Anti-PD-L1 nanobody

Abstract

Provided herein are affinity mature single domain anti-PD-L1 antibodies and polypeptides, such as multispecific antibodies and chimeric antigen receptors, including these single domain antibodies. These antibodies, including their humanized counterparts, exhibit excellent activity and are suitable for use in a variety of multispecific antibody forms. Methods of treating and diagnosing diseases, such as cancer and infectious diseases, using the antibodies or polypeptides are also provided.

Description

Background Art

[0001] Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Like full antibodies, it is capable of selectively binding to a specific antigen. The molecular weight of single-domain antibodies is only 12 - 15 kDa, much smaller than that of ordinary antibodies (150 - 160 kDa). Given their small size and single-chain nature, single-domain antibodies can be particularly suitable for inclusion as fragments in other proteins, such as bispecific antibodies.

[0002] Antibodies specific to programmed death ligand 1 (PD-L1) (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) are being used in cancer treatment and other clinical applications. PD-L1 is a 40 kDa type I transmembrane protein that is thought to play a major role in suppressing the immune system during specific events such as pregnancy, tissue allotransplantation, autoimmune diseases, and other disease states (such as hepatitis). The binding of PD-L1 to PD-1 or B7.1 transmits an inhibitory signal that reduces the proliferation of CD8+ T cells in lymph nodes; in addition, PD-1 is also able to control the accumulation of exogenous antigen-specific T cells in lymph nodes through apoptosis, which is further mediated by the downregulation of the Bcl-2 gene.

[0003] In addition to treating cancer, PD-L1 inhibition has also shown potential in treating infectious diseases. In a mouse model of intracellular infection, Listeria monocytogenes (L. monocytogenes) induces the expression of PD-L1 protein in T cells, NK cells, and macrophages. PD-L1 blockade (e.g., using a blocking antibody) leads to an increased mortality rate in infected mice. PD-L1 blockade reduces the production of TNFα and nitric oxide by macrophages, reduces the production of granzyme B by NK cells, and decreases the proliferation of Listeria monocytogenes antigen-specific CD8 T cells (but not CD4 T cells). This evidence indicates that PD-L1 acts as a positive costimulatory molecule in intracellular infection. Summary of the Invention

[0004] The present disclosure provides novel single-domain antibodies and affinity-matured counterparts that target the human PD-L1 protein. Despite their small size, these single-domain antibodies exhibit excellent binding affinity and biological functions. When included in various different forms of multispecific antibodies, some of the resulting multispecific antibodies exhibit excellent properties.

[0005] One embodiment of the present disclosure provides a single-domain antibody or a polypeptide comprising the single-domain antibody, wherein the single-domain antibody has binding specificity for the human PD-L1 protein and comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein:

[0006] (1) CDR1 contains the amino acid sequence of SEQ ID NO: 54 or 95;

[0007] CDR2 contains the amino acid sequence of SEQ ID NO: 55 or 96; and

[0008] CDR3 contains the amino acid sequence of SEQ ID NO: 56, 97, 98, 99, 100, 101, 102 or 103, wherein said CDR1, CDR2 and CDR3 do not contain the sequences of SEQ ID NO: 54 - 56 respectively; or

[0009] (2) CDR1 contains the amino acid sequence of SEQ ID NO: 57 or 90;

[0010] CDR2 contains the amino acid sequence of SEQ ID NO: 58 or 91; and

[0011] CDR3 contains the amino acid sequence of SEQ ID NO: 59, 92, 93 or 94, wherein said CDR1, CDR2 and CDR3 do not contain the sequences of SEQ ID NO: 57 - 59 respectively.

[0012] In some embodiments, said CDR1, CDR2 and CDR3 respectively contain:

[0013] (1) SEQ ID NO: 90, 58 and 59;

[0014] (2) SEQ ID NO: 57, 91 and 59;

[0015] (3) SEQ ID NO: 57, 58 and 92;

[0016] (4) SEQ ID NO: 57, 58 and 93;

[0017] (5) SEQ ID NO: 90, 58 and 92;

[0018] (6) SEQ ID NO: 90, 58 and 93;

[0019] (7) SEQ ID NO: 57, 91 and 92;

[0020] (8) SEQ ID NO: 57, 91 and 93;

[0021] (9) SEQ ID NO: 57, 58 and 94;

[0022] (10) SEQ ID NO: 90, 91 and 94;

[0023] (11) SEQ ID NO: 90, 91, and 92; or

[0024] (12) SEQ ID NO: 90, 91, and 93.

[0025] In some embodiments, the antibody or polypeptide comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 53. In some embodiments, the antibody or polypeptide comprises the framework region of SEQ ID NO: 53.

[0026] In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO: 57, 91, and 93, respectively. In some embodiments, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO: 67.

[0027] In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO: 90, 91, and 92, respectively. In some embodiments, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO: 70.

[0028] In some embodiments, CDR1, CDR2, and CDR3 comprise, respectively:

[0029] (13) SEQ ID NO: 95, 55, and 56;

[0030] (14) SEQ ID NO: 54, 96, and 56;

[0031] (15) SEQ ID NO: 54, 55, and 97;

[0032] (16) SEQ ID NO: 54, 55, and 98;

[0033] (17) SEQ ID NO: 54, 55, and 99;

[0034] (18) SEQ ID NO: 95, 55, and 97;

[0035] (19) SEQ ID NO: 95, 55, and 98;

[0036] (20) SEQ ID NO: 95, 55, and 99;

[0037] (21) SEQ ID NO: 54, 96, and 97;

[0038] (22) SEQ ID NO: 54, 96, and 98;

[0039] (23)SEQ ID NO: 54, 96, and 99;

[0040] (24)SEQ ID NO: 95, 96, and 97;

[0041] (25)SEQ ID NO: 95, 96, and 98;

[0042] (26)SEQ ID NO: 95, 96, and 99;

[0043] (27)SEQ ID NO: 95, 96, and 100;

[0044] (28)SEQ ID NO: 54, 55, and 101;

[0045] (29)SEQ ID NO: 54, 55, and 102; or

[0046] (30)SEQ ID NO: 54, 55, and 103.

[0047] In some embodiments, the antibody or polypeptide comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 42. In some embodiments, the antibody or polypeptide comprises the framework region of SEQ ID NO: 42.

[0048] In some embodiments, CDR1, CDR2, and CDR3 respectively comprise SEQ ID NO: 95, 96, and 100. In some embodiments, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO: 86.

[0049] In one embodiment, there is also provided a multispecific antibody comprising the antibody of the present disclosure and a second antibody or antigen-binding fragment, wherein the second antibody or antigen-binding fragment has binding specificity for a target antigen that is not PD-L1. In some embodiments, the target antigen is a tumor-associated antigen. In some embodiments, the second antibody is a full-size Fab antibody.

[0050] In another aspect, the present disclosure provides a polynucleotide encoding the antibody or polypeptide of the present application or the multispecific antibody of the present application.

[0051] In another aspect, the present disclosure provides a vector comprising the polynucleotide of the present application.

[0052] In another aspect, there is provided a cell comprising the polynucleotide or vector of the present application.

[0053] In another aspect, the present disclosure provides a composition comprising: (1) an antibody or polypeptide, multispecific antibody, or polynucleotide of the present application, and (2) a pharmaceutically acceptable carrier.

[0054] In another aspect, the present disclosure provides a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an antibody or polypeptide, multispecific antibody, or polynucleotide of the present application. In another aspect, the present disclosure provides the use of an antibody or polypeptide, multispecific antibody, or polynucleotide of the present application in the preparation of a medicament for treating cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from 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, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Results of ELISA binding of affinity matured nanobodies are shown.

[0056] Figure 2 Results of cell-based PD-L1 binding on PDL-1 overexpressing cells are shown.

[0057] Figure 3 Results of a PD-L1 ELISA blocking assay are shown.

[0058] Figure 4 Results of a PD-1 / PD-L1 functional reporter gene assay are shown.

[0059] Figure 5 Results of PDL-1 nanobody-mediated human IL-2 release in a mixed lymphocyte reaction (MLR) assay are shown.

[0060] Figure 6 Results of the in vivo anti-tumor effect of affinity matured nanobodies are shown. DETAILED DESCRIPTION

[0061] Definition

[0062] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" is to be understood as representing one or more antibodies. Thus, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein.

[0063] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence, which means that when aligned, that percentage of bases (or amino acids) is the same in the comparison of the two sequences. Such alignment and the percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One alignment program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBankCDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are those polynucleotides having the specified percentage of homology as described above and encoding polypeptides having the same or similar biological activity.

[0064] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a certain degree of homology or sequence identity with the nucleotide sequence of a certain nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that has a certain degree of homology with a certain nucleic acid or its complement. In one aspect, the nucleic acid homolog is capable of hybridizing with the nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a certain 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, compared to the reference polypeptide or polynucleotide, the equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt-bridge) of the reference sequence.

[0065] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a full antibody and any antigen-binding fragment or its single chain. Thus, the term "antibody" includes any molecule containing a protein or peptide that comprises at least a portion of an immunoglobulin molecule and has the biological activity of binding to an antigen. Examples of such include, but are not limited to, complementarity-determining regions (CDRs) of heavy or light chains or ligand-binding portions thereof, variable regions of heavy or light chains, constant regions of heavy or light chains, framework (FR) regions or any portion thereof, or at least a portion of a binding protein.

[0066] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc.). Regardless of structure, an antibody fragment binds to the same antigen recognized by the full antibody. The term "antibody fragment" includes aptamer, spiegelmer, and diabody. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0067] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (V H ) and light chain (V L ) of an immunoglobulin. In some aspects, these regions are linked by a short linker peptide of 10 to about 25 amino acids. For flexibility, the linker can be rich in glycine, and for solubility, the linker can be rich in serine or threonine, and the linker can link the N-terminus of V H to the C-terminus of V L , or vice versa. Despite the removal of the constant regions and the introduction of a linker, such a protein still retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019.

[0068] The term antibody encompasses a wide variety of biochemically distinguishable polypeptide classes. Those skilled in the art will understand that heavy chains can be classified as gamma, mu, alpha, delta, or epsilon (g, m, a, d, e), and there are also some subclasses within these classes (e.g., g1-g4). The nature of such chains determines the "class" of the antibody to be IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specificity. Given the present disclosure, those skilled in the art can readily identify modified versions of each of these classes and isotypes, and thus these modified versions are within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the IgG class of immunoglobulins will generally be discussed hereinafter. In the case of IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000 - 70,000. The four chains are generally linked by disulfide bonds in a "Y" configuration, where the light chains start from the opening of the "Y" shape, sandwich the heavy chains, and extend to the variable regions.

[0069] The antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab′, and F(ab′)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies of the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of the present 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 molecules.

[0070] "Specifically binds" or "is specific for" generally means that an antibody binds to an epitope through its antigen-binding domain, and the binding requires 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 a particular epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity of a particular antibody for binding to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B", or it can be said that antibody "A" has a higher specificity for binding to epitope "C" than its specificity for a related epitope "D".

[0071] As used herein, the term "treat" or "treatment" refers to therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (mitigate) an undesired physiological change or condition, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the degree of disease, stabilization of the disease state (i.e., not getting worse), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete), whether detectable or not. "Treatment" can also mean an extended survival as compared to the expected survival of a subject not receiving treatment. Subjects in need of treatment include those who already have a condition or disorder, those who are susceptible to a condition or disorder, or those in whom a condition or disorder is to be prevented.

[0072] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject in need of diagnosis, prognosis, or treatment, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0073] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects who would benefit from administration of the antibodies or compositions of the present disclosure (e.g., for detection, for diagnostic procedures, and / or for treatment), such as mammalian subjects.

[0074] Single-domain anti-PD-L1 antibody

[0075] The present disclosure provides single-chain anti-PD-L1 antibodies and humanized versions thereof that have high affinity for the human PD-L1 protein. Some of the best antibodies were further affinity matured, and some of the affinity-matured candidates exhibited excellent performance. These new antibodies exhibit potent binding and inhibitory activities and can be used for therapeutic and diagnostic purposes. Equally importantly, when integrated as one of the targeting units into various different forms of multispecific antibodies, certain resulting multispecific antibodies exhibit outstanding properties, establishing an additional utility of these single-domain anti-PD-L1 antibodies.

[0076] Accordingly, in one embodiment of the present disclosure, single-domain antibodies and polypeptides comprising such single-domain antibodies are provided. In some embodiments, the polypeptide is a bispecific antibody, a trispecific antibody, or a multispecific antibody.

[0077] One embodiment of the present disclosure provides a single-domain antibody or a polypeptide comprising the single-domain antibody, wherein the single-domain antibody has binding specificity for the human PD-L1 protein and comprises complementarity determining region 1 (CDR1), CDR2, and CDR3.

[0078] In some embodiments, the affinity matured single-domain anti-PD-L1 antibody is derived from the parental antibody 112-VH47 (SEQ ID NO: 53). Thus, in some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 54 or 95; CDR2 comprises the amino acid sequence of SEQ ID NO: 55 or 96; and CDR3 comprises the amino acid sequence of SEQ ID NO: 56, 97, 98, 99, 100, 101, 102, or 103, wherein CDR1, CDR2, and CDR3 do not comprise the sequences of SEQ ID NOs: 54-56, respectively.

[0079] In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 90, 58, and 59, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 57, 91, and 59, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 57, 58, and 92, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 57, 58, and 93, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 90, 58, and 92, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 90, 58, and 93, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 57, 91, and 92, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 57, 91, and 93, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 57, 58, and 94, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 90, 91, and 94, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 90, 91, and 92, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 90, 91, and 93, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 55, and 56, respectively.

[0080] In some embodiments, the antibody or polypeptide comprising the above CDRs comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:53. In some embodiments, the antibody or polypeptide comprises the framework region of SEQ ID NO:53.

[0081] In some embodiments, CDR1, CDR2 and CDR3 comprise SEQ ID NOs:57, 91 and 93, respectively. In some embodiments, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO:67.

[0082] In some embodiments, CDR1, CDR2 and CDR3 comprise SEQ ID NOs:90, 91 and 92, respectively. In some embodiments, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO:70.

[0083] In some embodiments, the affinity matured single domain anti-PD-L1 antibody is derived from the parental antibody 93_VH-6 (SEQ ID NO:42). Thus, in some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:57 or 90; CDR2 comprises the amino acid sequence of SEQ ID NO:58 or 91; and CDR3 comprises the amino acid sequence of SEQ ID NO:59, 92, 93 or 94, wherein CDR1, CDR2 and CDR3 do not comprise the sequences of SEQ ID NOs:57-59, respectively.

[0084] In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 96, and 56, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 55, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 55, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 55, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 55, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 55, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 55, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 96, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 96, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 96, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 96, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 96, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 96, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 96, and 100, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 55, and 101, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 55, and 102, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:54, 55, and 103, respectively.

[0085] In some embodiments, an antibody or polypeptide comprising the above CDRs comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:42. In some embodiments, the antibody or polypeptide comprises the framework region of SEQ ID NO:42.

[0086] In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NO:95, 96, and 100, respectively. In some embodiments, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO:86.

[0087] In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that compete with any of the antibodies disclosed herein for binding to human PD-L1 are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments that bind to the same epitope as any of the antibodies disclosed herein are also provided. In some embodiments, anti-PD-L1 antibodies and antigen-binding fragments are also provided that comprise the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 of the antibodies disclosed herein.

[0088] Compositions are also provided that comprise an antibody or polypeptide and a pharmaceutically acceptable carrier.

[0089] One of ordinary skill in the art will also understand that the antibodies disclosed herein can be modified such that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein can be similar, e.g., having a certain percentage identity to the starting sequence, e.g., it can 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.

[0090] Bispecific antibodies

[0091] Bispecific and multispecific antibodies are also provided that comprise one, two, three, or four units of the single-domain anti-PD-L1 antibodies disclosed herein and one or more other specificities (non-PD-L1).

[0092] In some embodiments, the second specificity is directed against a tumor-associated antigen (TAA) or an immune checkpoint protein. Non-limiting examples of tumor-associated antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, and claudin 18.2.

[0093] Non-limiting examples of immune checkpoint proteins include PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, and CD47.

[0094] Multispecific antibodies can include constant regions from any IgG type, such as IgG1 and IgG4.

[0095] Compositions are also provided that include an antibody or polypeptide and a pharmaceutically acceptable carrier.

[0096] Those of ordinary skill in the art will understand that the antibodies disclosed herein can be modified such that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a designated protein can be similar, e.g., having a certain percentage identity with the starting sequence, e.g., it can 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.

[0097] Polynucleotides encoding antibodies and methods of making antibodies

[0098] The present disclosure also provides isolated polynucleotides or nucleic acid molecules that encode the antibodies, variants, or derivatives of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, its variants, or derivatives on the same polynucleotide molecule or on different polynucleotide molecules. Additionally, the polynucleotides of the present disclosure can encode portions of the heavy chain variable region and the light chain variable region of the antigen-binding polypeptide, its variants, or derivatives on the same polynucleotide molecule or on different polynucleotide molecules.

[0099] Methods of making antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be prepared 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 upon antigen challenge, but whose endogenous loci have been inactivated. Exemplary techniques for preparing such antibodies are described in U.S. Patents: 6,150,584; 6,458,592; 6,420,140, which are incorporated herein by reference in their entirety.

[0100] Cancer treatment

[0101] As described herein, the antibodies, multispecific antibodies, polypeptides, variants or derivatives of the present disclosure can be used in certain therapeutic and diagnostic methods.

[0102] The present disclosure also relates to antibody-based therapies, which involve administering an antibody of the present disclosure to a patient (such as an animal, mammal, and human) to treat one or more of the diseases or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including their variants and derivatives as described herein).

[0103] The antibodies of the present disclosure can also be used to treat or inhibit cancer. It has been reported that PD-L1 is overexpressed in tumor cells. Tumor-derived PD-L1 can bind to PD-1 on immune cells, thereby restricting anti-tumor T cell immunity. The results of small molecule inhibitors or monoclonal antibodies targeting PD-L1 in mouse tumor models indicate that PD-L1-targeted therapy is an important and achievable alternative for effectively controlling tumor growth. As shown in the experimental examples, anti-PD-L1 antibodies activate the adaptive immune response mechanism, which can lead to an increase in the survival rate of cancer patients.

[0104] Accordingly, in some embodiments, a method of treating cancer in a patient in need thereof is provided. In one embodiment, the method involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one cancer cell (such as a stromal cell) of the patient expresses, overexpresses, or is induced to express PD-L1. For example, PD-L1 expression can be induced by administering a tumor vaccine or radiotherapy.

[0105] Tumors expressing the PD-L1 protein include tumors of 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, kidney cancer, and small cell lung cancer. Accordingly, the antibodies presently disclosed can be used to treat any one or more of such cancers.

[0106] Compositions

[0107] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (such as an immune checkpoint inhibitor).

[0108] In a specific embodiment, the term "pharmaceutically acceptable" refers to being approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for animals (more particularly for humans). Additionally, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation adjuvant.

[0109] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with a therapeutic agent. 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, etc. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Aqueous solutions of salts and aqueous solutions of dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition can also contain small amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and osmotic pressure regulators, 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, etc. The composition can be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, and a suitable amount of carrier in order to provide a form suitable for administration to a patient. The formulation should be suitable for the mode of administration. Parenteral formulations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0110] In one embodiment, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to humans according to conventional methods. Generally, compositions for intravenous administration are solutions in sterile, isotonic, aqueous buffers. If necessary, the composition may also include solubilizing agents and local anesthetics (such as lidocaine) to reduce the pain at the injection site. Generally, the components may be provided individually or may be provided in admixture in unit dosage form, for example as a dry lyophilized powder or an anhydrous concentrate, in a hermetically sealed container such as an ampoule or sachet, and labeled with the content of the active agent. In the case of administering the composition by infusion, it may be provided in an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water or saline for injection may be provided so that the components can be mixed before administration.

[0111] Example

[0112] Example 1. Generation of alpaca single-domain antibodies against human PD-L1

[0113] This example shows how to generate anti-human-PD-L1 single-domain antibodies by immunizing alpacas, followed by constructing a phage library and screening.

[0114] Antigen : Recombinant human PD-L1 / hFc fusion protein was used as an immunogen to generate anti-human PD-L1 antibodies. A fusion protein containing the entire extracellular region of human PD-L1 fused to the human immunoglobulin Fc domain was used as an immunogen.

[0115] Immunization

[0116] On day 0, an alpaca was first immunized subcutaneously (SC) with a 1:1 mixture of 600 mg of murine PD-L1 and Freund's complete adjuvant, immunized with 250 mg of murine PD-L1 and Freund's incomplete adjuvant on day 21, and immunized with 250 mg of human PD-L1 and Freund's incomplete adjuvant on day 42. The immune response was monitored by measuring the titer of anti-PD-L1 binding.

[0117] Library construction and screening

[0118] Alpaca PBMCs were collected, and an antibody phage display library was generated by extracting RNA, reverse transcribing cDNA, PCR amplifying, and cloning into a phage display vector. Then, the library was subjected to one round of liquid panning and one round of solid-phase panning. Generally, the library was 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 screening.

[0119] The sequences of the binders were amplified from 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.

[0120] Table 1. Antibody Sequences

[0121]

[0122]

[0123]

[0124] Example 2. Binding and Blocking Activities of Llama Human PD-L1 Monoclonal Antibodies

[0125] The binding and blocking properties of some antibodies were characterized by Gator. First, the anti-his probe was loaded onto the chip, and then the antigen was captured by human PD-L1-his. Then, the antibody was injected to record the binding curve. Finally, human PD1 / hFc was injected to determine whether the antibody could block the interaction between PD-1 and PD-L1. ALP-Tan-3p-112, ALP-Tan-3p-93, and ASP-30-46 could all effectively block the interaction between PD-1 and PD-L1 (Table 2). The affinity was further confirmed by Biacore T200.

[0126] Table 2. Affinity

[0127] Kon (1 / Ms) Koff (1 / s) KD (M) ALP-Tan-3p-112 <![CDATA[4.9X10 5 > 0.000235 <![CDATA[4.79X10 -10 > ALP-Tan-3p-93 <![CDATA[1.54X10 5 > <![CDATA[7.8X10 -5 > <![CDATA[4.59X10 -10 > ASP-3p-46 <![CDATA[7.1X10 5 > 0.00128 <![CDATA[1.81X10 -9 >

[0128] Example 3. Humanization of Llama Anti-PD-L1 Monoclonal Antibodies.

[0129] Generating humanized mAbs using the variable region genes of monoclonal antibodies (mAbs) ALP-Tan-3p-93 and ALP-Tan-3p-112. In the first step of this process, the amino acid sequences of ALP-Tan-3p-93 and ALP-Tan-3p-112 were compared with the database of existing human Ig gene sequences to find the human germline Ig gene sequences that were the best overall match. For ALP-Tan-3p-93, the closest human match was the IGHV3-23*04 gene. Then, humanized variable domain sequences were designed in which the CDR1, 2, and 3 of ALP-Tan-3p-93 were grafted onto the framework sequence of the IGHV3-23*04 gene. For ALP-Tan-3p-112, the closest human match was the IGHV3-48*03 gene. Then, humanized variable domain sequences were designed in which the CDR1, 2, and 3 of ALP-Tan-3p-112 were grafted onto the framework sequence of the IGHV3-48*03 gene. At the same time, a residue mutation (N34Q, Kabat numbering) was introduced into CDR1 to reduce the risk of post-translational modification. Then, 3D models were generated to determine if there were any framework positions where replacing alpaca amino acids with human amino acids might affect binding and / or CDR conformation.

[0130] Table 3. Humanized antibodies and revertant mutations

[0131]

[0132]

[0133] Table 3A. CDR sequences

[0134]

[0135] Example 4. Complete kinetic analysis of the humanized anti-PD-L1 monoclonal antibody.

[0136] To explore the binding kinetics of the humanized antibodies, in this example, the binding and dissociation between various doses of antigen (100 nM, 50 nM, 25 nM, 12.5 nM, 6.15 nM, 3.125 nM, 1.5625 nM) and different monoclonal antibodies were monitored by Biacore to further perform a complete kinetic affinity test. As shown in Table 4, the affinity of 112-VHH5-PTM was comparable to that of the ALP-Tan-3p-112 chimeric antibody. The affinities of 93VH-4, 93VH-6, and 93VH-8 were comparable to those of the ALP-Tan-3p-93 chimeric antibody.

[0137] Table 4. Affinity

[0138] Kon (1 / Ms) Koff (1 / s) KD (M) ALP-Tan-3p-112 (chimeric) <![CDATA[2.14X10 6 > <![CDATA[5.66X10 -4 > <![CDATA[2.64X10 -10 > 112-VHH3-PTM <![CDATA[2.07X10 6 > <![CDATA[4.98X10 -3 > <![CDATA[2.40X10 -9 > 112-VHH5-PTM <![CDATA[1.62X10 6 > <![CDATA[1.49X10 -3 > <![CDATA[9.20X10 -10 > 112-VHH7-PTM <![CDATA[1.69X10 6 > <![CDATA[3.93X10 -3 > <![CDATA[2.32X10 -9 > ALP-Tan-3p-93 (chimeric) <![CDATA[1.92X10 5 > <![CDATA[1.07X10 -4 > <![CDATA[5.59X10 -10 > 93-VH-2 <![CDATA[6.15X10 4 > <![CDATA[1.62X10 -3 > <![CDATA[2.63X10 -8 > 93-VH-3 <![CDATA[4.86X10 5 > <![CDATA[1.15X10 -3 > <![CDATA[2.36X10 -9 > 93-VH-4 <![CDATA[8.05X10 4 > <![CDATA[1.47X10 -4 > <![CDATA[1.82X10 -9 > 93-VH-5 <![CDATA[4.52X10 5 > <![CDATA[1.25X10 -3 > <![CDATA[2.77X10 -9 > 93-VH-6 <![CDATA[9.59X10 4 > <![CDATA[1.97X10 -4 > <![CDATA[2.06X10 -9 > 93-VH-7 <![CDATA[8.76X10 4 > <![CDATA[3.39X10 -4 > <![CDATA[3.87X10 -9 > 93-VH-8 <![CDATA[1.33X10 5 > <![CDATA[2.39X10 -4 > <![CDATA[1.80X10 -9 > 93-VH-9 <![CDATA[1.40X10 5 > <![CDATA[3.10X10 -4 > <![CDATA[2.20X10 -9 >

[0139] Example 5. Binding Characteristics of Humanized Anti-PD-L1 Antibody

[0140] First, the binding characteristics of the humanized anti-PD-L1 antibody of the present application were evaluated by ELISA assay. Briefly, 100 μl of anti-PD-L1 antibodies 93-VH6 or 112-VH47 at different concentrations 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. Exemplary anti-PD-L1 antibodies 93-VH6 and 112-VH47 both showed specific binding to human PD-L1 in a dose-dependent manner.

[0141] The binding ability of the anti-PD-L1 antibody of the present application was further evaluated by using Raji cells overexpressing human PD-L1. Briefly, 50 μl of Raji cells overexpressing human PD-L1 were seeded into a 96-well plate at a concentration of 2*10 5 cells / well. 50 μl of anti-PD-L1 antibodies 93-VH6 or 112-VH47 at different concentrations were added to each well and incubated with the cells on ice for 1 hour. Then the cells were washed twice with FACS buffer and 100 μl of PE-anti hu IgG was added, followed by incubation on ice for 1 hour. After incubation, the cells in each well were collected and resuspended in 65 μl of FACS buffer for analysis by flow cytometry. Exemplary anti-PD-L1 antibodies 93-VH6 and 112-VH47 both showed specific binding to Raji cells overexpressing human PD-L1 in a dose-dependent manner.

[0142] Example 6. T Cell Activation Bioassay (NFAT)

[0143] To test the ability of anti-PD-L1 antibodies to stimulate T cell responses, Jurkat cells expressing hPD-1 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 the 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) was used to stimulate TCR signaling. In this system, ectopically expressed huPD-L1 can inhibit NFAT-luciferase activity in SEE-stimulated Jurkat cells, while anti-PD-L1 antibodies can reverse NFAT-luciferase activity. Briefly, APCs (2.5×10 4) Co-cultured with Jurkat T cells expressing PD-1 (1×10 5 ) in the presence of SEE stimulation. Anti-PD-L1 antibody was added at the start of the culture. After 6 hours, the luciferase activity of the resulting cells was evaluated.

[0144] All tested anti-PD-L1 antibodies blocked the PD-1 / PD-L1 interaction, thereby enhancing NFAT-mediated luciferase activity.

[0145] Example 7. Affinity maturation of 93-VH6 and 112-VH47

[0146] Affinity maturation was performed on two anti-PD-L1 single domain antibodies (sdAbs), 93-VH6 and 112-VH47. One, two, or three residues in each CDR were selected for mutation (see the mutated CDRs in Table 5A). A total of 30 candidate antibodies were designed and prepared using synthetic cDNA. Their sequences and the mutated CDRs are listed in Tables 5B-C below.

[0147] Table 5A. CDRs for affinity maturation

[0148]

[0149]

[0150] Table 5B. Affinity maturation

[0151]

[0152]

[0153] Table 5C. CDR sequences

[0154]

[0155]

[0156] Example 8. Testing of affinity matured antibodies

[0157] Various tests were performed on the affinity matured antibodies for their binding affinity to the target PD-L1 protein.

[0158] ELISA test results are in Figure 1Shown in. Briefly, 100 μl of anti-PD-L1 antibodies at different concentrations 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 the analysis was performed by the chromogenic reaction of HRP with its substrate. The affinity-matured anti-PD-L1 antibodies showed specific binding to human PD-L1 in a dose-dependent manner.

[0159] To explore the binding kinetics of the affinity-matured antibodies, the binding and dissociation between various doses of antigen (50 nM, 25 nM, 12.5 nM, 6.15 nM, 3.125 nM, 1.5625 nM) and different monoclonal antibodies were monitored by Biacore, and a complete kinetic affinity test was further carried out. Compared with the parental antibody, the affinity-matured antibodies showed similar or improved binding to human PD-L1.

[0160] The activities of these antibodies in binding to PD-L1 expressed on CHO or Raji cells were also tested. As Figure 2 shown, all of these antibodies showed good binding effects on cell surface PD-L1. The experiment was carried out by a method similar to that in Example 5.

[0161] The ability of the antibodies to block the binding of PD-L1 and PD-1 was tested by an ELISA blocking assay. Briefly, 100 μl of anti-PD-L1 antibodies at different concentrations and Biotin-PD-1 were co-incubated in each well of a 96-well plate pre-coated with human His-PD-L1, then streptavidin-HRP was added and the analysis was performed by the chromogenic reaction of HRP with its substrate. As Figure 3 shown, all of these antibodies retained high blocking activity.

[0162] The biological functions of these affinity-matured antibodies were tested by a PD-1 / PD-L1 blocking bioassay. Briefly, CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein were used as target cells, and Jurkat T cells expressing human PD-1 and a luciferase reporter gene driven by a NFAT-responsive element (NFAT-RE) were used as effector cells. Different concentrations of anti-PD-L1 antibodies were incubated with these two types of cells at 37 °C for 6 hours. After 6 hours, the luciferase activity of the resulting cells was evaluated. As Figure 4 shown in A-C, all the antibodies were biologically active. The NFAT T cell activation assay described in Example 6 was also carried out and similar results were obtained, as Figure 4As shown in D. All tested affinity-matured anti-PD-L1 antibodies blocked the PD-1 / PD-L1 interaction, thereby enhancing NFAT-mediated luciferase activity. And the affinity-matured antibodies, such as R21448516, R21560108, R21560111, showed improved effects compared to their parental molecules 93-VH6 and 112-VH47.

[0163] Based on these data, two affinity-matured antibodies, R21560108 and R21560111, were selected for further study. In a mixed lymphocyte reaction (MLR) assay with two donor pairs, the anti-PDL-1 antibodies could induce human IL-2 production in a dose-dependent manner, as Figure 5 shown.

[0164] Example 9. In Vivo Antitumor Effects of Affinity-Matured Antibodies

[0165] The MC38-PD-L1 syngeneic model with tumor cells expressing human PD-L1 was used to test the antitumor effects of the affinity-matured antibodies R21560102, R21560108, and R21448516. As Figure 6 shown, compared to the control group (vehicle), all three tested affinity-matured anti-PD-L1 antibodies showed effective tumor growth inhibition.

[0166] ***

[0167] The scope of the present disclosure is not limited by the specific embodiments described, which are only individual examples of various aspects of the present disclosure, and any composition or method that is functionally equivalent is within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they fall within the scope of the appended claims and their equivalents.

[0168] All publications and patent applications mentioned in this specification are incorporated herein by reference to the 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 complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein: (1) CDR1 comprises the amino acid sequence of SEQ ID NO: 54 or 95; CDR2 comprises the amino acid sequence of SEQ ID NO: 55 or 96; and CDR3 comprises the amino acid sequence of SEQ ID NO: 56, 97, 98, 99, 100, 101, 102, or 103, wherein said CDR1, CDR2, and CDR3 do not respectively comprise the sequences of SEQ ID NO: 54-56; or (2) CDR1 comprises the amino acid sequence of SEQ ID NO: 57 or 90; CDR2 comprises the amino acid sequence of SEQ ID NO: 58 or 91; and CDR3 comprises the amino acid sequence of SEQ ID NO: 59, 92, 93, or 94, wherein said CDR1, CDR2, and CDR3 do not respectively comprise the sequences of SEQ ID NO: 57-59.

2. The antibody or polypeptide of claim 1, wherein said CDR1, CDR2, and CDR3 respectively comprise: (1) SEQ ID NO: 90, 58, and 59; (2) SEQ ID NO: 57, 91, and 59; (3) SEQ ID NO: 57, 58, and 92; (4) SEQ ID NO: 57, 58, and 93; (5) SEQ ID NO: 90, 58, and 92; (6) SEQ ID NO: 90, 58, and 93; (7) SEQ ID NO: 57, 91, and 92; (8) SEQ ID NO: 57, 91, and 93; (9) SEQ ID NO: 57, 58, and 94; (10) SEQ ID NO: 90, 91, and 94; (11) SEQ ID NO: 90, 91, and 92; or (12) SEQ ID NO: 90, 91, and 93.

3. The antibody or polypeptide of claim 2, which comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

53.

4. The antibody or polypeptide of claim 2, which comprises the framework region of SEQ ID NO:

53.

5. The antibody or polypeptide of any one of claims 2-4, wherein said CDR1, CDR2, and CDR3 respectively comprise SEQ ID NO: 57, 91, and 93.

6. The antibody or polypeptide of claim 5, which comprises the amino acid sequence of SEQ ID NO:

67.

7. The antibody or polypeptide of any one of claims 2-4, wherein said CDR1, CDR2, and CDR3 respectively comprise SEQ ID NO: 90, 91, and 92.

8. The antibody or polypeptide of claim 7, which comprises the amino acid sequence of SEQ ID NO:

70.

9. The antibody or polypeptide of claim 1, wherein said CDR1, CDR2, and CDR3 respectively comprise: (13) SEQ ID NO: 95, 55, and 56; (14) SEQ ID NO: 54, 96, and 56; (15) SEQ ID NO: 54, 55, and 97; (16) SEQ ID NO: 54, 55, and 98; (17) SEQ ID NO: 54, 55, and 99; (18) SEQ ID NO: 95, 55, and 97; (19) SEQ ID NO: 95, 55, and 98; (20) SEQ ID NO: 95, 55, and 99; (21) SEQ ID NO: 54, 96, and 97; (22) SEQ ID NO: 54, 96, and 98; (23) SEQ ID NO: 54, 96, and 99; (24) SEQ ID NO: 95, 96, and 97; (25) SEQ ID NO: 95, 96, and 98; (26) SEQ ID NO: 95, 96, and 99; (27) SEQ ID NO: 95, 96, and 100; (28) SEQ ID NO: 54, 55, and 101; (29) SEQ ID NO: 54, 55, and 102; or (30) SEQ ID NO: 54, 55, and 103.

10. The antibody or polypeptide of claim 9, which comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

42.

11. The antibody or polypeptide of claim 9, which comprises the framework region of SEQ ID NO:

42.

12. The antibody or polypeptide of any one of claims 9 - 11, wherein said CDR1, CDR2, and CDR3 respectively comprise SEQ ID NO: 95, 96, and 100.

13. The antibody or polypeptide of claim 12, which comprises the amino acid sequence of SEQ ID NO:

86.

14. The antibody or polypeptide of claim 10, wherein said antibody is humanized.

15. The antibody or polypeptide of any one of claims 1 - 14, wherein said polypeptide is a multispecific antibody, and said multispecific antibody further has binding specificity for an antigen different from PD - L1.

16. A multispecific antibody, which comprises the antibody of any one of claims 1 - 14 and a second antibody or antigen - binding fragment, and said second antibody or antigen - binding fragment has binding specificity for a target antigen that is not PD - L1.

17. The multispecific antibody of claim 16, wherein said target antigen is a tumor - associated antigen.

18. The multispecific antibody of claim 16 or 17, wherein said second antibody is a full - size Fab antibody.

19. A polynucleotide, which encodes the antibody or polypeptide of any one of claims 1 - 15 or the multispecific antibody of any one of claims 16 - 18.

20. A vector, which comprises the polynucleotide of claim 19.

21. A cell, which comprises the polynucleotide of claim 19 or the vector of claim 20.

22. A composition, which comprises: (1) An antibody or polypeptide according to any one of claims 1-15, a multispecific antibody according to any one of claims 16-18, a polynucleotide according to claim 19, a vector according to claim 20 or a cell according to claim 21, and (2) a pharmaceutically acceptable carrier.

23. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an antibody or polypeptide according to any one of claims 1-15, a multispecific antibody according to any one of claims 16-18, a polynucleotide according to claim 19, a vector according to claim 20 or a cell according to claim 21.

24. Use of an antibody or polypeptide according to any one of claims 1-15, a multispecific antibody according to any one of claims 16-18, a polynucleotide according to claim 19, a vector according to claim 20 or a cell according to claim 21 in the manufacture of a medicament for the treatment of cancer.

25. The method of claim 23 or the use of claim 24, wherein the cancer is a solid tumor.

26. The method of claim 23 or the use of claim 24, wherein the cancer is selected from 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, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer.

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