PD-L1 binding agonists and their use

Abstract

The present disclosure provides PD-L1-binding agents (e.g., antibodies, including multispecific antibodies such as bispecific antibodies) and uses thereof. Such antibodies, in some embodiments, compete for binding to human PD-L1 with antibodies having heavy chain variable regions and light chain variable regions described herein (e.g., in Tables 1-3). The present disclosure also provides compositions comprising the PD-L1-binding agents. The present disclosure also provides methods of treating, preventing, or alleviating T cell dysfunction diseases, disorders, or conditions, including one or more symptoms of the T cell dysfunction disease, disorder, or condition, with a PD-L1-binding agent or a composition comprising the agent, including a PD-L1-binding agent or a composition comprising the agent.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 061,109 filed 4 August 2020, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence List This application incorporates, by reference, a sequence listing file submitted with this application as a text file titled 14529-007-228_SEQ_LISTING.txt, created on July 29, 2021, with a size of 52,941 bytes.

[0003] field This disclosure generally relates to binding agents such as antibodies that bind to PD-L1, including human PD-L1, and methods of using the same. [Background technology]

[0004] background Programmed death ligand 1 (PD-L1) is a cell surface glycoprotein ligand that specifically binds to programmed death receptor 1 (PD-1), an important immune checkpoint receptor. PD-1 is upregulated on activated T cells, B cells, and monocytes, mediating immunosuppression. PD-L2, the other PD-1 ligand, is primarily expressed on activated antigen-presenting cells (APCs), while PD-L1 is widely expressed in hematopoietic cells such as activated T cells, B cells, monocytes, dendritic cells, and macrophages, as well as in peripheral tissues including the heart, skeletal muscle, placenta, lungs, kidneys, and liver.

[0005] The binding of PD-L1 to PD-1 is a negative checkpoint that activates downstream signaling of the PD-1 receptor in T cells, thereby inhibiting T cell proliferation, cytokine production and release, and cytotoxicity. Such inhibition of T cell activation and effector cytokine secretion can prevent autoimmunity and chronic infections.

[0006] However, many tumor cells use this mechanism to protect themselves from immune attack, resulting in tumor immune evasion (e.g., tumor immunity). Many cancers overexpress PD-L1, and this overexpression is often associated with a poor prognosis. In cancer, the PD-1 / PD-L1 interaction stimulates downstream signaling to suppress T cell activation, leading to tumor cell survival.

[0007] Blocking the interaction between its ligand and PD-1 has been proposed as an immunotherapeutic method to enhance the T-cell immune response against tumor cells. While current strategies of PD-1 / PD-L1-based immunotherapy have shown efficacy in treating some advanced cancers, their effects have been limited in many solid tumors and in certain PD-L1 functions. Therefore, there remains an urgent need in the art for agonists that can block or prevent PD-1 / PD-L1 interactions and / or target PD-L1 to treat, prevent or alleviate T-cell dysfunction diseases, disorders, or conditions, including those involving PD-L1-expressing tumor cells. The PD-L1-binding agonists, compositions, and methods provided herein satisfy such needs and offer relevant advantages. [Overview of the project] [Means for solving the problem]

[0008] Abstract This disclosure provides PD-L1 binding agonists, including human PD-L1 binding agonists. Such agonists include antibodies that bind to PD-L1, such as monospecific or multispecific (e.g., bispecific) antibodies that bind to PD-L1. In some embodiments, such antibodies compete for binding to human PD-L1 with antibodies having heavy-chain and light-chain variable regions as described herein (e.g., Tables 1-3).

[0009] This disclosure also provides compositions comprising PD-L1-binding activators. Such compositions include, in some embodiments, antibodies that bind to PD-L1, such as monospecific or multispecific (e.g., bispecific) antibodies that bind to PD-L1. Such compositions include, in some embodiments, antibodies that have essentially the same affinity as and / or compete with antibodies having heavy-chain and light-chain variable regions described herein (e.g., Tables 1-3) for binding to human PD-L1.

[0010] This disclosure also provides methods for treating, preventing, or mitigating T-cell dysfunction disorders, disorders, or conditions, including one or more symptoms of such disorders, disorders, or conditions, using a PD-L1-binding agonist or a composition containing such agonist. Such compositions include an antibody that binds to PD-L1, for example, a monospecific or multispecific (e.g., bispecific) antibody that binds to PD-L1. [Brief explanation of the drawing]

[0011] [Figure 1-1] Figures 1A-1C illustrate exemplary results obtained from the Octet binding assay, which is further described in Example 2. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0012] [Figure 2-1] Figures 2A-2C illustrate exemplary results obtained from the cell binding assay further described in Example 3. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0013] [Figure 3-1] Figures 3A-3C illustrate exemplary results obtained from the PD-L1 / PD-1 inhibition assay further described in Example 4. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0014] [Figure 4-1] Figures 4A-4C illustrate exemplary results obtained from SEC chromatography, which is further described in Example 5. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0015] [Figure 5-1] Figures 5A to 5C illustrate exemplary results obtained from HIC chromatography, which is further described in Example 5. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0016] [Figure 6-1] Figures 6A-6C illustrate exemplary results obtained from SMAC chromatography, which is further described in Example 5. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0017] [Figure 7-1] Figures 7A–7B show the sequence alignments of the heavy-chain and light-chain variable regions of P22, P24, and P31.2, including the consensus sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The CDR boundaries are indicated by Kabat, AbM, Chothia, Contact, and IMGT numbering. [Figure 7-2] Same as above. [Modes for carrying out the invention]

[0018] Detailed explanation This disclosure provides PD-L1-binding agonists. Such agonists include antibodies that bind to human PD-L1 (e.g., multispecific antibodies, including monospecific or bispecific antibodies). Such binding agonists are useful in compositions and in methods for treating, preventing, or mitigating T-cell dysfunction diseases, disorders, or conditions, including one or more symptoms of the disease, disorder, or condition. T-cell dysfunction diseases, disorders, and conditions include, but are not limited to, tumor immunology and related cancers, including any cancer in which tumor cells express or overexpress PD-L1. Such PD-L1-expressing tumor cells may help tumor cells escape immune surveillance and clearance (e.g., tumor immunology). In addition, PD-L1-binding agonists described herein, such as PD-L1-binding antibodies (e.g., multispecific antibodies, including monospecific or bispecific antibodies), are useful for inhibiting PD-1 signaling and / or enhancing T-cell function, and therefore for enhancing immune surveillance and tumor cell elimination. The PD-L1-binding agonists described herein, such as PD-L1-binding antibodies (e.g., multispecific antibodies including monospecific or bispecific antibodies), are useful in compositions and in methods for enhancing T cell function, including the upregulation of cell-mediated immune responses.

[0019] The terms “Programmed Cell Death Ligand-1 (PD-L1),” “Programmed Cell Death Ligand-1,” “PD-1 Ligand 1,” or similar terms refer, unless otherwise specified, to polypeptides ("polypeptide" and "protein" are used interchangeably herein) or any native PD-L1 derived from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats). PD-L1, also known as surface antigen classification 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene in humans. PD-L1 is one of two naturally occurring cell surface glycoprotein ligands for PD-1 (the other being PD-L2). Like PD-1, PD-L1 belongs to the immunoglobulin superfamily and consists of two extracellular Ig domains, an N-terminal V domain, and a C-terminal constant domain. It is known in the art that PD-L1, after binding to PD-1, downregulates T cell activation and cytokine secretion. The term PD-L1 encompasses "full-length" PD-L1, as well as any form of PD-L1 or any fragment thereof resulting from cellular processing. The term PD-L1 also encompasses naturally occurring variants of PD-L1, such as SNP variants, splice variants, and allele variants. The full-length amino acid sequence of human PD-L1 is shown below (exemplary extracellular domain = underlined text): [ka]

[0020] Other related PD-L1 polypeptides similarly encompassed by the term PD-L1 include fragments, derivatives (e.g., substitution, deletion, shortening, and insertion variants), fusion polypeptides, and interspecies homologs sufficient to retain PD-L1 activity and / or generate an anti-PD-L1 immune response. As will be understood by those skilled in the art, the PD-L1-binding agonists (e.g., antibodies) described herein can bind to PD-L1 polypeptides, PD-L1 polypeptide fragments, PD-L1 antigens, and / or PD-L1 epitopes. Epitopes can be part of a larger PD-L1 antigen, antigens can be part of a larger PD-L1 polypeptide fragment, and fragments can be part of a larger PD-L1 polypeptide. PD-L1 can exist in its native or denatured form. The PD-L1 polypeptides described herein can be isolated from various sources, such as human tissue types, or from other sources, or can be prepared by recombinant or synthetic methods. PD-L1 polypeptides may include polypeptides having the same amino acid sequence as the corresponding naturally occurring PD-L1 polypeptide. Orthologs for PD-L1 polypeptides are also well known in the art.

[0021] The terms “Programmed Cell Death-1 (PD-1),” “Programmed Cell Death-1,” “PD-1 Receptor,” or similar terms refer, unless otherwise specified, to polypeptides ("polypeptide" and "protein" are used interchangeably herein) or any native PD-1 derived from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats). PD-1, also known as CD279 (surface antigen classification 279), is an immunosuppressive receptor belonging to the CD28 family. PD-1 is predominantly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. PD-1 belongs to the immunoglobulin superfamily and consists of two extracellular Ig domains, an N-terminal V domain, and a C-terminal constant domain. PD-1 contains two cytoplasmic tyrosine-based signaling motifs: an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The term PD-1 encompasses any form of PD-1 or any fragment thereof resulting from cellular processing, along with "full-length" PD-1. The term PD-1 also encompasses naturally occurring variants of PD-1, such as SNP variants, splice variants, and allele variants. It is known in the art that, after T cell stimulation, PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif in its cytoplasmic tail, resulting in the dephosphorylation of effector molecules such as CD3 zeta, PKC theta, and ZAP70, among others, involved in the CD3 T cell signaling cascade (Carter et al. (2002) Eur J Immunol 32:634-43). The full-length amino acid sequence of human PD-1 is presented below: [ka]

[0022] As used herein, the term “binding agonist” or its grammatical equivalent refers to a molecule (e.g., an antibody) having one or more antigen-binding sites that bind to an antigen. In some embodiments, the PD-L1 binding agonists described herein are molecules based on antibodies, antibody fragments, or other peptides that bind to PD-L1, such as human PD-L1.

[0023] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and in the broadest sense, specifically encompassing, for example, polyclonal antibodies having full-length heavy and / or light chains, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multi-epitope specificity or single-epitope specificity, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies. This disclosure also includes antibody fragments (and / or polypeptides containing antibody fragments) that retain the characteristic of binding to PD-L1. Non-limiting examples of antibody fragments include the antigen-binding region and / or effector region of an antibody, e.g., Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate region antibodies, single variable region antibodies, linear antibodies, V region, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fv (dsFv), single-domain antibodies (e.g., nanobody) or other fragments (e.g., fragments consisting of a heavy-chain variable region and a light-chain variable region coupled by non-covalent bonds). Generally speaking, the variable (V) region domain can be an immunoglobulin heavy-chain (VH) and / or light-chain (VL) variable domain in any suitable arrangement. For example, this disclosure also includes two heavy-chain molecules and two light-chain molecules, antibody light-chain monomers, and tetrameric antibodies containing an antibody heavy-chain monomer. Therefore, for example, the V region domain may be a dimer and may contain VH-VH, VH-VL, or VL-VL dimers that bind to PD-L1. If desired, the VH and VL chains may be covalently coupled directly or through a linker to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein in the category of “antibody fragments.” Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody.CDRs (also known as “minimal recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides that encode the desired CDR. Such polynucleotides are prepared, for example, by using mRNA from antibody-producing cells as a template and synthesizing the variable region using polymerase chain reaction (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments can be incorporated into single-domain antibodies, maxibodies, minibodies, intracellular antibodies, diabodies, triabodies, tetrabodies, variable domains (v-NARs) of novel antigen receptors, and bis-single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). In some embodiments, the binding agonist contains one or more constant regions including light chain and / or heavy chain constant regions, e.g., one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, the antibody may contain any of the above epitope-binding fragments.The antibodies described herein may be any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies may be agonist antibodies or antagonist antibodies.

[0024] As used herein, the term "single-specific" refers, when used in reference to a binding agonist (e.g., an antibody), to a binding agonist having one or more binding sites, each binding to the same epitope of the same antigen.

[0025] When the term "bispecificity" is used in reference to a binding agent (e.g., an antibody), it means that the binding agent is capable of specifically binding to at least two distinct antigenic determinants, for example, two binding sites that bind to different antigens or different epitopes on the same antigen, each formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL). Such a bispecific binding agent (e.g., an antibody) may have a 1+1 format. Other bispecific binding agent (e.g., an antibody) formats may be a 2+1 or 1+2 format (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). If a bispecific binding agent (e.g., an antibody) contains two antigen-binding sites, each may bind to a different antigenic determinant. Such bispecific binding agents (e.g., antibodies) can bind to two different epitopes on the same antigen (e.g., epitopes on PD-L1).

[0026] The terms "identical" or "percent "identical" refer to two or more sequences or subsequences that, in the context of two or more nucleic acids or polypeptides, are identical or have a specific percentage of the same nucleotide or amino acid residues when compared and aligned (with gaps introduced if necessary) to maximize correspondence, without considering any conserved amino acid substitutions as part of sequence identity. Percent identical can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variations. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning that when compared and aligned to maximize correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, and 99% nucleotide or amino acid residue identity, as measured by using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40–60 residues, at least about 60–80 residues, or any integer value in between. In some embodiments, the identity exists over a region longer than 60–80 residues, e.g., at least about 80–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequence being compared, e.g., the coding region of a target protein or antibody. In some embodiments, identity exists over a region of nucleotide sequence having a length of at least about 10 bases, at least about 20 bases, at least about 40–60 bases, at least about 60–80 bases, or any integer value in between.In some embodiments, the identity exists over a region longer than 60–80 bases, for example, at least about 80–1000 bases or longer, and in some embodiments, the sequence is substantially identical over the entire length of the sequence being compared, for example, the nucleotide sequence encoding the protein of interest.

[0027] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain with similar chemical characteristics. Families of amino acid residues with similar side chains are commonly defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine in phenylalanine is a conservative substitution. Generally, conserved substitutions in the sequences of polypeptides, soluble proteins, and / or antibodies of this disclosure do not prevent binding of the polypeptide, soluble protein, or antibody containing that amino acid sequence to a target binding site. Methods for identifying conserved amino acid substitutions that do not preclude binding are well known in the art.

[0028] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, or may contain non-amino acids (e.g., may be interrupted by non-amino acids). The term also includes amino acid polymers that have been modified naturally or by intervention; for example, by the formation of disulfide bonds, glycosylation, lipid addition, acetylation, phosphorylation, or any other manipulation or modification, such as linkage or conjugation (directly or indirectly) with parts such as labeling components. For example, polypeptides containing one or more analogues of amino acids (e.g., non-natural amino acids) and other modifications known in the art are also included in this definition. Since the polypeptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it will be understood that in some embodiments, polypeptides may exist as single chains.

[0029] As used herein, “antigen” is a portion or molecule containing an epitope to which a binding agent (e.g., an antibody) can bind. Thus, an antibody can bind to an antigen. In some embodiments, the antigen to which the binding agent (e.g., an antibody) described herein binds is PD-L1 (e.g., human PD-L1) or a fragment thereof.

[0030] As used herein, “epitope” is a term used in the art and refers to a localized region of an antigen to which an antibody can bind. Epitopes can be linear epitopes or structural, nonlinear, or discontinuous epitopes. In the case of polypeptide antigens, for example, an epitope may be a sequence of amino acids in a polypeptide (a “linear” epitope), or it may consist of amino acids from two or more discontinuous regions of a polypeptide, such as PD-L1 (a “structural,” “nonlinear,” or “discontinuous” epitope). Generally, it will be understood by those skilled in the art that linear epitopes may or may not depend on the secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids, whether or not they are folded into a native three-dimensional protein structure. In other embodiments, for an antibody to recognize and bind to an epitope, the amino acid residues must constitute the epitope in such a way that it exhibits a specific conformation (e.g., bending, twisting, turning, or folding).

[0031] An antibody binds to an "epitope," or to an "essentially identical epitope" or "same epitope" as a reference antibody, when the two antibodies recognize epitopes that are identical, overlapping, or adjacent in three-dimensional space. The most widely used and rapid method for determining whether two antibodies bind to identical, overlapping, or adjacent epitopes in three-dimensional space is a competitive assay, which can be constructed in several different formats, for example, by using either a labeled antigen or a labeled antibody. In some assays, the antigen is immobilized in a 96-well plate or expressed on the cell surface, and the ability of an unlabeled antibody to block the binding of a labeled antibody is measured using radiolabeling, fluorescent labeling, or enzymatic labeling.

[0032] Epitope binning is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition characteristics of different antibodies by combining competitive assays with computer processes to cluster antibodies based on their epitope recognition characteristics and identify antibodies with distinct binding specificities.

[0033] As used herein, the terms “specifically bind,” “specifically recognize,” “immunospecifically bind,” “selectively bind,” “immunospecifically recognize,” and “immunospecific” are synonymous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope), and such binding is understood by those skilled in the art. In some embodiments, “specifically bind” means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with higher affinity, or in some combination of the above, than with alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity, as determined by immunoassays, Biacore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds to or specifically binds to an antigen if, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), it binds to the antigen with a higher affinity than any cross-reactive antigen. Typically, specific or selective reactions are at least twice the background signal or noise, and may exceed 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In some embodiments, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA, the degree of binding of an antibody or antigen-binding domain to a “non-target” protein is less than about 10% of the binding of the antibody or antigen-binding domain to its particular target antigen. In some embodiments, a molecule that specifically binds to an antigen binds to that antigen with a Ka of at least 2log, 2.5log, 3log, 4log, or greater than the Ka of the molecule when it binds to another antigen. In some embodiments, molecules that specifically bind to the antigen do not cross-react with other proteins.In other specific embodiments, molecules that specifically bind to an antigen do not cross-react with other non-PD-L1 proteins. In some embodiments, “specifically binding” means, for example, that a polypeptide or molecule binds to a protein or target with a KD of about 0.1 mM or less, but typically less than 1 μM. In some embodiments, “specifically binding” means that a polypeptide or molecule binds to a target with a KD of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. Due to sequence identity between homologous proteins in different species, specific binding may include polypeptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of polypeptide sequences of different proteins, specific binding may include polypeptides or molecules that recognize more than one protein or target. In some embodiments, it is understood that a polypeptide or molecule that specifically binds to a first target may also specifically bind to a second target, or it may not specifically bind to the second target. Therefore, “specific binding” does not necessarily require exclusive binding, e.g., binding to a single target (although it may include such binding). Thus, a polypeptide or molecule may, in some embodiments, specifically bind to more than one target. In some embodiments, the same antigen-binding site of a polypeptide or molecule may bind to multiple targets. For example, an antibody may, in certain cases, contain two identical antigen-binding sites, each specifically binding to the same epitope of two or more proteins. In certain alternative embodiments, an antibody may be bispecific, containing at least two antigen-binding sites with different specificities. Generally, though not always, references to “binding” mean “specific binding.”

[0034] "Binding affinity" generally refers to the strength of the overall non-covalent interaction between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the endogenous binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally expressed by the dissociation constant (K). D Affinity can be expressed by ). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this disclosure. In one embodiment, "K D " or "K D The value can be measured, for example, by biolayer interferometry (BLI) using the OctetQK384 system (ForteBio, Menlo Park, CA). Alternatively, K D This can also be measured, for example, by an radiolabeled antigen-binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen (Chen, et al., (1999) J. Mol Biol 293: 865-881), or by using a surface plasmon resonance (SPR) assay with Biacore, for example, using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). The term "on-rate" or "rate of association" or "association rate" or "k" can also be used. on ", and "off-rate" or "rate of dissociation" or "dissociation rate" or "k offThis can also be determined using the same SPR or BLI techniques as described above, for example, by using the OctetQK384 system (ForteBio, Menlo Park, CA) or BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), respectively.

[0035] When used in the context of PD-L1 binding agonists (e.g., antibodies), the term "competitive" refers to binding agonists competing for the same epitope or binding site on the target, including competition among such agonists, as determined by assays in which the specific binding of a reference molecule (e.g., a reference antibody, or a reference ligand or reference antigen-binding protein) to a common antigen (e.g., PD-L1) is prevented or inhibited. Numerous types of competitive binding assays can be used to determine whether a test binding agonist competes with a reference molecule for binding to PD-L1 (e.g., human PD-L1). Examples of assays that can be used include solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619); solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using 1-125 labeling (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., See (1990) Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82).Typically, such assays involve the use of purified antigen (e.g., PD-L1, such as human PD-L1) bound to a solid surface or cell having either an unlabeled test antigen-binding protein (e.g., test PD-L1 antibody) or a labeled reference antigen-binding protein (e.g., reference PD-L1 antibody). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and / or antibodies that bind to an adjacent epitope proximal to the epitope bound by the reference antibody (e.g., a similar or overlapping epitope) to a degree sufficient to cause steric hindrance. Additional details regarding methods for determining competitive binding are described herein, as shown in Example 6. Typically, when competing antibodies are present in excess, the specific binding of the reference antibody to the common antigen is inhibited by at least 20%, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, or 97%, 98%, 99%, or more.

[0036] As used herein, the terms “constant region” or “constant domain” are well-known antibody terminology and refer to a portion of an antibody, for example, the carboxyl-terminal portion of the light and / or heavy chain, which is not directly involved in the antibody’s binding to an antigen but may exhibit various effector functions, such as interaction with Fc receptors. This term generally includes portions of immunoglobulin molecules that have a more conserved amino acid sequence compared to immunoglobulin variable domains.

[0037] Antibody "effector function" refers to the biological activity resulting from the antibody's Fc region (e.g., the Fc region of the native sequence or the Fc region of an amino acid sequence variant), and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated injury (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0038] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, the native sequence Fc region, recombinant Fc region, and variant Fc region. While the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is often defined as a continuum from the amino acid residue at position Cys226 (according to the EU numbering scheme) or from Pro230 (according to the EU numbering scheme) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering scheme) may be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are shown below (CH2 domain = bold; CH3 domain = underlined): [ka]

[0039] A "functional Fc region" possesses the "effector function" of the native sequence's Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated injury (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various disclosed assays.

[0040] A “native sequence Fc region” contains the same amino acid sequence as a naturally occurring Fc region and has not been manipulated, modified, and / or altered by humans (e.g., isolated, purified, selected, included in or combined with other sequences such as variable region sequences). Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A allotype and A allotype); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as their naturally occurring variants.

[0041] A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the Fc region of the native sequence by a modification (e.g., substitution, addition, or deletion) of at least one amino acid, preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, within the Fc region of the native sequence or the Fc region of the parent polypeptide, compared to the Fc region of the native sequence or the Fc region of the parent polypeptide. The variant Fc regions described herein may have at least about 80% homology with the Fc region of the native sequence and / or the Fc region of the parent polypeptide, or at least about 90% homology with them, for example, at least about 95% homology with them. The variant Fc regions described herein may be loss of effector function (e.g., silent Fc). An example variant Fc region ("silent Fc") sequence is shown below (CH2 domain = bold, amino acid changes are underlined; CH3 domain = underlined text): [ka]

[0042] As used herein, the term “heavy chain,” when used in reference to antibodies, refers to a polypeptide chain of about 50–70 kDa, where the amino-terminal portion comprises a variable region of about 120–130 or more amino acids, and the carboxy-terminal portion comprises one or more constant regions. “Heavy chain” may refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG (including subclasses, e.g., IgG1, IgG2, IgG3, and IgG4) and IgM classes, respectively.

[0043] As used herein, the term “light chain,” when used in reference to antibodies, may refer to a polypeptide chain of approximately 25 kDa, where the amino-terminal portion contains a variable region of approximately 100 to 110 or more amino acids, and the carboxy-terminal portion contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, two distinct types exist, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0044] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to a portion of an antibody that interacts with an antigen and contains amino acid residues (e.g., CDRs) that confer specificity and affinity to the antigen to the binding fragment, domain, or region. “Antigen-binding fragment,” as used herein, includes an “antibody fragment” that contains a portion of an antibody, such as an antigen-binding region or variable region of the antibody, which contains one or more CDRs.

[0045] The antibodies described herein are not limited to, but include synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv(scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies, and any of the epitope-binding fragments described above.

[0046] In some embodiments, the antibodies described herein include an immunoglobulin molecule and an immunoactive portion of the immunoglobulin molecule, which includes a molecule containing one or more antigen-binding sites that bind to the PD-L1 antigen.

[0047] The antibody may be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a, or IgG2b). In some embodiments, the antibody described herein is an IgG antibody (e.g., human IgG), or a class thereof (e.g., human IgG1, IgG2, IgG3, or IgG4), or a subclass thereof.

[0048] In some embodiments, the antibody is a four-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, where the amino acid sequences of the H chains and L chains are identical. In some embodiments, the H chains and L chains include a constant region, e.g., a human constant region. In some embodiments, the L chain constant region of such an antibody is a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of such an antibody includes a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In some embodiments, such an antibody includes an IgG constant region, e.g., a human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant regions).

[0049] An antibody or fragment thereof can preferentially bind to PD-L1, such as human PD-L1, meaning that the antibody or fragment binds to PD-L1 with a higher affinity than it binds to an unrelated control protein, and / or to human PD-L1 with a higher affinity than it binds to an unrelated control protein. For example, an antibody or fragment thereof can specifically recognize and bind to PD-L1 or a portion thereof. "Specific binding" means that the antibody or fragment binds to PD-L1 with an affinity at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times higher than its affinity to an unrelated control protein (e.g., chicken egg white lysozyme). In some embodiments, the antibody or fragment thereof can bind to PD-L1 substantially exclusively (e.g., PD-L1 can be distinguished from other known polypeptides based on, for example, a measurable difference in binding affinity). In some embodiments, a PD-L1-binding agonist (e.g., an antibody) can react with PD-L1 sequences other than human PD-L1 sequences (e.g., cynomolgus monkey PD-L1 sequences).

[0050] The term "variable region" or "variable domain" generally refers to a portion of an antibody's light or heavy chain located at the amino terminus of the light or heavy chain, with a heavy chain length of approximately 120–130 amino acids and a light chain length of approximately 100–110 amino acids, used for the binding and specificity of each particular antibody to its specific antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that the sequence of a particular segment of the variable region varies widely among antibodies. The V region mediates binding to the antigen and determines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed across the 110-amino acid length of the variable region. Instead, the V region consists of a stretch of approximately 15–30 amino acids called the framework region (FR), which is less variable (e.g., relatively invariant), separated by a shorter region called the "hypervariable region" or "complementarity-determining region," which is more variable (e.g., extremely variable). The variable regions of the heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4), which primarily adopt a β-sheet configuration and are connected by three hypervariable regions that form loops connecting the β-sheet structure, and in some cases forming part of it. The hypervariable regions within each chain are held together in very close proximity by the FRs, and hypervariable regions from other chains contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant region does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). The sequences of the variable regions vary widely among different antibodies. Sequence variability is concentrated in the CDR, while less variable regions within the variable region are called framework regions (FRs).The CDRs of the light and heavy chains are primarily responsible for the antibody's interaction with the antigen. In certain embodiments, the variable region is a human variable region.

[0051] The terms “hypervariable region,” “HVR,” “HV,” “complementarity-determining region,” or “CDR,” as used herein, refer to regions of antibody variable regions where the sequence is hypervariable and / or forms structurally defined loops. Generally, antibodies contain six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several descriptions of hypervariable regions have been used and are included in the present invention. Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia, on the other hand, refers to the location of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the loop length (this is because insertions are made at H35A and H35B in the Kabat numbering scheme; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region is a compromise between Kabat CDRs and Chothia structural loops and is used in Oxford Molecular's AbM antibody modeling software (see, for example, Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable region is based on the analysis of available complex crystal structures. The residues from each of these hypervariable regions or CDRs are listed below.

[0052] A universal numbering scheme, ImMunoGeneTics (IMGT) Information System (registered trademark) (Lefranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003)), has been developed and is widely adopted. IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC) of human and other vertebrates. Herein, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. The “location” of CDRs within the structure of immunoglobulin variable domains is conserved across species and can be easily identified by a structure referred to as a loop, indicated by a numbering scheme that aligns the variable domain sequence according to structural features, CDRs, and framework residues. This information can be used for grafting and substitution of CDR residues from immunoglobulins of one species to acceptor frameworks, typically derived from human antibodies. An additional numbering scheme (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309: 657-670 (2001). For example, correspondences between numbering schemes, including Kabat numbering and the IMGT unique numbering scheme, are well known to those skilled in the art (see, e.g., Kabat, above; Chothia and Lesk, above; Martin, above; Lefranc et al., above), and are also illustrated below. The exemplary scheme shown herein combines Kabat and Chothia. [Table A]

[0053] The hypervariable region may include the "hypervariable region extensions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "hypervariable region," "HVR," "HV," "complementarity determination region," or "CDR" are used interchangeably.

[0054] The term "vector" refers to a substance used to carry or contain nucleic acid sequences, including, for example, those intended to introduce nucleic acid sequences into host cells. Applicable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or markers that can be activated for stable integration into the chromosomes of host cells. Furthermore, a vector may contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes that may be included may, for example, confer resistance to antibiotics or toxins, compensate for nutritional requirement deficiencies, or supply critical nutrients that are not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcription enhancers, transcription terminators, etc., which are well known in the art. When co-expressing two or more nucleic acid molecules (e.g., both antibody heavy and light chains or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector, or into separate expression vectors. With regard to expression by a single vector, the encoding nucleic acid can be operationally ligated to one common expression regulatory sequence, or to different expression regulatory sequences, such as one inductive promoter and one constitutive promoter. The introduction of the nucleic acid molecule into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for the expression of gene products, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., the PD-L1 binding agonist described herein), and further, that the expression level can be optimized using methods well known in the art to obtain sufficient expression.

[0055] In the context of immunodeficiency, "dysfunction" refers to a state of immunosuppressive response to antigenic stimuli. This term encompasses a common element of both depletion and / or anergy, where antigen recognition may occur, but the subsequent immune response is ineffective in controlling infection or tumor growth.

[0056] The terms "T-cell dysfunction disorder," "T-cell dysfunction impairment," and "T-cell dysfunction state" are used interchangeably and refer to any disease, disorder, or state of T cells characterized by reduced responsiveness to antigenic stimuli. T-cell dysfunction disorders include any disease, disorder, or state that is entirely or partially caused by or a result of PD-L1 or the interaction between PD-L1 and PD-1, and / or, instead, any disease, disorder, or state in which it is desirable to inhibit the in vivo effects of the interaction between PD-L1 and PD-1. In some embodiments, a T-cell dysfunction disorder is a disease, disorder, or state (contition) specifically associated with inadequately increased signaling via PD-1. In some embodiments, a T-cell dysfunction disorder is a disease in which T cells are anergistic or have reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In some embodiments, reduced responsiveness results in ineffective control of a pathogen or tumor, including but not limited to tumors expressing PD-L1. Examples of T-cell dysfunction disorders characterized by T-cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity (e.g., originating from any cancer, including but not limited to cancers expressing or overexpressing PD-L1).

[0057] "Tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated and the tumor is recognized and attacked by the immune system. Thus, such treatment includes treatment of any cancer. Examples of tumor recognition include binding to the tumor, tumor shrinkage (strinkage), and tumor clearance.

[0058] "T cell enhancement" means inducing, causing, or stimulating T cells to have sustained or increased biological function, or to regenerate or reactivate depleted or inactive T cells. An example of T cell enhancement is CD8 compared to pre-intervention levels. + Enhancements include increased secretion of cytokines (e.g., TNFα, IFNγ) from T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor cell removal). In some embodiments, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Methods of measuring this enhancement are known to those skilled in the art.

[0059] An "effective dose" is generally a quantity sufficient to reduce the severity and / or frequency of symptoms resulting from or associated with a disease, disorder, or condition; to eliminate symptoms and / or their underlying causes; to prevent the appearance of symptoms and / or their underlying causes; and / or to improve or correct damage. In some embodiments, the effective dose is a therapeutic effective dose or a preventive effective dose.

[0060] When used herein, the term “therapeutic dose” refers to the amount of an agonist (e.g., the antibody described herein or any other agonist described herein) sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder, or condition and / or symptoms thereof. The therapeutic dose of an agonist, including an agonist, may be the amount necessary to (i) reduce or improve the progression or exacerbation of a given disease, disorder, or condition; (ii) reduce or improve the recurrence, occurrence, or onset of a given disease, disorder, or condition; and / or (iii) improve or enhance the preventive or therapeutic effect of another treatment (e.g., a treatment other than the administration of the antibody described herein). The “therapeutic dose” of a substance / molecule / agonist in this disclosure (e.g., PD-L1 antibody) may vary depending on factors such as the individual’s condition, age, sex, and weight, as well as the substance / molecule / agonist’s ability to elicit a desired response in the individual. A therapeutically effective dose encompasses the amount of a substance / molecule / agent in which the therapeutically beneficial effects outweigh any toxic or adverse effects. In certain embodiments, the term “therapeutically effective dose” refers to the amount of an antibody or other agonist (e.g., drug) that is effective in “treating” a disease, disorder, or condition in a subject or mammal.

[0061] The "prophylactic effective dose" is the amount of a pharmaceutical composition that, when administered to a subject, has the intended preventive effect, for example, preventing or delaying the onset (or recurrence) of a disease, disorder, or condition, or reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms(s). The complete therapeutic or prophylactic effect is not necessarily achieved by a single dose, but may only occur after a series of doses. Therefore, the therapeutic or prophylactic effective dose may be administered in one or more doses.

[0062] When used herein, the term "pharmaceutically acceptable" means that a product is approved by a federal or state regulatory authority for use in animals, more specifically in humans, or is listed in the United States Pharmacopeia, the European Pharmacopeia, or any other generally accepted pharmacopoeia.

[0063] When used herein, “carrier” includes carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the dosage and concentration used. Often, the carrier is an aqueous pH buffer. Examples of carriers include buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugar alcohols, e.g., mannitol or sorbitol; salt-forming counterions, e.g., sodium; and / or nonionic surfactants, e.g., TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The term "carrier" may also refer to diluents, adjuvants (e.g., Freund's adjuvants (complete or incomplete)), excipients, or vehicles with which the therapeutic agent is administered. Such carriers may be sterile liquids, such as water, and oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is a typical carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly as injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, grain flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol. The composition may also contain, if desired, trace amounts of wetting agents, emulsifiers, or pH buffers. The composition may take the form of a liquid, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like.Oral compositions containing the formulation may include, for example, standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA. Compositions containing the pharmaceutical compound may contain a prophylactic or therapeutically effective dose of a PD-L1-binding agonist (e.g., an antibody), for example, in an isolated or purified form, together with an appropriate amount of carrier to provide a form for appropriate administration to a subject (e.g., a patient). The formulation should be adapted to the dosage form.

[0064] In some embodiments, this disclosure provides PD-L1-binding agonists that can be used as therapeutic agents herein. Such agonists include antibodies that bind to PD-L1 (e.g., multispecific antibodies, including monospecific or bispecific antibodies). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies, and heteroconjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0065] In some embodiments, PD-L1-binding agonists (e.g., antibodies) that bind to PD-L1, including PD-L1 polypeptides, PD-L1 polypeptide fragments, PD-L1 peptides, or PD-L1 epitopes, are described herein. In some embodiments, the PD-L1-binding agonist is a human antibody or humanized antibody (e.g., including a human constant region) that binds to PD-L1, including PD-L1 polypeptides, PD-L1 polypeptide fragments, PD-L1 peptides, or PD-L1 epitopes. In some embodiments, a PD-L1-binding agonist (e.g., an antibody), such as a human PD-L1-binding agonist, can bind to PD-L1 expressed on the surface of mammalian (e.g., human) cells, including PD-L1-expressing tumor cells. In some embodiments, a PD-L1-binding agonist (e.g., an antibody) binds to an exposed PD-L1 extracellular epitope (e.g., a PD-L1 epitope) on a cell, such as a tumor cell. In some embodiments, PD-L1-binding agonists (e.g., antibodies) that bind to PD-L1, such as human PD-L1 or a portion thereof, are described herein. In some embodiments, PD-L1 is human PD-L1. In some embodiments, the PD-L1-binding agonist is a human PD-L1-binding agonist (e.g., an antibody that binds to human PD-L1). An exemplary amino acid sequence of human PD-L1 is described herein.

[0066] In some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein compete for binding to PD-L1 such as human PD-L1 with any one of the antibodies described herein that includes the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 amino acid sequences shown in Tables 1-3. Therefore, in some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein compete with PD-L1 binding agonists (e.g., antibodies) comprising one, two, and / or three VH CDRs and / or one, two, and / or three VL CDRs from (a) an antibody designated as P22, (b) an antibody designated as P24; or (c) an antibody designated as P31.2, as shown in Tables 1-3, for binding to PD-L1 such as human PD-L1. In some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein compete with PD-L1 binding agonists (e.g., antibodies) that include one, two, and / or three VH CDRs and one, two, and / or three VL CDRs from the antibodies (a) referred to as P22, (b) referred to as P24, or (c) referred to as P31.2, as shown in Tables 1-3, for binding to PD-L1 such as human PD-L1.In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies) compete for binding to PD-L1 such as human PD-L1 with PD-L1 binding agonists (e.g., antibodies) comprising (a) a VH region containing the amino acid sequence of SEQ ID NO: 25 and a VL region containing the amino acid sequence of SEQ ID NO: 26, (b) a VH region containing the amino acid sequence of SEQ ID NO: 51 and a VL region containing the amino acid sequence of SEQ ID NO: 52, or (c) a VH region containing the amino acid sequence of SEQ ID NO: 77 and a VL region containing the amino acid sequence of SEQ ID NO: 78.

[0067] In some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein include the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, such as the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 amino acid sequences shown in Tables 1-3. Accordingly, in some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein include one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from the antibodies shown in Tables 1-3, namely (a) an antibody referred to as P22; (b) an antibody referred to as P24; or (c) an antibody referred to as P31.2. In some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein include one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs from (a) an antibody designated as P22; (b) an antibody designated as P24; or (c) an antibody designated as P31.2, as shown in Tables 1-3.

[0068] In some embodiments, the PD-L1 binding agonist (e.g., antibody) comprises a VH region including VH CDR1, VH CDR2, and / or VH CDR3 of any one of the binding agonists described herein, and a VL region including VL CDR1, VL CDR2, and / or VL CDR3 (see, for example, Tables 1, 2, and 3). Thus, in some embodiments, the PD-L1 binding agonist (e.g., antibody) described herein comprises one, two, and / or three heavy chain CDRs from Table 1, and / or one, two, and / or three light chain CDRs. In some embodiments, the PD-L1 binding agonist described herein comprises one, two, and / or three heavy chain CDRs from Table 2, and / or one, two, and / or three light chain CDRs. In some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein include one, two, and / or three heavy chain CDRs from Table 3, and / or one, two, and / or three light chain CDRs. In some embodiments, the PD-L1 binding agonists (e.g., antibodies) described herein are bispecific and include a first binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from Table 1, Table 2, or Table 3, and a second binding domain comprising one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs from a binding agonist that binds to a second target antigen other than PD-L1.

[0069] The antibody referred to as P22 contains the VH sequence, which is sequence number 25, and the VL sequence, which is sequence number 26.

[0070] The antibody referred to as P24 contains the VH sequence, which is sequence number 51, and the VL sequence, which is sequence number 52.

[0071] The antibody referred to as P31.2 contains the VH sequence, which is sequence number 77, and the VL sequence, which is sequence number 78. Table 1: Antibody P22 [Table 1] Table 2: Antibody P24 [Table 2] Table 3: Antibody P31.2 [Table 3]

[0072] In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, includes a VH region or VH domain. In other embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, includes a VL region or VL domain. In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, has (i) a VH domain or VH region; and / or (ii) a combination of a VL domain or VL region.

[0073] In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonists described herein, comprises a heavy chain having a combination of (i) a VH domain described in any one of Tables 1-3 and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). An exemplary IgG heavy chain comprises any VH domain described herein, as well as the following CH1, hinge, CH2, and CH3 amino acid sequences:

[0074] [ka] Another exemplary IgG heavy chain comprises any VH domain described herein, as well as the following CH1, hinge, CH2, and CH3 amino acid sequences: [ka] [ka]

[0075] In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonists described herein, comprises a light chain having a combination of (i) a VL domain described in any one of Tables 1-3 and (ii) a light chain constant domain (CL). An exemplary light chain (e.g., for pairing with an IgG heavy chain) comprises any VL as the domain described herein and the following CL amino acid sequence: [ka]

[0076] In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, comprises (a) a heavy chain having a combination of (i) a VH domain described in any one of Tables 1 to 3 and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3); and (b) a light chain having a combination of (i) a VL domain described in any one of Tables 1 to 3 and (ii) a light chain constant domain (CL1) in IgG format. An exemplary PD-L1 binding agonist (e.g., an antibody) comprises an IgG heavy chain having any VH domain described herein and the amino acid sequence of SEQ ID NO: 96 or 98, and a light chain having any VL domain described herein and the amino acid sequence of SEQ ID NO: 97.

[0077] In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, comprises one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as identified in Table 1. In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, comprises one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3, as identified in Table 2. In some embodiments, the PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, comprises one or more CDRs, including, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 3.

[0078] In some embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, comprises one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 1. In other embodiments, a PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, comprises one or more CDRs, including three CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 1. In further embodiments, the PD-L1 binding agonist (e.g., an antibody such as a bispecific antibody), including the human PD-L1 binding agonist described herein, comprises one or more CDRs including three VH CDRs, e.g., VH CDR1, VH CDR2, and VH CDR3 listed in Table 1, and one or more CDRs including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VL CDR3 listed in Table 1.

[0079] In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) include one or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) include two or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) include three or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) include four or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) include five or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76. In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) include six or more complementarity-determining regions (CDRs) containing amino acid sequences selected from the group consisting of SEQ ID NOs: 1-24, 27-50, and 53-76.

[0080] In some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3. In other embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-3. In yet another embodiment, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3, and one or more VL CDRs listed in Tables 1-3. Accordingly, in some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes VH CDR1 having any one amino acid sequence of SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, and 70. In some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes VH CDR2 having any one amino acid sequence of SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, and 76. In some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) includes VH CDR3 having any one amino acid sequence of SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, and 72. In some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises VH CDR1 and / or VH CDR2 and / or VH CDR3 independently selected from VH CDR1, VH CDR2, and VH CDR3, which are represented by any one of the amino acid sequences shown in Tables 1 to 3. In some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises VL CDR1 having any one of the amino acid sequences of SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, and 73.In another embodiment, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises VL CDR2 having any one amino acid sequence of SEQ ID NOs. 5, 11, 22, 31, 37, 48, 57, 63, and 74. In some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises VL CDR3 having any one amino acid sequence of SEQ ID NOs. 6, 17, 23, 32, 43, 49, 58, 69, and 75. In some embodiments, the PD-L1 binding agonist described herein (e.g., an antibody such as a bispecific antibody) comprises VL CDR1 and / or VL CDR2 and / or VL CDR3 independently selected from VL CDR1, VL CDR2, and VL CDR3, which are represented by any one of the amino acid sequences shown in Tables 1-3.

[0081] In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) are: (1) VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 1, 27, or 53, (ii) SEQ ID NOs: 7, 33, or 59, (iii) SEQ ID NOs: 12, 38, or 64, (iv) SEQ ID NOs: 13, 39, or 65, and (v) SEQ ID NOs: 18, 44, or 70; (2) VH having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 2, 28, or 54, (ii) SEQ ID NOs: 8, 34, or 60, (iii) SEQ ID NOs: 14, 40, or 66, (iv) SEQ ID NOs: 19, 45, or 71, and (v) SEQ ID NOs: 24, 50, or 76. A heavy chain variable (VH) region comprising a VH CDR3 having an amino acid sequence selected from the group consisting of (3)(i)SEQ ID NOs: 3, 29, or 55, (ii)SEQ ID NOs: 9, 35, or 61, (iii)SEQ ID NOs: 15, 41, or 67, and (iv)SEQ ID NOs: 20, 46, or 72, and / or a VL CDR1 having an amino acid sequence selected from the group consisting of (1)(i)SEQ ID NOs: 4, 30, or 56, (ii)SEQ ID NOs: 10, 36, or 62, (iii)SEQ ID NOs: 16, 42, or 68, and (iv)SEQ ID NOs: 21, 47, or 73, and a VL CDR1 having an amino acid sequence selected from the group consisting of (2)(i)SEQ ID NOs: 5, 31, or 57, (ii)SEQ ID NOs: 11, 37, or 63, and (iii)SEQ ID NOs: 22, 48, or 74 The device comprises a CDR2 and a light chain variable (VL) region including a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 6, 32, or 58, (ii) SEQ ID NOs: 17, 43, or 69, and (iii) SEQ ID NOs: 23, 49, or 75.

[0082] In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies such as bispecific antibodies) are: (1) VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 1, 27, or 53, (ii) SEQ ID NOs: 7, 33, or 59, (iii) SEQ ID NOs: 12, 38, or 64, (iv) SEQ ID NOs: 13, 39, or 65, and (v) SEQ ID NOs: 18, 44, or 70; (2) VH having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 2, 28, or 54, (ii) SEQ ID NOs: 8, 34, or 60, (iii) SEQ ID NOs: 14, 40, or 66, (iv) SEQ ID NOs: 19, 45, or 71, and (v) SEQ ID NOs: 24, 50, or 76. The CDR2 and the heavy chain variable (VH) region comprising a VH CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 3, 29, or 55, (ii) SEQ ID NOs: 9, 35, or 61, (iii) SEQ ID NOs: 15, 41, or 67, and (iv) SEQ ID NOs: 20, 46, or 72.

[0083] In some embodiments, the PD-L1 binding activator described herein (e.g., an antibody such as a bispecific antibody) comprises a light chain variable (VL) region including (1) VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 4, 30, or 56, (ii) SEQ ID NO: 10, 36, or 62, (iii) SEQ ID NO: 16, 42, or 68, and (iv) SEQ ID NO: 21, 47, or 73; (2) VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 5, 31, or 57, (ii) SEQ ID NO: 11, 37, or 63, and (iii) SEQ ID NO: 22, 48, or 74; and (3) VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 6, 32, or 58, (ii) SEQ ID NO: 17, 43, or 69, and (iii) SEQ ID NO: 23, 49, or 75.

[0084] PD-L1-binding agonists (e.g., antibodies such as bispecific antibodies) comprising one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-3, and one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-3 are also described herein. In particular, VH CDR1 (sequences 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70) and VL CDR1 (sequences 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) listed in Tables 1-3; VH CDR1 (sequences 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70) and VL CDR2 (sequences 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR1 (sequences 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR2(sequences 5, 11, 22, 31, 37, 48, 57, 63, or 74);VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74);VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75);VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), and VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR3(SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR3; (Sequence codes 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR2 (Sequence codes 5, 11, 22, 31, 37, 48, 57, 63, or 74) and VL CDR3 (Sequence codes 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR2 (Sequence codes 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (Sequence codes 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73), and VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74) and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75);VH CDR1 (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, or 70), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); VH CDR2 (SEQ ID NOs: 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, or 76), VH CDR3 (SEQ ID NOs: 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72), VL CDR1 (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, or 73), VL CDR2 (SEQ ID NOs: 5, 11, 22, 31, 37, 48, 57, 63, or 74), and VL CDR3 (SEQ ID NOs: 6, 17, 23, 32, 43, 49, 58, 69, or 75); or these VH PD-L1 binding agonists (e.g., antibodies such as bispecific antibodies) comprising any combination of CDRs (SEQ ID NOs: 1, 7, 12, 13, 18, 27, 33, 38, 39, 44, 53, 59, 64, 65, 70, 2, 8, 14, 19, 24, 28, 34, 40, 45, 50, 54, 60, 66, 71, 76, 3, 9, 15, 20, 29, 35, 41, 46, 55, 61, 67, or 72) and VL CDRs (SEQ ID NOs: 4, 10, 16, 21, 30, 36, 42, 47, 56, 62, 68, 73, 5, 11, 22, 31, 37, 48, 57, 63, 74, 6, 17, 23, 32, 43, 49, 58, 69, or 75) are described herein. ;

[0085] In some embodiments, an antibody or fragment that binds to PD-L1, comprising: (a) (1) VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 1, 27, or 53, (ii) SEQ ID NO: 7, 33, or 59, (iii) SEQ ID NO: 12, 38, or 64, (iv) SEQ ID NO: 13, 39, or 65, and (v) SEQ ID NO: 18, 44, or 70; (2) VH having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 2, 28, or 54, (ii) SEQ ID NO: 8, 34, or 60, (iii) SEQ ID NO: 14, 40, or 66, (iv) SEQ ID NO: 19, 45, or 71, and (v) SEQ ID NO: 24, 50, or 76 A heavy chain variable (VH) region comprising a VH CDR3 having an amino acid sequence selected from the group consisting of (3)(i)SEQ ID NO: 3, 29, or 55, (ii)SEQ ID NO: 9, 35, or 61, (iii)SEQ ID NO: 15, 41, or 67, (iv)SEQ ID NO: 20, 46, or 72, and / or (b)(1)(i)SEQ ID NO: 4, 30, or 56, (ii)SEQ ID NO: 10, 36, or 62, (iii)SEQ ID NO: 16, 42, or 68, (iv)SEQ ID NO: 21, 47, or 73, and (2)(i)SEQ ID NO: 5, 31, or 57, (ii)SEQ ID NO: 11, 37, or 63, (iii)SEQ ID NO: 22, 48, or 74 An antibody or fragment thereof is described herein, comprising a CDR2 and a light chain variable (VL) region including a VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 6, 32, or 58, (ii) SEQ ID NOs: 17, 43, or 69, and (iii) SEQ ID NOs: 23, 49, or 75.

[0086] In some embodiments, an antibody or fragment thereof that binds to PD-L1, comprising: (1) VH CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 1, 27, or 53, (ii) SEQ ID NOs: 7, 33, or 59, (iii) SEQ ID NOs: 12, 38, or 64, (iv) SEQ ID NOs: 13, 39, or 65, and (v) SEQ ID NOs: 18, 44, or 70; (2) VH having an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 2, 28, or 54, (ii) SEQ ID NOs: 8, 34, or 60, (iii) SEQ ID NOs: 14, 40, or 66, (iv) SEQ ID NOs: 19, 45, or 71, and (v) SEQ ID NOs: 24, 50, or 76 The following antibodies or fragments thereof are described herein, comprising a heavy chain variable (VH) region including CDR2 and a VH CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 3, 29, or 55, (ii) SEQ ID NO: 9, 35, or 61, (iii) SEQ ID NO: 15, 41, or 67, and (iv) SEQ ID NO: 20, 46, or 72.

[0087] In some embodiments, antibodies or fragments thereof that bind to PD-L1 are described herein, comprising a light chain variable (VL) region including (1) VL CDR1 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 4, 30, or 56, (ii) SEQ ID NO: 10, 36, or 62, (iii) SEQ ID NO: 16, 42, or 68, and (iv) SEQ ID NO: 21, 47, or 73, (2) VL CDR2 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 5, 31, or 57, (ii) SEQ ID NO: 11, 37, or 63, and (iii) SEQ ID NO: 22, 48, or 74, and (3) VL CDR3 having an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 6, 32, or 58, (ii) SEQ ID NO: 17, 43, or 69, and (iii) SEQ ID NO: 23, 49, or 75.

[0088] In some embodiments, antibodies or fragments thereof that bind to PD-L1 are described herein, comprising: an antibody referred to as P22, comprising the VH sequence of SEQ ID NO: 25 and the VL sequence of SEQ ID NO: 26; an antibody referred to as P24, comprising the VH sequence of SEQ ID NO: 51 and the VL sequence of SEQ ID NO: 52; or an antibody referred to as P31.2, comprising all three heavy chain complementarity determining regions (CDRs) and / or all three light chain CDRs from an antibody referred to as P31.2. In some embodiments, the antibody or fragment comprises all three heavy chain CDRs and / or all three light chain CDRs from an antibody referred to as P22. In some embodiments, the antibody or fragment comprises all three heavy chain CDRs and / or all three light chain CDRs from an antibody referred to as P24. In some embodiments, the antibody or fragment comprises all three heavy chain CDRs and / or all three light chain CDRs from an antibody referred to as P31.2.

[0089] In some embodiments, antibodies or fragments thereof that bind to PD-L1 are described herein, comprising (a) a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences shown in Tables 1-3, and / or (b) a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences shown in Tables 1-3. In some embodiments, the antibody comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences shown in Tables 1-3. In some embodiments, the antibody comprises a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences shown in Tables 1-3.

[0090] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) a VH CDR1 having an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 1, 7, 12, 13, and 18, (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24, and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20, and a light chain variable (VL) region comprising (b) a VL CDR1 having an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 4, 10, 16, and 21, (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

[0091] In some embodiments, antibodies are described herein that include (a) a heavy chain variable (VH) region comprising (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0092] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 7, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 8, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 9, and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0093] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 12, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0094] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 13, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 16, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0095] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 18, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 19, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 20, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 22, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 23.

[0096] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 24, and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable (VL) region comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0097] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) a VH CDR1 having an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 27, 33, 38, 39, and 44, (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 34, 40, 45, and 50, and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 35, 41, and 46, and a light chain variable (VL) region comprising (b) a VL CDR1 having an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 30, 36, 42, and 47, (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 37, and 48, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 43, and 49.

[0098] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 27, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 29, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 30, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 31, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 32.

[0099] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 33, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 34, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 35, and a light chain variable (VL) region comprising (b) (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 36, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 37, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 32.

[0100] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 38, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 29, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 30, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 31, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 32.

[0101] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 39, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 40, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 41, and a light chain variable (VL) region comprising (b) (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 42, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 37, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 43.

[0102] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 44, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 45, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 46, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 47, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 48, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 49.

[0103] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 27, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 50, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 29, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 30, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 31, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 32. In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) a VH CDR1 having an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 53, 59, 64, 65, and 70, (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 60, 66, 71, and 76, and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 61, 67, and 72, and a light chain variable (VL) region comprising (b) a VL CDR1 having an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 56, 62, 68, and 73, (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 63, and 74, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 69, and 75.

[0104] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 53, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 54, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 55, and a light chain variable (VL) region comprising (b) (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 56, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 57, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 58.

[0105] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 59, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 60, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 61, and a light chain variable (VL) region comprising (b) (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 62, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 63, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 58.

[0106] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 64, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 54, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 55, and a light chain variable (VL) region comprising (b) (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 56, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 57, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 58.

[0107] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 65, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 66, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 67, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 68, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 63, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 69.

[0108] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 70, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 71, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 72, and (b) a light chain variable (VL) region comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 73, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 74, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 75.

[0109] In some embodiments, antibodies are described herein that include a heavy chain variable (VH) region comprising (a) (1) VH CDR1 having the amino acid sequence of SEQ ID NO: 53, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 76, and (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 55, and a light chain variable (VL) region comprising (b) (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 56, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 57, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 58.

[0110] In some embodiments, antibodies comprising the VH region and / or VL region described herein, wherein the VH region and / or VL region further comprises a human framework sequence, are described herein. In some embodiments, the VH region and / or VL region further comprises framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

[0111] In some embodiments, the antibodies described herein are monoclonal antibodies. In some embodiments, the monoclonal antibodies are humanized antibodies, human antibodies, or chimeric antibodies. In some embodiments, the antibodies described herein are multispecific antibodies formed from Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate region antibodies, single variable region antibodies, linear antibodies, V regions, or antibody fragments.

[0112] In some embodiments, the CDRs disclosed herein include consensus sequences derived from a group of relevant antibodies (see, for example, Tables 1-3). As described herein, “consensus sequence” refers to an amino acid sequence having conserved amino acids common among several sequences and variable amino acids that vary within a given amino acid sequence. The provided CDR consensus sequences include CDRs corresponding to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. Consensus sequences of CDRs for PD-L1 binding agonists (e.g., antibodies such as bispecific antibodies) are shown in Figures 7A and 7B. Therefore, in some embodiments, the PD-L1 binding activators described herein (e.g., antibodies such as bispecific antibodies) are: (a) (1) VH CDR1 having the amino acid sequence GFTFX1X2YYIH (SEQ ID NO: 83) (wherein X1 and X2 are independently naturally occurring amino acids); (2) VH CDR2 having the amino acid sequence X1IX2X3X4GX5X6TX7YADSVKG (SEQ ID NO: 84) (wherein X1, X2, X3, X4, X5, X6, and X7 are independently naturally occurring amino acids); and (3) VH having the amino acid sequence X1X2X3X4X5X6X7X8LDY (SEQ ID NO: 85) (wherein X1, X2, X3, X4, X5, X6, X7, and X8 are independently naturally occurring amino acids). The material comprises a heavy chain variable (VH) region containing CDR3 and / or (b) a light chain variable (VL) region containing (1) VL CDR1 having the amino acid sequence RASQSVSSAVA (SEQ ID NO: 86), (2) VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO: 87), and (3) VL CDR3 having the amino acid sequence QQX1X2X3X4PX5T (SEQ ID NO: 88) (wherein X1, X2, X3, X4, and X5 are independently naturally occurring amino acids). In some embodiments, the VH CDR1 of the PD-L1 binding activator described herein has the amino acid sequence GFTFX1X2YYIH (SEQ ID NO: 101) (wherein X1 is D or S and X2 is Q or S).In some embodiments, the PD-L1 binding activator VH CDR2 described herein has the amino acid sequence X1IX2X3X4GX5X6TX7YADSVKG (SEQ ID NO: 89) (wherein X1 is E, W, or T; X2 is Y, T, or S; X3 is P, or S; X4 is A, H, or G; X5 is S, Y, or G; X6 is Y, S, or F; and X7 is Y, or K). In some embodiments, the VH CDR3 of the PD-L1 binding activator described herein has the amino acid sequence X1X2X3X4X5X6X7X8LDY (SEQ ID NO: 90) (wherein X1 is G or D, X2 is P, S or Y, X3 is Y, V or T, X4 is S, I or L, X5 is V, Y or T, X6 is R, G or P, X7 is Y or V (or absent), and X8 is A (or absent)). In some embodiments, the VL CDR1 of the PD-L1 binding activator described herein has the amino acid sequence RASQSVSSAVA (SEQ ID NO: 86). In some embodiments, the VL CDR2 of the PD-L1 binding activator described herein has the amino acid sequence SASSLYS (SEQ ID NO: 87). In some embodiments, the VL CDR3 PD-L1 binding activator described herein has the amino acid sequence QQX1X2X3X4PX5T (SEQ ID NO: 91) (wherein X1 is V, Y, or F; X2 is S, Y, or G; X3 is Y, T, or A; X4 is S, or E; and X5 is Y, or I).

[0113] In some embodiments, binding agents are described herein that bind to essentially the same epitope as one of the antibodies or fragments described herein. In some embodiments, binding agents are described herein that compete with one of the antibodies or fragments described herein for binding to human PD-L1. In some embodiments, the binding agent is an antibody or fragment thereof.

[0114] In certain embodiments, the CDR of PD-L1-binding agonists (e.g., antibodies), including human PD-L1-binding agonists, can be determined according to the Kabat method (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0115] In certain embodiments, the CDR of PD-L1-binding agonists (e.g., antibodies), including human PD-L1-binding agonists, can be determined according to the Chothia formula, which is referred herein as the "Chothia CDR" (see, for example, Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol. 215(1):175-82; and U.S. Patent No. 7,709,226).

[0116] In certain embodiments, the CDR of PD-L1-binding agonists (e.g., antibodies), including human PD-L1-binding agonists, can be determined according to the ImMunoGeneTics (IMGT) method, as described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 ("IMGT CDR").

[0117] In certain embodiments, the CDR of PD-L1-binding agonists (e.g., antibodies), including human PD-L1-binding agonists, can be determined according to the AbM method, as described, for example, MacCallum et al., 1996, J. Mol. Biol., 262:732-745, which is referred herein to as "AbM CDR". See also, for example, Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0118] In certain embodiments, the CDR of PD-L1-binding agonists (e.g., antibodies), including human PD-L1-binding agonists, can be determined according to the Contact method, which is referred to herein as "Contact CDR" (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5: 732-745). Contact CDR is based on the analysis of available complex crystal structures.

[0119] In some embodiments, the positions of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of a PD-L1-binding agonist (e.g., an antibody), including the human PD-L1-binding agonists described herein, may vary by only one, two, three, four, five, or six amino acid positions, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the position defining the CDR in either Table 1 or 2 may be varied by shifting the N-terminal and / or C-terminal boundary of the CDR by 1, 2, 3, 4, 5, or 6 amino acids compared to the current CDR position, as long as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of a PD-L1-binding agonist (e.g., an antibody), including the human PD-L1-binding agonists described herein, may vary by 1, 2, 3, 4, 5, or more amino acids (e.g., may be shorter or longer), insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).For example, in some embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be 1, 2, 3, 4, 5, or more amino acids shorter than one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be 1, 2, 3, 4, 5 or more amino acids longer than one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50 or 53-76, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino-terminuses of VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be elongated by 1, 2, 3, 4, 5, or more amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the carboxyl termini of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be extended by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50 or 53-76, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In other embodiments, the amino-terminuses of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24, 27-50 or 53-76, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxyl terminus of VH and / or VL CDR1, CDR2 and / or CDR3 described herein may be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-50, or 53-76, insofar as binding to PD-L1 (e.g., human PD-L1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Whether binding to PD-L1 (e.g., human PD-L1) is maintained can be determined using any method known in the art, for example, the binding assays and conditions described in the “Examples” section herein. For example, Example 2 described herein describes an assay for measuring binding to PD-L1 (e.g., human PD-L1).

[0120] In other embodiments, PD-L1-binding agonists (e.g., antibodies), including human PD-L1-binding agonists presented herein, include conservative sequence modifications. With respect to polypeptides that are PD-L1-binding agonists (e.g., antibodies), such as human PD-L1-binding agonists, conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the Art. Such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in some embodiments, non-essential amino acid residues predicted in PD-L1 are replaced with other amino acid residues from the same side chain family. Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate binding to antigens are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conservative sequence modifications described herein modify the amino acid sequence of a PD-L1 binding agonist (e.g., an antibody), including a human PD-L1 binding agonist, by only 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.In some embodiments, nucleotide and amino acid sequence modifications refer to at most one, two, three, four, five, or six amino acid substitutions for the CDRs listed in Table 1, Table 2, or Table 3. Thus, for example, each of such CDRs may contain up to five conservative amino acid substitutions, e.g., up to four (not more than four) conservative amino acid substitutions, e.g., up to three (not more than three) conservative amino acid substitutions, e.g., up to two (not more than two) conservative amino acid substitutions, or not more than one conservative amino acid substitution.

[0121] This disclosure provides a PD-L1-binding agonist (e.g., an antibody) having a shielded portion and / or cleavable portion, wherein one or more of the PD-L1-binding domains of the PD-L1-binding agonist (e.g., an antibody) are shielded (e.g., by a shielded portion) and / or activatable (e.g., by a cleavable portion). Techniques for shielding PD-L1-binding agonists (e.g., antibodies) are well known in the art and include SAFE body shielding techniques (see, for example, U.S. Patent Application Publication 2019 / 0241886) and Probody shielding techniques (see, for example, U.S. Patent Application Publication 2015 / 0079088). Using such techniques, shielded and / or activatable PD-L1-binding agonists (e.g., antibodies) can be produced. Such shielded and / or activatable PD-L1-binding agonists (e.g., antibodies) are useful in the preparation of conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), shielded ADCs, and activatable antibody-drug conjugates (AADCs), which include any one of the PD-L1-binding agonists (e.g., antibodies), such as the human PD-L1-binding agonists of this disclosure, including those directly or indirectly linked to another agonist, such as a drug. For example, a PD-L1-binding agonist (e.g., antibody), such as the human PD-L1-binding agonist of this disclosure, can be covalently linked to one or more agonists, such as a drug, by a synthetic linker.

[0122] If desired, PD-L1 binding agonists (e.g., antibodies), including human PD-L1 binding agonists, may be linked or conjugated (directly or indirectly) to a portion having effector functions such as cytotoxic activity (e.g., a chemotherapeutic portion or a radioisotope) or immune mobilization activity. The linked or conjugated portion (directly or indirectly) may be in the form of a shielded conjugate and may be a cytotoxic drug (e.g., a toxin such as aurostatin) or a non-cytotoxic drug (e.g., a signal transduction modulator such as a kinase, or a shielding portion that shields one or more binding domains of a PD-L1 binding agonist (e.g., an antibody), or a cleavable portion that enables activation of the PD-L1 binding agonist by cleaving a cleavable portion to remove shielding of one or more binding domains of the PD-L1 binding agonist (e.g., an antibody) in the tumor microenvironment). Other antigen-binding agonists, such as viral proteins that selectively bind to cells of the innate immune system, may be used as immune mobilization-promoting portions. Alternatively, or in addition to the above, a PD-L1 binding agonist (e.g., an antibody), including a human PD-L1 binding agonist, may be linked or conjugated (directly or indirectly) to a moiety that facilitates isolation from the mixture (e.g., a tag) or a moiety having reporter activity (e.g., a detection label or reporter protein), as needed. It will be noted that the features of the PD-L1 binding agonists (e.g., antibodies), including the human PD-L1 binding agonist described herein, can also be extended to polypeptides containing PD-L1 binding agonist fragments.

[0123] In some embodiments, a PD-L1-binding agonist (e.g., an antibody), including the PD-L1-binding agonists described herein that bind to human PD-L1, can be linked or conjugated (directly or indirectly) with a polypeptide that can result in the production of an activatable antibody. In some embodiments, a PD-L1-binding agonist (e.g., an antibody), including a human PD-L1-binding agonist, is linked or conjugated (directly or indirectly) with an agonist. In some embodiments, the agonist is a drug, resulting in an ADC, or an AADC if the antibody of the ADC includes a shielding portion and a cleavable portion.

[0124] In some embodiments, PD-L1 binding agonists (e.g., antibodies), including the human PD-L1 binding agonists described herein, are conjugated or recombinantly linked (directly or indirectly) with therapeutic agents (e.g., cytotoxic agonists) or diagnostic or detection agents. Conjugated or recombinantly linked antibodies, including shielded or activatable conjugates, may be useful for treating or preventing diseases, disorders, or conditions, such as PD-L1-mediated diseases, disorders, or conditions. Conjugated or recombinantly linked PD-L1 binding agonists (e.g., antibodies), including shielded or activatable conjugates, may be useful for monitoring or prognosing the onset, development, exacerbation, and / or severity of T-cell dysfunction diseases, disorders, or conditions, for example.

[0125] Such diagnostic and detection methods include, for example, PD-L1-binding agonists (e.g., antibodies), enzymes including, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups including, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent materials including, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dancylkloride, or phycoerythrin; luminescent materials including, but not limited to, luminol; bioluminescent materials including, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent materials including, but not limited to, acridinium-based compounds or HALOTAG; and iodine. (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La This can be achieved by coupling with radioactive materials including 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, or 117Sn; positron-emitting metals using various positron emission tomography techniques; and detectable materials including non-radioactive paramagnetic metal ions.

[0126] PD-L1-binding agonists (e.g., antibodies) that synthesize fusion proteins by recombination or conjugation (direct or indirect covalent or noncovalent conjugation) with heterologous proteins or polypeptides (or fragments thereof), for example, polypeptides (e.g., those with about 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 amino acids), and their uses are also described herein. In particular, fusion proteins comprising antigen-binding fragments (e.g., CDR1, CDR2, and / or CDR3 of VH and / or VL) of PD-L1-binding agonists (e.g., antibodies), including the human PD-L1-binding agonists described herein, and heterologous proteins, polypeptides, or peptides are described herein. In some embodiments, heterologous proteins, polypeptides, or peptides that conjugate to PD-L1-binding agonists (e.g., antibodies) are useful for targeting the PD-L1-binding agonists to specific cells (e.g., PD-L1-expressing cells, including tumor cells).

[0127] Furthermore, PD-L1 binding agonists (e.g., antibodies), including the human PD-L1 binding agonists described herein, can be linked (directly or indirectly) to marker or "tag" sequences, such as peptides, to facilitate purification. In some embodiments, the marker or tag amino acid sequence is, among other things, a hexahistidine peptide, such as a tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, hemagglutinin ("HA") tags corresponding to epitopes derived from influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and "FLAG" tags.

[0128] Methods (directly or indirectly) for linking or conjugating a portion (including a polypeptide) with an antibody are well known in the art, and any one of these can be used to produce the antibody-drug conjugate or fusion protein described herein.

[0129] In some embodiments, the PD-L1 binding agonist (e.g., antibody) described herein is a fusion protein. The term "fusion protein," as used herein, refers to a polypeptide comprising the amino acid sequence of a binding agonist (e.g., antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not typically part of an antibody (e.g., a non-PD-L1 binding antibody)). In certain embodiments, the fusion protein retains the biological activity of the PD-L1 binding agonist. In certain embodiments, the fusion protein comprises a VH region, a VL region, a VH CDR (one, two, or three VH CDRs), and / or a VL CDR (one, two, or three VL CDRs) of a PD-L1 antibody, and the fusion protein binds to a PD-L1 epitope, a PD-L1 fragment, and / or a PD-L1 polypeptide.

[0130] Fusion proteins can be generated, for example, by techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modify the activity of PD-L1 binding agonists (e.g., antibodies), including the human PD-L1 binding agonists described herein, which include PD-L1 binding agonists having higher affinity and lower dissociation rates (e.g., U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patenten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques See 24(2):308-13). In some embodiments, PD-L1 binding agonists, including human PD-L1 binding agonists, can be modified prior to recombination by error-prone PCR, random nucleotide insertion, or random mutagenesis by other means. The polynucleotides encoding the PD-L1 binding agonists described herein can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0131] PD-L1-binding agonists (e.g., antibodies), including the human PD-L1-binding agonists described herein, can also be conjugated to solid supports useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0132] A PD-L1-binding agonist (e.g., an antibody), including the human PD-L1-binding agonist described herein, can also be linked or conjugated (directly or indirectly) with a second antibody to form an antibody heteroconjugate.

[0133] A linker may be a “cleavable portion” that facilitates the intracellular release of a linked or conjugated activator, but non-cleavable linkers are also intended herein. Linkers for use in the conjugates of this disclosure (e.g., antibody-drug conjugates) include, but are not limited to, acid-unstable linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids, e.g., valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photosensitive linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid resistance mediated by multidrug transporters.

[0134] Antibody-agonist conjugates, including cases where the agonist is a drug for preparing ADCs or AADCs, can be prepared using various bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoic acid). This disclosure further intends that antibody-agonist conjugates, including cases where the agonist is a drug for preparing ADCs or AADCs, can be prepared using any suitable method disclosed in the Art (see, for example, Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0135] Antibody-agonist conjugation strategies, including those where the agonist is a drug for preparing ADCs or AADCs, have traditionally relied on random conjugation chemistry resulting in heterogeneous conjugates involving ε-amino groups on Lys residues or thiol groups on Cys residues. Recently developed techniques enable site-specific conjugation with antibodies, thereby resulting in a uniform load and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics. These techniques involve "thiomab" manipulations, which include cysteine ​​substitutions at positions on the heavy and light chains that yield reactive thiol groups without disrupting immunoglobulin folding and assembly or altering antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol. 26: 925-32). Alternatively, selenocysteine ​​can be inserted into the antibody sequence in a manner that allows for co-translation, by recoding the stop codon UGA from termination to selenocysteine ​​insertion, thereby enabling site-directed covalent conjugation of selenocysteine ​​at the nucleophilic selenool group in the presence of other native amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105: 12451-56; and Hofer et al., 2009, Biochemistry 48(50): 12047-57).

[0136] In some embodiments, a PD-L1 binding agonist (e.g., an antibody), including the human PD-L1 binding agonists described herein, is conjugated with a cytotoxic agonist. In some embodiments, a PD-L1 binding agonist (e.g., an antibody), including the human PD-L1 binding agonists disclosed herein, may be optionally conjugated with one or more cytotoxic agonists disclosed herein or known in the art to produce an ADC or AADC. In some embodiments, the cytotoxic agonist is a chemotherapeutic agent, including, but not limited to, methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other inserts. In some embodiments, the cytotoxic agent is a toxin or fragment thereof of bacterial, fungal, plant, or animal origin that has enzymatic activity, including, but is not limited to, diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, geronin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene. In some embodiments, the cytotoxic agent is a radioisotope for creating a radioconjugate or a radioconjugated agent. However, various radionuclides, including but not limited to 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re, and 212Bi, can be used to create radioconjugated agents.Conjugates of polypeptides or molecules with one or more small molecule toxins, such as calitiamycin, maytansinoids, trichothene, and CC1065, as well as derivatives of these toxins having toxic activity, can also be used. Various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyl(pyridyi)dithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanic acid (e.g., toluene-2,6-diisocyanic acid), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), are used to create conjugates between polypeptides or molecules and cytotoxic agents.

[0137] In some embodiments, PD-L1-binding agonists (e.g., antibodies), including the human PD-L1-binding agonists described herein, are conjugated with drugs such as signal transduction modulators, apoptosis promoters, mitotic inhibitors, antitumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agonists, chemoprotective agents, hormones, antihormone agents, corticosteroids, phototherapeutic agents, oligonucleotides, radionuclear agonists, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors. In some embodiments, the mitotic inhibitors are dorastatin, auristatin, maytansinoids, and plant alkaloids. In some embodiments, the drugs are dorastatin, auristatin, maytansinoids, and plant alkaloids. Examples of auristatin are monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). Examples of meitansinoids include, but are not limited to, DM1, DM2, DM3, and DM4. In some embodiments, the antitumor antibiotic is selected from the group consisting of actinomycin, anthracyclines, calitiamycin, and duocalmycin. In some embodiments, actinomycin is pyrrolobenzodiazepine (PBD).

[0138] PD-L1 binding agonists (e.g., antibodies), including the human PD-L1 binding agonists described herein, may have monospecificity, bispecificity, tripspecificity, or higher-order multispecificity. Such agonists may include antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity to at least two different targets (e.g., antigens) or to two different epitopes on the same target (e.g., a bispecific antibody targeting PD-L1 having a first binding domain to a first epitope of PD-L1 and a second binding domain to a second epitope of PD-L1). In some embodiments, multispecific (e.g., bispecific) antibodies can be constructed based on the antibody sequences described herein, e.g., the CDR sequences listed in Tables 1, 2, and 3. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are mouse, chimeric, human, or humanized antibodies. In some embodiments, one binding specificity of a multispecific antibody is for PD-L1, and the other is for any other target (e.g., an antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may contain more than one target (e.g., antigen) binding domains, where different binding domains are specific to different targets (e.g., a first binding domain that binds to PD-L1 and a second binding domain that binds to another target (e.g., an antigen), such as an immune checkpoint regulator (e.g., a negative checkpoint regulator)). In some embodiments, a multispecific (e.g., bispecific) antibody molecule may bind to more than one (e.g., two or more) epitopes on the same target (e.g., an antigen).In some embodiments, one of the binding specificities is PD-L1, while the other is cytotoxic T lymphocyte antigen-4 (CTLA-4), CD80, CD86, programmed cell death 1 (PD-1), programmed cell death ligand 2 (PD-L2), lymphocyte activation gene-3 (LAG-3; also known as CD223), galectin-3, B and T lymphocyte attenuator (BTLA), T cell membrane protein 3 (TIM3), galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, Ig, and ITIM domains. This targets one or more of the following: T cell immune receptors (TIGIT / Vstm3 / WUCAM / VSIG9), V-domain Ig inhibitor of T cell activation (VISTA), glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0139] Methods for producing multispecific antibodies are known in the art, for example, by simultaneously expressing two immunoglobulin heavy-light chain pairs in which the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305: 537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0140] Exemplary structures of multispecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10: 1-18; Brinkman et al., 2017, MABS, 9:2, 182-212; Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276; and Spiess et al., 2015, Mol. Immunol. 67 95-106.

[0141] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with an additional antigen-binding moiety, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates. As a non-limiting example, BsIgG formats may include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-hole assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab.

[0142] In some embodiments, BslgG includes a heavy chain manipulated for heterodimerization. For example, the heavy chain can be manipulated for heterodimerization by using a "knobs-into-holes" strategy, a SEED platform, a common heavy chain (e.g., one in the κλ-body), and the use of a heterodimer Fc region. Strategies to avoid homodimer heavy chain pairing in BsIgG are known in the art, including knob-into-holes, duobody, azymetric, charge pairing, HA-TF, SEEDbody, and differential protein A affinity.

[0143] Another bispecific antibody format is IgG with an additional antigen-binding moiety. For example, monospecific IgG can be manipulated to be bispecific by adding an additional antigen-binding unit to monospecific IgG, for example, to the N-terminus or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single-domain antibodies (e.g., variable heavy or variable light chains), manipulated protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). Non-limiting examples of additional IgG formats include bivariable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67 (2015): 95-106. In some embodiments, exemplary antibody formats are the B-Body format for monospecific or multispecific (e.g., bispecific antibodies) described, for example, in International Patent Application Publication WO2018 / 075692 and U.S. Patent Application Publication 2018 / 0118811.

[0144] A bispecific antibody fragment (BsAb) is a format of bispecific antibody molecule that lacks some or all of the antibody constant domain. For example, some BsAbs lack the Fc region. In several embodiments, the bispecific antibody fragment comprises a heavy chain region and a light chain region linked by a peptide linker, thereby enabling efficient expression of the BsAb in a single host cell. Non-exclusive examples of bispecific antibody fragments include, but are not limited to, nanobodies, nanobodies-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intracellular antibodies.

[0145] Bispecific fusion proteins contain antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or functionality. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with high binding titers. For example, the serum half-life of an antibody fragment can be extended by fusing a bispecific antibody with an albumin-binding protein or human serum albumin. In several embodiments, BsAb molecules can be created using chemical conjugation, such as chemical conjugation of an antibody and / or antibody fragment. An exemplary bispecific antibody conjugate is the CovX-body format, in which a low molecular weight drug is site-specifically conjugated to a single reactive lysine within each Fab arm or antibody or fragment. In several embodiments, conjugation improves the serum half-life.

[0146] Methods for producing multispecific antibodies, including bispecific antibodies, are known in the art. For example, multispecific antibodies, including bispecific antibodies, can be produced by separately expressing constituent antibodies in different host cells and then purifying / assembling them, or by expressing constituent antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be carried out by various methods known in the art, including affinity chromatography.

[0147] In some embodiments, PD-L1 binding agonists (e.g., antibodies), including the human PD-L1 binding agonists disclosed herein, can be provided in any antibody format disclosed herein or known in the art. As a non-limiting example, in some embodiments, PD-L1 binding agonists (e.g., antibodies), including human PD-L1 binding agonists, include Fabs-in-tandem-1g (FIT-1g); DVD-1g; Hybrid Hybridoma (Quadroma or Tetradoma); Anticalin Platform (Pieris); Diabody; Single-chain Diabody; Tandem Single-chain Fv Fragment; TandAb, Triple-specific Ab (Affimed); Darts Biaffinity Retargeting (Macrogenics); Bi-specific Xmab (Xencor); Bi-specific T-cell Inducing Antibody (engager) (Bite; Amgen; 55kDa); Triplebody; Tribody = Fab-scFv Fusion Protein Multifunctional Recombinant Antibody Derivative (CreativeBiolabs); Duobody Platform (Genmab); Dock and Lock You can choose from the following platforms: lock platform; knobs-into-holes (KIH) platform; humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-lg = bivariable domain immunoglobulin (Abbott); copper lambda body; TBTI = tetravalent bispecific tandem Ig; and CrossMab (Roche).

[0148] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a PD-L1 binding domain and one or more additional binding domains that bind to one or more targets other than PD-L1. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a PD-L1 binding domain containing, for example, the VH and / or VL amino acid sequences of Table 1. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a PD-L1 binding domain containing the VH and / or VL amino acid sequences of Table 2. In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein include a PD-L1 binding domain containing the VH and / or VL amino acid sequences of Table 3.

[0149] In some embodiments, multispecific (e.g., bispecific) antibodies comprising binding domains that bind to PD-L1, including VH and VL CDR as listed in Table 1, are described herein. In some embodiments, multispecific (e.g., bispecific) antibodies comprising binding domains that bind to PD-L1, including (set for) VH and VL CDR as listed in Table 2, are described herein. In some embodiments, multispecific (e.g., bispecific) antibodies comprising binding domains that bind to PD-L1, including VH and VL CDR as listed in Table 3, are described herein.

[0150] Antibodies that bind to PD-L1 can be obtained by any suitable method, including but not limited to, immunization and antibody collection using whole tumor cells containing PD-L1, recombinant techniques, or screening of a library of antibodies or antibody fragments using PD-L1 extracellular domain epitopes. Monoclonal antibodies can be produced using various known techniques (see, for example, Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). One exemplary technique for generating monoclonal antibodies involves immunizing an animal with human PD-L1 antigen and generating a hybridoma from spleen cells isolated from this animal. The hybridoma can produce monoclonal antibodies or antibody fragments that bind to PD-L1.

[0151] In further embodiments, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques described, for example, in *Antibody Phage Display: Methods and Protocols*, PM O'Brien and R. Aitken, eds, Humana Press, Totawa NJ, 2002. In principle, synthetic antibody clones are selected by screening a phage library containing phages displaying various fragments of the antibody variable region (Fv) fused to a phage coat protein. Such phage libraries are then screened against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen adsorb to the antigen and are therefore separated from non-binding clones in the library. The binding clones can then be eluted from the antigen and further enriched by additional cycles of antigen adsorption / elution.

[0152] For example, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994), the variable domain can be functionally displayed on the phage either as a single-chain Fv(scFv) fragment in which VH and VL are covalently linked by a short, flexible peptide, or as a Fab fragment in which VH and VL are fused to a constant domain and interact noncovalently.

[0153] As described by Winter et al., the VH gene repertoire and the VL gene repertoire can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then searched for antigen-binding clones. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12: 725-734 (1993), cloning a naive repertoire can provide a single source of human antibodies against a wide range of non-self antigens and similarly against self antigens without any immunization. Finally, as described, for example, in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), naive libraries can also be synthetically created by cloning unreconstituted V gene segments derived from stem cells and using PCR primers containing random sequences, in order to encode a highly variable CDR3 region and to enable reconstitution in vitro.

[0154] Library screening can be achieved by various techniques known in the art. For example, PD-L1 (e.g., PD-L1 polypeptide, fragment, or epitope) can be used to coat wells of an adsorption plate, expressed in host cells attached to the adsorption plate, used in cell sorting, conjugated with biotin for capture by streptavidin-coated beads, or used in any other method for panning a display library. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be achieved by using long washes and monovalent phage displays, as described in Bass et al., Proteins, 8: 309-314 (1990) and WO92 / 09690, and can be facilitated by low antigen coating density, as described in Marks et al., Biotechnol., 10: 779-783 (1992).

[0155] PD-L1-binding agonists (e.g., antibodies) can be obtained by designing a suitable antigen screening procedure for selecting a phage clone of interest, and then constructing a full-length PD-L1-binding agonist (e.g., antibody) clone using VH and / or VL sequences (e.g., Fv sequences) or various CDR sequences derived from VH and VL sequences, as well as suitable constant region (e.g., Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0156] Similarly, human antibodies that bind to PD-L1 can be generated by any of several techniques, including, but not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells derived from immunized transgenic mice into which human immunoglobulin genes have been inserted, isolation from a human immunoglobulin V region phage library, or other procedures known in the art and based on this disclosure. Methods for obtaining human antibodies from transgenic animals are described, for example, in Bruggemann et al., Curr. Opin. Biotechnol., 8: 455 58, 1997; Jakobovits et al., Ann. NY Acad. Sci., 764: 525 35, 1995; Green et al., Nature Genet., 7: 13-21, 1994; Lonberg et al., Nature, 368: 856-859, 1994; Taylor et al., Int. Immun. 6: 579-591, 1994; and further in U.S. Patent No. 5,877,397.

[0157] For example, human antibodies that bind to PD-L1 can be obtained from transgenic animals engineered to produce specific human antibodies in response to antigenic attack. For instance, International Patent Publication WO98 / 24893 discloses a transgenic animal having a human Ig locus that does not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy and light chain loci. Transgenic non-primate mammalian hosts capable of initiating an immune response to an immunogen are also described, in which the antibody has primate constant and / or variable regions, and the endogenous immunoglobulin encoding the locus is substituted or inactivated. International Patent Publication WO96 / 30498 discloses the use of a Cre / Lox system to modify an immunoglobulin locus in a mammal, for example, by replacing all or part of the constant or variable region to form a modified antibody molecule. International Patent Publication WO94 / 02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus. U.S. Patent No. 5,939,598 discloses a method for producing a transgenic mouse that lacks an endogenous heavy chain and expresses an exogenous immunoglobulin locus expressing one or more heterologous constant regions. Transgenic animals, such as the transgenic animals described herein, can be used to induce an immune response to selected antigenic molecules, and antibody-producing cells can be isolated from the animals and used to produce hybridomas that secrete human-derived monoclonal antibodies. Immunization protocols, adjuvants, etc., are known in the art and have been used, for example, in the immunization of transgenic mice as described in International Patent Publication WO96 / 33735. Monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effects of the corresponding protein.

[0158] This disclosure provides humanized antibodies that bind to PD-L1, including human PD-L1. The humanized antibodies of this disclosure may comprise one or more CDRs shown in Tables 1-3. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues derived from a non-human source introduced into it. These non-human amino acid residues are often referred to as “implant” residues and are typically obtained from “implant” variable domains. Humanized antibodies that bind to PD-L1 can be prepared using techniques known to those skilled in the art (Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Patents No. 6,180,370; No. 6,054,927; No. 5,869,619; No. 5,861,155; No. 5,712,120; and No. 4,816,567, all of which are expressly incorporated herein by reference).

[0159] In some cases, humanized antibodies are constructed by CDR grafting, which involves grafting the amino acid sequences of six complementarity-determining regions (CDRs) of a parent non-human antibody (e.g., rodents) onto a human antibody framework. For example, Padlan et al. (FASEB J. 9: 133-139, 1995) determined that only about one-third of the residues within the CDR actually come into contact with the antigen; these are called "specificity-determining residues" or SDRs. In SDR grafting techniques, only the SDR residues are grafted onto the human antibody framework (see, for example, Kashmiri et al., Methods 36: 25-34, 2005).

[0160] The selection of human variable domains for both the light and heavy chains used in the production of humanized antibodies can be important for reducing antigenicity. For example, according to the so-called "best-fit" method, the sequences of the variable domains of non-human (e.g., rodent) antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent sequence can be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mol. Biol. 196: 901). In another method, a particular framework derived from the consensus sequence of human antibodies, where the light or heavy chain is of a particular subgroup, is used. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623). In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, V L 6 subgroup I (V L 6I) and V H subgroup III (V H III). In another method, human germline genes are used as the source for the framework region.

[0161] In an alternative paradigm based on comparison of CDRs, called "superhumanization", FR homology is irrelevant. This method consists of a comparison of non-human sequences with the functional human germline gene repertoire. Next, genes encoding the same or closely related canonical structures as the mouse sequence are selected. Then, among the genes sharing the canonical structure with the non-human antibody, the one with the highest homology within the CDR is selected as the FR donor. Finally, the non-human CDRs are grafted onto these FRs (see, for example, Tan et al., J. Immunol. 169: 1119-1125, 2002).

[0162] Furthermore, it is generally desirable that antibodies be humanized while retaining their affinity for their antigens and other favorable biological properties. To achieve this objective, according to one method, humanized antibodies are prepared by a process in which the parental sequence and various conceptual humanized products are analyzed using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display highly accurate three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Examination of these indications allows for the analysis of residues that are likely to play a role in the function of candidate immunoglobulin sequences, for example, residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and transfer sequences, resulting in the realization of desired antibody characteristics, such as increased affinity for the target antigen(s). Generally, hypervariable region residues are most directly and substantially involved in influencing antigen binding.

[0163] Another method for antibody humanization is based on a metric for antibody humanization called Human String Content (HSC). This method compares mouse sequences with a repertoire of human germline genes and scores the differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using an overall identity metric to generate a large number of diverse humanized variants (Lazar et al., Mol. Immunol. 44: 1986-1998, 2007).

[0164] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods include generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as by bacterial colony screening (see, for example, Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21: 163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17: 847-60, 2004).

[0165] In the FR library method, a collection of residue variants is introduced at specific positions in the FR, and then library selection is performed to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the "Vernier" residues from a more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997) or those identified as potentially contributing to the CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992).

[0166] In FR shuffling, instead of creating a combinatorial library of selected residue variants, the entire FR is combined with a non-human CDR (see, e.g., Dall'Acqua et al., Methods 36: 43-60, 2005). The library can be screened for binding in a two-step selection process, first for humanization of the VL, and then for VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than two-step screening because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and increased thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

[0167] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and involves sequentially replacing non-human fragments with human FR libraries and assessing binding. Starting with the CDR3 region of non-human VH and VL chains, other regions, including CDR1 and CDR2 of both VH and VL of the non-human antibody, are progressively replaced with human FR. This methodology typically leads to the identification of antibodies from multiple subclasses possessing epitope retention and distinct human V-segment CDRs. Humaneering makes it possible to isolate antibodies that are 91–96% homologous to human germline gene antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0168] Human engineering methods involve modifying non-human antibodies or antibody fragments, such as mouse or chimeric antibodies or antibody fragments, by making specific changes to the amino acid sequence of the antibody, thereby creating modified antibodies that retain the desired binding properties of the original non-human antibody, even though their immunogenicity in humans is reduced. Generally, this technique involves classifying amino acid residues in non-human (e.g., mouse) antibodies into "low-risk," "medium-risk," or "high-risk" residues. The classification is performed using a comprehensive risk / reward calculation that evaluates the expected benefit of making a particular substitution (e.g., in terms of immunogenicity in humans) against the impact of the substitution on the folding of the resulting antibody and / or the risk of substitution with human residues. Specific human amino acid residues (e.g., low or medium risk) to be substituted at a given position in the non-human (e.g., mouse) antibody sequence can be selected by aligning the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific or consensus human antibody sequence. Amino acid residues at low or medium-risk positions in the non-human sequence can be substituted with corresponding residues in the human antibody sequence according to the alignment. Techniques for creating human-engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994), U.S. Patents No. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Publication No. WO93 / 11794.

[0169] In some embodiments, the PD-L1 binding agonists described herein include non-antibody protein scaffolds. Non-limiting examples of such non-antibody protein scaffolds include fibronectin scaffolds, anticarin, adonectin, afibody, DARPin, finomer, afitin, afirin, avimer, cysteine-rich Nottin peptide, or engineered Knitz-type inhibitors. Methods for generating such non-antibody protein scaffolds are well known in the art, and any one of them can be used to generate PD-L1 binding agonists containing non-antibody protein scaffolds (see, for example, Simeon and Chen, Protein Cell, 9(1): 3-14 (2018); Yang et al., Annu Rev Anal Chem (Palo Alto Calif). 10(1): 293-320 (2017)).

[0170] Further materials are provided for generating PD-L1 binding agonists, such as human PD-L1 binding agonists and fragments thereof. For example, isolated cells (e.g., hybridomas) can produce PD-L1 binding agonists (e.g., antibodies or antibody fragments). In this regard, cells (e.g., isolated cells) can produce antibodies or fragments thereof containing VH and VL as shown in Tables 1, 2, or 3 for P22, P24, or P31.2, respectively. In some embodiments, the polynucleotides described herein may include one or more nucleic acid sequences encoding a PD-L1 binding agonist (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotides are isolated and / or recombinant polynucleotides. In various embodiments, the isolated polynucleotides include an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (V L It contains a nucleotide sequence that codes for V H and V L This includes the same complementarity determination region (CDR) as the CDR shown in Table 1, the CDR shown in Table 2, or the CDR shown in Table 3.

[0171] In some embodiments, one or more vectors (e.g., expression vectors) may contain one or more polynucleotides for expressing one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying polynucleotides in a host cell to produce a useful quantity thereof, and for expressing binding agents such as antibodies or antibody fragments using recombinant techniques.

[0172] In some embodiments, one or more vectors are expression vectors, where one or more polynucleotides are operatively ligated to one or more polynucleotides containing an expression regulatory sequence. Autonomous recombinant expression constructs, such as plasmids and viral DNA vectors, incorporating one or more polynucleotides encoding an antibody sequence that binds to PD-L1 are particularly intended. Expression regulatory DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression system in which the expression construct is used. Promoter and enhancer sequences are generally selected for their ability to increase gene expression, while operator sequences are generally selected for their ability to regulate gene expression. The expression construct may also include sequences encoding one or more selection markers that enable the identification of the host cell carrying the construct. The expression construct may also include sequences that facilitate, preferably promote, homologous recombination in the host cell. In some embodiments, the expression construct may also include sequences necessary for replication in the host cell.

[0173] Exemplary expression control sequences include promoter / enhancer sequences for expression in mammalian cells, such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29: 2494-2502, 1991; Boshart et al., Cell, 41: 521-530, 1985); the Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4: 151, 1993); the Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); the simian virus 40 promoter; DRA (downregulated in adenoma; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293: G923-G934, 2007); MCT1 (monocarboxylic acid transporter 1; Cuff et al., Am. J. Physiol. Gastrointet. Liver Physiol., G977-G979, 2005); and Math1 (mouse atonal homolog 1; Shroyer et al., Gastroenterology, 132: 2477-2478, 2007). The promoter is operatively linked upstream (e.g., 5') of the polypeptide coding sequence. In another variant, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide may also optionally include an appropriate polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0174] If desired, the one or more polynucleotides may also optionally include a nucleotide sequence encoding a secretion signal peptide fused in-frame with the polypeptide sequence. The secretion signal peptide directs the secretion of the antibody polypeptide by the cell expressing the one or more polynucleotides and is cleaved from the secreted polypeptide by the cell. The one or more polynucleotides may also optionally further include sequences that are intended to function only to facilitate the large-scale production of the vector. The production and administration of polynucleotides for gene therapy can be carried out using procedures described in the literature for various transgenes. See, for example, Isner et al., Circulation, 91: 2687-2692, 1995; and Isner et al., Human Gene Therapy, 7: 989-1011, 1996.

[0175] In some embodiments, the polynucleotide may further include additional sequences to facilitate uptake and expression of the antibody or fragment thereof (and / or any other peptide) by the host cell. In some embodiments, a "naked" transgene encoding an antibody or fragment thereof described herein (e.g., a transgene without a virus, liposome, or other vector to facilitate transfection) is used.

[0176] Using any suitable vector, one or more polynucleotides encoding an antibody or a fragment thereof can be introduced into a host. The exemplary vectors listed are not limited to those listed above, but include replication-deficient retroviral vectors, including lentiviral vectors (Kim et al., J. Virol., 72(1): 811-816, 1998; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46); parvovirus vectors, such as adeno-associated virus (AAV) vectors (US Patent Nos. 5,474,935; 5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390; 5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnatenko et al., J. Invest.). Med., 45: 87-98, 1997); Adenovirus (AV) vector (US Patent Nos. 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584, 1992; Stratford Perricaudet et al., J. Clin. Invest., 90: 626-630, 1992; and Rosenfeld et al., Cell, 68: 143-155, Examples include adenovirus adeno-associated virus chimeras (U.S. Patent No. 5,856,152) or vaccinia virus or herpesvirus vectors (U.S. Patents No. 5,879,934; No. 5,849,571; No. 5,830,727; No. 5,661,033; No. 5,328,688); lipofectin-mediated gene transfer (BRL); liposomal vectors (U.S. Patent No. 5,631,237); and combinations thereof.These expression vectors can all be prepared using standard recombinant DNA techniques described, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994). If necessary, replication deficiency can be conferred to the viral vector, for example, by deleting or disrupting select genes necessary for viral replication.

[0177] Other intended nonviral delivery mechanisms include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52: 456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10: 689-695, 1990), DEAE-dextran (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81: 7161-7165, 1984), and direct microinjection (Harland and Weintraub, J. Cell Biol., 101: 1094-1099, 1985, DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, 1979; Felgner, Sci Am., 276(6): 102-6, 1997; Felgner, Hum Gene Ther., 7(15): 1791-3, 1996), cell sonication (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84: 8463-8467, 1987), gene guns using high-speed microparticle guns (Yang et al., Examples include Proc. Natl. Acad. Sci USA, 87: 9568-9572, 1990, and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262: 4429-4432, 1987; Wu and Wu, Biochemistry, 27: 887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev., 12: 159-167, 1993).

[0178] Expression vectors (or antibodies or fragments thereof as discussed herein) can be encapsulated within liposomes. See, for example, Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al., Science, 275(5301): 810-814, 1997). Various commercial methods involving “lipofection” techniques are also intended. In some embodiments, liposomes can be complexed with Sendai virus (hemagglutinating virus) (HVJ). This has been shown to facilitate fusion with the cell membrane and promote the entry of liposome-encapsulated DNA into cells (Kaneda et al., Science, 243: 375-378, 1989). In some embodiments, liposomes are complexed with or used in combination with nuclear nonhistone chromosomal protein (HMG-1) (Kato et al., J. Biol. Chem., 266: 3361-3364, 1991). In some embodiments, liposomes are complexed with or used in combination with both HVJ and HMG-1. Such expression constructs have been successfully used for nucleic acid transfer and expression in vitro and in vivo. In some embodiments, PD-L1 binding agonists (e.g., antibodies), including human PD-L1 binding agonists, are incorporated into liposomes to target cells expressing PD-L1 on their surface (e.g., tumor cells).

[0179] Cells may contain one or more polynucleotides or one or more vectors. For example, cells may be transformed or transfected with one or more polynucleotides encoding a PD-L1 binding agonist (e.g., an antibody) including a human PD-L1 binding agonist, or with one or more vectors containing one or more polynucleotides. In some embodiments, cells express a PD-L1 binding agonist (e.g., an antibody) including a human PD-L1 binding agonist containing one or more CDRs including six having at least 75% identity with the P22 CDR (see, for example, Table 1). In some embodiments, cells express a V containing a CDR identical to the P24 CDR (see, for example, Table 2). H and V L The cells express PD-L1 binding agonists (e.g., antibodies) including human PD-L1 binding agonists containing the same CDR as P31.2 (see, for example, Table 3). H and V LThe cells express PD-L1-binding agonists (e.g., antibodies), including human PD-L1-binding agonists containing [specific ingredient]. The cells may be prokaryotic cells such as Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol., 178: 497-515, 1989), or eukaryotic cells such as animal cells (e.g., myeloma cells, Chinese hamster ovary (CHO) cells, or hybridoma cells), yeast (e.g., Saccharomyces cerevisiae), or plant cells (e.g., tobacco, maize, soybean, or rice cells). The use of mammalian host cells can provide post-translational modifications (e.g., glycosylation, truncation, lipid addition, and phosphorylation) that may be desirable for conferring optimal biological activity in the recombinant expression product. Similarly, polypeptides (including human PD-L1 binding agonists, such as antibodies) may be glycosylated or unglycosylated and / or covalently modified to contain one or more water-soluble polymer bonds, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0180] Methods for introducing DNA or RNA into host cells are well known, including transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Such host cells are useful for amplifying polynucleotides and for expressing polypeptides encoded by polynucleotides. In this regard, the process for producing PD-L1-binding agonists (e.g., antibodies) may involve culturing host cells and isolating the PD-L1-binding agonists. The transfer of naked DNA expression constructs into cells can be achieved using particle bombardment, which relies on the ability to accelerate DNA-coated particle guns to high velocities, allowing them to penetrate the cell membrane and enter the cell without killing the cell (Klein et al., Nature, 327: 70-73, 1987). Several devices for accelerating small particles have been developed. One such device relies on high-voltage discharge to generate an electric current, which in turn provides the driving force (Yang et al., Proc. Natl. Acad. Sci USA, 87: 9568-9572, 1990). The particulate gun used consisted of a biologically inert material such as tungsten or gold beads. Host cells can be isolated and / or purified. Host cells may also be cells transformed in vivo to induce transient or permanent expression of polypeptides in vivo. Host cells may also be isolated cells transformed ex vivo and then introduced after transformation, for example, to produce polypeptides in vivo for therapeutic purposes. Transgenic humans are explicitly excluded from the definition of host cells.

[0181] Various methods for producing antibodies from polynucleotides are generally well known. For example, basic molecular biology procedures are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). Furthermore, numerous publications describe techniques suitable for DNA manipulation, creation of expression vectors, and antibody preparation by transformation and culture of appropriate cells (see, for example, Mountain and Adair, Chapter 1 in Biotechnology and Genetic Engineering Reviews, Tombs ed., Intercept, Andover, UK, 1992; and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0182] PD-L1 binding agonists, including human PD-L1 binding agonists (e.g., antibodies), are prepared using any suitable method, for example, by isolating them from immunized animals, by recombinant or synthetic production, or by genetic engineering. Antibody fragments derived from antibodies are obtained, for example, by proteolytic hydrolysis of the antibody. For example, digestion of the whole antibody with papain or pepsin yields a 5S fragment designated F(ab')2 or two monovalent Fab and Fc fragments, respectively. F(ab')2 can be further cleaved using a thiol reducing agent to produce a 3.5S Fab monovalent fragment. Methods for generating antibody fragments are described, for example, in Edelman et al., Methods in Enzymology, 1: 422 Academic Press (1967); Nisonoff et al., Arch. Biochem. Biophys., 89: 230-244, 1960; Porter, Biochem. J., 73: 119-127, 1959; U.S. Patent No. 4,331,647; and Andrews, SM and Titus, JA in Current Protocols in Immunology (Coligan et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1, 2.8.10 and 2.10A.1, 2.10A.5.

[0183] PD-L1 binding agonists, including human PD-L1 binding agonists (e.g., antibodies), can be genetically engineered. For example, PD-L1 binding agonists, including human PD-L1 binding agonists (e.g., antibodies), include variable region domains generated, for example, by recombinant DNA manipulation techniques. In this regard, including the above, the variable region is modified as necessary by insertions, deletions, or changes in the amino acid sequence of the antibody in order to produce the desired antibody. The polynucleotide encoding the desired complementarity-determining region (CDR) is prepared, for example, by synthesizing the variable region using polymerase chain reaction with mRNA from antibody-producing cells as a template (see, for example, Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995); and Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106-110, 1991). Current antibody manipulation techniques make it possible to construct an manipulated variable region domain containing at least one CDR and optionally one or more framework amino acids from a first antibody, as well as the remaining variable region domain from a second antibody. Such techniques are used, for example, to humanize antibodies or to improve the affinity of antibodies to binding targets.

[0184] A "humanized antibody" is an antibody in which the heavy-chain and light-chain variable CDRs of a non-human immunoglobulin are transferred into the human variable domain. A constant region is not required, but if present, it is, if necessary, substantially identical to the constant region of human immunoglobulin, for example, in some embodiments, at least about 85-90%, about 95%, 96%, 97%, 98%, 99%, or more identical. Thus, in some cases, perhaps all parts of the humanized immunoglobulin other than the CDR are substantially identical to the corresponding parts of the native human immunoglobulin sequence. For example, a humanized antibody is a human immunoglobulin (e.g., a host antibody) in which the hypervariable region residues of the host antibody are replaced with hypervariable region residues derived from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capability.

[0185] In some embodiments, the PD-L1 binding agonists described herein (e.g., antibodies) are useful in compositions and in methods for treating, preventing, or mitigating T-cell dysfunction diseases, disorders, or conditions, including one or more symptoms of a disease, disorder, or condition. T-cell dysfunction diseases, disorders, and conditions include, but are not limited to, tumor immunology and related cancers, including any cancer in which tumor cells overexpress PD-L1. Such PD-L1 overexpressing tumor cells can help the tumor cells escape immune surveillance and elimination. In addition, the PD-L1 binding agonists described herein, such as PD-L1 binding antibodies (e.g., multispecific antibodies including monospecific or bispecific antibodies), are useful for inhibiting PD-1 signaling and / or enhancing T-cell function (e.g., secreting cytokines, proliferating, performing cytolytic activity), and therefore for enhancing immune surveillance and elimination of tumor cells.

[0186] In some embodiments, a method for treating tumor immunity in a subject is described herein, comprising the step of administering to the subject a pharmaceutical composition comprising a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a binding agonist (e.g., an antibody) described herein.

[0187] In some embodiments, a method for treating cancer or tumors in a subject is described herein, comprising the step of administering to the subject a pharmaceutical composition comprising a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a binding agonist (e.g., an antibody) described herein.

[0188] In some embodiments, the present invention describes a method for alleviating one or more symptoms associated with cancer or a tumor in a subject, comprising the step of administering to the subject a pharmaceutical composition comprising a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a binding agonist (e.g., an antibody) as described herein.

[0189] In some embodiments, a method for reducing the size of a tumor in a subject having a tumor is described herein, comprising the step of administering to the subject a pharmaceutical composition comprising a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a binding agonist (e.g., an antibody) described herein.

[0190] In some embodiments, a method for treating a T-cell dysfunction disorder, disorder, or condition in a subject is described herein, comprising the step of administering to the subject a pharmaceutical composition comprising a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a binding agonist (e.g., an antibody) as described herein. In some embodiments, the T-cell dysfunction disorder, disorder, or condition is tumor immunity.

[0191] In some embodiments, a method for enhancing tumor cell removal in a subject having a tumor is described herein, comprising the step of administering to the subject a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a pharmaceutical composition comprising a binding agonist (e.g., an antibody) as described herein.

[0192] In some embodiments, a method for enhancing T cell function in a subject is described herein, comprising the step of administering to the subject a pharmaceutical composition comprising a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a binding agonist (e.g., an antibody) described herein. In some embodiments, the T cell function is cytokine secretion. In some embodiments, the T cell function is tumor cell elimination. In some embodiments, the subject has been diagnosed with cancer or a tumor.

[0193] For subjects of the above method, one or more therapeutic agents described herein can be administered in combination with a PD-L1 binding agonist (e.g., an antibody) or a fragment thereof, or a pharmaceutical composition containing a binding agonist (e.g., an antibody) described herein.

[0194] In some embodiments, the antibody is a human antibody, including, but is not limited to, an antibody having a variable region in which both the framework and CDR region are derived from a human germline immunoglobulin sequence, as described, for example, Kabat et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. If the antibody contains a constant region, it is preferable that the constant region is also derived from a human germline immunoglobulin sequence. The human antibody may contain, for example, amino acid residues not encoded by a human germline immunoglobulin sequence to enhance antibody activity, but it does not contain CDRs derived from other species (e.g., mouse CDRs located within a human variable framework region).

[0195] In some embodiments, PD-L1-binding agonists (e.g., antibodies) increase T cell function in cell cultures and / or enhance the cytolytic activity of cells. Such cell cultures may contain tumor cells that express or overexpress PD-L1. Tumor cells include, but are not limited to, breast cancer cells, bladder cancer cells, melanoma cells, prostate cancer cells, mesothelioma cells, lung cancer cells, testicular cancer cells, thyroid cancer cells, squamous cell carcinoma cells, glioblastoma cells, neuroblastoma cells, uterine cancer cells, colorectal cancer cells, and pancreatic cancer cells.

[0196] In some embodiments, methods for enhancing the removal of tumor cells in a subject are described herein. For example, the method includes administering a PD-L1 binding agent (e.g., an antibody), such as a human PD-L1 binding agent described herein, in an amount effective to enhance the removal of tumor cells. In some embodiments, the method competes for binding to human PD-L1 with antibody P22, antibody P24, and / or antibody P31.2 (see, e.g., the CDRs and VH / VL of Tables 1, 2, and / or 3) and / or binds to the region of PD-L1 recognized by antibody P22, antibody P24, and / or antibody P31.2 (see, e.g., the CDRs and VH / VL of Tables 1, 2, and / or 3), and includes administering a PD-L1 binding agent (e.g., an antibody), including a PD-L1 binding agent that binds to the region of PD-L1, resulting in enhanced removal of tumor cells. In some embodiments, one or more of the above binding agents (e.g., antibodies), polynucleotides, vectors, and / or cells can be used in methods for enhancing the removal of tumor cells in vivo (e.g., methods for treating cancer in a subject).

[0197] Methods for modulating (e.g., inhibiting, reducing, or preventing) tumor growth in a subject are also provided. For example, a method may include administering to a subject a composition comprising a PD-L1-binding agonist (e.g., an antibody) in an amount effective to modulate tumor growth in the subject. "Tumor" refers to the growth or proliferation of any new cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" typically refer to or describe physiological conditions in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, breast cancer, colon cancer, kidney cancer, lung cancer, squamous cell myeloid leukemia, hemangioma, melanoma, astrocytoma, and glioblastoma, as well as, but are not limited to, other cell proliferative disorders including: heart: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma; lung: bronchogenic lung cancer (squamous cell, anaplastic small cell, anaplastic large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, inesothelioma; gastrointestinal tract: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (tubular adenocarcinoma, insoleosarcoma) Rinoma (insulinorma, glucagonoma, gastrinoma, carcinoid tumor, VIP-producing tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma, leiomyoma); urogenital system: kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia, renal cell carcinoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma of the prostate, sarcoma, small cell carcinoma), testes (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma); liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;Bone: Osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticuloma), malignant giant cell tumor, chordoma, osteochondrodyscoplasma, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor; Nervous system: Skull (osteoma, hemangioma, granuloma, xanthomas, osteoosteitis), Meninges (meningioma, meningiosarcoma, gliomas) ), Brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal glandoma), glioblastoma multiforme, oligobranch glioma, Schwann cell tumor, retinoblastoma, congenital tumor, spinal nerve fibroma, meningioma, glioma, sarcoma); Gynecology: Uterus (endometrial cancer), cervix (cervical cancer, pretumescent cervical dysplasia), ovaries (ovarian cancer) serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified cancer, granulosa cell tumor, Sertoli-Leydig cell tumor, undifferentiated embryonic cell tumor Cystoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, staphyloid sarcoma (embryonic rhabdomyosarcoma), fallopian tube (carcinoma); blood: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin lymphoma (malignant lymphoma); skin: malignant melanoma, basal cell carcinoma, Examples include squamous cell carcinoma, Kaposi's sarcoma, lentigo dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal neuroblastoma, as well as thyroid cancers, including medullary thyroid carcinoma. Methods of treating cancer are also provided, involving the administration of PD-L1 binding agonists (e.g., antibodies), such as human PD-L1 binding agonists, alone or in combination with other agonists, to the target requiring them.

[0198] "Enhancing" tumor cell removal does not require a 100% enhancement of removal; any enhancement of the removal rate is intended. Similarly, "modulating" tumor growth refers to reducing tumor size, slowing tumor growth, or inhibiting the growth of an existing tumor. Complete elimination of the tumor is not required; any reduction in tumor size or slowing of tumor growth constitutes a beneficial biological effect in the subject. In this regard, tumor cell removal may be enhanced by at least about 5%, at least about 10%, or at least about 20%, compared to the level of removal observed in the absence of the method (e.g., in a biologically balanced control subject or specimen not exposed to the agonist of the method). The effect is detected, for example, by reduction in tumor size, decrease or maintenance of tumor marker levels, or decrease or maintenance of the tumor cell population. In some embodiments, tumor cell removal is enhanced by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%) compared to tumor cell removal in the absence of a PD-L1 binding agonist (e.g., antibody) of the method.

[0199] Furthermore, PD-L1-binding agonists (e.g., antibodies) can be used to mitigate or reduce cancer-related side effects, such as bone deterioration, spinal depression, and paralysis. In one embodiment, a subject has bone metastases or is at risk of developing them, and a PD-L1-binding agonist (e.g., antibody) is administered in a dose that reduces the deterioration of the surrounding bone. Thus, in some embodiments, the PD-L1-binding agonist prevents bone deterioration due to bone metastases, where tumor cell proliferation is reduced or not reduced. In some embodiments, the PD-L1-binding agonist (e.g., antibody) performs both the prevention of bone deterioration due to bone metastases and the reduction of tumor cell proliferation. Generally, the effect on tumor cell proliferation (e.g., inhibition of proliferation or no effect on proliferation) depends on the specific metastatic microenvironment. For example, the proliferation of metastases located in a microenvironment with a substantial amount of type 1 collagen may be inhibited. In contrast, while the proliferation of metastases located in a microenvironment lacking a substantial amount of type 1 collagen may not be inhibited, bone deterioration near the metastases is nevertheless reduced or prevented.

[0200] A specific administration regimen of a PD-L1-binding agonist (e.g., an antibody) for a particular subject depends, in part, on the agonist used, the amount of the agonist administered, the route of administration, and the cause and severity of any side effects. The amount of the agonist (e.g., an antibody) administered to the subject (e.g., a mammal such as a human) should be sufficient to produce the desired response over a reasonable time frame. Accordingly, in some embodiments, the amount of the PD-L1-binding agonist (e.g., an antibody) or pharmaceutical composition described herein administered to the subject is an effective dose. In some embodiments, the amount of the PD-L1-binding agonist (e.g., an antibody) or pharmaceutical composition described herein administered to the subject is a therapeutically effective dose. In some embodiments, the method includes a step of administering, for example, an amount from about 0.1 μg / kg to about 100 mg / kg or more. In some embodiments, the dosage ranges from approximately 1 μg / kg to approximately 100 mg / kg; or from approximately 5 μg / kg to approximately 100 mg / kg; or from approximately 10 μg / kg to approximately 100 mg / kg; or from approximately 1 mg / kg to approximately 50 mg / kg; or from approximately 2 mg / kg to approximately 30 mg / kg; or from approximately 3 mg / kg to approximately 25 mg / kg; or from approximately 3 mg / kg to approximately 25 mg / kg; or from approximately 5 mg / kg to approximately 10 mg / kg; or from approximately 10 mg / kg to approximately 20 mg / kg; or from approximately 10 mg / kg to approximately 30 mg / kg. Some conditions or disease states require continuous treatment, which may or may not require the administration of doses of PD-L1 binding agonists (e.g., antibodies), including human PD-L1 binding agonists (e.g., antibodies), over multiple treatment periods (e.g., 3 days, 7 days, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 2 years, or longer), daily, 3 times a week, once a week, once every two weeks, and once a month.

[0201] Suitable routes of administration for compositions containing PD-L1-binding agonists (e.g., antibodies), such as human PD-L1-binding agonists (e.g., antibodies), are well known in the art. While agonists (e.g., antibodies) can be administered using more than one route, certain routes may provide a more rapid and effective response than others. Depending on the situation, compositions containing PD-L1-binding agonists (e.g., antibodies), such as human PD-L1-binding agonists, may be applied or administered by drop, absorbed through the skin or mucous membranes, taken orally, inhaled, and / or introduced into circulation. For example, it may be desirable to deliver a composition containing a PD-L1 binding agonist (e.g., an antibody), such as a human PD-L1 binding agonist, by injection via intravenous, subcutaneous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intrafocal, intramedullary, subarachnoid, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, enteral, local, sublingual, urethral, ​​vaginal, or rectal means, by a sustained-release system, or by an implantable device. If desired, the PD-L1 binding agonist (e.g., an antibody), such as a human PD-L1 binding agonist, may be locally administered via intraarterial or intravenous administration to deliver to the area of ​​interest, for example, via the hepatic artery for delivery to the liver. Alternatively, a PD-L1 binding agonist (e.g., an antibody), such as a human PD-L1 binding agonist, is administered locally via an implant made of a membrane, sponge, or other suitable material that has absorbed or encapsulated the binding agonist. When using an implantable device, in one embodiment, the device is implanted in any suitable tissue or organ, and the delivery of the PD-L1 binding agonist (e.g., an antibody), such as a human PD-L1 binding agonist, is made, for example, by diffusion, a time-acting bolus, or continuous administration. In other embodiments, the PD-L1 binding agonist (e.g., an antibody) is administered directly to exposed tissue during tumor resection or other surgical procedures.

[0202] This disclosure provides compositions such as pharmaceutical compositions comprising a PD-L1 binding agonist (e.g., an antibody), such as a human PD-L1 binding agonist, and a carrier (e.g., a pharmaceutically acceptable carrier). The specific carrier used may depend on chemistry-physical considerations such as solubility and lack of reactivity with the binding agonist or combination therapy, as well as the route of administration. Pharmaceutically acceptable carriers are well known in the art, and examples thereof are described herein. Exemplary pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. Injectable formulations are further described, for example, in Pharmaceuticals and Pharmacy Practice, JB Lippincott Co., Philadelphia. Pa., Banker and Chalmers. eds., pages 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986). A pharmaceutical composition containing a PD-L1 binding agonist (e.g., an antibody), such as a human PD-L1 binding agonist, is placed in a container, in one embodiment, together with packaging material that provides instructions for the use of such a pharmaceutical composition. Generally, such instructions include tangible representations that, along with the reagent concentration, in some embodiments, indicate the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be necessary to reconstitute the pharmaceutical composition.

[0203] In some embodiments, the methods described herein further include the step of administering one or more additional agonists, including a therapeutic agent, which may be present in a composition, administered together with a PD-L1 binding agonist (e.g., an antibody), such as a human PD-L1 binding agonist, or provided in separate compositions using the same or different routes of administration. One or more additional agonists, including a therapeutic agent, may be administered together with a PD-L1 binding agonist (e.g., an antibody) (e.g., in combination therapy), or separately (e.g., simultaneously or sequentially). Such additional therapeutic agents include, but are not limited to, therapeutic antibodies, immunotherapies and immunotherapeutic agents, cytotoxic agonists, chemotherapeutic agents, and inhibitors.

[0204] Therapeutic antibodies that can be used with the PD-L1 binding agonists (e.g., antibodies) described herein (e.g., for combination therapy) include, but are not limited to, trastuzumab; absiximab; daclizumab; BEC2; IMC-C22; vitaxin; Campath 1H / LDP-03; Smart M195; epratuzumab; bectumomab; vizilizumab; CM3, humanized anti-ICAM3 antibody; IDEC-1 14; ibritumomab tiuxetan; IDEC-131; IDEC-151; IDEC-152; SMART anti-CD3; eculizumab; adalimumab; certolizumab; IDEC-1 51; MDX-CD4; CD20-streptavidin; CDP571; LDP-02; OrthoClone Examples include OKT4A; ruplizumab; natalizumab; and lerdelimumab.

[0205] Immunotherapy and immunotherapeutic agents that can be used in conjunction with the PD-L1 binding agonists (e.g., antibodies) described herein (e.g., for combination therapy) are not limited to, but include, cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-l-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factor (including TNF-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma); aluminum hydroxide (Alam); Bacille Calmette-Guerin (BCG); keyhole limpet hemocyanin (KLH); and incomplete Freund's adjuvant (IFN). A); QS-21; DETOX; Levamisol; and dinitrophenyl (DNP), as well as combinations thereof, such as interleukins, e.g., IL-2, and other cytokines, e.g., IFN-alpha. In some embodiments, immunotherapies include immunotherapeutic agents that modulate the immune response, such as checkpoint inhibitors or checkpoint agonists. In some embodiments, the immunotherapeutic agent is an antibody modulator targeting PD-1, PD-L1, PD-L2, CEACAM (e.g., CEACAM-1, -3, and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF-beta, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, and / or BTNL2, among those known in the art. In some embodiments, the immunotherapeutic agent is an agent that increases natural killer (NK) cell activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits the suppression of the immune response. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppressor cells or suppressor cell activity.In some embodiments, the immunotherapy agent is an agonist or treatment that inhibits Treg activity. In some embodiments, the immunotherapy agent is an agonist that inhibits the activity of inhibitory immune checkpoint receptors.

[0206] In some embodiments, the immunotherapy agent includes a T cell modulator selected from a costimulatory molecule agonist or activator. In one embodiment, the costimulatory molecule agonist is selected from GITR, OX40, ICOS, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM-I, LFA-I (CD1 I / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, NKG2C, NKp80, CD160, B7-H3, or CD83 ligand agonists (e.g., agonist antibodies or their antigen-binding fragments, or soluble fusions). In other embodiments, the effector cell combination may include a bispecific T cell-inducing antibody (e.g., a bispecific antibody molecule that binds to CD3 and tumor antigens (e.g., EGFR, PSCA, PSMA, EpCAM, HER2, among others)).

[0207] Cytotoxic agents that can be used in conjunction with the PD-L1-binding agonists described herein (e.g., antibodies) (for example, for combination therapy) include substances that inhibit or prevent cellular function and / or cause cell death or destruction. Examples of cytotoxic agents, but not limited to, include radioisotopes (e.g., At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu radioisotopes); growth inhibitors; enzymes such as nucleases and their fragments; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants. Other exemplary cytotoxic agents can be selected from microtubule inhibitors, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, apoptosis promoters, LDH-A inhibitors; fatty acid biosynthesis inhibitors; cell cycle signaling inhibitors; HDAC inhibitors; proteasome inhibitors; and cancer metabolism inhibitors.

[0208] Examples of chemotherapeutic agents that can be used in conjunction with the PD-L1 binding agonists (e.g., antibodies) described herein (e.g., for combination therapy) include chemical compounds useful for treating cancer. Examples of chemotherapeutic agents, but not limited to, include erlotinib, bortezomib, disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, l7-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant, sunitinib, letrozole, imatinib mesylate, fmasunate, oxaliplatin, 5-FET (5-fluorouracil), leucovorin, rapamycin, lapatinib, ronafamib (SCH66336), sorafenib, and Bayer. Labs, gefitinib, AG1478; alkylating agents such as thiotepa and CYTOXAN®; cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and pigosulfan; aziridines such as benzodopa, carbocone, metsuredopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomellamine; acetogenins (especially bratacin and bratacinone); camptothecin (including topotecan and irinotecan); bryostatin; callistatin (cally statin); CC-1065 (including its synthetic analogues adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5-alpha-reductase including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat, dorastatin; aldesleukin, talc duocalmycin (including synthetic analogues, KW-2189, and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongistatin;Nitrogen mustards such as chlorambucil, chromafazin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembicin, fenestrine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; engine antibiotics (e.g., calitiamycin, especially calitiamycin gamma II and calitiamycin omega I) (Angew Chem. Inti. Ed. Engl. 1994 33: Antibiotics such as 183-186); dinemicin including dinemicin A; bisphosphonates such as clodronate; esperamicin, as well as neocardinostatin chromophores and related pigment proteins (endiin antibiotic chromophores), acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carabicin, kaminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin Mitomycins such as cin, idarubicin, marcelomycin, and mitomycin C; mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine;Androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folinic acid Folic acid supplements such as acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecoltin; diazicone; erhomitin; eruptinium acetate; epotilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; ronidynin; metansinoids such as mytansin and anthamitosin; mitogluzone; mitoxantrone; mopidamnol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Ore.); Lazoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic Acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecenes, especially T-2 toxin, Beraclin A, Loridine A and Anguidin; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside "Ara-C"; Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel, ABRAXANE® (Cremofol-free), Albumin-Modified Nanoparticle Formulations of Paclitaxel (American Pharmaceutical) Partners, Schaumberg, Ill.), and docetaxel / doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine; ibandronate; CPT-1; topoisomerase inhibitor RFS2000;Examples of chemotherapeutic agents include (i) antihormone agents that act to modulate or inhibit the hormonal effects on tumors, e.g., anti-estrogen agents and selective estrogen receptor modulators (SERMs), e.g., tamoxifen (including tamoxifen citrate), raloxifen, droloxifen, iodoxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene citrate; and (ii) estrogen in the adrenal gland. (iii) Aromatase inhibitors that inhibit the enzyme aromatase which regulates production, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestan, fadrozol, borozole, letrozole, and anastrozole; (iii) Antiandrogenic drugs, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, p (iv) Protein kinase inhibitors, (v) Lipid kinase inhibitors, (vi) Antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, e.g., PKC-alpha, Ralf, and H-Ras, (vii) Ribozymes, e.g., VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors, (viii) Vaccines, e.g., gene therapy vaccines, e.g., ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; Topoisomerase 1 inhibitors, e.g., LEIRTOTECAN®; ABARELIX®, and (ix) pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0209] As mentioned above, chemotherapy agents include alemtuzumab, bevacizumab, cetuximab, panitumumab, rituximab, pertuzumab, tocitumomab, and antibodies, including antibody-drug conjugates and gemtuzumab ozogamicin. Additional humanized monoclonal antibodies having therapeutic potential as agonists in combination with the PD-L1-binding agonists (e.g., antibodies) described herein include apolizumab, aselizumab, atlizumab, bapineozumab, vibatuzumab meltansine, cantuzumab meltansine, sedelizumab, certolizumab pegol, sidofcituzumab, sidotuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erulizumab, felbizumab, fontrizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, and rabetsi. Zumab, lintuzumab, matsuzumab, mepolizumab, motabizumab, motobizumab, natalizumab, nimotuzumab, nivolumab, norovizumab, numabizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecufcituzumab, pectuzumab, paxerizumab, larivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, loberizumab, luprizumab, cibrotuzumab, ciprizumab, sontuzumab, tacuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, tralizumab, tucotzuzumab Examples include cermoleukin, tucituzumab, umabizumab, urtoxazumab, ustekinumab, bicilizumab, and anti-interleukin-l2 (ABT-8744695, Wyeth Research and Abbott Laboratories), a recombinant, exclusively human full-length IgG1λ antibody genetically modified to recognize the interleukin-12p40 protein.Chemotherapy agents include dexamethasone, interferon, colchicine, methoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostin, arsenic trioxide, asparaginase, live BCG, becuzimab, besarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, and interferon alfa. -2b, lenalidomide, levamisol, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelbequin, parifermin, pamidronate, pegademase, pegasparagase, pegfilgrastim, pemetrexed sodium, plicamycin, porfimer sodium, quinacrine, rasburicase, salglamostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, barrubicin, zoledronate, and zoledronic acid, as well as pharmaceutically acceptable salts thereof.

[0210] Chemotherapy agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, thixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone l7-butyrate, hydrocortisone 17-valerate, alclomethasone propionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone l7-butyrate, and clobetasol l 7-propionic acid ester, fluocortron caproate ester, fluocortron pivalate ester, and flupredniden acetate ester; immunoselective anti-inflammatory peptides (ImSAIDs), e.g., phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN BioTherapeutics, LLC; Antirheumatic drugs, e.g., azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salt, hydroxychloroquine, leflunomidominocycline, sulfasalazine; Tumor necrosis factor alpha (TNF-alpha) blockers, e.g., etanercept, infliximab, adalimumab, certolizumab pegol, golimumab (Simponi), etc.; Interleukin-1 (IL-I) blockers such as anakinra; T-cell costimulatory blockers such as abatacept; Interleukin-6 (IL-6) blockers such as tocilizumab; Interleukin-13 (IL-I3) blockers, e.g., lebrikizumab; Interferon-alpha (IFN) blockers, e.g., lontalizumab; Beta-7 integrin blockers, e.g., rhuMAb Beta7; IgE pathway blockers, e.g., Anti-Ml Prime, etc.; secretory homotrimer LTa3 and membrane-bound heterotrimer LTa / l32 blockers, e.g., antilymphotoxin alfa (LTa); various investigational drugs, e.g., thioplatin, PS-341, phenylbutyrate, ET-I8-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832);Polyphenols, e.g., quercetin, resveratrol, picetanol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and their derivatives; autophagy inhibitors, e.g., chloroquine; delta-9-tetrahydrocannabinol (dronabinol), etc.; beta-lapacon; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopoletin, and 9-aminocamptothecin; podophyllotoxin; tegafur; bexarotene; bisphosphonates, e.g., clodronate, etidronate, NE-58095, zoledronate Electrolyte / zoledronate, alendronate, pamidronate, tildronate, or risedronate, etc.; and epidermal growth factor receptor (EGF-R); vaccines, e.g., THERATOPE® vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifamib (R11577); olafenib, ABT510; Bcl-2 inhibitors, e.g., oblimersen sodium pixantrone, etc.; farnesyltransferase inhibitors, e.g., ronafamib (SCH Other examples include 6636), etc.; as well as any pharmaceutically acceptable salts, acids, or derivatives of the above, and combinations of two or more of the above, for example, CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is an abbreviation for a treatment regimen using oxaliplatin in combination with 5-FU and leucovorin. Other chemotherapeutic agents include poly-ADP-ribose polymerase (PARP) inhibitors: olaparib, rucaprib, niraparib, and tarzoparib.

[0211] Inhibitors that can be used with the PD-L1 binding agonists (e.g., antibodies) described herein (e.g., for combination therapy) are not limited to, but include, imatinib, baricitinib, gefitinib, erlotinib, sorafenib, dasatinib, sunitinib, lapatinib, nilotinib, pirfenidone, pazopanib, crizotinib, vemurafenib, vandetanib, luxolitinib, axitinib, bosutinib, regorafenib, tofacitinib, cabozantinib, ponatinib, trametinib, dabrafenib, afatinib, ibrutinib, ceritinib, idelalisib, nintedanib, palbociclib, lenvatinib, and ko Examples of kinase inhibitors include bimetinib, abemaciclib, acalabrutinib, alectinib, binimetinib, brigatinib, encorafenib, erdafitinib, everolimus, hostamatinib, giltel, lalotrectinib, lorlatinib, netaludil, osimertinib, pemigatinib, pexidartinib, ribociclib, temsirolimus, XL-092, XL-147, XL-765, XL-499, and XL-880. In some embodiments, the compounds described herein may be used in combination with HSP90 inhibitors (e.g., XL888), liver X receptor (LXR) modulators, retinoid-related orphan receptor gamma (RORy) modulators, checkpoint inhibitors (e.g., CK1 inhibitors or CK1α inhibitors), Wnt pathway inhibitors (e.g., SST-215), or mineralocorticoid receptor inhibitors (e.g., esaxerenone) or XL-888 for treating diseases disclosed herein, such as cancer.In some embodiments, the PD-L1 binding agonists disclosed herein (e.g., antibodies) can be combined with one or more inhibitors of the following kinases for treating cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, 1NS-R, IGF-1R, IR-R, PDGFαR, PDGFβ / R, CSFIR, KIT, FLK-II, KDR / FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt2, Flt4, EphAl, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYR, FRK, JAK (JAK1 and / or JAK2), ABL, ALK, CDK7, CDK12, KRAS, and B-Raf.

[0212] For example, additional non-limiting examples of inhibitors that can be used with (for example, for combination therapy) the PD-L1 binding agonists (e.g., antibodies) described herein for the treatment of cancer include FGFR inhibitors (FGFR1, FGFR2, FGFR3, or FGFR4, e.g., pemigatinib), EGFR inhibitors (also known as ErB-1 or HER-1; e.g., erlotinib, gefitinib, vande), Tanib, osimertinib, cetuximab, nesitumumab, or panitumumab), VEGFR inhibitors or pathway blockers (e.g., bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, regorafenib, ponatinib, vandetanib, ramucirumab, lenvatinib, ziv-aflibercept), PARP inhibitors (e.g., olaparib, lucaparib, veliparib, or niraparib), J AK inhibitors (e.g., ruxolitinib, baricitinib, itacitinib), IDO inhibitors (e.g., epacadostat, NLG919, or BMS-986205, MK7162), LSD1 inhibitors, TDO inhibitors, PI3K-delta inhibitors (e.g., parsaclisib), PI3K-gamma inhibitors such as PI3K-gamma selective inhibitors, Pim inhibitors, CSF1R inhibitors, TAM This includes receptor tyrosine kinases (Tyro-3, Axl, and Mer), adenosine receptor antagonists (e.g., A2a / A2b receptor antagonists), HPK1 inhibitors, chemokine receptor inhibitors (e.g., CCR2 or CCR5 inhibitors), SHP1 / 2 phosphatase inhibitors, histone deacetylase inhibitors (HDACs) such as HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromo and extra-terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors), or combinations thereof.

[0213] In some embodiments, the PD-L1 binding agonists disclosed herein (e.g., antibodies) are replaced with PD-1 inhibitors or PD-L1 inhibitors, such as anti-PD-1 monoclonal antibodies or anti-PD-L1 monoclonal antibodies, such as nivolumab (Opdivo), pembrolizumab (Keytruda, MK-3475), atezolizumab, avelumab, semiprimab, and spartalizumab. Mab, camrelizumab, cetrerimab, tripalimab, cintilimab, AB122, JTX-4014, BGB-108, BCD-100, BAT1306, LZM009, AK105, HLX10, and TSR-042, AMP-224, AMP-514, PDR001, durvalumab, pizilizumab (Imfinzi®, CT-011), CK-301, BMS It can be used in combination with 936559, MPDL3280A, tislerizumab, BMS-935559, MEDI4736, FAZ053, KN035, CS1001, CBT-502, A167, STI-A101, BGB-A333, MSB-2311, HLX20, AUNP12, CA-170, BMS-986189, LY3300054, and MSB0010718C.

[0214] In some embodiments, the PD-L1-binding agonists (e.g., antibodies) disclosed herein can be used in combination with CTLA-4 inhibitors, such as anti-CTLA-4 antibodies, such as ipilimumab (Yervoy), tremelimumab, and AGEN1884; or in combination with phosphatidylserine inhibitors, such as bavituximab (PGN401); or in combination with antibodies against cytokines (IL-10, TGF-β, etc.); or in combination with bispecific antibodies that bind to PD-L1 and CTLA-4 (e.g., AK104) or bispecific antibodies that bind to PD-1 and CTLA-4; or in combination with other anticancer agents such as semiprimab.

[0215] Additional activators may be pharmaceutically acceptable salts, esters, amides, hydrates, and / or prodrugs of any of the above-mentioned therapeutic agents or other activators.

[0216] Modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the scope of the subject matter definitions described herein. Accordingly, the following examples are illustrative and not limiting to the present disclosure. [Examples]

[0217] (Example 1) antibody generation To obtain a binder against human PD-L1, antibody discovery was performed by phage display of a human Fab library using a standard protocol. Extracellular domains of human PD-L1 were purchased from Acro Biosystems (human PD-L1-His tag Acro Cat.No.PD1H5229, biotinylated human PD-L1-His Avitag Acro Cat.No.PDL-H82E4). EZ-Link NHS-PEG was performed using the standard protocol as appropriate. 12 - The non-biotinized extracellular domain of PD-L1 was biotinylated using biotin (ThermoScientific Cat. No. 21312). Phage clones were screened for their ability to bind to biotinylated human PD-L1 by phage ELISA using a standard protocol. Briefly, a Fab-formatted phage library was constructed using expression vectors (also called phagemids) that could be replicated and expressed in phages. Both the heavy and light chains were encoded in the same expression vector, and the heavy chain was fused with a truncated variant of the phage coat protein pIII. The light chain and heavy chain-pIII fusions were expressed as separate polypeptides and assembled in a bacterial periplasm, where disulfide bonds could be formed by redox potential to create the antigen-binding domain (Fab) of the candidate antibody.

[0218] Libraries were created using sequences derived from specific human heavy chain variable domains (VH3-23) and specific human light chain variable domains (Vk-1). The light chain variable domains in the diversified libraries to be screened were introduced into VL CDR3 (L3), while the light chains VL CDR1 (L1) and CDR2 (L2) were left as human germline sequences. For the libraries to be screened, all three CDRs of the VH domain were diversified to match the positional amino acid frequencies per length of CDRs found in the human antibody repertoire. The phage display heavy chain (SEQ ID NO: 92) and light chain (SEQ ID NO: 93) scaffolds used in the libraries are listed below, where lowercase "x" represents a CDR amino acid that was varied to create the library, and bold italics represent a constant CDR sequence.

[0219] The array at sequence number 92 is, [ka] The sequence of sequence number 93 is: [ka] That is the case.

[0220] As described in more detail in Kunkel, TA (PNAS January 1, 1985. 82(2) 488-492), which is incorporated herein by reference, diversity was created by introducing diversity into VL CDR3 and VH CDR1(H1), CDR2(H2), and CDR3(H3) using mutagenesis with degenerate DNA oligonucleotide primers to mimic the diversity found in the natural antibody repertoire. Briefly, single-stranded circular DNA incorporating uracil was prepared from isolated phages using standard procedures, and diversity was introduced into the four CDRs by Kunkel mutagenesis. The chemosynthesized DNA was then introduced into TG1 cells by electroporation and subsequently recovered. The recovered cells were subcultured and infected with the M13K07 helper phage to construct a phage library.

[0221] Phage panning was performed using a standard procedure. Briefly, the first round of phage panning was performed using targets immobilized on streptavidin magnetic beads, yielding approximately 1 × 10⁶ molecules from the prepared library in a volume of 1 mL in PBST-2% BSA. 12 The phages were subjected to the treatment. After 1 hour of incubation, the phages bound to the beads were separated from the supernatant using a magnetic stand. The beads were washed three times to remove nonspecifically bound phages, and then approximately 0.6 OD was added to ER2738 cells (5 mL). 600 The cells were added with 2xYT+ ampicillin and M13K07 helper phage (final concentration approximately 1x10⁻¹⁶). After incubation at room temperature for 20 minutes, the infected cells were treated with 2xYT+ ampicillin and M13K07 helper phage (final concentration approximately 1x10⁻¹⁶). 10The cells were subcultured in 25 mL of pfu / ml and grown overnight at 37°C with vigorous shaking. The following day, phages were prepared by PEG precipitation using standard procedure. Panning was performed after preliminary clearance of phages specific to SAV-coated beads. A second round of panning was performed using a KingFisher magnetic bead handler with PD-L1 antigen immobilized on 50 or 25 nM beads using standard procedure (50 nM PD-L1 in round 3, and 25 nM PD-L1 in round 4). A total of 3-4 rounds of phage panning were performed to enrich phages displaying Fab specific to the target antigen. Target-specific enrichment was confirmed using polyclonal ELISA, and further validation was performed by isolating individual clones and performing monoclonal phage ELISA. The CDRs of isolated Fab clones containing candidate antibodies were sequenced using DNA sequencing.

[0222] The genes encoding the heavy and light chain variable domains of the candidate antibody were separately cloned into mammalian expression vectors to express them as full-length IgG in mammalian cells.

[0223] For full-length IgG, the heavy chain constant region (e.g., CH1 = standard lettering; hinge = italicized lettering; CH2 = bold; and CH3 = underlined lettering) contained the following amino acid sequence: [ka]

[0224] For full-length IgG, the light chain constant region (e.g., CL) contained the following amino acid sequence: [ka]

[0225] IgG antibodies were purified from the culture supernatant using protein A resin. (Example 2) Screening and selection

[0226] Antibodies against PD-L1 were generated by phage display, for example, as described in Example 1. For example, to determine qualitative binding, specific interactions between the antigen and the candidate antibody obtained in Example 1 were confirmed using biolayer interferometry (BLI).

[0227] The divalent interaction of a binder to biotinylated human PD-L1 (see Example 1), immobilized on a streptavidin biosensor, was monitored using the Octet (Pall ForteBio) instrument. To monitor monovalent interactions, full-length IgG molecules were immobilized on an Fc capture biosensor, and their interaction with soluble antigens was monitored using the Octet instrument.

[0228] Simply put, Octet in monovalent conjugation format is used to accurately measure affinity for higher PD-L1 antibodies (K D Values ​​were obtained. First, antibodies were immobilized on an Fc capture sensor. In the next association step, the binding interaction of PD-L1 (Acro Cat.No.PD1H5229 (PD-L1-His tag)) to the immobilized IgG was measured. To obtain accurate kinetic constants, a dilution series (approximately 10-20 × K) with at least seven concentrations of analytes was performed. D From 0.1 × K D The values ​​(2-fold dilution) were measured in the association step. In the subsequent dissociation step, the sensor was immersed in a buffer solution that did not contain the analyte, thereby dissociating the bound analyte on the surface of the sensor. Using Octet kinetics analysis software, the kinetics and equilibrium binding constants were calculated from the rates of the association and dissociation curves. The analysis was performed using overall curve fitting, which allowed the kinetic constants to be simultaneously derived from the total analyte concentrations present in the experiment.

[0229] Table 4 shows the results of the bivalent qualitative Octet assay. For qualitative binding affinity using Octet, strong binding is indicated by "+++", moderate binding by "++", and weak binding by "+". "ND" indicates that antibody binding could not be determined. "NB" indicates that antibody binding was not detected. The results show that 24 antibodies exhibited strong binding affinity (P1, P2, P3, P4, P5, P9, P10, P18, P19, P21, P22, P24, P26, P31.2, P33, P34, P35, P36, P38, P39, P45, P47, P53, and P54), 12 antibodies exhibited moderate cell binding affinity (P6, P12, P17, P23, P28, P30, P37, P40, P41, P44, P46, and P49), and 9 antibodies exhibited weak cell binding affinity (P20, P25, P27, P29, P32, P43, P51, P52, and P55). Table 4. Bivalent qualitative binding affinity and mPD-L1 cross-reactivity by Octet. [Table 4-1] [Table 4-2] [Table 4-3]

[0230] Exemplary sensograms regarding monovalent antibody binding are shown in Figures 1A-1C. Qualitative PD-L1 binding and monovalent K D The results are shown in Table 5. K D The values ​​are listed, and there is a strong bond, <2 × 10 -8 M (<20nM) is denoted by the symbol "+++", indicating a moderate bond, 2 × 10⁻⁶ -8 ~2×10 -7 M (20~200nM) is indicated by "++", representing a weak bond, >2×10 -7M (>200nM) is indicated by "+". "ND" indicates that antibody binding could not be determined. The results show that eight antibodies showed strong binding (P2, P6, P9, P10, P22, P24, P31.2, and P39), seven antibodies showed moderate binding (P21, P23, P34, P35, P36, P45, and P47), five antibodies showed weak binding (P18, P26, P28, P30, and P54), and two antibodies showed no binding (P7 and P8). No binding was detected for P7 and P8. Table 5. Monovalent K D [Table 5-1] [Table 5-2]

[0231] Binding to mouse PD-L1 (mPD-L1) was characterized by assessing cell binding to mouse cells. Specifically, antibody binding to mouse cells was determined by flow cytometry. Antibodies were incubated with the indicated cell lines at the indicated concentrations and subsequently labeled with fluorescently labeled secondary antibodies. Mouse PD-L1 was obtained from Acro Biosystems (Acro Cat.No.PD1-M5220) and biotinylated using a standard protocol. Free biotin was removed by large-scale dialysis with PBS. Biotinylated antigen (PD-L1) was immobilized on a streptavidin sensor. Cross-reactive antibodies were identified by association with mouse PD-L1 using Octet.

[0232] The results of mouse cross-reactivity are shown in Table 6. Mouse cross-reactivity is indicated by "Y," and no mouse cross-reactivity is indicated by "X." Antibody cross-reactivity that could not be determined is indicated by "ND." Table 6. Mouse PD-L1 cross-reactivity [Table 6-1] [Table 6-2] [Table 6-3] (Example 3) Additional screening and selection

[0233] For example, antibodies selected for their binding to PD-L1, such as the antibody described in Example 2, were evaluated for their binding to PD-L1-expressing cells. For instance, antibodies were tested for binding to PD-L1 overexpressing CHO-K1 cells (BPS Bioscience PD-L1 / TCR activator - CHO strain, catalog number 30536) with approximately 674,000 surface PD-L1 copies using flow cytometry.

[0234] Cells were collected at 70-90% concentration on the day of the assay. Cells were collected by centrifugation at 200×g for 5 minutes, and the culture medium was removed. Cells were then placed in cold PBS at a rate of 2×10 cells per 1 mL. 6 The cells were resuspended individually. To cover the predicted binding affinity of the antibody to be tested, an 8-point antibody dilution series (2× concentration) was prepared in PBS. 50 μL of antibody dilution per well was plated into a 96-well V-bottom plate (Costar 3897). 50 μL of cell suspension was added per well. The plates were incubated at 4°C for 45–60 minutes.

[0235] Cells were collected by centrifugation at 400×g for 7 minutes, and primary antibodies were removed. 50 μL of AF488 goat anti-human IgG Fab (Jackson Immuno Research 109-547-003) was added per well at a dilution of 1:100. The plates were left at 4°C for 30 minutes.

[0236] Cells were collected by centrifugation at 400 × g for 7 minutes, and secondary antibodies were removed. Cells were resuspended in 50 μL of PBS per well and analyzed by flow cytometry. Binding curves were calculated using the mean fluorescence intensity (MFI) of the FITC fluorescence signal in the cells.

[0237] Exemplary binding curves are shown in Figures 2A-2C. Qualitative binding affinity to cells and the effective concentration (EC) of the maximum half of the amount of binding to cells for the assayed antibody. 50 Table 7 summarizes the findings regarding qualitative binding affinity. Strong binding, <2 × 10⁻¹⁰ -8 M (<20nM) is denoted by the symbol "+++", indicating a moderate bond, 2 × 10⁻⁶ -8 ~2×10 -7 M (20~200nM) is indicated by "++", representing a weak bond, >2×10 -7 A value of M (>200nM) is indicated by "+". "ND" indicates that antibody binding could not be determined. The results show that 29 antibodies exhibited strong cell binding affinity (P2, P3, P6, P7, P8, P9, P10, P11, P12, P13, P14, P18, P19, P21, P22, P23, P24, P26, P28, P31.2, P34, P35, P36, P37, P38, P39, P45, P47, and P54), while 21 antibodies showed weak binding (P1, P4, P5, P15, P17, P27, P29, P30, P32, P33, P40, P41, P42, P43, P46, P49, P50, P51, P52, P53, and P55). Table 7. Qualitative binding affinity and EC of antibody binding to CHO-K1 cells 50 [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] (Example 4) Functional assay

[0238] For example, antibodies selected for their binding to PD-L1, such as the antibodies described in Examples 2 and 3, were evaluated for their inhibition of PD-1 / PD-L1 signaling in PD-L1 overexpressing CHO-K1 cells.

[0239] To test the ability of antibodies to block PD-1 / PD-L1 signaling, the antibodies were assayed using a PD-1 / PD-L1 signaling bioassay kit (93-1104Y19-00117, Eurofins DiscoverX) following the manufacturer's protocol. In this assay, Jurkat cells were engineered to express PD1 tagged with β-galactosidase ProLink and enzyme acceptor SH1. The engineered Jurkat cells were co-cultured with a CD47 / PD-L1 expressing cell line. When PD-L1 binds to PD1, enzyme complementation occurs, and the β-gal signal is high. In the presence of the PD-L1 blocking antibody, SHP-1 / 2 is not recruited, enzyme complementation does not occur, and the β-gal signal is low.

[0240] Briefly, 40 μL of target cells (30,000 cells per 40 μL) were added to each well of a white 96-well plate in cell plating reagent 0 (CP0). The plate was incubated overnight at 37°C. The PathHunter PD-1 Jurkat cells were thawed overnight by adding 9.6 mL of preheated cell plating reagent (CP0) to a T25 flask, removing two cryovials of PD-1 Jurkat cells from liquid nitrogen, and immediately adding 1 mL of preheated CP0 from the T25 flask to the cryovials to thaw the pellet. The cells were mixed by gently pipetting several times to break up all aggregates. The cell suspension was transferred to a T25 flask containing the remaining CP0, and all remaining medium / suspension was removed from the cryovials to ensure complete recovery of all cells from the vials. The cells were incubated overnight at 37°C.

[0241] After incubation, 20 μL of antibody was added to each well, and the plate was incubated at 37°C for 1 hour. 40 μL of PathHunter PD-1 Jurkat cells in CP0 were added to each well of a 96-well plate, and the plate was incubated at room temperature in the dark for 1 hour. Next, 10 μL of PathHunter bioassay detection reagent 1 was added to each well, and the plate was incubated at room temperature in the dark for 15 minutes. Then, 40 μL of PathHunter bioassay detection reagent 2 was added to each well of the assay plate, and the plate was incubated at room temperature in the dark for 3 hours. The plates were analyzed using a ClarioStar plate reader (BMG Labtech).

[0242] Exemplary results of the PD-1 / PD-L1 checkpoint signaling assay in PD-L1 MDA-MB-231 cells are shown in Figures 3A-3C. IQ of the assay 50 The values ​​are shown in Table 3. Strong blocking, <2 × 10 -8 M (<20nM) is indicated by the symbol "+++", representing moderate blocking, 2 × 10 -8 ~2×10 -7 M(20~200nM) is indicated by "++", representing weak blocking, >2×10 -7 A value of M (>200nM) is indicated by a "+". "ND" indicates that the blockade of the interaction was not determined.

[0243] The results show that 15 antibodies exhibited potent blockade of the PD-1 / PD-L1 interaction (P2, P7, P8, P18, P22, P24, P26, P28, P31.2, P34, P35, P36, P39, P45, and P47), and 11 antibodies exhibited moderate blockade (P3, P6, P9, P10, P15, P21, P23, P30, P37, P41, and P54). Other antibodies tested exhibited weak blockade. "Low blockade" and "no blockade" indicate that the IC50 could not be determined due to low or undetectable blockade in the assay. Table 8. PD-1 / PD-L1 interaction blockade reporter assay [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] (Example 5) Feasibility assay

[0244] For example, antibodies selected for binding to PD-L1, such as those described in Examples 2 and 3, were tested using various developmental feasibility methods. For instance, various chromatographic methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and stand-up monolayer adsorption chromatography (SMAC), were used to assess developmental feasibility factors such as monomer percentage, solubility, and antibody aggregation or precipitation.

[0245] Size exclusion chromatography (SEC) analysis was performed using an Agilent 1100 HPLC with a 7.8 mm ID × 30 cm TSKgel G3000SWXL column (Tosoh Bioscience LLC, PN 08541). The antibody was tested in Dulbecco's PBS (pH 7.4, Ca 2+ / Mg 2+ The mixture was standardized to a concentration of 1 mg / mL (without accompanying iontophoresis) and clarified by centrifugation until the particles had pelletized but soluble aggregates were still retained. The mobile phase buffer was Dulbecco's PBS (pH 7.4, Ca 2+ / Mg 2+(without accompanying) was observed. For each sample, 10 μL was loaded and eluted at a uniform concentration of 1.0 mL / min over 20 minutes. Absorbance was monitored at 280 nm. Percentage uniformity and retention time were determined by integrating the chromatographic peaks. The choice of mobile phase, along with the column stationary phase, supports hydrophobic interactions in addition to molecular size classification (hydrophobic interactions are much milder compared to SMAC). Data analysis was performed using Agilent ChemStation B.04.03.

[0246] Exemplary SEC results are shown in Figures 4A-4C and summarized in Table 9. Strong development potential is indicated by the symbol "+++", moderate development potential by "++", and weak development potential by "++". "ND" indicates that development potential could not be determined using SEC. The results show that two antibodies, P11 and P26, have weak development potential based on the SEC assay, while all other antibodies tested have strong or moderate development potential. The results indicate that the majority of the antibodies tested show low antibody aggregation and strong development potential based on the SEC results. Table 9. Development Feasibility - Size Exclusion Chromatography [Table 9-1] [Table 9-2] [Table 9-3]

[0247] Hydrophobic interaction chromatography (HIC) analysis was performed using an Agilent 1100 HPLC with a 4.6 mm ID × 3.5 cm TSKgel Butyl-NPR column (Tosoh Bioscience LLC, PN 14947). The antibody was standardized to a concentration of 2 mg / mL in dPBS (pH 7.4) and then diluted to a final protein concentration of 1 mg / mL using an equal volume of mobile phase buffer B. The column was equilibrated with 100% mobile phase buffer B (2 M ammonium sulfate / 20 mM sodium phosphate, pH 7.0) at a flow rate of 1 mL / min. For each sample, 10 μL was loaded and eluted over 15 minutes at a flow rate of 1.0 mL / min using a gradient from 100% mobile phase buffer B to 100% mobile phase buffer A (20 mM sodium phosphate, pH 7.0). The column was washed by holding 100% A for 3 minutes and then returned to 100% B over 2 minutes for equilibration. Absorbance was monitored at 280 nm. Sample retention time was calculated and compared with a set of standard controls to identify antibodies that showed increased retention time (increased hydrophobicity).

[0248] Exemplary HIC results are shown in Figures 5A-5C and summarized in Table 10. Strong development potential is indicated by the symbol "+++", moderate development potential by "++", and weak development potential by "++". "ND" indicates that development potential could not be determined using HIC. The results indicate that 23 antibodies had strong development potential based on HIC results (P2, P4, P6, P7, P9, P11, P14, P15, P16, P21, P22, P23, P24, P28, P30, P31.2, P34, P35, P36, P37, P39, P45, and P47), 8 antibodies had moderate development potential (P3, P8, P10, P12, P13, P17, P18, and P54), and 3 antibodies had weak development potential (P1, P5, and P26). Antibody hydrophobicity can affect antibody aggregation, solubility, and viscosity. The results indicate that these antibodies have a low tendency to aggregate and precipitate. Table 10. Development Potential - Hydrophobic Interaction Chromatography [Table 10-1] [Table 10-2] [Table 10-3]

[0249] Stand-up monolayer adsorption chromatography (SMAC) analysis was performed using an Agilent 1100 HPLC with a 4.6 mm ID × 300 mm Zenix SEC 300 column (Sepax Technologies, PN 213300P-4630). Antibodies were standardized to a concentration of 1 mg / mL in dPBS (pH 7.4) and clarified by centrifugation until pelletized particles were formed. The mobile phase buffer was dPBS (pH 7.4, without calcium and magnesium). For each sample, 10 μL was loaded and eluted at a uniform concentration of 0.25 mL / min over 32 minutes. Absorbance was monitored at 280 nm. Sample retention times were calculated and antibodies with increased retention times (increased tendency to form aggregates) were identified compared to a standard control set.

[0250] Exemplary SMAC results are shown in Figures 6A-6C and summarized in Table 11. Strong development potential is indicated by the symbol "+++", moderate development potential by "++", and weak development potential by "++". "ND" indicates that development potential could not be determined using SMAC. The results show that 14 of the tested antibodies had good retention times, indicating good colloidal stability and a low tendency to aggregate (P4, P6, P9, P11, P14, P15, P16, P21, P24, P30, P31.2, P34, P35, and P45), 6 antibodies had moderate development potential (P8, P12, P17, P22, P36, and P39), and 14 antibodies had weak development potential based on SMAC (P1, P2, P3, P5, P7, P10, P13, P18, P23, P26, P28, P37, P47, and P54). Table 11. Development Feasibility - Stand-up Monolayer Adsorption Chromatography [Table 11-1] [Table 11-2] [Table 11-3]

[0251] The in vivo stability of antibodies was assessed using numerous assays with UNcle (Unchained Labs) analytical instruments. The stability of manipulated antibody variants was evaluated by measuring the polydispersity index (PDI), hydrodynamic diameter (Z-ave D), melting temperature (Tm), and aggregation temperature (Tagg).

[0252] The antibody was administered at concentrations ranging from 1 to 20 mg / mL in Dulbecco's PBS (pH 7.4, Ca 2+ / Mg 2+ The sample was formulated in a mixture (without fertilizer), clarified by centrifugation until large particles pelletized but soluble aggregates were still retained, and then assayed. The sample was dispensed into UNcle's 9 μL quartz capillary cuvette device (Uni) and sealed. PDI and hydrodynamic diameter were measured by DLS at 15°C. The temperature was gradient from 15°C to 95°C at 0.5°C per minute, during which Tm and Tagg were measured by fluorescence and SLS (266 nm, filter 4; 473 nm, filter 3), respectively. Data were analyzed using UNcle Analysis Software v3.1 or v3.2.

[0253] Table 12 shows exemplary in vivo stabilization results. Table 12. Assessment of biocompatibility [Table 12-1] [Table 12-2] [Table 12-3] (Example 6) Competitive binding assays and epitope binning

[0254] A competitive binding assay was used to determine whether antibodies competed for the same binding region of human PD-L1. Using a competitive immunoassay, if the binding of one antibody to the antigen prevented the binding of another antibody, these two antibodies were considered to bind to the same or similar (e.g., overlapping) epitopes and were placed in the same epitope bin. If the binding of one antibody did not interfere with the binding of another, these antibodies were considered to bind to distinct epitopes of PD-L1 and were placed in different epitope bins.

[0255] A tandem assay format based on octet was used for cross-competitive assays to establish competitive binding data and epitope binning. For these assays, 100 nM biotinylated antigen was immobilized on a streptavidin sensor in 10 × kinetic buffer (ForteBio). Antibody binding to PD-L1 was monitored by immersing the sensor in a series of steps into wells containing two competing (or non-competitive) antibodies at saturation concentrations. If binding was not blocked by saturation of the first antibody (indicated by a further increase in the BLI signal), these antibodies were considered to have bound to separate, non-overlapping epitopes and belonged to different bins.

[0256] The results show that P22 and P31.2 will be placed in the same bin (data not shown). These will be placed in separate bins from P24.

[0257] Throughout this application, various publications, patents, patent applications, and other documents have been referenced. The entirety of these publications, patents, patent applications, and other documents is thus incorporated into this application by reference for any purpose, including to provide a more detailed description of the state of the art relating to the subject matter disclosed herein. While the disclosed subject matter has been described with reference to the embodiments provided above, it should be understood that various modifications can be made without departing from the essence of the disclosed subject matter. A close examination of this specification will reveal many variations to those skilled in the art. The present invention provides, for example, the following items: (Item 1) (i) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; (ii) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; or (iii) An antibody that is essentially the same as an antibody containing a heavy chain variable region having the amino acid sequence of SEQ ID NO: 77 and a light chain variable region having the amino acid sequence of SEQ ID NO: 78, with monovalent binding affinity (K) to human PD-L1. D An antibody or fragment thereof having ) (Item 2) An antibody or fragment thereof that binds to PD-L1, (a) (1) (i) Sequence IDs 1, 27, or 53, (ii) Sequence IDs 7, 33, or 59, (iii) Sequence IDs 12, 38, or 64, (iv) Sequence IDs 13, 39, or 65, and (v) Sequence IDs 18, 44, or 70 V having an amino acid sequence selected from the group consisting of the following H CDR1; (2) (i) Sequence IDs 2, 28, or 54, (ii) Sequence IDs 8, 34, or 60, (iii) Sequence IDs 14, 40, or 66, (iv) Sequence IDs 19, 45, or 71, and (v) Sequence IDs 24, 50, or 76 V having an amino acid sequence selected from the group consisting of the following H CDR2; along (3) (i) Sequence IDs 3, 29, or 55, (ii) Sequence IDs 9, 35, or 61, (iii) Sequence IDs 15, 41, or 67, and (iv) Sequence IDs 20, 46, or 72 V having an amino acid sequence selected from the group consisting of the following H CDR3; Heavy chain variable (V) H ) region and, (b) (1) (i) Sequence IDs 4, 30, or 56, (ii) Sequence IDs 10, 36, or 62, (iii) Sequence IDs 16, 42, or 68, and (iv) Sequence IDs 21, 47, or 73 V having an amino acid sequence selected from the group consisting of the following L CDR1; (2) (i) Sequence IDs 5, 31, or 57, (ii) Sequence IDs 11, 37, or 63, and (iii) Sequence IDs 22, 48, or 74 V having an amino acid sequence selected from the group consisting of the following L CDR2; along (3) (i) Sequence IDs 6, 32, or 58, (ii) Sequence IDs 17, 43, or 69, and (iii) Sequence IDs 23, 49, or 75 V having an amino acid sequence selected from the group consisting of the following L CDR3 Light chain variable (V) L ) region and Antibodies or fragments thereof, including those containing antibodies. (Item 3) An antibody or fragment thereof that binds to PD-L1, (1) (i) Sequence IDs 1, 27, or 53, (ii) Sequence IDs 7, 33, or 59, (iii) Sequence IDs 12, 38, or 64, (iv) Sequence IDs 13, 39, or 65, and (v) Sequence IDs 18, 44, or 70 V having an amino acid sequence selected from the group consisting of the following H CDR1; (2) (i) Sequence IDs 2, 28, or 54, (ii) Sequence IDs 8, 34, or 60, (iii) Sequence IDs 14, 40, or 66, (iv) Sequence IDs 19, 45, or 71, and (v) Sequence IDs 24, 50, or 76 V having an amino acid sequence selected from the group consisting of the following H CDR2; along (3) (i) Sequence IDs 3, 29, or 55, (ii) Sequence IDs 9, 35, or 61, (iii) Sequence IDs 15, 41, or 67, and (iv) Sequence IDs 20, 46, or 72 V having an amino acid sequence selected from the group consisting of the following H CDR3 Heavy chain variable (V) H An antibody or fragment thereof containing the ) region. (Item 4) An antibody or fragment thereof that binds to PD-L1, (1) (i) Sequence IDs 4, 30, or 56, (ii) Sequence IDs 10, 36, or 62, (iii) Sequence IDs 16, 42, or 68, and (iv) Sequence IDs 21, 47, or 73 V having an amino acid sequence selected from the group consisting of the following L CDR1; (2) (i) Sequence IDs 5, 31, or 57, (ii) Sequence IDs 11, 37, or 63, and (iii) Sequence IDs 22, 48, or 74 V having an amino acid sequence selected from the group consisting of the following L CDR2; along (3) (i) Sequence IDs 6, 32, or 58, (ii) Sequence IDs 17, 43, or 69, and (iii) Sequence IDs 23, 49, or 75 V having an amino acid sequence selected from the group consisting of the following L CDR3 Light chain variable (V) L An antibody or fragment thereof containing the ) region. (Item 5) An antibody or fragment thereof that binds to PD-L1, From an antibody called P22, containing the VH sequence (sequence number 25) and the VL sequence (sequence number 26); From an antibody called P24, containing the VH sequence, which is sequence number 51, and the VL sequence, which is sequence number 52; or From the antibody called P31.2, which contains the VH sequence (sequence number 77) and the VL sequence (sequence number 78) An antibody or fragment thereof containing all three heavy chain complementarity-determining regions (CDRs) or all three light chain CDRs. (Item 6) The antibody or fragment thereof as described in item 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody referred to as P22. (Item 7) The antibody or fragment thereof as described in item 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody referred to as P24. (Item 8) The antibody or fragment thereof as described in item 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody referred to as P31.2. (Item 9) (a) V shown in Tables 1-3 H CDR1, V H CDR2 and V H Heavy chain variable (V) containing CDR3 amino acid sequence H )region or (b) V shown in Tables 1-3 L CDR1, V L CDR2 and V L Light chain variable (V) containing CDR3 amino acid sequence L )region An antibody or fragment thereof that binds to PD-L1, including [the specified element]. (Item 10) (a) V shown in Tables 1-3 H CDR1, V H CDR2 and V H Heavy chain variable (V) containing CDR3 amino acid sequence H )region Furthermore (b) V shown in Tables 1-3 L CDR1, V L CDR2 and V L Light chain variable (V) containing CDR3 amino acid sequence L )region The antibody or fragment thereof, including those described in item 9. (Item 11) V shown in Tables 1-3 H CDR1, V H CDR2 and V H Heavy chain variable (V) containing CDR3 amino acid sequence H The antibody or fragment thereof, including the region described in item 9. (Item 12) V shown in Tables 1-3 L CDR1, V L CDR2 and V L Light chain variable (V) containing CDR3 amino acid sequence L The antibody or fragment thereof, including the region described in item 9. (Item 13) (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18 H CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19 and 24 H CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15 and 20 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21 L CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 5, 11, and 22 L CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 9. (Item 14) (a) (1) V having the amino acid sequence of Sequence ID No. 1 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 2 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 4 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, as described in item 13, including the antibody or fragment thereof. (Item 15) (a) (1) V having the amino acid sequence of SEQ ID NO: 7 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 8 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 9 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 10 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 13. (Item 16) (a) (1) V having the amino acid sequence of Sequence ID No. 12 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 2 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 4 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 13. (Item 17) (a) (1) V having the amino acid sequence of SEQ ID NO: 13 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 14 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 15 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO. 16 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 17 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 13. (Item 18) (a) (1) V having the amino acid sequence of SEQ ID NO: 18 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 19 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 20 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 21 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 22 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 23 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 13. (Item 19) (a) (1) V having the amino acid sequence of Sequence ID No. 1 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 24 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 4 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 13. (Item 20) (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 33, 38, 39, and 44H CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 34, 40, 45, and 50 H CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 35, 41, and 46 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, 42, and 47 L CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 37, and 48 L CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 43, and 49 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 9. (Item 21) (a) (1) V having the amino acid sequence of SEQ ID NO: 27 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 30 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 20. (Item 22) (a) (1) V having the amino acid sequence of SEQ ID NO: 33 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 34 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 35 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 36 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 37 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 20. (Item 23) (a) (1) V having the amino acid sequence of SEQ ID NO: 38 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 30 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 20. (Item 24) (a) (1) V having the amino acid sequence of Sequence ID No. 39 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 40 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 41 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 42 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 37 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 43 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 20. (Item 25) (a) (1) V having the amino acid sequence of SEQ ID NO: 44 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 45 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 46 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 47 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 48 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 49 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 20. (Item 26) (a) (1) V having the amino acid sequence of SEQ ID NO: 27 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 50 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 30 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 20. (Item 27) (a) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 53, 59, 64, 65, and 70 H CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 54, 60, 66, 71, and 76 H CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 55, 61, 67, and 72 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 56, 62, 68, and 73 L CDR1, (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 57, 63, and 74 L CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs. 58, 69, and 75 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 9. (Item 28) (a) (1) V having the amino acid sequence of SEQ ID NO: 53 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 55 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 56 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 27. (Item 29) (a) (1) V having the amino acid sequence of SEQ ID NO: 59 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 60 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 61 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 62 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 27. (Item 30) (a) (1) V having the amino acid sequence of SEQ ID NO: 64 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 55 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 56 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 27. (Item 31) (a) (1) V having the amino acid sequence of SEQ ID NO. 65 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 66 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 67 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO. 68 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 69 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 27. (Item 32) (a) (1) V having the amino acid sequence of SEQ ID NO: 70 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 71 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 72 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 73 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 74 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 75 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 27. (Item 33) (a) (1) V having the amino acid sequence of SEQ ID NO: 53 H CDR1, (2) V having the amino acid sequence of SEQ ID NO: 76 H CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 55 H CDR3 Heavy chain variable (V) H ) region and, (b) (1) V having the amino acid sequence of SEQ ID NO: 56 L CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 L CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 58 L CDR3 Light chain variable (V) L ) region and The antibody or fragment thereof, including those described in item 27. (Item 34) The aforementioned V H Area or V L An antibody or fragment thereof according to any one of items 9 to 33, wherein the region further comprises a human framework sequence. (Item 35) The antibody or fragment thereof according to item 34, wherein the VH region and VL region further comprise a human framework sequence. (Item 36) The aforementioned V H Area or V L An antibody or fragment thereof as described in any one of items 9 to 33, wherein the region further comprises the framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences. (Item 37) The antibody or fragment thereof according to item 36, wherein the VH region and VL region further comprise the framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and framework 4 (FR4) sequences. (Item 38) A monoclonal antibody or a fragment thereof, as described in any one of items 1 to 37. (Item 39) The antibody or fragment thereof as described in item 38, wherein the monoclonal antibody is a humanized antibody, a human antibody, or a chimeric antibody. (Item 40) Fab, Fab', F(ab') 2 , Fv, scFv, (scFv) 2 An antibody or fragment thereof, as described in any one of items 1 to 39, which is a multispecific antibody formed from a single-chain antibody molecule, a bivariate region antibody, a single variable region antibody, a linear antibody, a V region, or an antibody fragment. (Item 41) An antibody or fragment thereof as described in any one of items 1 to 40, which is conjugated with a diagnostic agent, detection agent or therapeutic agent, or is recombinantly fused with such an antibody or fragment thereof. (Item 42) The antibody or fragment thereof as described in item 41, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxic agent, or a drug. (Item 43) A binding agent that binds to essentially the same epitope as the antibody or fragment described in any one of items 1 through 42. (Item 44) A binding agent as described in item 43, which is an antibody or a fragment thereof. (Item 45) A binding agent as described in item 43, including a non-antibody protein scaffold. (Item 46) The binding agent according to item 45, wherein the non-antibody protein scaffold comprises a fibronectin scaffold, antikalin, adonectin, afibody, DARPin, finomer, afitin, afirin, avimer, cysteine-rich Nottin peptide, or a modified Knitz-type inhibitor. (Item 47) A binding agent that competes with any one of the antibodies or fragments described in item 1 to 42 for binding to human PD-L1. (Item 48) A binding agent, as described in item 47, which is an antibody or a fragment thereof. (Item 49) One or more vectors comprising one or more polynucleotides encoding an antibody or fragment thereof as described in any one of items 1 to 42. (Item 50) A pharmaceutical composition comprising an antibody or fragment thereof as described in any one of items 1 to 42, and a pharmaceutically acceptable carrier. (Item 51) A method for treating cancer or tumor in a subject, comprising the step of administering to the subject an antibody or fragment thereof as described in any one of items 1 to 42, or a pharmaceutical composition as described in item 50. (Item 52) A method for alleviating one or more symptoms associated with cancer or a tumor in a subject, comprising the step of administering to the subject an antibody or fragment thereof as described in any one of items 1 to 42, or a pharmaceutical composition as described in item 50. (Item 53) A method for reducing the size of a tumor in a subject having a tumor, comprising the step of administering to the subject an antibody or fragment thereof as described in any one of items 1 to 42, or a pharmaceutical composition as described in item 50. (Item 54) A method for enhancing tumor cell removal in a subject having a tumor, comprising the step of administering to the subject an antibody or fragment thereof as described in any one of items 1 to 42, or a pharmaceutical composition as described in item 50. (Item 55) A method for treating a T-cell dysfunction disorder, impairment, or condition in a subject, comprising the step of administering to the subject an antibody or fragment thereof as described in any one of items 1 to 42, or a pharmaceutical composition as described in item 50. (Item 56) The method according to item 55, wherein the T-cell dysfunction disorder, disorder, or condition is tumor immunity. (Item 57) A method for enhancing T cell function in a subject, comprising the step of administering to the subject an antibody or fragment thereof as described in any one of items 1 to 42, or a pharmaceutical composition as described in item 50. (Item 58) The method according to item 57, wherein the T cell function is cytokine secretion. (Item 59) The method according to item 57, wherein the T cell function is the removal of tumor cells. (Item 60) The method described in any one of items 57 to 59, wherein the subject has been diagnosed with cancer or a tumor. (Item 61) The method according to any one of items 51 to 60, comprising administering one or more therapeutic agents to the subject in combination with the antibody or a fragment thereof or the pharmaceutical composition.

Claims

1. An antibody that binds to PD-L1 or an antigen-binding fragment thereof, It includes a heavy chain variable (VH) region and a light chain variable (VL) region, The antibody or its antigen-binding fragment is From an antibody containing the VH sequence, which is sequence number 77, and the VL sequence, which is sequence number 78; From an antibody containing the VH sequence, which is SEQ ID NO: 25, and the VL sequence, which is SEQ ID NO: 26; or From an antibody containing the VH sequence (SEQ ID NO: 51) and the VL sequence (SEQ ID NO: 52) It includes all three heavy chain complementarity-determining regions (CDRs) and all three light chain CDRs. The three heavy chain CDRs and three light chain CDRs are antibodies or antigen-binding fragments defined by exemplary numbering systems, IMGT numbering systems, Kabat numbering systems, Chothia numbering systems, Contact numbering systems, or AbM numbering systems.

2. (i) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 53 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 55 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 56 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 including, (ii) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 59 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 60 Ｈ CDR2, and (3)V having the amino acid sequence of SEQ ID NO: 61 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 62 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 including, (iii) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 64 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 54 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 55 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 56 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 including, (iv) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 65 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 66 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 67 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 68 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 63 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 69 Ｌ CDR3 including, (v) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 70 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 71 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 72 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 73 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 74 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 75 Ｌ CDR3 including, or (vi) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 53 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 76 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 55 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 56 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 57 Ｌ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 58 Ｌ CDR3 The antibody or antigen-binding fragment thereof according to claim 1, comprising:

3. (i) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 1 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 2 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 4 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 including, (ii) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 7 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 8 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 9 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 10 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 including, (iii) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 12 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 2 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 4 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 including, (iv) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 13 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 14 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 15 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 16 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 11 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 17 Ｌ CDR3 including, (v) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 18 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 19 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 20 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 21 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 22 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 23 Ｌ CDR3 including, or (vi) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 1 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 24 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 3 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 4 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 5 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 6 Ｌ CDR3 The antibody or antigen-binding fragment thereof according to claim 1, comprising:

4. (i) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 27 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 30 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 including, (ii) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 33 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 34 Ｈ CDR2, and (3) V having the amino acid sequence of Sequence ID No. 35 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 36 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 37 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 including, (iii) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 38 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 28 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 30 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 Ｌ CDR2, and (3)V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 including, (iv) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 39 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 40 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 41 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 42 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 37 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 43 Ｌ CDR3 including, (v) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 44 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 45 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 46 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 47 Ｌ CDR1, (2) V having the amino acid sequence of Sequence ID No. 48 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 49 Ｌ CDR3 including, or (vi) (a) The VH region is (1) V having the amino acid sequence of SEQ ID NO: 27 Ｈ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 50 Ｈ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 29 Ｈ CDR3 Includes, (b) the V L region is (1) V having the amino acid sequence of SEQ ID NO: 30 Ｌ CDR1, (2) V having the amino acid sequence of SEQ ID NO: 31 Ｌ CDR2, and (3) V having the amino acid sequence of SEQ ID NO: 32 Ｌ CDR3 The antibody or antigen-binding fragment thereof according to claim 1, comprising:

5. The aforementioned V Ｈ Area and / or V Ｌ The antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the region further comprises (i) a human framework sequence and / or (ii) framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

6. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO:

78.

7. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises VH containing the amino acid sequence of SEQ ID NO: 25 and VL containing the amino acid sequence of SEQ ID NO:

26.

8. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises VH containing the amino acid sequence of SEQ ID NO: 51 and VL containing the amino acid sequence of SEQ ID NO:

52.

9. The antibody or its antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody.

10. The antibody or its antigen-binding fragment is Fab, Fab', F(ab') ２ , Fv, scFv, (scFv) ２ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which is a multispecific antibody formed from a single-chain antibody molecule, a bivariate region antibody, a linear antibody, a V region, or an antibody fragment.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or its antigen-binding fragment is conjugated with a diagnostic agent, detection agent, or therapeutic agent, or is fused with it by recombination.

12. The antibody or its antigen-binding fragment according to claim 11, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxic agent, or a drug.

13. One or more polynucleotides encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12.

14. One or more vectors comprising one or more polynucleotides encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12.

15. A cell comprising one or more polynucleotides according to claim 13, or one or more vectors according to claim 14.

16. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

17. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 16 for use in a method for treating cancer or a tumor in a subject, alleviating one or more symptoms associated with cancer or a tumor in a subject, reducing the size of a tumor in a subject having a tumor, enhancing tumor cell removal in a subject having a tumor, treating a T cell dysfunction disorder, disorder or condition in a subject, or enhancing T cell function in a subject, wherein the method comprises the step of administering the composition or the pharmaceutical composition to the subject.

18. The composition for use according to claim 17, wherein the subject is administered in combination with one or more therapeutic agents, or the composition or pharmaceutical composition.

19. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 16, in the manufacture of a pharmaceutical for treating cancer or a tumor in a subject, alleviating one or more symptoms associated with cancer or a tumor in a subject, reducing the size of a tumor in a subject having a tumor, enhancing tumor cell removal in a subject having a tumor, treating a T cell dysfunction disorder, impairment or condition in a subject, or enhancing T cell function in a subject, wherein the pharmaceutical is administered to the subject.

20. The use according to claim 19, wherein the subject is administered in combination with one or more therapeutic agents.

Images