Anti-PD-L1 nanobodies

Abstract

Provided are affinity-matured single-domain anti-PD-L1 antibodies and polypeptides (e.g., multispecific antibodies and chimeric antigen receptors) comprising the single-domain antibodies. The antibodies, including their humanized counterparts, exhibit excellent activity and are suitable for use in a variety of multispecific antibody formats. Also provided are methods of using the antibodies or polypeptides to treat and diagnose diseases such as cancer and infectious diseases. The present disclosure provides novel single-domain antibodies and affinity-matured counterparts that target the human PD-L1 protein.

Description

[Background technology]

[0001] background Single domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of only one monomeric variable antibody domain. Like intact antibodies, single domain antibodies can selectively bind to specific antigens. With molecular weights of only 12-15 kDa, single domain antibodies are much smaller than typical antibodies (150-160 kDa). Given their small size and single-chain nature, single domain antibodies may be particularly suitable for inclusion as fragments in other proteins (e.g., bispecific antibodies).

[0002] Various antibodies specific for programmed death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), are 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 allotransplantation, autoimmune diseases, and other disease states (e.g., hepatitis). When PD-L1 binds to PD-1 or B7.1, it transmits an inhibitory signal that reduces CD8+ T cell proliferation in lymph nodes. In addition, PD-1 can also control the accumulation of foreign antigen-specific T cells in lymph nodes through apoptosis, which is further mediated by downregulation of the gene Bcl-2.

[0003] 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 in infected mice. PD-L1 blockade reduced TNFα and nitric oxide production by macrophages, reduced granzyme B production by NK cells, and reduced the proliferation of L. monocytogenes antigen-specific CD8 T cells (but not CD4 T cells). This evidence suggests that PD-L1 acts as a positive costimulatory molecule in intracellular infection. Summary of the Invention [Means for solving the problem]

[0004] overview This disclosure provides novel single-domain antibodies and affinity-matured counterparts that target the human PD-L1 protein. These single-domain antibodies exhibited excellent binding affinity and biological function despite their small size. When included in a variety of different multispecific antibody formats, some of the resulting multispecific antibodies exhibited excellent properties.

[0005] One embodiment of the present disclosure provides a single domain antibody, or a polypeptide comprising a single domain antibody, wherein the single domain antibody has binding specificity for human PD-L1 protein and comprises complementarity determining region 1 (CDR1), CDR2, and CDR3; (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; CDR3 comprises the amino acid sequence of SEQ ID NO: 56, 97, 98, 99, 100, 101, 102, or 103, and CDR1, CDR2, and CDR3 do not comprise the sequences of SEQ ID NOs: 54 to 56, respectively; 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; CDR3 comprises the amino acid sequence of SEQ ID NO: 59, 92, 93, or 94, and CDR1, CDR2, and CDR3 do not comprise the sequences of SEQ ID NOs: 57 to 59, respectively.

[0006] In some embodiments, CDR1, CDR2, and CDR3 are each (1) SEQ ID NOs: 90, 58, and 59; (2) SEQ ID NOs: 57, 91, and 59; (3) SEQ ID NOs: 57, 58, and 92; (4) SEQ ID NOs: 57, 58, and 93; (5) SEQ ID NOs: 90, 58, and 92; (6) SEQ ID NOs: 90, 58, and 93; (7) SEQ ID NOs: 57, 91, and 92; (8) SEQ ID NOs: 57, 91, and 93; (9) SEQ ID NOs: 57, 58, and 94; (10) SEQ ID NOs: 90, 91, and 94; (11) SEQ ID NOs: 90, 91, and 92; or (12) SEQ ID NOs: 90, 91, and 93 Includes.

[0007] In some embodiments, the antibody or polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 53. In some embodiments, the antibody or polypeptide comprises the framework region of SEQ ID NO:53.

[0008] 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.

[0009] 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.

[0010] In some embodiments, CDR1, CDR2, and CDR3 are each (13) SEQ ID NOs: 95, 55, and 56; (14) SEQ ID NOs: 54, 96, and 56; (15) SEQ ID NOs: 54, 55, and 97; (16) SEQ ID NOs: 54, 55, and 98; (17) SEQ ID NOs: 54, 55, and 99; (18) SEQ ID NOs: 95, 55, and 97; (19) SEQ ID NOs: 95, 55, and 98; (20) SEQ ID NOs: 95, 55, and 99; (21) SEQ ID NOs: 54, 96, and 97; (22) SEQ ID NOs: 54, 96, and 98; (23) SEQ ID NOs: 54, 96, and 99; (24) SEQ ID NOs: 95, 96, and 97; (25) SEQ ID NOs: 95, 96, and 98; (26) SEQ ID NOs: 95, 96, and 99; (27) SEQ ID NOs: 95, 96, and 100; (28) SEQ ID NOs: 54, 55, and 101; (29) SEQ ID NOs: 54, 55, and 102; or (30) SEQ ID NOs: 54, 55, and 103 Includes.

[0011] In some embodiments, the antibody or polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 42. In some embodiments, the antibody or polypeptide comprises the framework region of SEQ ID NO:42.

[0012] In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 96, and 100, respectively. In some embodiments, the antibody or polypeptide comprises the amino acid sequence of SEQ ID NO:86.

[0013] Also provided is, in one embodiment, a multispecific antibody comprising an antibody of the present disclosure and a second antibody or antigen-binding fragment thereof having binding specificity for a target antigen other than 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.

[0014] In another aspect, provided herein are polynucleotides encoding the antibodies or polypeptides of the present application, or the multispecific antibodies of the present application.

[0015] In another aspect, provided herein is a vector comprising the polynucleotide of the present application.

[0016] In another aspect, provided herein is a cell comprising the polynucleotide or vector of the present application.

[0017] In another aspect, provided herein are compositions comprising (1) the antibody or polypeptide, multispecific antibody, or polynucleotide of the present application, and (2) a pharmaceutically acceptable carrier.

[0018] 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, polypeptide, multispecific antibody, or polynucleotide of the present application. In another aspect, provided herein is use of an antibody, polypeptide, multispecific antibody, or polynucleotide of the present application for 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 the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. [Brief explanation of the drawings]

[0019] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the ELISA binding results of affinity matured nanobodies.

[0020] [Figure 2] Figure 2 shows the results of cell-based PD-L1 binding to PDL-1-overexpressing cells.

[0021] [Figure 3] Figure 3 shows the results of a PD-L1 ELISA blocking assay.

[0022] [Figure 4] Figure 4 shows the results of the PD-1 / PD-L1 functional reporter assay.

[0023] [Figure 5] Figure 5 shows the results of PDL-1 nano-mediated human IL-2 release in a mixed lymphocyte reaction (MLR) assay.

[0024] [Figure 6]Figure 6 shows the in vivo antitumor efficacy of affinity matured nanobodies. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description definition It should be noted that the term "a" or "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 terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0026] 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" to another sequence; this means that when aligned, that percentage of bases (or amino acids) are the same when comparing these two sequences. This alignment and percent homology or percent sequence identity can be determined using software programs known in the art, for example, using the software programs described in Ausubel et al. (eds.) (2007) Current Protocols in Molecular Biology. 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 specified percent homology above and encode polypeptides having the same or similar biological activity.

[0027] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that shares a degree of homology or sequence identity with the nucleotide sequence of a nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that shares 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 that shares 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 polynucleotide. In some aspects, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0028] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. Antibodies include whole antibodies and any antigen-binding fragments thereof or single chains thereof. Thus, the term "antibody" includes any protein or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, a heavy or light chain complementarity-determining region (CDR) or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0029] 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 that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0030] "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 scFv molecule can be linked to the C-terminus of the original immunoglobulin, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. Various scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0031] The term antibody encompasses a wide variety of biochemically distinguishable polypeptide classes. 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 (e.g., γ1-γ4) within them. It is the nature of this chain that determines the "class" of an antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), 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 those skilled in the art in light of the present disclosure and, therefore, are within the scope of the present disclosure. While all immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule comprises 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. These 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.

[0032] Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present 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 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 molecule.

[0033] By "specifically binds" or "having specificity for," it is generally meant that an antibody binds to an epitope via its antigen-binding domain and that such 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 via 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 higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with greater specificity than it has for related epitope "D."

[0034] 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) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., non-worsening) state of disease, delay or slowing of disease progression, improvement or palliation of the disease state, and "Treatment" includes, but is not limited to, improvement and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those with a condition or already with the disorder, as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0035] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject for whom 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, etc.

[0036] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, e.g., mammalian subjects, who would benefit from the administration of an antibody or composition of the present disclosure used, e.g., for detection, for diagnostic procedures, and / or for treatment.

[0037] Single-domain PD-L1 antibodies This 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 top antibodies were further subjected to affinity maturation, and some of the affinity-matured candidates showed excellent performance. These novel antibodies exhibited potent binding and inhibitory activity and are useful for therapeutic and diagnostic applications. Importantly, when incorporated as one of the targeting units in multispecific antibodies of various different formats, certain of the resulting multispecific antibodies exhibited remarkable properties, establishing the additional utility of this single-domain anti-PD-L1 antibody.

[0038] Thus, 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.

[0039] One embodiment of the present disclosure provides a single domain antibody, or a polypeptide comprising a single domain antibody, wherein the single domain antibody has binding specificity for human PD-L1 protein and comprises complementarity determining region 1 (CDR1), CDR2, and CDR3.

[0040] In some embodiments, the affinity-matured single domain anti-PD-L1 antibody is derived from the parent 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, and CDR1, CDR2, and CDR3 do not comprise the sequences of SEQ ID NOs: 54-56, respectively.

[0041] 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.

[0042] 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: 53. In some embodiments, the antibody or polypeptide comprises the framework region of SEQ ID NO: 53.

[0043] 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.

[0044] 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.

[0045] In some embodiments, the affinity-matured single domain anti-PD-L1 antibody is derived from the parent 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, and CDR1, CDR2, and CDR3 do not comprise the sequences of SEQ ID NOs: 57-59, respectively.

[0046] In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 96, and 56, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 55, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 55, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 55, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 55, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 55, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 55, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 96, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 96, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 96, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 96, and 97, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 96, and 98, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 96, and 99, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 96, and 100, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 55, and 101, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 55, and 102, respectively. In some embodiments, CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 54, 55, and 103, respectively.

[0047] 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.

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

[0049] 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 that include the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 of an antibody disclosed herein are also provided.

[0050] Compositions including the antibody or polypeptide and a pharmaceutically acceptable carrier are also provided.

[0051] It will also be understood by those skilled in the art that antibodies as disclosed herein can 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 can be similar to the starting sequence, e.g., have a certain percent identity to the starting sequence, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequences.

[0052] multispecific antibodies Also provided are bispecific and multispecific antibodies comprising one, two, three, or four units of the single domain anti-PD-L1 antibodies disclosed herein and one or more other specificities (other than PD-L1).

[0053] In some embodiments, the second specificity is directed against a tumor-associated antigen (TAA) or 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.

[0054] 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.

[0055] Multispecific antibodies can comprise constant regions from any IgG type, such as IgG1 and IgG4.

[0056] Compositions including the antibody or polypeptide and a pharmaceutically acceptable carrier are also provided.

[0057] It will also be understood by those skilled in the art that antibodies as disclosed herein can 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 can be similar to the starting sequence, e.g., have a certain percent identity to the starting sequence, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequences.

[0058] Polynucleotides encoding antibodies and methods for preparing antibodies The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the surface of the same polynucleotide molecule or on the surface of separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the surface of the same polynucleotide molecule or on the surface of separate polynucleotide molecules.

[0059] Various methods for producing antibodies are widely known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be produced 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 in which the endogenous gene locus has been disabled. Exemplary techniques that can be used to produce 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).

[0060] Cancer treatment As described herein, the antibodies, multispecific antibodies, polypeptides, variants or derivatives of the disclosure may be used in certain treatment and diagnostic methods.

[0061] The present disclosure further relates to antibody-based therapies that involve administering antibodies of the present disclosure to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0062] The antibodies of the present disclosure can also be used to treat or inhibit cancer. PD-L1 has been reported to be overexpressed in tumor cells. Tumor-derived PD-L1 can limit anti-tumor T cell immunity by binding to PD-1 on immune cells. Results from small molecule inhibitors or monoclonal antibodies targeting PD-L1 in mouse tumor models indicate that targeted PD-L1 therapy is an important alternative and practical approach to effectively control tumor growth. As demonstrated in experimental examples, anti-PD-L1 antibodies activate adaptive immune response mechanisms, which can lead to improved survival in cancer patients.

[0063] 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, overexpresses, or is induced to express PD-L1. Induction of PD-L1 expression can be achieved, for example, by administering a tumor vaccine or radiation therapy.

[0064] 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, stomach cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer. Accordingly, the presently disclosed antibodies can be used to treat any one or more of such cancers.

[0065] composition 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 (e.g., an immune checkpoint inhibitor).

[0066] In specific embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly, for use in humans. Furthermore, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any kind.

[0067] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a 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, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents (e.g., acetates, citrates, or phosphates). Antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can also be formulated as suppositories with traditional binders and carriers (e.g., 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 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, 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.

[0068] 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 contain a solubilizing agent and a local anesthetic (such as lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a hermetically sealed container (such as an ampoule or sachet) indicating the amount of active ingredient. When the composition is to be administered by injection, the composition can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration. [Example]

[0069] Example 1. Generation of alpaca single-domain antibodies 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.

[0070] 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.

[0071] 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. Immune responses were monitored by measuring titers for anti-PD-L1 binding.

[0072] 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. Typically, the libraries were incubated in immunotubes or beads coated with biotinylated PD-L1. Unbound phages were removed by washing 5 to 20 times with PBST. A total of three rounds of panning were performed for each selection.

[0073] The sequences of the binders were amplified from the antigen-binding positive phages by PCR and confirmed by DNA sequencing. The sequences of the specific antibodies and their CDR regions are provided in the table below. [Table 1-1] [Table 1-2] [Table 1-3]

[0074] 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 using 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. ALP-Tan-3p-112, ALP-Tan-3p-93, and ASP-30-46 all effectively blocked the interaction between PD-1 and PD-L1 (Table 2). The affinity was further confirmed using Biacore T200. [Table 2]

[0075] Example 3. Humanization of anti-PD-L1 alpaca monoclonal antibody. Humanized mAbs were generated using the variable region genes of 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 against available databases of human Ig gene sequences to find the best overall match for the human germline Ig gene sequence. For ALP-Tan-3p-93, the closest human match was IGHV3-23. * The CDR1, CDR2, and CDR3 of ALP-Tan-3p-93 were then cloned into IGHV3-23. * We designed a humanized variable domain sequence that was grafted onto the framework sequence of the O4 gene. For ALP-Tan-3p-112, the closest human match was IGHV3-48. * The CDR1, CDR2, and CDR3 of ALP-Tan-3p-112 were then cloned into IGHV3-48 *We designed a humanized variable domain sequence grafted onto the framework sequence of the O3 gene. Meanwhile, a single residue mutation (N34Q, Kabat numbering) was introduced into CDR1 to reduce the risk of post-translational modification. We then generated a 3D model to determine whether there were any framework positions where replacing an alpaca amino acid with a human amino acid could affect binding and / or CDR conformation. [Table 3-1] [Table 3-2] [Table 3A]

[0076] 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 (100 nM, 50 nM, 25 nM, 12.5 nM, 6.15 nM, 3.125 nM, 1.5625 nM) for various monoclonal antibodies using Biacore. As shown in Table 4, the affinity of 112-VHH5-PTM was comparable to that of the ALP-Tan-3p-112 chimeric antibody. The affinities of 93VH-4, 93VH-6, and 93VH-8 were comparable to that of the ALP-Tan-3p-93 chimeric antibody. [Table 4]

[0077] Example 5. Binding characteristics of humanized anti-PD-L1 antibodies The binding properties of the present humanized anti-PD-L1 antibodies were first assessed by ELISA assay. Briefly, 100μl of anti-PD-L1 antibody (93-VH6 or 112-VH47) at different concentrations was incubated in each well of a 96-well plate pre-coated with human His-PD-L1, followed by the addition of goat anti-human IgG Fc HRP and analysis by the color reaction of HRP with its substrate. Both exemplary anti-PD-L1 antibodies, 93-VH6 and 112-VH47, exhibited specific binding to human PD-L1 in a dose-dependent manner.

[0078] 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 cells / mL. * 10 5 Cells were seeded into a 96-well plate at a concentration of 100 μl / well. 50 μl of anti-PD-L1 antibody (93-VH6 or 112-VH47) at different concentrations 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 100 μl 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 65 μl of FACS buffer for flow cytometric analysis. 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.

[0079] 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 the human PD-1 gene via lentivirus were used as responder cells. Raji-PD-L1 cells were used as antigen-presenting cells (APCs). Staphylococcal enterotoxin E (SEE) was used to stimulate TCR signaling. In this system, ectopically expressed huPD-L1 can suppress SEE-stimulated NFAT-luciferase activity in Jurkat cells, while anti-PD-L1 antibodies can reverse NFAT-luciferase activity. Briefly, APCs (2.5 × 10 4 ) were incubated with PD-1-expressing Jurkat T cells (1 × 10 5 The cells were co-cultured with PD-L1 antibodies (anti-PD-L1 antibodies) at the initiation of culture. After 6 hours, the resulting cells were assessed for luciferase activity.

[0080] All anti-PD-L1 antibodies tested blocked the PD-1 / PD-L1 interaction and therefore enhanced NFAT-mediated luciferase activity.

[0081] Example 7. Affinity Maturation of 93-VH6 and 112-VH47 Two anti-PD-L1 single domain antibodies (sdAbs), 93-VH6 and 112-VH47, were subjected to affinity maturation. One, two, or three residues in each CDR were selected for mutation (see mutated CDRs in Table 5A). A total of 30 candidate antibodies were designed and generated using synthetic cDNA. Their sequences and mutated CDRs are listed in Tables 5B-5C below. [Table 5A-1] [Table 5A-2] [Table 5B-1] [Table 5B-2] [Table 5C]

[0082] Example 8. Testing of affinity matured antibodies The affinity-matured antibodies were subjected to various tests for their binding affinity to the target PD-L1 protein.

[0083] The ELISA test results are shown in Figure 1. Briefly, 100μl of anti-PD-L1 antibodies at various concentrations were incubated in each well of a 96-well plate pre-coated with human His-PD-L1, followed by the addition of goat anti-human IgG Fc HRP and analysis by the color reaction of HRP with its substrate. The affinity-matured anti-PD-L1 antibodies demonstrated specific binding to human PD-L1 in a dose-dependent manner.

[0084] To investigate the binding kinetics of the affinity matured antibodies, full kinetic affinity studies were further performed by monitoring the association and dissociation of various doses of antigen (50 nM, 25 nM, 12.5 nM, 6.15 nM, 3.125 nM, 1.5625 nM) to various monoclonal antibodies by Biacore. The affinity matured antibodies had similar or improved binding to human PD-L1 compared to the parental antibodies.

[0085] The activity of these antibodies was also tested for binding to PD-L1 expressed on CHO cells or Raji cells. As shown in Figure 2, all of these antibodies exhibited good binding effects on cell surface PD-L1. The experiment was performed in a similar manner to that described in Example 5.

[0086] The ability of antibodies to block the binding of PD-L1 and PD-1 was tested by ELISA blocking assay. Briefly, 100μl of various concentrations of anti-PD-L1 antibodies and biotin-PD-1 were co-incubated in each well of a 96-well plate pre-coated with human His-PD-L1, followed by the addition of streptavidin-HRP and analysis by the color reaction of HRP with its substrate. As shown in Figure 3, all of these antibodies retained high blocking activity.

[0087] The biological function of these affinity-matured antibodies was tested using a PD-1 / PD-L1 Blockade bioassay. Briefly, CHO-K1 cells expressing human PD-L1 and engineered cell surface proteins were used as target cells, and Jurkat T cells expressing human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE) were used as effector cells. Various concentrations of anti-PD-L1 antibodies were incubated with these two types of cells at 37°C for 6 hours. After 6 hours, the resulting cells were assessed for luciferase activity. As shown in Figures 4A-4C, all antibodies were biologically active. The NFAT T cell activation assay described in Example 6 was also performed, yielding similar results, as shown in Figure 4D. All affinity-matured anti-PD-L1 antibodies tested blocked the PD-1 / PD-L1 interaction and thus enhanced NFAT-mediated luciferase activity. Additionally, affinity matured antibodies such as R21448516, R21560108, and R21560111 showed improved efficacy over their parental molecules 93-VH6 and 112-VH47.

[0088] 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 were able to induce human IL-2 production in a dose-dependent manner, as shown in Figure 5.

[0089] Example 9. In vivo antitumor effects of affinity matured antibodies The MC38-PD-L1 syngeneic model, which harbors tumor cells expressing human PD-L1, was used to test the anti-tumor efficacy of affinity-matured antibodies R21560102, R21560108, and R21448516. As shown in Figure 6, all three affinity-matured anti-PD-L1 antibodies tested demonstrated effective tumor growth inhibition compared to the control group (vehicle). * * *

[0090] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure; any functionally equivalent compositions or methods are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Accordingly, the present disclosure is intended to cover such modifications and variations of the present disclosure, provided they fall within the scope of the appended claims and their equivalents.

[0091] 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 complementarity determining region 1 (CDR1), CDR2, and CDR3; (1) the CDR1 comprises the amino acid sequence of SEQ ID NO: 54 or 95; the CDR2 comprises the amino acid sequence of SEQ ID NO: 55 or 96; the CDR3 comprises the amino acid sequence of SEQ ID NO: 56, 97, 98, 99, 100, 101, 102, or 103, and the CDR1, CDR2, and CDR3 do not comprise the sequences of SEQ ID NOs: 54-56, respectively; or (2) the CDR1 comprises the amino acid sequence of SEQ ID NO: 57 or 90; the CDR2 comprises the amino acid sequence of SEQ ID NO: 58 or 91; An antibody or polypeptide wherein the CDR3 comprises the amino acid sequence of SEQ ID NO: 59, 92, 93, or 94, and the CDR1, CDR2, and CDR3 do not comprise the sequences of SEQ ID NOs: 57-59, respectively.

2. the CDR1, CDR2, and CDR3 are each (1) SEQ ID NOs: 90, 58, and 59; (2) SEQ ID NOs: 57, 91, and 59; (3) SEQ ID NOs: 57, 58, and 92; (4) SEQ ID NOs: 57, 58, and 93; (5) SEQ ID NOs: 90, 58, and 92; (6) SEQ ID NOs: 90, 58, and 93; (7) SEQ ID NOs: 57, 91, and 92; (8) SEQ ID NOs: 57, 91, and 93; (9) SEQ ID NOs: 57, 58, and 94; (10) SEQ ID NOs: 90, 91, and 94; (11) SEQ ID NOs: 90, 91, and 92; or (12) SEQ ID NOs: 90, 91, and 93 The antibody or polypeptide of claim 1, comprising:

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

53.

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

53.

5. The antibody or polypeptide of any one of claims 2 to 4, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 57, 91, and 93, respectively.

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

67.

7. The antibody or polypeptide of any one of claims 2 to 4, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 90, 91, and 92, respectively.

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

70.

9. the CDR1, CDR2, and CDR3 are each (13) SEQ ID NOs: 95, 55, and 56; (14) SEQ ID NOs: 54, 96, and 56; (15) SEQ ID NOs: 54, 55, and 97; (16) SEQ ID NOs: 54, 55, and 98; (17) SEQ ID NOs: 54, 55, and 99; (18) SEQ ID NOs: 95, 55, and 97; (19) SEQ ID NOs: 95, 55, and 98; (20) SEQ ID NOs: 95, 55, and 99; (21) SEQ ID NOs: 54, 96, and 97; (22) SEQ ID NOs: 54, 96, and 98; (23) SEQ ID NOs: 54, 96, and 99; (24) SEQ ID NOs: 95, 96, and 97; (25) SEQ ID NOs: 95, 96, and 98; (26) SEQ ID NOs: 95, 96, and 99; (27) SEQ ID NOs: 95, 96, and 100; (28) SEQ ID NOs: 54, 55, and 101; (29) SEQ ID NOs: 54, 55, and 102; or (30) SEQ ID NOs: 54, 55, and 103 The antibody or polypeptide of claim 1, comprising:

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

42.

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

42.

12. The antibody or polypeptide of any one of claims 9 to 11, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 95, 96, and 100, respectively.

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

86.

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

15. The antibody or polypeptide according to any one of claims 1 to 14, wherein the polypeptide is a multispecific antibody having additional binding specificity for an antigen other than PD-L1.

16. A multispecific antibody comprising the antibody of any one of claims 1 to 14 and a second antibody or antigen-binding fragment having binding specificity for a target antigen other than PD-L1.

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

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

19. A polynucleotide encoding an antibody or polypeptide according to any one of claims 1 to 15, or a multispecific antibody according to any one of claims 16 to 18.

20. A vector comprising the polynucleotide of claim 19.

21. 21. A cell comprising the polynucleotide of claim 19 or the vector of claim 20.

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

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

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

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

26. 25. The method of claim 23 or the use 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, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

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