-determining region 1 (CDR1), CDR2, and CDR3 selected from the group consisting of SEQ ID NOs: 1 to 36, SEQ ID NOs: 114 to 122, and SEQ ID NOs: 123 to 130, or a polypeptide comprising the single-domain antibody. In some embodiments, the single-domain antibody has binding specificity to the human PD-L1 protein and comprises one complementarity-determining region 1 (CDR1), CDR2, and CDR3 selected from the group consisting of SEQ ID NOs: 1 to 36, SEQ ID NOs: 114 to 122, and SEQ ID NOs: 123 to 130, and CDR1, CDR2, and CDR3 follow a Kabat numbering scheme.

[0009] In some embodiments of the antibody or polypeptide, CDR1, CDR2, and CDR3 each contain (1) the amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, or (2) the amino acid sequences of SEQ ID NO: 113, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.

[0010] In some embodiments, CDR1 contains the amino acid sequence of SEQ ID NO: 55, CDR2 contains the amino acid sequence of SEQ ID NO: 56, and CDR3 contains the amino acid sequence of SEQ ID NO: 57. In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody contains one or more reverse mutations selected from the group consisting of 37F, 47F, 49A, 78V, and 94A, according to Kabat numbering. In some embodiments, the humanized antibody contains the reverse mutations 37F, 47F, 49A, 78V, and 94A, according to Kabat numbering. In some embodiments, the antibody contains any one amino acid sequence selected from the group consisting of SEQ ID NOs: 114 to 122. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO: 119.

[0011] In some embodiments, CDR1 contains the amino acid sequence of SEQ ID NO: 113, CDR2 contains the amino acid sequence of SEQ ID NO: 49, and CDR3 contains the amino acid sequence of SEQ ID NO: 50. In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody contains one or more reverse mutations selected from the group consisting of 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the humanized antibody contains the reverse mutations 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the antibody contains any one amino acid sequence selected from the group consisting of SEQ ID NOs: 123 to 130. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 130.

[0012] In some embodiments, the polypeptide is a bispecific antibody having binding specificity to an antigen different from PD-L1.

[0013] In another aspect, this specification provides a bispecific antibody comprising the antibody of the present application and a second antibody or antigen-binding fragment having binding specificity to a target antigen other than PD-L1.

[0014] In another aspect, the Specified provides a bispecific antibody comprising an anti-PD-L1 moiety having binding specificity to human PD-L1 protein and an anti-TIGIT moiety having binding specificity to human TIGIT protein, wherein the anti-TIGIT moiety comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 171 and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 172.

[0015] In some embodiments of the bispecific antibody, the anti-TIGIT moiety comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 171, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 172, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 follow the Kabat numbering scheme. In some embodiments, the anti-TIGIT moiety comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, where VH CDR1, VH CDR1, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 173 to 178, respectively. In some embodiments, the anti-TIGIT moiety comprises a VH containing the amino acid sequence of SEQ ID NOs. 171 and a VL containing the amino acid sequence of SEQ ID NOs. 172.

[0016] In some embodiments of the bispecific antibody, the anti-PD-L1 antigen-binding portion comprises a full-length antibody, Fab, F(ab’)2, scFv, scFv-Fc or a single-domain antibody. In some embodiments, the anti-PD-L1 antigen-binding portion comprises a single-domain antibody. In some embodiments, the single-domain antibody comprises any one CDR1, CDR2 and CDR3 selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 36, SEQ ID NO: 114 to SEQ ID NO: 122 and SEQ ID NO: 123 to SEQ ID NO: 130. In some embodiments, the single-domain antibody comprises any one CDR1, CDR2 and CDR3 selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 36, SEQ ID NO: 114 to SEQ ID NO: 122 and SEQ ID NO: 123 to SEQ ID NO: 130, and CDR1, CDR2 and CDR3 follow the Kabat numbering scheme. In some embodiments, the anti-PD-L1 portion comprises a single-domain antibody comprising complementarity-determining region 1 (CDR1), CDR2 and CDR3, and CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, or (2) respectively comprise the amino acid sequences of SEQ ID NO: 113, SEQ ID NO: 49 and SEQ ID NO: 50.

[0017] In some embodiments of the bispecific antibody, the anti-PD-L1 portion comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 55, CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR3 comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the anti-PD-L1 portion comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 113, CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0018] In some embodiments of the bispecific antibody, the anti-PD-L1 portion is humanized. In some embodiments, the anti-PD-L1 portion comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 114 to SEQ ID NO: 122 and SEQ ID NO: 123 to SEQ ID NO: 130. In some embodiments, the anti-PD-L1 portion comprises the amino acid sequence of 119 or 130.

[0019] In some embodiments of the bispecific antibody, the anti-PD-L1 moiety is fused to the C-terminus of the heavy chain of the anti-TIGIT moiety. In some embodiments, the anti-PD-L1 moiety is fused to the N-terminus of the heavy chain of the anti-TIGIT moiety. In some embodiments, the anti-PD-L1 moiety is fused to the C-terminus of the light chain of the anti-TIGIT moiety. In some embodiments, the anti-PD-L1 moiety is fused to the N-terminus of the light chain of the anti-TIGIT moiety.

[0020] In some embodiments of the bispecific antibody, the bispecific antibody is a homodimer. In some embodiments, the bispecific antibody comprises two anti-PD-L1 moieties. In some embodiments, each of the two anti-PD-L1 moieties is fused to the C-terminus of the heavy chain of the anti-TIGIT moiety. In some embodiments, the bispecific antibody comprises four anti-PD-L1 moieties.

[0021] In some embodiments of the bispecific antibody, the bispecific antibody comprises (1) a heavy chain component comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 184, and (2) a light chain component comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 180 and SEQ ID NO: 183. In some embodiments, the bispecific antibody comprises a heavy chain component comprising the amino acid sequence of SEQ ID NO: 179 and a light chain component comprising the amino acid sequence of SEQ ID NO: 180. In some embodiments, the bispecific antibody comprises a heavy chain component comprising the amino acid sequence of SEQ ID NO: 184 and a light chain component comprising the amino acid sequence of SEQ ID NO: 180.

[0022] In another aspect, the Specified provides a bispecific antibody comprising an anti-PD-L1 moiety having binding specificity to human PD-L1 protein and an anti-CD47 moiety having binding specificity to human CD47 protein, wherein the anti-CD47 moiety comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 131 or SEQ ID NO: 133, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 132 or SEQ ID NO: 134.

[0023] In some embodiments of the bispecific antibody, the anti-CD47 moiety comprises a heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 131 or 133, and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 132 or 134, where VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 follow the Kabat numbering scheme.

[0024] In some embodiments, the anti-CD47 moiety comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, where VH CDR1, VH CDR1, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 135 to 140, respectively. In some embodiments, the anti-CD47 moiety comprises a VH containing the amino acid sequence of SEQ ID NOs. 131 and a VL containing the amino acid sequence of SEQ ID NOs. 132.

[0025] In some embodiments, the anti-CD47 moiety comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, where VH CDR1, VH CDR1, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 141 to 146, respectively. In some embodiments, the anti-CD47 moiety comprises a VH containing the amino acid sequence of SEQ ID NOs. 133 and a VL containing the amino acid sequence of SEQ ID NOs. 134.

[0026] In some embodiments of the bispecific antibody, the anti-PD-L1 antigen-binding moiety comprises a full-length antibody, Fab, F(ab')2, scFv, scFv-Fc, or a single-domain antibody. In some embodiments, the anti-PD-L1 antigen-binding moiety comprises a single-domain antibody. In some embodiments, the single-domain antibody comprises one of CDR1, CDR2, and CDR3 selected from the group consisting of SEQ ID NOs: 1 to 36, SEQ ID NOs: 114 to 122, and SEQ ID NOs: 123 to 130, where CDR1, CDR2, and CDR3 follow the Kabat numbering scheme. In some embodiments, the anti-PD-L1 moiety comprises a single-domain antibody including complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 each contain (1) the amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, or (2) the amino acid sequences of SEQ ID NO: 113, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.

[0027] In some embodiments of the bispecific antibody, the anti-PD-L1 moiety comprises CDR1 containing the amino acid sequence of SEQ ID NO: 55, CDR2 containing the amino acid sequence of SEQ ID NO: 56, and CDR3 containing the amino acid sequence of SEQ ID NO: 57. In some embodiments, the anti-PD-L1 moiety comprises CDR1 containing the amino acid sequence of SEQ ID NO: 113, CDR2 containing the amino acid sequence of SEQ ID NO: 49, and CDR3 containing the amino acid sequence of SEQ ID NO: 50.

[0028] In some embodiments of the bispecific antibody, the anti-PD-L1 moiety is humanized. In some embodiments, the anti-PD-L1 moiety includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 114 to 122 and SEQ ID NOs: 123 to 130. In some embodiments, the anti-PD-L1 moiety includes the amino acid sequence 119 or 130.

[0029] In some embodiments of the bispecific antibody, the anti-PD-L1 moiety is fused to the C-terminus of the heavy chain of the anti-CD47 moiety. In some embodiments, the anti-PD-L1 moiety is fused to the N-terminus of the heavy chain of the anti-CD47 moiety. In some embodiments, the anti-PD-L1 moiety is fused to the C-terminus of the light chain of the anti-CD47 moiety. In some embodiments, the anti-PD-L1 moiety is fused to the N-terminus of the light chain of the anti-CD47 moiety.

[0030] In some embodiments of bispecific antibodies, the bispecific antibody is a homodimer. In some embodiments, the bispecific antibody contains two anti-PD-L1 moieties. In some embodiments, each of the two anti-PD-L1 moieties is fused to the C-terminus of the heavy chain of the anti-CD47 moiety. In some embodiments, the bispecific antibody contains four anti-PD-L1 moieties.

[0031] In some embodiments of the bispecific antibody, the bispecific antibody comprises (1) a heavy chain component comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 166, 167, 169, and 170, and (2) a light chain component comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 148, 150, 152, 154, 156, 158, 160, 162, 165, and 168.

[0032] In another context, this specification provides polynucleotides encoding the antibody or polypeptide of the present application or the bispecific antibody of the present application.

[0033] In another context, vectors comprising the polynucleotides of the present application are provided herein.

[0034] In another aspect, cells comprising the polynucleotide or vector of the present application are provided herein.

[0035] In another aspect, the Specified herein provides compositions comprising (1) an antibody or polypeptide, a bispecific antibody, or a polynucleotide of the present application, and (2) a pharmaceutically acceptable carrier.

[0036] In another aspect, this specification provides a method for treating cancer in a patient who requires treatment of cancer, comprising administering an effective amount of the antibody or polypeptide, bispecific antibody, or polynucleotide of the Application to the patient. In another aspect, this specification provides the use of the antibody or polypeptide, bispecific antibody, or polynucleotide of the Application for preparing a pharmaceutical product for treating cancer. In some embodiments, cancer is a solid tumor. In some embodiments, cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. [Brief explanation of the drawing]

[0037] [Figure 1A-1B] Figure 1A illustrates that the exemplary anti-PD-L1 antibody of the present invention effectively blocks the interaction between PD-1 and PD-L1; Figure 1B illustrates the specific binding of the exemplary anti-PD-L1 antibody of the present invention to human PD-L1; and Figure 1C illustrates the specific binding of the exemplary anti-PD-L1 antibody of the present invention to Raji cells overexpressing human PD-L1. [Figure 1C] Figure 1A illustrates that the exemplary anti-PD-L1 antibody of the present invention effectively blocks the interaction between PD-1 and PD-L1; Figure 1B illustrates the specific binding of the exemplary anti-PD-L1 antibody of the present invention to human PD-L1; and Figure 1C illustrates the specific binding of the exemplary anti-PD-L1 antibody of the present invention to Raji cells overexpressing human PD-L1.

[0038] [Figure 2A] Figures 2A to 2C illustrate that blocking the PD-1 / PD-L1 interaction by the exemplary anti-PD-L1 antibody of the present invention was able to enhance NFAT-mediated luciferase activity in a dose-dependent manner. [Figure 2B-2C]Figures 2A to 2C illustrate that blocking the PD-1 / PD-L1 interaction by the exemplary anti-PD-L1 antibody of the present invention was able to enhance NFAT-mediated luciferase activity in a dose-dependent manner.

[0039] [Figure 3A-3F] Figures 3A to 3F illustrate exemplary forms of the anti-CD47 / PD-L1 bispecific antibody of this application.

[0040] [Figure 4A] Figures 4A and 4B illustrate how the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention blocked the CD47 / SIRPα interaction in a dose-dependent manner. [Figure 4B] Figures 4A and 4B illustrate how the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention blocked the CD47 / SIRPα interaction in a dose-dependent manner.

[0041] [Figure 5A] Figures 5A and 5B illustrate how the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention effectively blocks PD-1 / PD-L1-mediated NF-AT-luciferase activity. [Figure 5B] Figures 5A and 5B illustrate how the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention effectively blocks PD-1 / PD-L1-mediated NF-AT-luciferase activity.

[0042] [Figure 6A] Figures 6A to 6C illustrate the ADCP efficacy of the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention. [Figure 6B-6C] Figures 6A to 6C illustrate the ADCP efficacy of the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention.

[0043] [Figures 7A-7B] Figures 7A and 7B illustrate the RKO binding ability of the exemplary antibodies of this invention.

[0044] [Figure 8A-8B] Figures 8A and 8B illustrate that the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention exhibits minimal or no RBC binding, and Figure 8C illustrates the in vivo antitumor efficacy of the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention. [Figure 8C] Figures 8A and 8B illustrate that the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention exhibits minimal or no RBC binding, and Figure 8C illustrates the in vivo antitumor efficacy of the exemplary anti-CD47 / PD-L1 bispecific antibody of the present invention.

[0045] [Figure 9] Figure 9 illustrates an exemplary form of the anti-TIGIT / PD-L1 bispecific antibody of the present invention.

[0046] [Figure 10A-10C] Figures 10A to 10C illustrate the binding properties of the exemplary anti-TIGIT / PD-L1 bsAb of this application to the human PD-L1 protein.

[0047] [Figure 11] Figure 11 illustrates that blocking the PD-1 / PD-L1 interaction by the exemplary anti-TIGIT / PD-L1 bsAb of the present invention was able to enhance NFAT-mediated luciferase activity in a dose-dependent manner.

[0048] [Figures 12A-12B] Figures 12A and 12B illustrate the specific binding of the present exemplary anti-TIGIT / PD-L1 bsAb to the human TIGIT protein.

[0049] [Figure 13] Figure 13 illustrates that effective blockade of the TIGIT / CD155 interaction by the exemplary anti-TIGIT / PD-L1 bsAb of the present invention was able to enhance NFAT-mediated luciferase activity in a dose-dependent manner.

[0050] [Figure 14] Figure 14 illustrates the antagonistic activity of the present invention's exemplary anti-TIGIT / PD-L1 bsAb in a Jurkat cell-based bifunctional assay.

[0051] [Figure 15] Figure 15 illustrates that the exemplary anti-TIGIT / PD-L1 bsAb of the present invention significantly enhanced IFN-γ production in human primary CD8+ T cells in a concentration-dependent manner. [Modes for carrying out the invention]

[0052] Detailed explanation definition It should be noted that the terms "a" or "an" refer to one or more such entities. For example, "an antibody" is understood to refer to one or more antibodies. In this specification, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably.

[0053] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of “sequence identity” with respect to another sequence; this means that when aligned, that percentage of bases (or amino acids) are the same when comparing these two sequences. This alignment and percentage homology or percentage sequence identity can be determined using software programs known in the art, for example, using the software program described in Ausubel et al. (2007), Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program that uses default parameters is BLAST. In particular, the programs BLASTN and BLASTP use the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sort order = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. A bioequivalent polynucleotide is a polynucleotide that has the specified percentage homology above and encodes a polypeptide having the same or similar biological activity.

[0054] The term “equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence that exhibits some degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that exhibits some degree of homology with it or its complement. In one aspect, homologs of nucleic acids can hybridize to that nucleic acid or its complement. Similarly, “equivalent polypeptide” refers to a polypeptide that exhibits some degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof when compared to a reference polypeptide or reference polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0055] As used herein, “antibody” or “antigen-binding polypeptide” refers to a polypeptide or polypeptide complex that specifically recognizes and specifically binds to an antigen. Antibodies may include complete antibodies, any antigen-binding fragment thereof, or a single chain thereof. Therefore, the term “antibody” includes any protein molecule or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having biological activity to bind to an antigen. Examples of such include, but are not limited to, the complementarity-determining region (CDR) or its ligand-binding portion of a heavy or light chain, the heavy or light chain variable region, the heavy or light chain constant region, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0056] The terms “antibody fragment” or “antigen-binding fragment,” as used herein, refer to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen recognized by an intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically modified protein that acts like an antibody by binding to a specific antigen and forming a complex.

[0057] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V) of immunoglobulins. H ) and light chain (V L This shows a fusion protein of the variable region of ). In some aspects, these regions are linked by short linker peptides of 10 to about 25 amino acids. The linkers can be rich in glycine for flexibility, and similarly, they can also be rich in serine or threonine for solubility. H The N-terminus of V L It can be linked to the C-terminus of and vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Various scFv molecules are known in this art, for example, described in U.S. Patent No. 5,892,019.

[0058] The term antibody encompasses a broad range of polypeptide classes that can be biochemically distinguished. Those skilled in the art will understand that the heavy chain is classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4). It is the properties of this chain that determine the “class” of an antibody, such as IgG, IgM, IgA, IgG, or IgE. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and known to confer functional specialization. Various modifications of each of these classes and isotypes are readily identifiable to those skilled in the art in light of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are clearly within the scope of this disclosure, and the following discussion generally focuses on the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule consists of two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy-chain polypeptides with a molecular weight of 53,000–70,000. These four chains are typically linked by disulfide bonds in a Y-shaped configuration, with the light chains acting as arms, supporting the heavy chains that begin at a Y-shaped opening and continue through a variable region.

[0059] The antibodies, antigen-binding polypeptides, variants, or derivatives of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-like antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv(scFv), single-chain antibodies, disulfide-linked Fv(sdFv), fragments containing either a VK domain or a VH domain, fragments produced by Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibody disclosed herein). The immunoglobulin molecules or antibody molecules of the present disclosure may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0060] The terms "specifically bind" or "have specificity for" generally mean that an antibody binds to an epitope via its antigen-binding domain, and that this binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to limit the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with higher specificity than it has for the relevant epitope "D."

[0061] As used herein, the terms “to treat” or “treatment” refer to both therapeutic treatment and preventive or deterrent measures aimed at preventing or slowing (mitigating) unwanted physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, whether detectable or undetectable, relief of symptoms, reduction of disease spread, a stabilized (i.e., non-worsening) state of the disease, delay or slowing of disease progression, improvement or reduction of the disease state, etc. This includes, but is not limited to, remission (whether partial or complete). "Treatment" can also mean extending survival compared to the expected survival if no treatment was received. Those requiring treatment have a medical condition. This also includes persons who already have a disability, as well as persons who are prone to developing a condition or disability, or who will be prevented from developing a condition or disability.

[0062] The term "subject," "individual," "animal," "patient," or "mammal" refers to any subject for which diagnosis, prognosis, or treatment is desired, particularly mammalian subjects. Mammalian subjects include humans, domesticated animals, farm animals, and zoo animals, sports animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, and dairy cows.

[0063] Where used herein, phrases such as “to patients in need of treatment” or “subjects in need of treatment” include subjects, e.g., mammalian subjects, who would benefit from the administration of the antibodies or compositions of this disclosure used, for example, for detection, for diagnostic procedures, and / or for treatment.

[0064] Single-domain PD-L1 antibody This disclosure provides a single-chain anti-PD-L1 antibody exhibiting high affinity for the human PD-L1 protein. The antibody demonstrates potent binding and inhibitory activity, making it useful for therapeutic and diagnostic applications. Importantly, when incorporated as one of the targeting units in various different forms of bispecific antibodies, certain resulting bispecific antibodies exhibited remarkable properties that establish the further utility of these single-domain anti-PD-L1 antibodies.

[0065] Accordingly, one embodiment of the present disclosure provides a single-domain antibody and a polypeptide containing such a single-domain antibody. In some embodiments, the polypeptide is a bispecific antibody, a triplicate antibody, or a multispecific antibody.

[0066] In some embodiments, the single-domain antibody has binding specificity to the human PD-L1 protein and includes complementarity-determining regions 1 (CDR1), 2, and 3. In some embodiments, CDR1, 2, and 3 each contain the amino acid sequences of SEQ ID NO: 55, 56, and 57, respectively. In some embodiments, CDR1, 2, and 3 each contain the amino acid sequences of SEQ ID NO: 113, 49, and 50, respectively. In some embodiments, CDR1, 2, and 3 each contain one of the CDR1, CDR2, and CDR3 from the antibodies provided in Table 1 (e.g., SEQ ID NOs: 1 to 36).

[0067] In one embodiment, in the antibody, CDR1 contains the amino acid sequence of SEQ ID NO: 55, CDR2 contains the amino acid sequence of SEQ ID NO: 56, and CDR3 contains the amino acid sequence of SEQ ID NO: 57. SEQ ID NOs: 55, 56, and 57 are CDRs of the antibody ALP-Tan-3p-93 and its humanized counterparts 93_VH-1 to 93_VH-9. In some embodiments, CDR1, CDR2, and CDR3 each contain SEQ ID NOs: 55, 56, and 57, except that they involve the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitutions are conservative substitutions.

[0068] In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody includes one or more revertant mutations that are tested to improve the properties of the grafted antibody. In some embodiments, the revertant mutations are selected from the group consisting of 37F, 47F, 49A, 78V, and 94A according to Kabat numbering. In some embodiments, the humanized antibody includes all of the revertant mutations 37F, 47F, 49A, 78V, and 94A according to Kabat numbering.

[0069] Exemplary humanized antibodies include 93_VH-1, 93_VH-2, 93_VH-3, 93_VH-4, 93_VH-5, 93_VH-6, 93_VH-7, 93_VH-8, and 93_VH-9. Exemplary sequences are SEQ ID NOs. 114 to 122. In some embodiments, the humanized antibody has the amino acid sequence of SEQ ID NO. 119.

[0070] In some embodiments, the antibody CDR1 contains the amino acid sequence of SEQ ID NO: 113, CDR2 contains the amino acid sequence of SEQ ID NO: 49, and CDR3 contains the amino acid sequence of SEQ ID NO: 50. SEQ ID NOs: 113, 49, and 50 are CDRs of the humanized antibodies 112-VHH1-PTM, 112-VHH2-PTM, 112-VHH3-PTM, 112-VHH4-PTM, 112-VHH5-PTM, 112-VHH6-PTM, or 112-VHH7-PTM. Compared to the original antibody ALP-Tan-3p-112, these humanized antibodies contain an N34Q substitution (Kabat numbering) in CDR1 (SEQ ID NO: 48) to prevent post-translational modification. In some embodiments, CDR1, CDR2, and CDR3 include SEQ ID NOs: 113, 49, and 50, respectively, except that they involve the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitutions are conservative substitutions.

[0071] In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody contains one or more reverse mutations selected from the group consisting of 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the humanized antibody contains all of the reverse mutations 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 123 to 130. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO. 127. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO. 130.

[0072] In some embodiments, the antibodies CDR1, CDR2, and CDR3 each contain one of the antibodies provided in Table 1 (e.g., SEQ ID NOs. 1 to 36). In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 38, and 39. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 42. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 43, 44, and 45. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47.

[0073] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 48, 49, and 50, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 51, and 52, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 53, and 54, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 47, respectively.

[0074] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 55, 56, and 57, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 58, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 53, and 39, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 59, and 60, respectively.

[0075] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 61, 62, and 63, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 64, 65, and 66, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 67, 68, and 69, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 70, 71, and 72, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 73, and 74, respectively.

[0076] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 75, and SEQ ID NO: 76. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 64, SEQ ID NO: 77, and SEQ ID NO: 66. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 78, and SEQ ID NO: 79. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 83, and SEQ ID NO: 47.

[0077] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 85, and 86, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 64, 87, and 66, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 88, and 89, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 90, and 91, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 92, 93, and 94, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 98, and SEQ ID NO: 99, respectively.

[0078] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 106, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively.

[0079] In some embodiments, the antibody contains an amino acid sequence selected from SEQ ID NOs: 1 to 36.

[0080] In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that compete with any of the antibodies disclosed herein for binding to human PD-L1 are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that bind to the same epitope as any of the antibodies disclosed herein are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments comprising VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of the antibodies disclosed herein are also provided.

[0081] Compositions comprising an antibody or polypeptide and a pharmaceutically acceptable carrier are also provided.

[0082] It will also be understood by those skilled in the art that antibodies such as those disclosed herein may be modified, resulting in the antibody having a different amino acid sequence from the naturally occurring conjugated polypeptide from which the antibody is derived. For example, a polypeptide or amino acid sequence derived from a given protein may be similar to the starting sequence, for example, having a certain percentage of identity to the starting sequence, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the modified antibody or fragment retains a given CDR sequence.

[0083] Also provided are bispecific and multispecific antibodies comprising one, two, three, or four units of a single-domain anti-PD-L1 antibody as disclosed herein, and one or more other specificities (other than PD-L1).

[0084] This disclosure provides bispecific and multispecific antibodies having binding specificity to at least human PD-L1 protein and human CD47 protein. PD-L1 is a crucial "don't find me" signal to the adaptive immune system, while CD47 transmits an antiphatic "don't eat me" signal to the innate immune system. They are often overexpressed on the surface of human tumors. Therefore, dually targeting both innate and adaptive immune checkpoints would likely maximize antitumor therapeutic efficacy and induce a longer-lasting response.

[0085] In some embodiments, bispecific and multispecific antibodies include an anti-PD-L1 moiety containing at least a single-domain anti-PD-L1 antibody. As will be apparent, single-chain anti-PD-L1 antibodies have high affinity for the human PD-L1 protein. These antibodies exhibit potent binding and inhibitory activity, making them useful for therapeutic and diagnostic applications.

[0086] In some embodiments, the single-domain antibody has binding specificity to the human PD-L1 protein and includes complementarity-determining regions 1 (CDR1), 2, and 3. In some embodiments, CDR1, 2, and 3 each contain the amino acid sequences of SEQ ID NO: 55, 56, and 57, respectively. In some embodiments, CDR1, 2, and 3 each contain the amino acid sequences of SEQ ID NO: 113, 49, and 50, respectively. In some embodiments, CDR1, 2, and 3 each contain one of the CDR1, CDR2, and CDR3 from the antibodies provided in Table 1 (e.g., SEQ ID NOs: 1 to 36).

[0087] In one embodiment, in the antibody, CDR1 contains the amino acid sequence of SEQ ID NO: 55, CDR2 contains the amino acid sequence of SEQ ID NO: 56, and CDR3 contains the amino acid sequence of SEQ ID NO: 57. SEQ ID NOs: 55, 56, and 57 are CDRs of the antibody ALP-Tan-3p-93 and its humanized counterparts 93_VH-1 to 93_VH-9. In some embodiments, CDR1, CDR2, and CDR3 each contain SEQ ID NOs: 55, 56, and 57, except that they involve the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitutions are conservative substitutions.

[0088] In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody includes one or more revertant mutations that are tested to improve the properties of the grafted antibody. In some embodiments, the revertant mutations are selected from the group consisting of 37F, 47F, 49A, 78V, and 94A according to Kabat numbering. In some embodiments, the humanized antibody includes all of the revertant mutations 37F, 47F, 49A, 78V, and 94A according to Kabat numbering.

[0089] Exemplary humanized antibodies include 93_VH-1, 93_VH-2, 93_VH-3, 93_VH-4, 93_VH-5, 93_VH-6, 93_VH-7, 93_VH-8, and 93_VH-9. Exemplary sequences are SEQ ID NOs. 114 to 122. In some embodiments, the humanized antibody has the amino acid sequence of SEQ ID NO. 119.

[0090] In some embodiments, the antibody CDR1 contains the amino acid sequence of SEQ ID NO: 113, CDR2 contains the amino acid sequence of SEQ ID NO: 49, and CDR3 contains the amino acid sequence of SEQ ID NO: 50. SEQ ID NOs: 113, 49, and 50 are CDRs of the humanized antibodies 112-VHH1-PTM, 112-VHH2-PTM, 112-VHH3-PTM, 112-VHH4-PTM, 112-VHH5-PTM, 112-VHH6-PTM, or 112-VHH7-PTM. Compared to the original antibody ALP-Tan-3p-112, these humanized antibodies contain an N34Q substitution (Kabat numbering) in CDR1 (SEQ ID NO: 48) to prevent post-translational modification. In some embodiments, CDR1, CDR2, and CDR3 include SEQ ID NOs: 113, 49, and 50, respectively, except that they involve the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitutions are conservative substitutions.

[0091] In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody contains one or more reverse mutations selected from the group consisting of 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the humanized antibody contains all of the reverse mutations 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 123 to 130. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO. 127. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO. 130.

[0092] In some embodiments, the antibodies CDR1, CDR2, and CDR3 each contain one of the antibodies provided in Table 1 (e.g., SEQ ID NOs. 1 to 36). In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 38, and 39. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 42. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 43, 44, and 45. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47.

[0093] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 48, 49, and 50, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 51, and 52, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 53, and 54, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 47, respectively.

[0094] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 55, 56, and 57, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 58, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 53, and 39, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 59, and 60, respectively.

[0095] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 61, 62, and 63, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 64, 65, and 66, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 67, 68, and 69, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 70, 71, and 72, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 73, and 74, respectively.

[0096] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 75, and SEQ ID NO: 76. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 64, SEQ ID NO: 77, and SEQ ID NO: 66. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 78, and SEQ ID NO: 79. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 83, and SEQ ID NO: 47.

[0097] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 85, and 86, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 64, 87, and 66, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 88, and 89, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 90, and 91, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 92, 93, and 94, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 98, and SEQ ID NO: 99, respectively.

[0098] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 106, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively.

[0099] In some embodiments, the antibody contains an amino acid sequence selected from SEQ ID NOs: 1 to 36.

[0100] In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that compete with any of the antibodies disclosed herein for binding to human PD-L1 are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that bind to the same epitope as any of the antibodies disclosed herein are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments comprising VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of the antibodies disclosed herein are also provided.

[0101] In some embodiments, the anti-CD47 moiety of a bispecific or multispecific antibody has one pair (or two pairs in some embodiments) of heavy chain variable regions (VH) and light chain variable regions (VL). The VH may include VH CDR1, VH CDR, and VH CDR3. The VL may include VL CDR1, VL CDR2, and VL CDR3.

[0102] In some embodiments, VH CDR1, VH CDR, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 141 to 146, respectively. These CDRs are derived from the parental anti-CD47 antibody 34C5. In some embodiments, VH contains the amino acid sequence of SEQ ID NO. 133, and VL contains the amino acid sequence of SEQ ID NO. 134 (Table 5).

[0103] In some embodiments, VH CDR1, VH CDR, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 135 to 140, respectively. These CDRs are derived from the parental anti-CD47 antibody 13H3. In some embodiments, VH contains the amino acid sequence of SEQ ID NO. 131, and VL contains the amino acid sequence of SEQ ID NO. 132 (Table 5).

[0104] Bispecific antibodies can take any form, including the form illustrated in Figure 3. In one embodiment, the bispecific antibody is symmetrical. An example is provided in Figure 3A, in which two single-domain anti-PD-L1 antibodies are fused to the respective N-terminuses of the heavy chain of an anti-CD47 antibody, optionally via a linker. In the example in Figure 3B, a single-domain anti-PD-L1 antibody is fused to the respective N-terminuses of the light chain of an anti-CD47 antibody, optionally via a linker.

[0105] In the example in Figure 3C, a single-domain anti-PD-L1 antibody is fused to the C-terminus of each light chain (constant region) of the anti-CD47 antibody via a linker as needed. In the example in Figure 3D, a single-domain anti-PD-L1 antibody is fused to the C-terminus of the Fc portion of the heavy chain of the anti-CD47 antibody via a linker as needed.

[0106] Bispecific antibodies can also be asymmetric, for example, as illustrated in Figures 3E-3F. In Figure 3E, two single-domain anti-PD-L1 antibodies are ligated in series to the N-terminus of one Fc chain. The other Fc chain is ligated with anti-CD47 The Fab unit is fused to the N-terminus. Slightly different, in Figure 3F, the anti-CD47 portion contains a single-chain fragment (scFv).

[0107] Bispecific antibodies may contain constant regions from either IgG type (e.g., IgG1 and IgG4).

[0108] Compositions comprising an antibody or polypeptide and a pharmaceutically acceptable carrier are also provided.

[0109] It will also be understood by those skilled in the art that antibodies such as those disclosed herein may be modified, resulting in the antibody having a different amino acid sequence from the naturally occurring conjugated polypeptide from which the antibody is derived. For example, a polypeptide or amino acid sequence derived from a given protein may be similar to the starting sequence, for example, having a certain percentage of identity to the starting sequence, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the modified antibody or fragment retains a given CDR sequence.

[0110] This disclosure provides bispecific and multispecific antibodies having binding specificity to at least human PD-L1 protein and human TIGIT protein. PD-L1 is a crucial "don't find me" signal to the adaptive immune system, while TIGIT helps tumor and infected cells evade the immune response. They are often overexpressed on the surface of human tumors. Therefore, dually targeting both innate and adaptive immune checkpoints would likely maximize antitumor therapeutic efficacy and induce a longer-lasting response.

[0111] In some embodiments, bispecific and multispecific antibodies include an anti-PD-L1 moiety containing at least a single-domain anti-PD-L1 antibody. As will be apparent, single-chain anti-PD-L1 antibodies have high affinity for the human PD-L1 protein. These antibodies exhibit potent binding and inhibitory activity, making them useful for therapeutic and diagnostic applications.

[0112] In some embodiments, the single-domain antibody has binding specificity to the human PD-L1 protein and includes complementarity-determining regions 1 (CDR1), 2, and 3. In some embodiments, CDR1, 2, and 3 each contain the amino acid sequences of SEQ ID NO: 55, 56, and 57, respectively. In some embodiments, CDR1, 2, and 3 each contain the amino acid sequences of SEQ ID NO: 113, 49, and 50, respectively. In some embodiments, CDR1, 2, and 3 each contain one of the CDR1, CDR2, and CDR3 from the antibodies provided in Table 1 (e.g., SEQ ID NOs: 1 to 36).

[0113] In one embodiment, in the antibody, CDR1 contains the amino acid sequence of SEQ ID NO: 55, CDR2 contains the amino acid sequence of SEQ ID NO: 56, and CDR3 contains the amino acid sequence of SEQ ID NO: 57. SEQ ID NOs: 55, 56, and 57 are CDRs of the antibody ALP-Tan-3p-93 and its humanized counterparts 93_VH-1 to 93_VH-9. In some embodiments, CDR1, CDR2, and CDR3 each contain SEQ ID NOs: 55, 56, and 57, except that they involve the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitutions are conservative substitutions.

[0114] In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody includes one or more revertant mutations that are tested to improve the properties of the grafted antibody. In some embodiments, the revertant mutations are selected from the group consisting of 37F, 47F, 49A, 78V, and 94A according to Kabat numbering. In some embodiments, the humanized antibody includes all of the revertant mutations 37F, 47F, 49A, 78V, and 94A according to Kabat numbering.

[0115] Exemplary humanized antibodies include 93_VH-1, 93_VH-2, 93_VH-3, 93_VH-4, 93_VH-5, 93_VH-6, 93_VH-7, 93_VH-8, and 93_VH-9. Exemplary sequences are SEQ ID NOs. 114 to 122. In some embodiments, the humanized antibody has the amino acid sequence of SEQ ID NO. 119.

[0116] In some embodiments, the antibody CDR1 contains the amino acid sequence of SEQ ID NO: 113, CDR2 contains the amino acid sequence of SEQ ID NO: 49, and CDR3 contains the amino acid sequence of SEQ ID NO: 50. SEQ ID NOs: 113, 49, and 50 are CDRs of the humanized antibodies 112-VHH1-PTM, 112-VHH2-PTM, 112-VHH3-PTM, 112-VHH4-PTM, 112-VHH5-PTM, 112-VHH6-PTM, or 112-VHH7-PTM. Compared to the original antibody ALP-Tan-3p-112, these humanized antibodies contain an N34Q substitution (Kabat numbering) in CDR1 (SEQ ID NO: 48) to prevent post-translational modification. In some embodiments, CDR1, CDR2, and CDR3 include SEQ ID NOs: 113, 49, and 50, respectively, except that they involve the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitutions are conservative substitutions.

[0117] In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody contains one or more reverse mutations selected from the group consisting of 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the humanized antibody contains all of the reverse mutations 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. In some embodiments, the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 123 to 130. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO. 127. In some embodiments, the antibody contains the amino acid sequence of SEQ ID NO. 130.

[0118] In some embodiments, the antibodies CDR1, CDR2, and CDR3 each contain one of the antibodies provided in Table 1 (e.g., SEQ ID NOs. 1 to 36). In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 38, and 39. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 42. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 43, 44, and 45. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47.

[0119] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 48, 49, and 50, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 51, and 52, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 53, and 54, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 47, respectively.

[0120] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 46, and 47, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 55, 56, and 57, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 40, 41, and 58, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 53, and 39, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 59, and 60, respectively.

[0121] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 61, 62, and 63, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 64, 65, and 66, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 67, 68, and 69, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 70, 71, and 72, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 37, 73, and 74, respectively.

[0122] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 75, and SEQ ID NO: 76. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 64, SEQ ID NO: 77, and SEQ ID NO: 66. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 78, and SEQ ID NO: 79. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 83, and SEQ ID NO: 47.

[0123] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 85, and 86, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 64, 87, and 66, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 88, and 89, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 84, 90, and 91, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 92, 93, and 94, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NOs. 95, 96, and 97, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 98, and SEQ ID NO: 99, respectively.

[0124] In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 106, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively. In one embodiment, CDR1, CDR2, and CDR3 each contain the amino acid sequences of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively.

[0125] In some embodiments, the antibody contains an amino acid sequence selected from SEQ ID NOs: 1 to 36.

[0126] In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that compete with any of the antibodies disclosed herein for binding to human PD-L1 are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that bind to the same epitope as any of the antibodies disclosed herein are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments comprising VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of the antibodies disclosed herein are also provided.

[0127] In some embodiments, the anti-TIGIT moiety of a bispecific or multispecific antibody has one pair (or two pairs in some embodiments) of heavy chain variable regions (VH) and light chain variable regions (VL). The VH may include VH CDR1, VH CDR, and VH CDR3. The VL may include VL CDR1, VL CDR2, and VL CDR3.

[0128] In some embodiments, VH CDR1, VH CDR, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 173 to 178, respectively. In some embodiments, VH contains the amino acid sequence of SEQ ID NO. 171, and VL contains the amino acid sequence of SEQ ID NO. 172 (Table 7).

[0129] The bispecific antibody can take any form, including the form illustrated in Figure 9. In one embodiment, the bispecific antibody is preferably symmetrical. In one embodiment, the single-domain anti-PD-L1 antibody is located at the C-terminus of the anti-TIGIT moiety.

[0130] An example configuration is provided in Figure 9A, in which two single-domain anti-PD-L1 antibodies are fused to the respective C-terminuses of the heavy chain constant region of an anti-TIGIT antibody, via a linker as needed. In the example in Figure 9B, each heavy chain contains two copies of the single-domain anti-PD-L1 antibody.

[0131] In the example shown in Figure 9C, a single-domain anti-PD-L1 antibody is fused to the C-terminus of each light chain (constant region) of the anti-TIGIT antibody via a linker as needed.

[0132] Bispecific antibodies may contain constant regions from either IgG type (e.g., IgG1 and IgG4).

[0133] Compositions comprising an antibody or polypeptide and a pharmaceutically acceptable carrier are also provided.

[0134] It will also be understood by those skilled in the art that antibodies such as those disclosed herein may be modified, resulting in the antibody having a different amino acid sequence from the naturally occurring conjugated polypeptide from which the antibody is derived. For example, a polypeptide or amino acid sequence derived from a given protein may be similar to the starting sequence, for example, having a certain percentage of identity to the starting sequence, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the modified antibody or fragment retains a given CDR sequence.

[0135] Method for preparing polynucleotides encoding antibodies and antibodies. The Disclosure also provides isolated polynucleotides or nucleic acid molecules encoding antibodies, variants thereof, or derivatives thereof. The polynucleotides of the Disclosure may encode the entire heavy chain variable region and light chain variable region of an antigen-binding polypeptide, its variant, or derivative on the surface of the same polynucleotide molecule or on the surface of a separate polynucleotide molecule. In addition, the polynucleotides of the Disclosure may encode a portion of the heavy chain variable region and light chain variable region of an antigen-binding polypeptide, its variant, or derivative on the surface of the same polynucleotide molecule or on the surface of a separate polynucleotide molecule.

[0136] Various methods for producing antibodies are widely known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of this disclosure are fully human. Fully human antibodies can be produced using the techniques described in the art and as described herein. For example, a fully human antibody against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such an antibody in response to an antigen challenge, but in which the endogenous locus has been deactivated. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140 (all of which are incorporated by reference).

[0137] Cancer treatment As described herein, the antibodies, bispecific antibodies, polypeptides, variants, or derivatives of this disclosure may be used in certain therapeutic and diagnostic methods.

[0138] This disclosure further relates to antibody-based therapies, which involve administering the antibodies of this disclosure to a patient, for example, an animal, mammal, or human, to treat one or more of the disorders or conditions described herein. The therapeutic compounds of this disclosure include, but are not limited to, the antibodies of this disclosure (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding the antibodies of this disclosure (including their variants and derivatives as described herein).

[0139] The antibodies of this disclosure can also be used to treat or inhibit cancer. PD-L1 can be overexpressed in tumor cells. Tumor-derived PD-L1 can bind to PD-1 on the surface of immune cells, thereby limiting anti-tumor T cell immunity. Results from the use of PD-L1-targeting small molecule inhibitors or monoclonal antibodies in mouse tumor models indicate that targeted PD-L1 therapy is an important alternative and practical approach for effective control of tumor growth. As revealed in the experimental examples, anti-PD-L1 antibodies activated adaptive immune response mechanisms that can lead to improved survival in cancer patients.

[0140] Accordingly, in some embodiments, methods are provided for treating cancer in patients who require treatment of cancer. In one embodiment, the method involves administering an effective amount of the antibody of this disclosure to the patient. In some embodiments, at least one cancer cell (e.g., stromal cell) in the patient expresses, overexpresses, or is induced to express PD-L1. Induction of PD-L1 expression can be carried out, for example, by administration of a tumor vaccine or by radiotherapy.

[0141] Tumors expressing the PD-L1 protein include bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, and small cell lung cancer. Therefore, the antibodies disclosed at this time can be used to treat any one or more of such cancers.

[0142] composition This disclosure also provides pharmaceutical compositions comprising an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0143] In specific embodiments, the term “pharmaceutically acceptable” means that it has been approved by a federal or state regulatory agency, or that it has been listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more specifically for use in humans. Furthermore, “pharmaceutically acceptable carrier” generally refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any kind of compounding aid.

[0144] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as water, and oils (including oils of petroleum, animal, plant, or synthetic origin), such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Salt solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. The composition may also contain small amounts of wetting agents or emulsifiers, or pH buffering agents (e.g., acetates, citrates, or phosphates), if desired. Antimicrobial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity, such as sodium chloride or dextrose, are also conceivable. These compositions can take the form of liquids, suspensions, emulsions, tablets, pills, capsules, powders, and sustained-release formulations. The compositions can be formulated as suppositories with conventional binders and carriers (e.g., triglycerides). Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin (which is incorporated herein by reference). Such compositions preferably contain a therapeutically effective amount of antigen-binding polypeptide in a purified form, along with an appropriate amount of carrier that provides a form suitable for administration to a patient.The formulation should be suitable for the mode of administration. Parental preparations can be enclosed in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0145] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, the composition for intravenous administration is a liquid in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the components are supplied separately or mixed together in unit dosage forms, for example, as a dry lyophilized powder or a water-free concentrate in an airtight container (e.g., an ampoule or sachet) indicating the amount of the active agent. If the composition is to be administered by injection, the composition can be administered in an injection bottle containing sterile pharmaceutical-grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the components may be mixed before administration. [Examples]

[0146] Example 1. Generation of alpaca single-domain antibody against human PD-L1 This example demonstrates how an anti-human PD-L1 single-domain antibody was generated using alpaca immunization, followed by phage library construction and selection.

[0147] Antigen: Recombinant human PD-L1 / hFc fusion protein was used as an immunogen to produce anti-human PD-L1 antibodies. The fusion protein, which includes the entire extracellular domain of human PD-L1 fused to the human immunoglobulin Fc domain, was used as the immunogen.

[0148] immunity Alpacas were initially immunized subcutaneously (SC) on day 0 with a 1:1 mixture of 600 μg of mouse PD-L1 and complete Freudian adjuvant, on day 21 with 250 μg of mouse PD-L1 with incomplete Freudian adjuvant, and on day 42 with 250 μg of human PD-L1 with incomplete Freudian adjuvant. The immune response was monitored by measuring the titer for anti-PD-L1 binding.

[0149] Library construction and screening Alpaca PBMCs were collected, and antibody phage display libraries were generated by RNA isolation, cDNA reverse transcription, PCR amplification, and cloning into phage display vectors. The libraries were then subjected to one liquid-phase panning and one solid-phase panning. Generally, the libraries were incubated in immunotubes or beads coated with biotinylated PD-L1. Unbound phages were removed by washing with PBST 5 to 20 times. Each selection was followed by a total of three panning cycles.

[0150] The binding agent sequence was amplified from antigen-positive phages by PCR and confirmed by DNA sequencing. Specific antibodies and their CDR region sequences are provided in the table below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1A-1] [Table 1A-2] [Table 1A-3]

[0151] Example 2. Binding and blocking activity of alpaca monoclonal antibody against human PD-L1. Several binding and blocking properties of the antibodies of the present invention were characterized by Gator. An anti-his probe was first loaded onto the tip, followed by human PD-L1-his to capture the antigen. The antibodies were then injected, and binding curves were recorded. Finally, human PD1 / hFc was injected to determine if the antibodies could block the interaction between PD-1 and PD-L1. As shown in Figure 1, ALP-Tan-3p-112, ALP-Tan-3p-93, and ASP-30-46 all effectively blocked the interaction between PD-1 and PD-L1. Affinity was further confirmed by Biacore T200. [Table 2] Example 3. Humanization of anti-PD-L1 alpaca monoclonal antibody. Humanized mAbs were created using the variable region genes ALP-Tan-3p-93 and ALP-Tan-3p-112. In the first step of this process, the amino acid sequences of ALP-Tan-3p-93 and ALP-Tan-3p-112 were compared against available databases of human immunoglobulin gene sequences to find the human germline immunoglobulin gene sequence that best matched overall. For ALP-Tan-3p-93, the closest match to a human gene was IGHV3-23. * It was the 04 gene. Subsequently, CDR1, CDR2, and CDR3 of ALP-Tan-3p-93 were identified as IGHV3-23. * We designed humanized variable domain sequences transplanted into the framework sequence of gene 04. For ALP-Tan-3p-112, the closest match to a human is IGHV3-48. * It was gene 03. Subsequently, CDR1, CDR2, and CDR3 of ALP-Tan-3p-112 were identified as IGHV3-48. *We designed a humanized variable domain sequence transplanted into the framework sequence of gene 03. Meanwhile, a single-residue mutation (N34Q, Kabat numbering) was introduced into CDR1 to reduce the risk of post-translational modification. Subsequently, a 3D model was generated to determine whether there are any arbitrary framework positions where the substitution of alpaca amino acids with human amino acids could affect binding and / or the CDR conformation. [Table 3-1] [Table 3-2] [Table 3A]

[0152] Example 4. Detailed kinetics of humanized anti-PD-L1 monoclonal antibody. To investigate the binding kinetics of humanized antibodies, this example further performed a complete kinetic affinity study by monitoring the association and dissociation of various doses of antigen (100 nM, 50 nM, 25 nM, 12.5 nM, 6.15 nM, 3.125 nM, 1.5625 nM) with various monoclonal antibodies using Biacore. As shown in Table 4, the affinity of 112-VHH5-PTM was comparable to that of the ALP-Tan-3p-112 chimeric antibody. The affinities of 93VH-4, 93VH-6, and 93VH-8 were comparable to those of the ALP-Tan-3p-93 chimeric antibody. [Table 4]

[0153] Example 5. Binding characteristics of humanized anti-PD-L1 antibody The binding characteristics of the humanized anti-PD-L1 antibody of the present application were first evaluated by an ELISA assay. Briefly, 100 μl of anti-PD-L1 antibody (93-VH6 or 112-VH47) at different concentrations as shown in Figure 1B was incubated in each well of a 96-well plate pre-coated with human His-PD-L1, and then goat anti-human IgG Fc HRP was added and analyzed by the color reaction of HRP with its substrate. As shown in Figure 1B, both the exemplary anti-PD-L1 antibodies 93-VH6 and 112-VH47 exhibited specific binding to human PD-L1 in a dose-dependent manner.

[0154] The binding ability of the anti-PD-L1 antibody of the present application was further evaluated by using Raji cells overexpressing human PD-L1. Briefly, 50 μl of Raji cells overexpressing human PD-L1 were seeded in a 96-well plate at a concentration of 2 * 10 5 cells / well. 50 μl of anti-PD-L1 antibody (93-VH6 or 112-VH47) at different concentrations as shown in Figure 1C was added to each well and incubated with the cells for 1 hour on ice. Then, the cells were washed twice with FACS buffer, supplemented with 100 μl of PE-anti hu IgG, and subsequent incubation was carried out for 1 hour on ice. After incubation, the cells in each well were collected for analysis by flow cytometry and resuspended in 65 μl of FACS buffer. As shown in Figure 1C, both the exemplary anti-PD-L1 antibodies 93-VH6 and 112-VH47 exhibited specific binding to Raji cells overexpressing human PD-L1 in a dose-dependent manner.

[0155] Example 6. T cell activation bioassay (NFAT) To test the ability of anti-PD-L1 antibodies to stimulate T cell responses, hPD-1-expressing Jurkat cells were used. Jurkat is a human T-cell leukemia cell line capable of activating NFAT-mediated luciferase expression upon TCR stimulation. In this assay, Jurkat cells transfected with the human PD-1 gene via lentivirus were used as responder cells. Raji-PD-L1 cells were used as antigen-presenting cells (APCs). Staphylococcal enterotoxin E (SEE) was used to stimulate TCR signaling. In this system, ectopically expressed hPD-L1 could suppress SEE-stimulated NF-AT-luciferase activity in Jurkat cells, while anti-PD-L1 antibodies could reverse NFAT-luciferase activity. In short, APCs (2.5 × 10⁻⁶ 4 (1 × 10) PD-1 expressing Jurkat T cells (1 × 10) in the presence of SEE stimulation. 5 The cells were co-cultured with (1) cells. Anti-PD-L1 antibody was added at the start of the culture. After 6 hours, the luciferase activity of the obtained cells was evaluated.

[0156] As shown in Figures 2A to 2C, all of the anti-PD-L1 antibodies tested blocked the PD-1 / PD-L1 interaction and therefore enhanced NFAT-mediated luciferase activity.

[0157] Example 7. Generation of PD-L1 / CD47 bispecific antibodies Two previously identified anti-CD47 antibodies, 13H3 and 34C5, and the anti-PD-L1 antibody, 93-VH-6, were selected to generate anti-CD47 / PD-L1 bispecific antibodies in "2:2" and "1:2" configurations (structures illustrated in Figure 3).

[0158] Figure 3A illustrates a bispecific antibody molecule in a "2-to-2" symmetrical configuration. Such a bispecific antibody may include two anti-PD-L1 single-domain antibodies, each linked via a GS linker to the heavy chain of an anti-CD47 Fab, which is linked to the Fc of either IgG1 or IgG4.

[0159] Figure 3B illustrates another "2-to-2" symmetrical form of bispecific antibody molecule. Such a bispecific antibody may include two anti-PD-L1 single-domain antibodies, each linked via a GS linker to the light chain of an anti-CD47 Fab, which is linked to the Fc of IgG1 or IgG4.

[0160] Figure 3C illustrates another "2-to-2" symmetrical form of bispecific antibody molecule. Such a bispecific antibody may include two anti-PD-L1 single-domain antibodies, each linked via a GS linker to the CL of the Fc of IgG1 or IgG4, respectively, linked to an anti-CD47 Fab.

[0161] Figure 3D illustrates another "2-to-2" symmetrical form of bispecific antibody molecule. This bispecific antibody includes an anti-CD47 Fab and an anti-PD-L1 single-domain antibody linked to CH3 via a GS linker, with either one IgG1 Fc or one IgG4 Fc.

[0162] Figure 3E shows a "2:1" asymmetrical bispecific antibody molecule. This bispecific antibody contains two tandem anti-PD-L1 single-domain antibodies linked by a single GS linker to the Fc of either IgG1 or IgG4, to which the anti-CD47 Fab is also linked. The Fc portion includes a knob-in-hole mutation at CH3 to reduce mispairing.

[0163] Figure 3F shows another "2:1" asymmetric form of a bispecific antibody molecule. This bispecific antibody contains two tandem anti-PD-L1 single-domain antibodies linked by a single GS linker to the Fc of either IgG1 or IgG4, to which anti-CD47 scFv is also linked. The Fc portion includes a knob-in-hole mutation at CH3 to reduce mispairing.

[0164] These bispecific antibodies were purified from 100 mL of transiently transfected supernatant of HEK293F cells using a protein A affinity column. The purity of each bispecific antibody was tested by HPLC and SDS-PAGE. [Table 5] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0165] Example 8. Anti-CD47 / PD-L1 bispecific antibody that blocks CD47 from binding to SIRPα. The assay was performed according to the instructions on the CD47 / SIRPα conjugation assay kit (Cisbio). Briefly, serially diluted antibodies, namely Tag1-CD47 and Tag2-SIRPa, were pre-mixed and incubated at room temperature for 15 minutes. Then, pre-mixed anti-Tag1-Tb3 and anti-Tag2-XL665 were added and incubated at RT for 1 hour. Fluorescence data were read using a PerkinElmer Envision plate reader with a laser light source. Anti-CD47 antibody (13H3 or 34C5) was used as a positive control in this assay.

[0166] The results, shown in Figures 4A and 4B, demonstrate that 34C5 exhibited stronger blocking activity than 13H3. Furthermore, 34C5-IgG1-93VH-6, 93VH6-13H3-H-IgG1, and 93VH6-13H3-L-IgG1 showed some loss of activity compared to their parental anti-CD47 monoclonal antibodies. However, the remaining PD-L1 / CD47 bispecific antibodies exhibited similar or even stronger SIRPα blocking activity compared to their parental anti-CD47 antibodies.

[0167] Example 9. T cell activation bioassay (NFAT) To test the ability of an anti-CD47 / PD-L1 bispecific antibody to stimulate a T cell response, hPD-1 expressing Jurkat cells were used. Jurkat is a human T-cell leukemia cell line capable of activating NF-AT activated luciferase expression upon TCR stimulation. In this assay, Jurkat cells transfected with a lentivirus containing the human PD-1 gene were used as responder cells. Raji-PD-L1 cells were used as antigen-presenting cells (APCs). Staphylococcal enterotoxin E (SEE) was used to stimulate TCR signaling. In this system, ectopically expressed huPD-L1 could suppress SEE-stimulated NF-AT-luciferase activity in Jurkat cells, while the anti-PD-L1 antibody could reverse NF-AT-luciferase activity. In short, APCs (2.5 × 10⁻⁶) 4 (1 × 10) PD-1 expressing Jurkat T cells (1 × 10) in the presence of SEE stimulation. 5 The cells were co-cultured with (1) cells. Anti-PD-L1 antibody was added at the start of the culture. After 6 hours, the luciferase activity of the obtained cells was evaluated.

[0168] As shown in Figure 5, all bispecific antibodies in a 2:2 configuration exhibited comparable or stronger efficacy in blocking PD-1 / PD-L1-mediated NF-AT-luciferase activity compared to the parental PD-L1 monoclonal antibody 93-VH-6.

[0169] Example 10. Anti-CD47 / PD-L1 bispecific antibody showed increased phagocytosis of tumor cells by human macrophages (MΦ). Monocytes were isolated from human blood and differentiated into macrophages in the presence of hGCSF for 6 days. Monocyte-derived macrophages (MDMs) were scraped, replated onto 24-well dishes, and adhered for 24 hours. Human tumor cell line RKO was selected as the target cell line and labeled with 1 mM CellTrace-Far Red for 20 minutes. MDMs were labeled with 1 mM CellTrace-Violet for 20 minutes, and then mixed with tumor cells in a 3:1 ratio per phagocytic cell. Anti-CD47 / PD-L1 bispecific antibody and corresponding control mAbs and combinations were added in various doses. After a 3-hour incubation, phagocytosis of the target cells was analyzed by flow cytometry. Phagocytosis was measured by gating macrophages and then evaluating the percentage of bi-positive cells.

[0170] As shown in Figures 6A and 6B, the anti-CD47 / PD-L1 bispecific antibody demonstrated higher ADCP efficacy than combination therapy with the parental monoclonal antibody and the clinical benchmark antibody.

[0171] Another study was conducted to further investigate whether isotype contributes to ADCP efficacy. This study compared the differences in ADCP assays between bispecific antibodies possessing hIgG1 Fc and hIgG4 Fc, respectively. The results are shown in Figure 6C. The ADCP efficacy of anti-CD47 / PD-L1 bispecific antibodies was generally similar when the isotype changed from hIgG1 to hIgG4.

[0172] Example 11. Binding of anti-CD47 / PD-L1 bispecific antibody to RKO cells. RKO cells are a human colon cancer cell line that expresses endogenous levels of human CD47 and human PD-L1 on its surface. RKO cells were incubated with serially diluted anti-CD47 / PD-L1 bispecific antibody, parental CD47 monospecific antibody, or PD-L1 monospecific antibody at 4°C for 30 minutes. The cells were then washed three times with FACS buffer, followed by incubation with APC-labeled secondary antibody at 4°C for 30 minutes. The cells were then washed three times with FACS buffer. Binding was measured by flow cytometry.

[0173] As shown in Figures 7A and 7B, the anti-CD47 / PD-L1 bispecific antibody in symmetric form exhibited either stronger or comparable binding ability to the parental PD-L1 monospecific antibody.

[0174] Example 12. RBC binding and RBC agglutination tests of anti-CD47 / PD-L1 bispecific antibodies 10.1 RBC Binding Assay Human RBCs were diluted to 1% in PBS and incubated with anti-CD47 / PD-L1 bispecific antibody (antibody dose was set starting at 200 nM and decreasing 3-fold increments) at 4°C for 1 hour, followed by the addition of PE-conjugated secondary antibody at 4°C for 30 minutes. Binding of the anti-CD47 / PD-L1 antibody to human RBCs was examined by flow cytometry.

[0175] As shown in Figure 8A, 93VH6-13H3-H-IgG1, 93VH6-13H3-L-IgG1, and 13H3-L-93VH6-IgG1 showed minimal or no RBC binding, similar to the 13H3 antibody. Of all the antibodies tested, the parental CD47 antibody 34C5 showed the strongest RBC binding.

[0176] 10.2 RBC Agglutination Assay Human RBCs were diluted to 1% in PBS and incubated in a round-bottom 96-well plate with dose-adjusted anti-CD47 / PD-L1 antibody (antibody dose was adjusted starting at 200 nM and decreasing 3-4 times increments) at room temperature for 2 hours. Evidence of hemagglutination is revealed by the presence of non-precipitating RBCs, which appear as a cloudy appearance compared to punctuate red dots in non-hemagglutinating RBCs.

[0177] As shown in Figure 8B, 93VH6-13H3-H-IgG1 and 93VH6-13H3-L-IgG1 were similar to the parent CD47 antibody 13H3, but showed no evaluable RBC agglutination. The reference antibody 5F9 showed RBC agglutination at four tested concentrations. Example 13. In vivo antitumor efficacy of anti-CD47 / PD-L1 bispecific antibody.

[0178] Twelve NOG mice were individually injected with human PBMCs in 0.2 mL of DPBS (iv, 5 × 10⁻¹⁰⁻¹ 6 (Individual / mouse). After 8 days, 1 x 10 6 RKO cells were subcutaneously inoculated into the right flank of mice. The average tumor size was 57 mm. 3 When the mice reached a certain stage, those with tumors were randomly divided into three groups (four mice per group), and each group was administered intraperitoneally with PBS, 93VH6-13H3-L-IgG1 (12 mg / kg), or 93VH6-13H3-L-IgG4 (12 mg / kg), respectively. Tumor volume and body weight were measured and recorded twice a week. On day 19, the animals were euthanized. As shown in Figure 8C, 93VH6-13H3-L-IgG1 treatment and 93VH6-13H3-L-IgG4 treatment showed significantly inhibited tumor growth compared to PBS, and from this, it can be concluded that the anti-CD47 / PD-L1 of this invention... The study suggested that bsAb has potent antitumor efficacy.

[0179] Example 14. Generation of PD-L1 / TIGIT bispecific antibodies Exemplary anti-PD-L1 single-domain antibodies (sdAbs), 93-VH6 and 112-VH47, were selected to generate anti-PD-L1 / TIGIT bispecific antibodies in various forms (structures exemplified in Figure 9).

[0180] In one configuration, two PD-L1 sdAbs are fused to the C-terminus of the heavy chain of the anti-TIGIT moiety via a G4S linker (represented as TIGIT-Fc-PD-L1), or to the C-terminus of the light chain of the anti-TIGIT moiety (represented as TIGIT-CL-PD-L1). Alternatively, four PD-L1 sdAbs, i.e., two in each tandem group, are linked to the C-terminus of the heavy chain of the TIGIT moiety via a G4S linker (represented as TIGIT-Fc-PD-L1). * (As shown as 2).

[0181] These bispecific antibodies were transiently transfected into HEK293F cell cultures. 100 mL of the supernatant was purified using a protein A affinity column. The purity of each bispecific antibody was confirmed by HPLC and SDS-PAGE. [Table 7] [Table 8-1] [Table 8-2] [Table 8-3]

[0182] Example 15. Binding characteristics of PD-L1 / TIGIT bsAb to PD-L1. The binding affinity of PD-L1 / TIGIT bsAb to recombinant human his-tagged PD-L1 protein was tested using BIACORE®. PD-L1 / TIGIT bsAb molecules or parental anti-PD-L1 sdAb were captured using a Protein A tip. A series of dilutions of human PD-L1 protein (6.25 nM to 100 nM) were injected onto the captured antibody at a flow rate of 10 μL / min. The antigen was associated for 180 seconds and dissociated for 1200 seconds. All experiments were performed using Biacore T200. Data analysis was performed using Biacore T200 evaluation software.

[0183] The data shows that the PD-L1 binding affinity of the PD-L1 / TIGIT bsAb molecule was not impaired compared to its parent anti-PD-L1 antibody (Table 9). [Table 9]

[0184] The binding of anti-PD-L1 / TIGIT bsAb molecules to human PD-L1 was further analyzed by ELISA. Briefly, 100 μl of anti-PD-L1 / TIGIT bsAb at different concentrations, as shown in Figures 10A and 10B, were analyzed using TIGIT-Fc-93-VH6 and TIGIT-Fc-93-VH6. * 2. TIGIT-CL-93-VH6 and TIGIT-Fc-112-VH47 were incubated in the respective wells of a 96-well plate pre-coated with human His-PD-L1, and the binding between anti-PD-L1 / TIGIT bsAb and human His-PD-L1 was then analyzed via goat anti-human IgG Fc HRP. As shown in Figures 10A and 10B, TIGIT-Fc-93-VH6, TIGIT-Fc-93-VH6 * 2. All PD-L1 / TIGIT bsAbs tested, including TIGIT-CL-93-VH6 and TIGIT-Fc-112-VH47, exhibited dose-dependent specific binding to human PD-L1.

[0185] Furthermore, the binding ability of the anti-PD-L1 / TIGIT bsAbs of this invention to PD-L1-expressing cells was analyzed using Raji-PD-L1 cells. Briefly, 50 μl of Raji cells overexpressing human PD-L1 were used to bind to 2 * 10 5 Cells were seeded in 96-well plates at a rate of cells / well. 50 μl of anti-PD-L1 antibody, either 112-VH47 or TIGIT-Fc-112-VH47, at different concentrations as shown in Figure 10C, was added to each well and incubated with the cells on ice for 1 hour. The cells were then washed twice with FACS buffer, supplemented with 100 μl of PE-anti-hu IgG, and incubated again on ice for 1 hour. After incubation, cells in each well were collected for flow cytometry analysis and resuspended in 65 μl of FACS buffer. As shown in Figure 10C, TIGIT-Fc-112-VH47 exhibited dose-dependent specific binding to human PD-L1-expressing Raji cells.

[0186] Example 16. PD-L1 antagonist activity of the PD-L1 / TIGIT bsAb molecule. To evaluate the PD-L1 antagonistic activity of the PD-L1 / TIGIT bsAb molecule, a functional assay based on PD-L1 cells was performed as described in Example 6.

[0187] As shown in Figure 11, TIGIT-Fc-93-VH6 * The 2 bsAb molecule showed comparable antagonistic activity to the parent 93-VH6 sdAb. TIGIT-Fc-93-VH6 showed an enhanced maximal effect compared to anti-93-VH6 sdAb, but exhibited a decreased EC50 in PD-L1 antagonist activity. TIGIT-CL-93-VH6 bsAb showed a comparable maximal effect compared to 93-VH6 sdAb, but exhibited a decreased EC50 in PD-L1 antagonist activity.

[0188] Example 17. Binding characteristics of PD-L1 / TIGIT bsAb to TIGIT. The binding of PD-L1 / TIGIT bsAb and its parent TIGIT antibody to recombinant His-tagged human TIGIT-ECD protein was investigated using Biacore T200. Antibodies were captured using a Protein A tip. A range of concentrations of His-tagged human TIGIT-ECD protein (0.78 nM to 12.5 nM) were injected onto the captured antibody at a flow rate of 10 μl / min. The association phase was 180 seconds, and the dissociation phase was 1200 seconds.

[0189] The results are shown in Table 10 below. The Biacore results for PD-L1 / TIGIT antibodies indicate that these bispecific antibodies are highly affinity binders for human TIGIT. As shown in the table, these PD-L1 / TIGIT antibodies had similar affinity to their parent TIGIT antibodies. [Table 10]

[0190] To evaluate the binding ability to the TIGIT protein, PD-L1 / TIGIT bsAbs were subjected to an ELISA binding assay for His-tagged human TIGIT. As shown in Figure 12, all tested PD-L1 / TIGIT bsAbs exhibited specific binding to TIGIT in a dose-dependent manner.

[0191] Example 18. TIGIT antagonist activity of PD-L1 / TIGIT bsAb To evaluate the TIGIT-blocking function of PD-L1 / TIGIT bsAb antibodies, an in vitro functional assay based on Jurkat cells was used. Briefly, human TIGIT and its counter-receptor CD226 were simultaneously overexpressed on the surface of Jurkat T cells, while their colligand, human CD155, was overexpressed on the surface of Raji cells. When these two cell types were co-cultured in the presence of superantigens, Jurkat cell activation was inhibited by negative signaling delivered to the Jurkat cell surface via TIGIT-CD155 ligation. The activation state of Jurkat cells was evaluated using a luciferase reporting system similar to that used in the PD-L1 blockade assay. When serially diluted PD-L1 / TIGIT bsAb or anti-TIGIT antibodies were added to the culture system, the antibodies could dose-dependently enhance luciferase expression in Jurkat-TIGIT-CD226 cells.

[0192] This assay demonstrated superior efficacy in blocking TIGIT / CD155 signaling and enhancing Jurkat cell activation when comparing the TIGIT-Fc-93-VH6 bsAb molecule to its parental TIGIT antibody (Figure 13). The other two forms, namely TIGIT-Fc-93-VH6, were also tested. * 2 bsAb and TIGIT-CL-93-VH6 bsAb showed TIGIT blocking activity comparable to that of the parental TIGIT antibody.

[0193] Example 19. Synergistic effect of PD-L1 / TIGIT bsAb in vitro To evaluate the synergistic effect of PD-L1 / TIGIT bsAb in enhancing T cell activation, we established a cell-based, robust in vitro functional assay. Briefly, human TIGIT, along with CD226 and PD1, were simultaneously overexpressed on the surface of Jurkat T cells, while their individual ligands, CD155 and PD-L1, were overexpressed on the surface of Raji cells. When these two cell types were co-cultured in the presence of superantigens, Jurkat cell activation, as indicated by luciferase reporter gene expression, was synergistically inhibited by negative signaling delivered to the Jurkat cell surface via both TIGIT-CD155 interaction and PD-1-PD-L1 interaction.

[0194] As shown in Figure 14, when serially diluted TIGIT antibody or PD-L1 antibody was added to the culture system, the antibody was able to dose-dependently enhance luciferase expression in Jurkat-TIGIT-CD226-PD-1 cells. However, the combination of anti-TIGIT antibody and anti-PD-L1 antibody significantly enhanced luciferase production, demonstrating a potent synergistic effect between these two antibodies. It should be noted that TIGIT-Fc-93-VH6 and TIGIT-Fc-PD-93-VH6 are PD-L1 / TIGIT bsAb forms. * Compound 2 showed significantly enhanced T cell activation compared to combo treatment, while TIGIT-CL-93-VH6 bsAb showed T cell activation comparable to that of combo treatment.

[0195] To further confirm observations in a bifunctional assay based on Jurkat cell lines, the synergistic effect of PD-L1 / TIGIT bsAb on the activation of primary CD8+ T cells derived from human PBMCs was further investigated. Briefly, CHO-K1 cells (CHO-TCR-CD155-PD-L1 cells) constitutively expressing the engineered T cell receptor (TCR) activator human CD155 and PD-L1 were seeded at a density of 35,000 cells per well and incubated overnight. Purified CD8+ T cells isolated from two healthy donors were incubated with the CHO-TCR-CD155-PD-L1 cells at a density of 50,000 cells per well. Subsequently, serially diluted PD-L1 / TIGIT bsAb, anti-TIGIT, anti-PD-L1, or a combination of these two antibodies were added to the co-culture system over 3 days, and the culture medium was collected for IFN-γ measurement using a standard ELISA kit.

[0196] As shown in Figure 15, anti-TIGIT antibodies or anti-PD-L1 antibodies could barely stimulate IFN-γ production by primary CD8+ T cells, but the combination of these two antibodies significantly enhanced IFN-γ production in a concentration-dependent manner. Most importantly, TIGIT-Fc-93-VH6 bsAb showed significantly superior efficacy to the combo treatment in T cell activation-induced IFN-γ production: this revealed a strong synergistic effect of this PD-L1 / TIGIT bsAb format on primary CD8+ T cell activation in vitro. * * *

[0197] This disclosure is not limited in scope by the specific embodiments described, which are intended as single examples of individual aspects of this disclosure, and any functionally equivalent composition or method falls within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Accordingly, this disclosure is intended to encompass such modifications and variations of this disclosure, insofar as they fall within the scope of the appended claims and their equivalents.

[0198] All publications and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference. In certain embodiments, for example, the following are provided: (Item 1) A single-domain antibody having binding specificity to the human PD-L1 protein and containing one complementarity-determining region 1 (CDR1), CDR2, and CDR3 selected from the group consisting of SEQ ID NOs: 1 to 36, SEQ ID NOs: 114 to 122, and SEQ ID NOs: 123 to 130, or a polypeptide containing the single-domain antibody. (Item 2) The antibody or polypeptide described in item 1, wherein the single-domain antibody has binding specificity to the human PD-L1 protein and comprises one complementarity-determining region 1 (CDR1), CDR2, and CDR3 selected from the group consisting of SEQ ID NOs: 1 to 36, SEQ ID NOs: 114 to 122, and SEQ ID NOs: 123 to 130, and wherein the CDR1, CDR2, and CDR3 conform to the Kabat numbering scheme. (Item 3) The aforementioned CDR1, CDR2, and CDR3 are (1) The amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, or (2) The amino acid sequences of SEQ ID NO: 113, SEQ ID NO: 49 and SEQ ID NO: 50 Includes the antibodies or polypeptides listed in item 1 or 2. (Item 4) The antibody or polypeptide according to any one of items 1 to 3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 55, CDR2 comprises the amino acid sequence of SEQ ID NO: 56, and CDR3 comprises the amino acid sequence of SEQ ID NO: 57. (Item 5) The antibody or polypeptide described in item 4, which is humanized. (Item 6) The antibody or polypeptide described in item 5, wherein the humanized antibody contains one or more revertant mutations selected from the group consisting of 37F, 47F, 49A, 78V, and 94A according to Kabat numbering. (Item 7) The humanized antibody or polypeptide described in item 5, wherein the humanized antibody contains the reverse mutations 37F, 47F, 49A, 78V, and 94A according to Kabat numbering. (Item 8) The antibody or polypeptide described in item 5, wherein the antibody contains one amino acid sequence selected from the group consisting of SEQ ID NOs. 114 to SEQ ID NOs. 122. (Item 9) The antibody or polypeptide described in item 8, wherein the antibody contains the amino acid sequence of SEQ ID NO: 119. (Item 10) The antibody or polypeptide according to any one of items 1 to 3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 113, CDR2 comprises the amino acid sequence of SEQ ID NO: 49, and CDR3 comprises the amino acid sequence of SEQ ID NO: 50. (Item 11) The antibody or polypeptide described in item 10, which is humanized. (Item 12) The antibody or polypeptide described in item 11, wherein the humanized antibody contains one or more revertant mutations selected from the group consisting of 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. (Item 13) The humanized antibody or polypeptide described in item 11, wherein the humanized antibody contains the reverse mutations 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering. (Item 14) The antibody or polypeptide described in item 11, wherein the antibody contains any one amino acid sequence selected from the group consisting of SEQ ID NOs. 123 to 130. (Item 15) The antibody or polypeptide described in item 14, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 130. (Item 16) The antibody or polypeptide described in any one of items 1 to 15, wherein the polypeptide is a bispecific antibody having binding specificity to an antigen different from PD-L1. (Item 17) A bispecific antibody comprising an antibody described in any one of items 1 to 15 and a second antibody or antigen-binding fragment having binding specificity to a target antigen other than PD-L1. (Item 18) A polynucleotide encoding an antibody or polypeptide as described in any one of items 1 to 16, or a bispecific antibody as described in item 17. (Item 19) A vector containing the polynucleotides described in item 18. (Item 20) Cells containing the polynucleotides described in item 18 or the vectors described in item 19. (Item 21) (1) An antibody or polypeptide as described in any one of items 1 to 16, a bispecific antibody as described in item 17, or a polynucleotide as described in item 18, and (2) Medically acceptable careers A composition containing the following: (Item 22) A method for treating cancer in a patient who requires treatment of cancer, comprising administering to the patient an effective amount of an antibody or polypeptide described in any one of items 1 to 16, a bispecific antibody described in item 17, or a polynucleotide described in item 18. (Item 23) Use of an antibody or polypeptide as described in any one of items 1 to 16, a bispecific antibody as described in item 17, or a polynucleotide as described in item 18 for the preparation of a drug for treating cancer. (Item 24) The method described in item 22 or the use described in item 23, wherein the cancer is a solid tumor. (Item 25) The method described in item 22 or the use described in item 23, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

Claims

1. A single-domain antibody, wherein the single-domain antibody has binding specificity to human PD-L1 protein and comprises complementarity-determining regions 1 (CDR1), CDR2 and CDR3, (1) The amino acid sequences of SEQ ID NOs. 113, 49, and 50, respectively, or (2) The amino acid sequences of SEQ ID NOs. 55, 56, and 57, respectively. A single-domain antibody containing [the specified component].

2. The antibody according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 113, CDR2 comprises the amino acid sequence of SEQ ID NO: 49, and CDR3 comprises the amino acid sequence of SEQ ID NO:

50.

3. The antibody according to claim 2, wherein the antibody is humanized.

4. The antibody according to claim 3, wherein the humanized antibody comprises one or more reverse mutations selected from the group consisting of 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering.

5. The antibody according to claim 3, wherein the humanized antibody comprises the reverse mutations 37Y, 44Q, 45R, 49A, 68A, 93R, and 94V according to Kabat numbering.

6. The antibody according to claim 3, wherein the antibody comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 125, 127, and 129.

7. The antibody according to claim 6, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 127 or 130.

8. The antibody according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 55, CDR2 comprises the amino acid sequence of SEQ ID NO: 56, and CDR3 comprises the amino acid sequence of SEQ ID NO:

57.

9. The antibody according to claim 8, wherein the antibody is humanized.

10. The antibody according to claim 9, wherein the humanized antibody comprises one or more reverse mutations selected from the group consisting of 37F, 47F, 49A, 78V, and 94A according to Kabat numbering.

11. The antibody according to claim 9, wherein the humanized antibody comprises the reverse mutations 37F, 47F, 49A, 78V, and 94A according to Kabat numbering.

12. The antibody according to claim 9, wherein the antibody comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 116-118, and 120-122.

13. The antibody according to claim 12, wherein the antibody comprises the amino acid sequence of SEQ ID NO:

119.

14. A bispecific antibody comprising the antibody described in Claim 1 and a second antibody or antigen-binding fragment having binding specificity to a target antigen other than PD-L1.

15. A polynucleotide encoding the antibody according to Claim 1.

16. A polynucleotide encoding the bispecific antibody described in Claim 14.

17. A vector comprising the polynucleotide according to claim 15 or claim 16.

18. A cell comprising the polynucleotide according to claim 15 or claim 16.

19. A cell comprising the vector according to claim 17.

20. (1) an antibody according to any one of claims 1 to 13, a bispecific antibody according to claim 14, or a polynucleotide according to claim 15 or claim 16, and (2) Medically acceptable careers A composition containing the following:

21. A composition for treating cancer in a patient who requires treatment for cancer, comprising an antibody according to any one of claims 1 to 13, a bispecific antibody according to claim 14, or a polynucleotide according to claim 15 or claim 16.

22. Use of an antibody according to any one of claims 1 to 13, a bispecific antibody according to claim 14, or a polynucleotide according to claim 15 or claim 16 for preparing a pharmaceutical product for treating cancer.

23. The composition according to claim 21, wherein the cancer is a solid tumor.

24. The use according to claim 22, wherein the cancer is a solid tumor.

25. The composition according to claim 21, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

26. The use according to claim 22, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

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