Single domain PD-L1 antibodies

Abstract

Various single domain anti-PD-L1 antibodies, and polypeptides (e.g., bispecific antibodies and chimeric antigen receptors) comprising these single domain antibodies are provided. These antibodies, including their humanized counterparts, have demonstrated excellent activity and are therefore suitable for use in various bispecific antibody formats. Methods of using the antibodies or polypeptides of the 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 No. 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 No. 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 only one monomeric variable antibody domain. Like complete antibodies, single domain antibodies are capable of selectively binding to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than common antibodies (150-160 kDa). Given their small size and single-chain nature, single domain antibodies may be particularly suitable for inclusion as fragments in other proteins (such as bispecific antibodies).

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

[0006] In addition to 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 a blocking antibody) resulted in increased mortality for infected mice. Blockade reduced TNFα and nitric oxide production by macrophages, reduced granzyme B production by NK cells, and reduced proliferation of L. monocytogenes antigen-specific CD8 T cells (but not CD4 T cells). This evidence suggests that PD-L1 acts as a positive co-stimulatory molecule in intracellular infection. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention The present disclosure provides a variety of novel single domain antibodies targeting human PD-L1 protein. These single domain antibodies have demonstrated excellent binding affinity and biological function despite their small size. When included in a variety of different formats of bispecific antibodies, some of the resulting bispecific antibodies have demonstrated excellent properties.

[0008] In one aspect, there is provided a single domain antibody, or a polypeptide comprising said single domain antibody, which has binding specificity for human PD-L1 protein and comprises a complementarity determining region 1 (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 has binding specificity for human PD-L1 protein and comprises a complementarity determining region 1 (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, where CDR1, CDR2 and CDR3 are according to the Kabat numbering scheme.

[0009] In some antibody or polypeptide embodiments, CDR1, CDR2 and CDR3 comprise (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 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. In some embodiments, the antibody is humanized. In some embodiments, the humanized antibody comprises one or more back mutations selected from the group consisting of 37F, 47F, 49A, 78V, and 94A according to the Kabat numbering. In some embodiments, the humanized antibody comprises back mutations of 37F, 47F, 49A, 78V, and 94A according to the Kabat numbering. In some embodiments, the antibody comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO:114 to SEQ ID NO:122. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:119.

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

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

[0013] In another aspect, provided herein are bispecific antibodies comprising an antibody of the present application and a second antibody or antigen-binding fragment that has binding specificity for a target antigen that is not PD-L1.

[0014] In another aspect, provided herein is a bispecific antibody comprising an anti-PD-L1 portion that has binding specificity for human PD-L1 protein, and an anti-TIGIT portion that has binding specificity for human TIGIT protein, wherein the anti-TIGIT portion comprises a heavy chain variable region (VH) that comprises VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 171, and a light chain variable region (VL) that comprises VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 172.

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

[0016] In some embodiments of the bispecific antibody, the anti-PD-L1 antigen-binding portion comprises a full-length antibody, a 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 of CDR1, CDR2, and CDR3 selected from the group consisting of SEQ ID NO:1-SEQ ID NO:36, SEQ ID NO:114-SEQ ID NO:122, and SEQ ID NO:123-SEQ ID NO:130. In some embodiments, the single domain antibody comprises any one of CDR1, CDR2, and CDR3 selected from the group consisting of SEQ ID NO:1-SEQ ID NO:36, SEQ ID NO:114-SEQ ID NO:122, and SEQ ID NO:123-SEQ ID NO:130, wherein the CDR1, CDR2, and CDR3 are according to 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, wherein CDR1, CDR2, and CDR3 comprise (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.

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

[0018] In some bispecific antibody embodiments, 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 of the amino acid sequences 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 of the amino acid sequences 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, provided herein is a bispecific antibody comprising an anti-PD-L1 portion that has binding specificity for a human PD-L1 protein, and an anti-CD47 portion that has binding specificity for a human CD47 protein, wherein the anti-CD47 portion comprises a heavy chain variable region (VH) that comprises the VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO:131 or SEQ ID NO:133, and a light chain variable region (VL) that comprises the 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 portion comprises a heavy chain variable region (VH) comprising the VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NO: 131 or SEQ ID NO: 133, and a light chain variable region (VL) comprising the VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 132 or SEQ ID NO: 134, where VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are according to the Kabat numbering scheme.

[0024] In some embodiments, the anti-CD47 moiety comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, where VH CDR1, VH CDR1, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 135 to 140, respectively. In some embodiments, the anti-CD47 moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 131, and a VL comprising the amino acid sequence of SEQ ID NO: 132.

[0025] In some embodiments, the anti-CD47 moiety comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, where VH CDR1, VH CDR1, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 141 to 146, respectively. In some embodiments, the anti-CD47 moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 133, and a VL comprising the amino acid sequence of SEQ ID NO: 134.

[0026] In some embodiments of the bispecific antibody, the anti-PD-L1 antigen-binding portion comprises a full-length antibody, a 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 of CDR1, CDR2, and CDR3 selected from the group consisting of SEQ ID NO:1-SEQ ID NO:36, SEQ ID NO:114-SEQ ID NO:122, and SEQ ID NO:123-SEQ ID NO:130. In some embodiments, the single domain antibody comprises any one of CDR1, CDR2, and CDR3 selected from the group consisting of SEQ ID NO:1-SEQ ID NO:36, SEQ ID NO:114-SEQ ID NO:122, and SEQ ID NO:123-SEQ ID NO:130, wherein the CDR1, CDR2, and CDR3 are according to 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, wherein CDR1, CDR2, and CDR3 comprise (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 bispecific antibody embodiments, the anti-PD-L1 portion comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the anti-PD-L1 portion comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 113, a CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50.

[0028] In some bispecific antibody embodiments, 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.

[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 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-CD47 moiety. In some embodiments, the bispecific antibody comprises four anti-PD-L1 moieties.

[0031] In some embodiments of a bispecific antibody, the bispecific antibody comprises (1) a heavy chain component comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:169, and SEQ ID NO:170, and (2) a light chain component comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:165, and SEQ ID NO:168.

[0032] In another aspect, provided herein is a polynucleotide encoding the antibody or polypeptide of the present application, or the bispecific antibody of the present application.

[0033] In another aspect, provided herein is a vector comprising the polynucleotide of the present application.

[0034] In another aspect, provided herein is a cell comprising the polynucleotide or vector of the present application.

[0035] In another aspect, provided herein is a composition comprising (1) an antibody or polypeptide, bispecific antibody, or polynucleotide of the present application; and (2) a pharma- ceutically acceptable carrier.

[0036] In another aspect, provided herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of an antibody or polypeptide, bispecific antibody, or polynucleotide of the present application. In another aspect, provided herein is a use of an antibody or polypeptide, bispecific antibody, or polynucleotide of the present application for preparing a medicament for treating cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, 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, renal cancer, melanoma, prostate cancer, and thyroid cancer. [Brief description of the drawings]

[0037] [Figure 1A-1B] Figure 1A illustrates that an exemplary anti-PD-L1 antibody of the present application effectively blocked the interaction between PD-1 and PD-L1, Figure 1B illustrates the specific binding of an exemplary anti-PD-L1 antibody of the present application to human PD-L1, and Figure 1C illustrates the specific binding of an exemplary anti-PD-L1 antibody of the present application to Raji cells that overexpress human PD-L1. [Figure 1C] Figure 1A illustrates that an exemplary anti-PD-L1 antibody of the present application effectively blocked the interaction between PD-1 and PD-L1, Figure 1B illustrates the specific binding of an exemplary anti-PD-L1 antibody of the present application to human PD-L1, and Figure 1C illustrates the specific binding of an exemplary anti-PD-L1 antibody of the present application to Raji cells that overexpress human PD-L1.

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

[0039] [Figure 3A-3F] Figures 3A-3F illustrate exemplary formats of the anti-CD47 / PD-L1 bispecific antibodies of the present application.

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

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

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

[0043] [Figure 7A-7B] 7A and 7B illustrate the RKO binding ability of exemplary antibodies of the present application.

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

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

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

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

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

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

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

[0051] [Figure 15] Figure 15 illustrates that an exemplary anti-TIGIT / PD-L1 bsAb of the present application significantly enhanced IFN-γ production in human primary CD8+ T cells in a concentration-dependent manner. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Detailed Description definition It should be noted that the term "a" or the term "an" entity refers to one or more of that entity. For example, "an antibody" is understood to refer to one or more antibodies. As such, the term "a" (or "an"), the term "one or more," and the term "at least one" may be used interchangeably herein.

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

[0054] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. A double-stranded nucleic acid homolog is intended to include a nucleic acid having a nucleotide sequence with a degree of homology with it or with its complement. In one aspect, a nucleic acid homolog can hybridize to the nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a 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, an 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 can include complete antibodies, and any antigen-binding fragments thereof or single chains thereof. Thus, the term "antibody" includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Examples of such include, but are not limited to, the heavy or light chain complementarity determining regions (CDRs) or ligand-binding portions thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework (FR) regions, or any portion thereof, or at least a portion of a binding protein.

[0056] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0057] "Single-chain variable fragment" or "scFv" refers to a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L In some aspects, these regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility, as well as serine- or threonine-rich for solubility, and the V H N-terminus of V L The 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 the art and are described, for example, in U.S. Patent No. 5,892,019.

[0058] The term antibody encompasses a wide variety of classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with several subclasses within them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA, IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. Various modifications of each of these classes and isotypes are readily discernible to the skilled artisan in light of the present disclosure and are therefore within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons and two identical heavy polypeptide chains with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains acting as arms and supporting the heavy chains, which begin at the opening of the "Y" and continue through the variable region.

[0059] Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized 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 comprising either the VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibody disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of 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 molecule.

[0060] By "specifically bind" or "having specificity for" it is generally meant that an antibody binds to an epitope through its antigen-binding domain and that the 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 the epitope through its antigen-binding domain more readily than the antibody would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" may be said to bind epitope "C" with higher specificity than it has for related epitope "D".

[0061] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) undesired physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the spread of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or reduction of disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already with a condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0062] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, and the like.

[0063] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, e.g., mammalian subjects, who would benefit from administration of an antibody or composition of the disclosure used, e.g., for detection, for diagnostic procedures, and / or for treatment.

[0064] Single domain PD-L1 antibodies The present disclosure provides single-chain anti-PD-L1 antibodies with high affinity to human PD-L1 protein. The antibodies exhibited strong binding and inhibitory activity, making them useful for therapeutic and diagnostic applications. Importantly, when incorporated as one of the targeting units in a variety of different formats of bispecific antibodies, certain resulting bispecific antibodies showed remarkable properties that establish further utility of these single domain anti-PD-L1 antibodies.

[0065] Thus, in one embodiment of the disclosure, single domain antibodies and polypeptides comprising such single domain antibodies are provided. In some embodiments, the polypeptide is a bispecific, trispecific, or multispecific antibody.

[0066] In some embodiments, the single domain antibody has binding specificity for human PD-L1 protein and comprises complementarity determining region 1 (CDR1), CDR2, and CDR3. In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:113, SEQ ID NO:49, and SEQ ID NO:50, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise the CDR1, CDR2, and CDR3, respectively, of any one of the antibodies provided in Table 1 (e.g., SEQ ID NO:1-SEQ ID NO:36).

[0067] In one embodiment, in the antibody, 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. SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 are the CDRs of antibody ALP-Tan-3p-93 and its humanized counterparts 93_VH-1 to 93_VH-9. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, except with the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitution is a conservative substitution.

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

[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 NO:114 to SEQ ID NO:122. In some embodiments, the humanized antibody has the amino acid sequence of SEQ ID NO:119.

[0070] In some embodiments, in the antibody, 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. SEQ ID NO: 113, SEQ ID NO: 49, and SEQ ID NO: 50 are the CDRs of 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 contained an N34Q substitution (Kabat numbering) in CDR1 (SEQ ID NO: 48) to prevent post-translational modification. In some embodiments, CDR1, CDR2 and CDR3 comprise SEQ ID NO:113, SEQ ID NO:49 and SEQ ID NO:50, respectively, except with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the substitutions are conservative substitutions.

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

[0072] In some embodiments, in the antibody, the CDR1, CDR2, and CDR3 comprise the CDR1, CDR2, and CDR3, respectively, of any one of the antibodies provided in Table 1 (e.g., SEQ ID NO:1-SEQ ID NO:36). In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46, and SEQ ID NO:47, respectively.

[0073] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46 and SEQ ID NO:47, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:51 and SEQ ID NO:52, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:53 and SEQ ID NO:54, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:47, respectively.

[0074] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46 and SEQ ID NO:47, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:58, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:53 and SEQ ID NO:39, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:59 and SEQ ID NO:60, respectively.

[0075] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:73 and SEQ ID NO:74, respectively.

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

[0077] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:64, SEQ ID NO:87 and SEQ ID NO:66, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:88 and SEQ ID NO:89, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:90 and SEQ ID NO:91, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise 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 comprise 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 comprise 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 comprise 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 comprise 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 comprise 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 comprises an amino acid sequence selected from SEQ ID NO:1-SEQ ID NO:36.

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

[0081] Compositions including an antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided.

[0082] It will also be understood by those skilled in the art that antibodies as disclosed herein may be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar to the starting sequence, e.g., may have a certain percent identity to the starting sequence, e.g., may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequences.

[0083] Bispecific and multispecific antibodies that include 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) are also provided.

[0084] The present disclosure provides bispecific and multispecific antibodies having binding specificity for at least human PD-L1 protein and human CD47 protein. PD-L1 is a critical "don't find me" signal to the adaptive immune system, whereas CD47 transmits an anti-phagocytic "don't eat me" signal to the innate immune system. They are often overexpressed on the surface of human tumors. Therefore, dual targeting of both innate and adaptive immune checkpoints will likely maximize anti-tumor therapeutic effects and induce longer-lasting responses.

[0085] In some embodiments, the bispecific and multispecific antibodies include an anti-PD-L1 portion that includes at least a single domain anti-PD-L1 antibody. As demonstrated, the single chain anti-PD-L1 antibody has high affinity for human PD-L1 protein. The antibody exhibits strong binding and inhibitory activity, making it useful for therapeutic and diagnostic applications.

[0086] In some embodiments, the single domain antibody has binding specificity for human PD-L1 protein and comprises complementarity determining region 1 (CDR1), CDR2, and CDR3. In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:113, SEQ ID NO:49, and SEQ ID NO:50, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise the CDR1, CDR2, and CDR3, respectively, of any one of the antibodies provided in Table 1 (e.g., SEQ ID NO:1-SEQ ID NO:36).

[0087] In one embodiment, in the antibody, 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. SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 are the CDRs of antibody ALP-Tan-3p-93 and its humanized counterparts 93_VH-1 to 93_VH-9. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, except with the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitution is a conservative substitution.

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

[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 NO:114 to SEQ ID NO:122. In some embodiments, the humanized antibody has the amino acid sequence of SEQ ID NO:119.

[0090] In some embodiments, in the antibody, 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. SEQ ID NO: 113, SEQ ID NO: 49, and SEQ ID NO: 50 are the CDRs of 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 contained an N34Q substitution (Kabat numbering) in CDR1 (SEQ ID NO: 48) to prevent post-translational modification. In some embodiments, CDR1, CDR2 and CDR3 comprise SEQ ID NO:113, SEQ ID NO:49 and SEQ ID NO:50, respectively, except with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the substitutions are conservative substitutions.

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

[0092] In some embodiments, in the antibody, the CDR1, CDR2, and CDR3 comprise the CDR1, CDR2, and CDR3, respectively, of any one of the antibodies provided in Table 1 (e.g., SEQ ID NO:1-SEQ ID NO:36). In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46, and SEQ ID NO:47, respectively.

[0093] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46 and SEQ ID NO:47, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:51 and SEQ ID NO:52, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:53 and SEQ ID NO:54, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:47, respectively.

[0094] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46 and SEQ ID NO:47, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:58, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:53 and SEQ ID NO:39, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:59 and SEQ ID NO:60, respectively.

[0095] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:73 and SEQ ID NO:74, respectively.

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

[0097] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:64, SEQ ID NO:87 and SEQ ID NO:66, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:88 and SEQ ID NO:89, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:90 and SEQ ID NO:91, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise 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 comprise 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 comprise 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 comprise 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 comprise 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 comprise 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 comprises an amino acid sequence selected from SEQ ID NO:1-SEQ ID NO:36.

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

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

[0102] In some embodiments, VH CDR1, VH CDR, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 141-146, respectively. These CDRs are from the parent anti-CD47 antibody 34C5. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 133, and VL comprises 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 comprise the amino acid sequences of SEQ ID NO: 135 to 140, respectively. These CDRs are from the parent anti-CD47 antibody 13H3. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 131 and VL comprises the amino acid sequence of SEQ ID NO: 132 (Table 5).

[0104] Bispecific antibodies can take any format, including that illustrated in Figure 3. In one embodiment, bispecific antibodies are symmetric. An example is provided in Figure 3A, where two single domain anti-PD-L1 antibodies are fused, optionally via linkers, to the N-terminus of each of the heavy chains of an anti-CD47 antibody. In the example of Figure 3B, a single domain anti-PD-L1 antibody is fused, optionally via linkers, to the N-terminus of each of the light chains of an anti-CD47 antibody.

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

[0106] Bispecific antibodies can also be asymmetric, such as those illustrated in Figures 3E-3F. In Figure 3E, two single domain anti-PD-L1 antibodies are linked in tandem to the N-terminus of one of the Fc chains. For the other Fc chain, an anti-CD47 Fab unit is fused to the N-terminus. In a slight variation, in Figure 3F, the anti-CD47 portion comprises a single chain fragment (scFv).

[0107] Bispecific antibodies may include constant regions from either IgG type (eg, IgG1 and IgG4).

[0108] Compositions including an antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided.

[0109] It will also be understood by those skilled in the art that antibodies as disclosed herein may be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar to the starting sequence, e.g., may have a certain percent identity to the starting sequence, e.g., may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequences.

[0110] The present disclosure provides bispecific and multispecific antibodies having binding specificity for at least human PD-L1 protein and human TIGIT protein. PD-L1 is a very important "don't find me" signal to the adaptive immune system, whereas TIGIT helps tumor and infected cells to escape immune responses. They are often overexpressed on the surface of human tumors. Therefore, dual targeting of both innate and adaptive immune checkpoints will likely maximize antitumor therapeutic effects and induce longer-lasting responses.

[0111] In some embodiments, the bispecific and multispecific antibodies include an anti-PD-L1 portion that includes at least a single domain anti-PD-L1 antibody. As demonstrated, the single chain anti-PD-L1 antibody has high affinity for human PD-L1 protein. The antibody exhibits strong binding and inhibitory activity, making it useful for therapeutic and diagnostic applications.

[0112] In some embodiments, the single domain antibody has binding specificity for human PD-L1 protein and comprises complementarity determining region 1 (CDR1), CDR2, and CDR3. In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:113, SEQ ID NO:49, and SEQ ID NO:50, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise the CDR1, CDR2, and CDR3, respectively, of any one of the antibodies provided in Table 1 (e.g., SEQ ID NO:1-SEQ ID NO:36).

[0113] In one embodiment, in the antibody, 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. SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 are the CDRs of antibody ALP-Tan-3p-93 and its humanized counterparts 93_VH-1 to 93_VH-9. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, except with the addition, deletion, and / or substitution of one, two, or three amino acids. In some embodiments, the substitution is a conservative substitution.

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

[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 NO:114 to SEQ ID NO:122. In some embodiments, the humanized antibody has the amino acid sequence of SEQ ID NO:119.

[0116] In some embodiments, in the antibody, 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. SEQ ID NO: 113, SEQ ID NO: 49, and SEQ ID NO: 50 are the CDRs of 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 contained an N34Q substitution (Kabat numbering) in CDR1 (SEQ ID NO: 48) to prevent post-translational modification. In some embodiments, CDR1, CDR2 and CDR3 comprise SEQ ID NO:113, SEQ ID NO:49 and SEQ ID NO:50, respectively, except with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the substitutions are conservative substitutions.

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

[0118] In some embodiments, in the antibody, the CDR1, CDR2, and CDR3 comprise the CDR1, CDR2, and CDR3, respectively, of any one of the antibodies provided in Table 1 (e.g., SEQ ID NO:1-SEQ ID NO:36). In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively. In one embodiment, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46, and SEQ ID NO:47, respectively.

[0119] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46 and SEQ ID NO:47, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:51 and SEQ ID NO:52, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:53 and SEQ ID NO:54, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:47, respectively.

[0120] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:46 and SEQ ID NO:47, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:58, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:53 and SEQ ID NO:39, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:59 and SEQ ID NO:60, respectively.

[0121] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:37, SEQ ID NO:73 and SEQ ID NO:74, respectively.

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

[0123] In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:64, SEQ ID NO:87 and SEQ ID NO:66, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:88 and SEQ ID NO:89, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:84, SEQ ID NO:90 and SEQ ID NO:91, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, respectively. In one embodiment, CDR1, CDR2 and CDR3 comprise 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 comprise 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 comprise 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 comprise 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 comprise 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 comprise 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 comprises an amino acid sequence selected from SEQ ID NO:1-SEQ ID NO:36.

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

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

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

[0129] Bispecific antibodies can take any format, including that illustrated in Figure 9. In one embodiment, the bispecific antibody is preferably symmetric. In one embodiment, a single domain anti-PD-L1 antibody is located C-terminal to the anti-TIGIT moiety.

[0130] An exemplary format is provided in Figure 9A, where two single domain anti-PD-L1 antibodies are fused, optionally via a linker, to the C-terminus of each of the heavy chain constant regions of an anti-TIGIT antibody. In the example of Figure 9B, each heavy chain contains two copies of a single domain anti-PD-L1 antibody.

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

[0132] Bispecific antibodies may include constant regions from either IgG type (eg, IgG1 and IgG4).

[0133] Compositions including an antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided.

[0134] It will also be understood by those skilled in the art that antibodies as disclosed herein may be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar to the starting sequence, e.g., may have a certain percent identity to the starting sequence, e.g., may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequences.

[0135] Polynucleotides encoding antibodies and methods for preparing antibodies The present disclosure also provides an isolated polynucleotide or nucleic acid molecule encoding the antibody of the present disclosure, its variant or derivative. The polynucleotide of the present disclosure may encode the entire heavy and light chain variable regions of the 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 polynucleotide of the present disclosure may encode a part of the heavy and light chain variable regions of the 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 of making 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 the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but has an inactivated endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated by reference in their entirety.

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

[0138] The present disclosure further relates to antibody-based therapies that involve administering an antibody of the present disclosure to a patient, such as an animal, mammal, or human, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, an antibody of the present disclosure, including variants and derivatives thereof as described herein, and a nucleic acid or polynucleotide encoding an antibody of the present disclosure, including variants and derivatives thereof as described herein.

[0139] The antibody of the present 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. The results of using small molecule inhibitors or monoclonal antibodies targeting PD-L1 in mouse tumor models show that targeted PD-L1 therapy is an important alternative and practical approach to effectively control tumor growth. As demonstrated in the experimental examples, anti-PD-L1 antibodies activated adaptive immune response mechanisms that can lead to improved survival in cancer patients.

[0140] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. The method, in one embodiment, involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient expresses, or overexpresses, PD-L1, or is induced to express PD-L1. Induction of PD-L1 expression can be achieved, for example, by administration of a tumor vaccine or radiation therapy.

[0141] Tumors that express 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, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer. Thus, the presently disclosed antibodies can be used to treat any one or more of such cancers.

[0142] composition The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the compositions further comprise a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0143] In specific embodiments, the term "pharmaceutical acceptable" means approved by a regulatory agency of a federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly, for use in humans. Moreover, a "pharmaceutical acceptable carrier" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any kind.

[0144] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline 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, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents, such as benzyl alcohol or methyl parabens; 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 contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories with conventional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.The formulation should suit the mode of administration. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0145] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic (such as lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, mixed together as a dry lyophilized powder or water-free concentrate in a hermetically sealed container (such as an ampoule or sachet) indicating the amount of active agent. If the composition is to be administered by injection, the composition can be dispensed with an infusion 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 can be provided so that the ingredients may be mixed before administration. EXAMPLES

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

[0147] Antigen: Recombinant human PD-L1 / hFc fusion protein was used as the immunogen to generate anti-human PD-L1 antibodies. A fusion protein containing the entire extracellular domain of human PD-L1 fused to a human immunoglobulin Fc domain was used as the immunogen.

[0148] immunity Alpacas were first immunized subcutaneously (SC) with a 1:1 mixture of 600μg mouse PD-L1 and complete Freud's adjuvant on day 0, 250μg mouse PD-L1 with incomplete Freud's adjuvant on day 21, and 250μg human PD-L1 with incomplete Freud's adjuvant on day 42. The immune response was monitored by measuring titers 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 round of liquid-phase panning and one round of solid-phase panning. Generally, libraries were incubated in immunotubes or beads coated with biotinylated PD-L1. Unbound phages were removed by washing 5-20 times with PBST. A total of three rounds of panning were performed for each selection.

[0150] The sequences of the binders were amplified from the antigen-binding positive phages by PCR and confirmed by DNA sequencing. The sequences of the unique antibodies and their CDR regions 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 antibodies against human PD-L1 The binding and blocking properties of some of the antibodies of the present invention were characterized by Gator. An anti-his probe was first loaded onto the chip, followed by human PD-L1-his to capture the antigen. The antibodies were then injected and the binding curves were recorded. Finally, human PD1 / hFc was injected to determine whether 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. The affinity was further confirmed by Biacore T200. [Table 2] Example 3. Humanization of anti-PD-L1 alpaca monoclonal antibody. The variable region genes of mAbs ALP-Tan-3p-93 and ALP-Tan-3p-112 were used to generate humanized mAbs. 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 Ig gene sequences to find the overall best matching human germline Ig gene sequence. For ALP-Tan-3p-93, the closest human match was IGHV3-23. * 04 gene. Then, CDR1, CDR2, and CDR3 of ALP-Tan-3p-93 were cloned into IGHV3-23 * We designed a humanized variable domain sequence that was grafted onto the framework sequence of the 04 gene. For ALP-Tan-3p-112, the closest match in humans was IGHV3-48. * 03 gene. Then, CDR1, CDR2 and CDR3 of ALP-Tan-3p-112 were cloned into IGHV3-48 *We designed a humanized variable domain sequence that was grafted onto the framework sequence of the 03 gene. Meanwhile, a single residue mutation (N34Q, Kabat numbering) was introduced within CDR1 to reduce the risk of post-translational modification. Then, a 3D model was generated to clarify whether there were any framework positions where the replacement of alpaca amino acids with human amino acids could affect binding and / or CDR conformation. [Table 3-1] [Table 3-2] [Table 3A]

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

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

[0154] The binding ability of the present anti-PD-L1 antibodies was further assessed by using Raji cells overexpressing human PD-L1. Briefly, 50 μl of Raji cells overexpressing human PD-L1 were cultured in 2000× culture medium. * 10 5 The cells were seeded into a 96-well plate at a concentration of 100μl cells / well. 50μl 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 on ice for 1 hour. The cells were then washed twice with FACS buffer and supplemented with 100μl of PE-anti-hu IgG, followed by incubation on ice for 1 hour. After incubation, the cells in each well were collected and resuspended in 65μl of FACS buffer for analysis by flow cytometry. 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 that can activate NFAT-mediated luciferase expression upon TCR stimulation. In this assay, Jurkat cells transfected with human PD-1 gene by lentivirus were used as responder cells. Raji-PD-L1 cells were used as antigen-presenting cells (APCs). Staphylococcal enterotoxin E (SEE) is used to stimulate TCR signals. In this system, ectopically expressed huPD-L1 can suppress SEE-stimulated NF-AT-luciferase activity in Jurkat cells, while anti-PD-L1 antibodies can reverse NFAT-luciferase activity. Briefly, APCs (2.5 × 10 4 ) in the presence of SEE stimulation, PD-1-expressing Jurkat T cells (1 × 10 5 The cells were co-cultured with PD-L1 antibodies at the initiation of culture. After 6 hours, the resulting cells were assessed for their luciferase activity.

[0156] As shown in Figures 2A-C, all anti-PD-L1 antibodies tested blocked 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 a "2 vs. 2" and "1 vs. 2" format (structures exemplified in Figure 3).

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

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

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

[0161] Figure 3D illustrates another "2-for-2" symmetric format bispecific antibody molecule. The bispecific antibody includes an anti-CD47 Fab, one IgG1 Fc or one IgG4 Fc, and an anti-PD-L1 single domain antibody linked to CH3 via a GS linker.

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

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

[0164] These bispecific antibodies were purified from 100 mL of transiently transfected supernatant of HEK293F cells by protein A affinity column. The purity of each of the bispecific antibodies 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 Blocks CD47 Binding to SIRPα The assay was performed as described in the CD47 / SIRPα binding assay kit (Cisbio). Briefly, serially diluted antibodies, i.e., Tag1-CD47 and Tag2-SIRPa, were premixed and incubated at room temperature for 15 min, and then premixed anti-Tag1-Tb3 and anti-Tag2-XL665 were added and incubated at RT for 1 h. Fluorescence data was read on a PerkinElmer Envision plate reader using a laser as the light source. Anti-CD47 antibodies (13H3 or 34C5) were used as positive controls in this study.

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

[0167] Example 9. T cell activation bioassay (NFAT) To test the ability of anti-CD47 / PD-L1 bispecific antibodies to stimulate T cell responses, hPD-1-expressing Jurkat cells were used. Jurkat is a human T cell leukemia cell line that can activate NF-AT-activated luciferase expression upon TCR stimulation. In this assay, Jurkat cells transfected with human PD-1 gene by lentivirus were used as responder cells. Raji-PD-L1 cells were used as antigen-presenting cells (APCs). Staphylococcal enterotoxin E (SEE) is used to stimulate TCR signals. In this system, ectopically expressed huPD-L1 can suppress SEE-stimulated NF-AT-luciferase activity in Jurkat cells, while anti-PD-L1 antibodies can reverse NF-AT-luciferase activity. Briefly, APCs (2.5 × 10 4 ) in the presence of SEE stimulation, PD-1-expressing Jurkat T cells (1 × 10 5 The cells were co-cultured with PD-L1 antibodies at the initiation of culture. After 6 hours, the resulting cells were assessed for their luciferase activity.

[0168] As shown in Figure 5, all bispecific antibodies in the 2:2 format had similar or greater potency 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 antibodies exhibit 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 and replated in 24-well dishes and allowed to adhere for 24 hours. Human tumor cell line RKO was selected as target cells and labeled with 1 mM CellTrace-Far Red for 20 minutes, MDMs were labeled with 1 mM Cell Trace-Violet for 20 minutes, and then mixed at a ratio of 3:1 tumor cells per phagocyte, and anti-CD47 / PD-L1 bispecific antibody and corresponding control mAb and combinations were added at various doses. After 3 hours of incubation, phagocytosis of target cells was analyzed by flow cytometry. Phagocytosis was measured by gating on macrophages and then evaluating the percentage of double-positive cells.

[0170] As shown in Figure 6A and Figure 6B, the anti-CD47 / PD-L1 bispecific antibody demonstrated greater ADCP efficacy than the combination treatment of the parental monoclonal antibody and the clinical benchmark antibody.

[0171] Another study was performed to further test whether isotype affects ADCP efficacy. In this study, the differences in ADCP assays between bispecific antibodies with hIgG1 and hIgG4 Fc, respectively, were compared. The results are shown in Figure 6C. The ADCP efficacy of anti-CD47 / PD-L1 bispecific antibodies was generally similar when the isotype was changed from hIgG1 to hIgG4.

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

[0173] As shown in Figure 7A and Figure 7B, the anti-CD47 / PD-L1 bispecific antibodies in a symmetric format exhibited either stronger or similar binding affinity to the parent PD-L1 monospecific antibody.

[0174] Example 12. RBC binding and RBC agglutination assays 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 titration starting at 200 nM with 3-fold reductions) for 1 hour at 4° C., followed by addition of PE-conjugated secondary antibody for 30 minutes at 4° C. Binding of 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, comparable to the 13H3 antibody. Of all 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 with titrated anti-CD47 / PD-L1 antibodies (antibody titration starting at 200 nM in 3-4 fold increments) in round-bottom 96-well plates for 2 hours at room temperature. Evidence of hemagglutination is revealed by the presence of non-sedimented RBCs, which appear as a cloud compared to the punctuate red dots of non-hemagglutinated RBCs.

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

[0178] Twelve NOG mice were individually injected with human PBMCs in 0.2 mL of DPBS (iv, 5 × 10 6 cells / mouse). After 8 days, 1 × 10 6 RKO cells were inoculated subcutaneously into the right flank of mice. The average tumor size was 57 mm. 3 When tumor-bearing mice reached 10 days after tumor growth, they were randomly divided into three groups (four mice per group) and intraperitoneally administered PBS, 93VH6-13H3-L-IgG1 (12 mg / kg), or 93VH6-13H3-L-IgG4 (12 mg / kg), respectively. Tumor volumes and body weights were measured and recorded twice weekly. On day 19, the animals were euthanized. As shown in Figure 8C, 93VH6-13H3-L-IgG1 and 93VH6-13H3-L-IgG4 treatments showed significantly inhibited tumor growth when compared to PBS, suggesting the strong anti-tumor efficacy of the present anti-CD47 / PD-L1 bsAb.

[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 formats (structures exemplified in Figure 9).

[0180] In one format, two PD-L1 sdAbs were fused via G4S linkers to the C-terminus of the heavy chain of the anti-TIGIT moiety (denoted as TIGIT-Fc-PD-L1) or to the C-terminus of the light chain of the anti-TIGIT moiety (denoted as TIGIT-CL-PD-L1). Alternatively, four PD-L1 sdAbs, two in each tandem group, were linked via G4S linkers to the C-terminus of the heavy chain of the TIGIT moiety (denoted as TIGIT-Fc-PD-L1). * 2).

[0181] These bispecific antibodies were purified from 100 mL of supernatant of transiently transfected HEK293F cell cultures by Protein A affinity column. The purity of each of the bispecific antibodies 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 by BIACORE®. PD-L1 / TIGIT bsAb molecules or parental anti-PD-L1 sdAb were captured by Protein A chip. A dilution series of human PD-L1 protein (6.25nM-100nM) was injected over the captured antibody at a flow rate of 10μL / min. Antigen was allowed to associate for 180 seconds and dissociate for 1200 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 Evaluation software.

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

[0184] The binding of the anti-PD-L1 / TIGIT bsAb molecules to human PD-L1 was further analyzed by ELISA. Briefly, 100μl of different concentrations of anti-PD-L1 / TIGIT bsAb, 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 then the binding between anti-PD-L1 / TIGIT bsAb and human His-PD-L1 was analyzed via goat anti-human IgG Fc HRP. As shown in Figure 10A and Figure 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 specific binding to human PD-L1 in a dose-dependent manner.

[0185] Furthermore, the binding ability of the present anti-PD-L1 / TIGIT bsAbs to cells expressing PD-L1 was analyzed by using Raji-PD-L1 cells. Briefly, 50 μl of Raji cells overexpressing human PD-L1 were cultured for 2 h. * 10 5 Cells / well were seeded into a 96-well plate. 50μl of anti-PD-L1 antibody, 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. Then, the cells were washed twice with FACS buffer and supplemented with 100μl of PE-anti-hu IgG, followed by incubation on ice for 1 hour. After incubation, the cells in each well were collected and resuspended in 65μl of FACS buffer for analysis by flow cytometry. As shown in Figure 10C, TIGIT-Fc-112-VH47 exhibited specific binding to Raji cells expressing human PD-L1 in a dose-dependent manner.

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

[0187] As shown in Figure 11, TIGIT-Fc-93-VH6 * The two bsAb molecules showed comparable antagonistic activity to the parental 93-VH6 sdAb. TIGIT-Fc-93-VH6 showed enhanced maximal efficacy, but a reduced EC50 for PD-L1 antagonist activity, when compared to anti-93-VH6 sdAb. TIGIT-CL-93-VH6 bsAb showed comparable maximal efficacy, but a reduced EC50 for PD-L1 antagonist activity, when compared to 93-VH6 sdAb.

[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 examined by Biacore T200. The antibodies were captured by a Protein A chip. A series of concentrations of His-tagged human TIGIT-ECD protein (0.78 nM-12.5 nM) were injected over the captured antibody at a flow rate of 10 μl / min. The association phase was 180 s and the dissociation phase was 1200 s.

[0189] The results are shown below in Table 10. The Biacore results for the PD-L1 / TIGIT antibodies indicate that these bispecific antibodies are high affinity binders to 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 TIGIT protein, PD-L1 / TIGIT bsAbs were subjected to ELISA binding assay with His-tagged human TIGIT. As shown in Figure 12, all of the 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 bsAbs To evaluate the TIGIT blocking function of PD-L1 / TIGIT bsAb antibodies, we used a Jurkat cell-based in vitro functional assay. Briefly, human TIGIT and its counterreceptor CD226 were simultaneously overexpressed on the surface of Jurkat T cells, while their co-ligand human CD155 was overexpressed on the surface of Raji cells. When these two cell types were co-cultured in the presence of superantigen, negative signaling delivered to the Jurkat cell surface by TIGIT-CD155 ligation inhibited Jurkat cell activation. A luciferase reporting system similar to that used in the PD-L1 blocking assay was used to evaluate the activation status of Jurkat cells. When serially diluted PD-L1 / TIGIT bsAb or anti-TIGIT antibodies were added to the culture system, the antibodies could enhance the luciferase expression of Jurkat-TIGIT-CD226 cells in a dose-dependent manner.

[0192] This assay demonstrated that TIGIT-Fc-93-VH6 bsAb molecules exhibited superior potency in blocking TIGIT / CD155 signaling and enhancing Jurkat cell activation when compared to their parental TIGIT antibodies (Figure 13). * 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 promoting T cell activation, we established a robust cell-based in vitro bifunctional assay. Briefly, human TIGIT was simultaneously overexpressed on the surface of Jurkat T cells with CD226 and PD1, while their respective 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 superantigen, the negative signaling delivered to the Jurkat cell surface by both TIGIT-CD155 interaction and PD-1-PD-L1 interaction synergistically inhibited Jurkat cell activation, as indicated by luciferase reporter gene expression.

[0194] As shown in Figure 14, when serially diluted TIGIT or PD-L1 antibodies were added to the culture system, the antibodies could enhance the luciferase expression of Jurkat-TIGIT-CD226-PD-1 cells in a dose-dependent manner. However, the combination of anti-TIGIT and anti-PD-L1 antibodies significantly enhanced luciferase production, indicating the strong synergistic effect of these two antibodies. Of note, the PD-L1 / TIGIT bsAb formats TIGIT-Fc-93-VH6 and TIGIT-Fc-PD-93-VH6 * 2 showed significantly enhanced T cell activation compared to the combo treatment, whereas TIGIT-CL-93-VH6 bsAb showed similar T cell activation as the combo treatment.

[0195] To further confirm the observations in the Jurkat cell line-based bifunctional assay, we further investigated the synergistic effect of PD-L1 / TIGIT bsAb on the activation of primary CD8+ T cells derived from human PBMCs. Briefly, CHO-K1 cells constitutively expressing the engineered T cell receptor (TCR) activator human CD155 and PD-L1 (CHO-TCR-CD155-PD-L1 cells) 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 CHO-TCR-CD155-PD-L1 cells at a density of 50,000 cells per well. Then, 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 for 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 or anti-PD-L1 antibodies were barely able to stimulate IFN-γ production by primary CD8+ T cells, whereas 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 greater efficacy than combo treatment in IFN-γ production induced by T cell activation: this revealed a strong synergistic effect of this PD-L1 / TIGIT bsAb format on the activation of primary CD8+ T cells in vitro. * * *

[0197] The present disclosure is not limited in scope by the described specific embodiments, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It is clear to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover such modifications and variations of the present disclosure as long as they are within the scope of the appended claims and their equivalents.

[0198] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A single domain antibody, or a polypeptide comprising said single domain antibody, wherein said single domain antibody has binding specificity for human PD-L1 protein and comprises any one of complementarity determining region 1 (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.

2. 2. The antibody or polypeptide of claim 1, wherein the single domain antibody has binding specificity for human PD-L1 protein and comprises any one of complementarity determining region 1 (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 wherein the CDR1, CDR2 and CDR3 are according to the Kabat numbering scheme.

3. the CDR1, CDR2 and CDR3 being (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. The antibody or polypeptide of claim 1 .

4. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO:55, the CDR2 comprises the amino acid sequence of SEQ ID NO:56, and the CDR3 comprises the amino acid sequence of SEQ ID NO:

57.

5. The antibody or polypeptide of claim 4, wherein the antibody is humanized.

6. 6. The antibody or polypeptide of claim 5, wherein the humanized antibody contains one or more back mutations selected from the group consisting of 37F, 47F, 49A, 78V and 94A according to the Kabat numbering.

7. 6. The antibody or polypeptide of claim 5, wherein the humanized antibody comprises the following back mutations according to the Kabat numbering: 37F, 47F, 49A, 78V and 94A.

8. The antibody or polypeptide of claim 5, wherein the antibody comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO:114 to SEQ ID NO:

122.

9. The antibody or polypeptide of claim 8, wherein the antibody comprises the amino acid sequence of SEQ ID NO:

119.

10. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO:113, the CDR2 comprises the amino acid sequence of SEQ ID NO:49, and the CDR3 comprises the amino acid sequence of SEQ ID NO:

50.

11. The antibody or polypeptide of claim 10, wherein the antibody is humanized.

12. 12. The antibody or polypeptide of claim 11, wherein the humanized antibody comprises one or more back mutations selected from the group consisting of 37Y, 44Q, 45R, 49A, 68A, 93R and 94V according to the Kabat numbering.

13. 12. The antibody or polypeptide of claim 11, wherein the humanized antibody comprises the following back mutations according to the Kabat numbering: 37Y, 44Q, 45R, 49A, 68A, 93R and 94V.

14. The antibody or polypeptide of claim 11, wherein the antibody comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO:123 to SEQ ID NO:

130.

15. The antibody or polypeptide of claim 14, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO:

130.

16. The antibody or polypeptide of claim 1, wherein the polypeptide is a bispecific antibody having binding specificity for an antigen different from PD-L1.

17. A bispecific antibody comprising the antibody of claim 1 and a second antibody or antigen-binding fragment having binding specificity for a target antigen that is not PD-L1.

18. A polynucleotide encoding the antibody or polypeptide of claim 1.

19. A polynucleotide encoding the bispecific antibody described in claim 17.

20. A vector comprising the polynucleotide of claim 18 or claim 19.

21. A cell comprising the polynucleotide of claim 18 or claim 19.

22. A cell containing the vector described in claim 20.

23. (1) An antibody or polypeptide according to any one of claims 1 to 16, a bispecific antibody according to claim 17, or a polynucleotide according to claim 18 or claim 19, and (2) A pharma- ceutically acceptable carrier A composition comprising:

24. 20. A composition for treating cancer in a patient in need thereof, comprising an antibody or a polypeptide according to any one of claims 1 to 16, a bispecific antibody according to claim 17, or a polynucleotide according to claim 18 or claim 19.

25. Use of an antibody or a polypeptide according to any one of claims 1 to 16, a bispecific antibody according to claim 17, or a polynucleotide according to claim 18 or claim 19 for the preparation of a medicament for the treatment of cancer.

26. 25. The composition of claim 24, wherein the cancer is a solid tumor.

27. The use of claim 25, wherein the cancer is a solid tumor.

28. 25. The composition of claim 24, 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, renal cancer, melanoma, prostate cancer and thyroid cancer.

29. The use of claim 25, 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, renal cancer, melanoma, prostate cancer and thyroid cancer.

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