Anti-PD-l1 antibodies

Abstract

To provide a novel PD / L1 binding agent that binds to PD / L1 with high affinity and can be used for a drug that effectively inhibits PD / L1 / PD-1 signaling, a drug that can deliver a therapeutic payload to PD / L1 expressing cells, and the like.SOLUTION: Provided are programmed death-ligand 1 (PD-L1) binding agents having specific sequences, nucleic acids comprising the binding agents of the invention, vectors and cells comprising the nucleic acids of the invention, and compositions thereof. Also provided are methods of making the binding agents of the invention, methods of treating a disease, disorder, or condition in a mammal, and methods of enhancing or reducing or suppressing an immune response in a mammal.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 963,771, filed January 21, 2020, which is incorporated by reference in its entirety.

[0002] Incorporation by Reference of Electronically Filed Materials The computer readable nucleotide / amino acid sequence listing, filed concurrently herewith and identified as follows, is hereby incorporated by reference in its entirety: One 186,979 byte ASCII (text) file entitled "751374_ST25", dated January 21, 2021. [Background technology]

[0003] Programmed death-ligand 1 (PD-L1, cluster of differentiation 274, CD274, B7-homolog 1, or B7-H1) is a member of the B7 protein superfamily and a ligand for programmed cell death protein 1 (PD-1, PDCD1, cluster of differentiation 279, or CD279). PD-L1 also interacts with B7.1 (CD80), and this interaction is thought to inhibit T cell priming. The PD-L1 / PD-1 axis plays a major role in suppressing adaptive immune responses. More specifically, binding of PD-L1 to its receptor, PD-1, is thought to transduce signals that suppress T cell activation and proliferation. Drugs that bind to PD-L1 and block ligand binding to the PD-1 receptor prevent this immune suppression, thereby enhancing immune responses when needed, such as for the treatment of cancer and infectious diseases. The PD-L1 / PD-1 pathway also contributes to the prevention of autoimmunity, and therefore agonistic agents against PD-L1 or agents that deliver immunoinhibitory payloads may be useful in the treatment of autoimmune diseases. Summary of the Invention [Problem to be solved by the invention]

[0004] Several antibodies targeting PD-L1, such as atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), and avelumab (BAVENCIO™), have been developed for the treatment of cancer. However, there remains a need for novel PD-L1-binding agents, including agents that bind to PD-L1 with high affinity and effectively inhibit PD-L1 / PD-1 signaling, as well as agents that can deliver therapeutic payloads to PD-L1-expressing cells. Additionally, new PD-L1-binding agents are needed to treat autoimmune and infectious diseases. [Means for solving the problem]

[0005] Provided herein are PD-L1-binding agents comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide. In some embodiments, the PD-L1-binding agent comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 223-264, 324-334, or 361-365, or at least the CDRs thereof, and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 265-306, 335-344, or 366-370, or at least the CDRs thereof. In other embodiments, the PD-L1-binding agent binds to any one of SEQ ID NOs: 223-264, 324-334, or 361-365 and immunoglobulin heavy chain variable region polypeptides having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 265-306, 335-344, or 366-370. In yet other embodiments, the PD-L1-binding agent, the immunoglobulin heavy chain variable region polypeptide comprises a complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 1-23, 309, or 345, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 24-57, 310-314, or 346-349, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 58-95, 315-318, or 350-354; and / or the immunoglobulin light chain variable region polypeptide comprises a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 96-128, 319-323, 355, or 356, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 129-151, or 357-359, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 152-176, or 360. Also provided are nucleic acids encoding the PD-L1-binding agents, or their individual heavy and light chains; vectors and cells containing the nucleic acids; and compositions containing the binding agents or nucleic acids.

[0006] Also provided are methods of preparing the binding agents described herein, comprising expressing in a cell one or more nucleic acids encoding the heavy and light chain variable region polypeptides of the binding agent.

[0007] Further provided is a method of delivering a payload to a cell that expresses PD-L1, comprising administering to the cell, or a mammal comprising the cell, a PD-L1-binding agent provided herein conjugated to a payload.

[0008] Also provided are methods for enhancing or reducing or inhibiting an immune response in a mammal, and methods for treating a disease, disorder, or condition in a mammal that responds to PD-L1 inhibition, comprising administering to the mammal a binding agent, or conjugate thereof, as described herein.

[0009] Additional aspects and embodiments of the present invention are provided in the detailed description that follows. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows experimental data demonstrating the affinity of binding agents according to embodiments of the invention for human PD-L1 on the surface of JIMT-1 cells. [Figure 2] Figure 2 shows experimental data demonstrating the affinity of binding agents according to embodiments of the invention for human PD-L1 using JIMT-1 cells. [Figure 3] FIG. 3 shows experimental data demonstrating the level of intracellular entry of binding agents according to embodiments of the present invention. [Figure 4] Figure 4 shows experimental data demonstrating the affinity of binding agents according to embodiments of the invention for human PD-L1 on the surface of JIMT-1 cells. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Invention The invention provides PD-L1-binding agents comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide.

[0012] PD-L1-binding agents specifically bind to PD-L1, and the binding specificity of the agents allows them to target, for example, cells that express PD-L1 and deliver a therapeutic payload to such cells.

[0013] In some embodiments, the PD-L1-binding agent binds PD-L1 without substantially inhibiting or preventing PD-L1 from binding to its receptor, PD-1. However, in other embodiments, the PD-L1-binding agent can completely or partially block (inhibit or prevent) the binding of PD-L1 to its receptor, PD-1, such that antibodies can be used (e.g., for therapeutic purposes) to inhibit PD-L1 / PD-1 signaling.

[0014] Furthermore, in some embodiments, the PD-L1-binding agents provided herein, upon binding to PD-L1 on the cell surface, cause the internalization of PD-L1 or the PD-L1 / PD-L1-binding agent complex. Without wishing to be bound to any particular theory or mechanism of action, it is believed that the PD-L1-binding agents according to this embodiment cause the internalization of PD-L1 upon binding, and remain bound to PD-L1 during internalization, internalizing the binding agent along with PD-L1. The internalization of PD-L1 and the bound PD-L1-binding agent can be determined by any suitable method, such as by assaying persistence on the cell surface and / or detecting internalizing antibodies. In some embodiments, the PD-L1-binding agent is internalized such that at least about 20% or 25% (e.g., at least about 35%, at least about 50%, at least about 75%, or at least about 90%) of the PD-L1-binding agent that binds to PD-L1 on the cell surface are internalized (e.g., using a surface persistence assay, of the PD-L1-binding agent molecules bound to PD-L1 on the cell surface at the start of the assay, no more than about 75%, no more than about 65%, no more than about 50%, no more than about 35%, or no more than about 20% remain bound at the end of the assay). Persistence can be tested over an appropriate period of time long enough for internalization to occur. In some embodiments, internalization occurs within about 2 days, or within about 1 day (24 hours or less), or even within about 12 hours.

[0015] In some embodiments, the PD-L1-binding agent binds to human PD-L1, for example, a protein comprising SEQ ID NO: 307 (MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET). However, binding agents that bind to any PD-L1 homologue or paralogue are also included. In some embodiments, the PD-L1 protein comprises at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 307. In some embodiments, the binding agent binds to human PD-L1 and cynomolgus PD-L1; or binds to human, cynomolgus, and mouse PD-L1.

[0016] The "identity" of a nucleic acid or amino acid sequence referred to herein can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percentage of identity is the number of identical (i.e., identical) nucleotides or amino acid residues between an optimally aligned sequence of interest and a reference sequence, divided by the length of the longest sequence (i.e., the longer length of either the sequence of interest or the reference sequence). Sequence alignment and percent identity calculation can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for nucleic acid and amino acid sequence alignment), BLAST programs (e.g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms are also described, for example, in Altschul et al., J. Molecular Biol., 215(3) 403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7): 951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997); Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). The percentage (%) of sequence identity can be calculated, for example, as follows: 100 × [(identical positions) / min(TG A , T.G. B )], where TG A and T.G.B , T.G. A and T.G. B is the sum of the number of residues and internal gap positions of peptide sequences A and B in the alignment that minimizes (see, for example, Russell et al., J. Mol. Biol., 244: 332-350 (1994)).

[0017] The binding agent comprises Ig heavy and light chain variable region polypeptides that together form an antigen-binding site. Each of the heavy and light chain variable regions is a polypeptide that includes three complementarity-determining regions (CDR1, CDR2, and CDR3) connected by framework regions. The binding agent can be any of the various types of binding agents known in the art, including Ig heavy and light chains. For example, the binding agent can be an antibody, an antigen-binding antibody "fragment," or a T-cell receptor.

[0018] In some embodiments, the binding agent is a whole (or complete) antibody comprising an antigen-binding domain comprising Ig heavy and light variable domains and a fragment crystallizable (Fc) domain. An exemplary antibody structure is a tetramer comprising two pairs of polypeptide chains, each pair having one "light" chain (smaller, e.g., about 25 kDa) and one "heavy" chain (larger, e.g., about 50-70 kDa), typically connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains are divided into different categories based on size and function, e.g., light and heavy chain variable domains or regions (V and V, respectively). L and V H ), light and heavy chain constant domains or regions (C L and C H) at the N-terminus of each chain is a variable region, or antigen-binding domain, called the paratope, which is primarily responsible for antigen recognition and is usually defined by (but not limited to) about 100-110 or more amino acids. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. These classes are further divided into subclasses. For example, in humans, there are four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), named in order of abundance in serum (i.e., IgG1 is the most abundant).

[0019] In some embodiments, the binding agent is an antigen-binding antibody "fragment," which is a construct that comprises at least the antigen-binding region of an antibody, either alone or together with other components that together comprise an antigen-binding construct. For example, (i) V L , V H , C L , and C H (ii) a Fab fragment, which is a monovalent fragment consisting of one domain; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iv) a V fragment, which is a single arm of an antibody. L and V H (iv) Fab' fragments, which result from cleavage of the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) two domains of the Fv fragment joined by a synthetic linker (i.e., V) that allows the two domains to be synthesized as a single polypeptide chain. L and V H Many different types of antibody "fragments" are known in the art, including single-chain Fvs (scFvs), which are monovalent molecules consisting of a single chain Fv (scFv) and a single chain Fv (scFv).

[0020] Antibodies or antibody fragments can be part of larger constructs, e.g., conjugates or fusion constructs of antibody fragments with additional regions. For example, in some embodiments, antibody fragments can be fused to an Fc region as described herein. In other embodiments, antibody fragments (e.g., Fab or scFv) can be part of a chimeric antigen receptor or chimeric T cell receptor, e.g., by fusion to a transmembrane domain (optionally, an intervening linker or "stalk" (e.g., hinge region)) and optionally an intercellular signaling domain. For example, antibody fragments can be fused to the gamma and / or delta chains of a T cell receptor to provide a T cell receptor-like construct that binds to PD-L1. In yet another embodiment, the antibody fragment is part of a bispecific T cell engager (BiTE) comprising a CD1- or CD3-binding domain and a linker.

[0021] Antibodies or antigen-binding antibody fragments may be monospecific for PD-L1, or may be bispecific or multispecific. For example, in a bivalent or multivalent antibody or antibody fragment, the binding domains may be different, targeting different epitopes of the same antigen, or targeting different antigens. Methods for constructing multivalent binding constructs are known in the art. Bispecific and multispecific antibodies are known in the art. Additionally, diabodies, triabodies, or tetrabodies may be provided, which are composed of V and V-type antibodies on the same polypeptide chain. H and V L V due to a peptide linker that is too short to allow pairing between L V connected to H and dimers, trimers, or tetramers of polypeptide chains each containing a different V H -V LPairing between complementary domains on the polypeptide chains is driven, resulting in the generation of multimeric molecules with two, three, or four functional antigen-binding sites. Bis-scFv fragments, which are small scFv fragments with two different variable domains, can also be generated, resulting in bispecific bis-scFv fragments capable of binding to two different epitopes. Fab dimers (Fab2) and Fab trimers (Fab3) can be generated using genetic engineering methods to create multispecific constructs based on Fab fragments.

[0022] The PD-L1-binding agent may also be an antibody conjugate. In this regard, the PD-L1-binding agent may be a conjugate of (1) an antibody, alternative scaffold, or fragment thereof, and (2) a protein or non-protein structural moiety. For example, the PD-L1-binding agent may be conjugated to a peptide, a fluorescent molecule, a chemotherapeutic agent or other cytotoxic payload, an immunostimulatory agent, or an immunosuppressant.

[0023] The PD-L1-binding agent may be or be derived from a human antibody, a non-human antibody, a humanized antibody, or a chimeric antibody, or a corresponding antibody fragment. A "chimeric" antibody is typically an antibody or fragment thereof that comprises a human constant region and a non-human variable region. A "humanized" antibody is typically a monoclonal antibody that comprises a human antibody scaffold but has amino acids or sequences of non-human origin in at least one CDR (e.g., one, two, three, four, five, or all six CDRs).

[0024] Methods for producing such antibodies are known in the art and are described, for example, in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 9th Ed., Garland Publishing, New York, NY (2017). In certain embodiments, human or chimeric antibodies or antibody fragments can be produced using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes have been replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes have been effectively replaced with human antibody genes include, but are not limited to, Medarex HUMAB-MOUSE™, Kirin TC MOUSE™, and Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181:69-97 (2008)). Humanized antibodies can be produced using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), such as grafting non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144(1): 115-120 (2008)), and using phage display (see, e.g., Fellouse et al., Journal of Molecular Biology, 373(4): 924-940 (2007) and Glanville, et al., PNAS, 106(48).20216-20221 (2009)).

[0025] In embodiments, the PD-L1-binding agent comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 223-264, 324-334, or 361-365, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NOs: 223-264, 324-334, or 361-365, or at least about 90% identity to SEQ ID NOs: 223-264, 324-334, or 361-365. and / or an immunoglobulin light chain variable region of any one of SEQ ID NOs: 265-306, 335-344, or 366-370, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NOs: 265-306, 335-344, or 366-370, or at least the CDRs thereof.

[0026] By way of further example, the PD-L1-binding agent may include:

[0027] (1) An immunoglobulin heavy chain variable region of SEQ ID NO: 223, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 265, or at least its CDR.

[0028] (2) An immunoglobulin heavy chain variable region of SEQ ID NO: 224, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 266, or at least its CDR.

[0029] (3) An immunoglobulin heavy chain variable region of SEQ ID NO: 225, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 267, or at least its CDR.

[0030] (4) An immunoglobulin heavy chain variable region of SEQ ID NO: 226, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 268, or at least its CDR.

[0031] (5) An immunoglobulin heavy chain variable region of SEQ ID NO: 227, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 269, or at least its CDR.

[0032] (6) An immunoglobulin heavy chain variable region of SEQ ID NO: 228, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 270, or at least its CDR.

[0033] (7) An immunoglobulin heavy chain variable region of SEQ ID NO: 229, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 271, or at least its CDR.

[0034] (8) An immunoglobulin heavy chain variable region of SEQ ID NO: 230, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 272, or at least its CDR.

[0035] (9) An immunoglobulin heavy chain variable region of SEQ ID NO: 231, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 273, or at least its CDR.

[0036] (10) An immunoglobulin heavy chain variable region of SEQ ID NO: 232, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 274, or at least its CDR.

[0037] (11) An immunoglobulin heavy chain variable region of SEQ ID NO: 233, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 275, or at least its CDR.

[0038] (12) An immunoglobulin heavy chain variable region of SEQ ID NO: 234, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 276, or at least its CDR.

[0039] (13) An immunoglobulin heavy chain variable region of SEQ ID NO: 235, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 277, or at least its CDR.

[0040] (14) An immunoglobulin heavy chain variable region of SEQ ID NO: 236, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 278, or at least its CDR.

[0041] (15) An immunoglobulin heavy chain variable region of SEQ ID NO: 237, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 279, or at least its CDR.

[0042] (16) An immunoglobulin heavy chain variable region of SEQ ID NO: 238, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 280, or at least its CDR.

[0043] (17) An immunoglobulin heavy chain variable region of SEQ ID NO: 239, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0044] (18) An immunoglobulin heavy chain variable region of SEQ ID NO: 240, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 282, or at least its CDR.

[0045] (19) An immunoglobulin heavy chain variable region of SEQ ID NO: 241, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 283, or at least its CDR.

[0046] (20) An immunoglobulin heavy chain variable region of SEQ ID NO: 242, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 284, or at least its CDR.

[0047] (21) An immunoglobulin heavy chain variable region of SEQ ID NO: 243, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 285, or at least its CDR.

[0048] (22) An immunoglobulin heavy chain variable region of SEQ ID NO: 244, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 286, or at least its CDR.

[0049] (23) An immunoglobulin heavy chain variable region of SEQ ID NO: 245, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 287, or at least its CDR.

[0050] (24) An immunoglobulin heavy chain variable region of SEQ ID NO: 246, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 288, or at least its CDR.

[0051] (25) An immunoglobulin heavy chain variable region of SEQ ID NO: 247, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 289, or at least its CDR.

[0052] (26) An immunoglobulin heavy chain variable region of SEQ ID NO: 248, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 290, or at least its CDR.

[0053] (27) An immunoglobulin heavy chain variable region of SEQ ID NO: 249, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 291, or at least its CDR.

[0054] (28) An immunoglobulin heavy chain variable region of SEQ ID NO: 250, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 292, or at least its CDR.

[0055] (29) An immunoglobulin heavy chain variable region of SEQ ID NO: 251, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 293, or at least its CDR.

[0056] (30) An immunoglobulin heavy chain variable region of SEQ ID NO: 252, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 294, or at least its CDR.

[0057] (31) An immunoglobulin heavy chain variable region of SEQ ID NO: 253, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 295, or at least its CDR.

[0058] (32) An immunoglobulin heavy chain variable region of SEQ ID NO: 254, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 296, or at least its CDR.

[0059] (33) An immunoglobulin heavy chain variable region of SEQ ID NO: 255, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 297, or at least its CDR.

[0060] (34) An immunoglobulin heavy chain variable region of SEQ ID NO: 256, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 298, or at least its CDR.

[0061] (35) An immunoglobulin heavy chain variable region of SEQ ID NO: 257, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 299, or at least its CDR.

[0062] (36) An immunoglobulin heavy chain variable region of SEQ ID NO: 258, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 300, or at least its CDR.

[0063] (37) An immunoglobulin heavy chain variable region of SEQ ID NO: 259, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 301, or at least its CDR.

[0064] (38) An immunoglobulin heavy chain variable region of SEQ ID NO: 260, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 302, or at least its CDR.

[0065] (39) An immunoglobulin heavy chain variable region of SEQ ID NO: 261, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 303, or at least its CDR.

[0066] (40) An immunoglobulin heavy chain variable region of SEQ ID NO: 262, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 304, or at least its CDR.

[0067] (41) An immunoglobulin heavy chain variable region of SEQ ID NO: 263, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 305, or at least its CDR.

[0068] (42) An immunoglobulin heavy chain variable region of SEQ ID NO: 164, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 306, or at least its CDR.

[0069] (43) An immunoglobulin heavy chain variable region of SEQ ID NO: 324, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0070] (44) An immunoglobulin heavy chain variable region of SEQ ID NO: 325 (wherein X can be any amino acid), or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDRs.

[0071] (45) An immunoglobulin heavy chain variable region of SEQ ID NO: 326 (wherein X can be any amino acid), or at least the CDRs thereof, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least the CDRs thereof.

[0072] (46) An immunoglobulin heavy chain variable region of SEQ ID NO: 327, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0073] (47) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0074] (48) An immunoglobulin heavy chain variable region of SEQ ID NO: 329, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0075] (49) An immunoglobulin heavy chain variable region of SEQ ID NO: 329, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0076] (50) An immunoglobulin heavy chain variable region of SEQ ID NO: 330, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0077] (51) An immunoglobulin heavy chain variable region of SEQ ID NO: 331, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0078] (52) An immunoglobulin heavy chain variable region of SEQ ID NO: 332, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0079] (53) An immunoglobulin heavy chain variable region of SEQ ID NO: 333, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0080] (54) An immunoglobulin heavy chain variable region of SEQ ID NO: 334, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0081] (55) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 335, or at least its CDR.

[0082] (56) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 336, or at least its CDR.

[0083] (57) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 337, or at least its CDR.

[0084] (58) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 338, or at least its CDR.

[0085] (59) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 339, or at least its CDR.

[0086] (60) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 340, or at least its CDR.

[0087] (61) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 341, or at least its CDR.

[0088] (62) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 342, or at least its CDR.

[0089] (63) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 343, or at least its CDR.

[0090] (64) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 344, or at least its CDR.

[0091] (65) An immunoglobulin heavy chain variable region of SEQ ID NO: 328, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR.

[0092] (66) An immunoglobulin heavy chain variable region of SEQ ID NO: 361, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 366, or at least its CDR.

[0093] (67) An immunoglobulin heavy chain variable region of SEQ ID NO: 362, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 367, or at least its CDR.

[0094] (68) An immunoglobulin heavy chain variable region of SEQ ID NO: 363, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 368, or at least its CDR.

[0095] (69) An immunoglobulin heavy chain variable region of SEQ ID NO: 364, or at least its CDR, and / or an immunoglobulin light chain variable region of SEQ ID NO: 369, or at least its CDR.

[0096] (70) an immunoglobulin heavy chain variable region of SEQ ID NO: 365, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 370, or at least its CDRs; and / or

[0097] (71) An immunoglobulin heavy chain variable region of Table 4 and / or an immunoglobulin light chain variable region of Table 4, or at least the CDRs thereof.

[0098] The CDRs of a given heavy or light chain Ig sequence can be determined according to any of the various known Ig numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, ABM). In certain embodiments, the PD-L1-binding agent comprises one or more of the following CDRs:

[0099] an HCDR1 comprising or consisting of any one of SEQ ID NOs: 1-23, 309, or 345, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-23, 309, or 345;

[0100] an HCDR2 comprising or consisting of any one of SEQ ID NOs:24-57, 310-314, or 346-349, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs:24-57, 310-314, or 346-349;

[0101] an HCDR3 comprising or consisting of any one of SEQ ID NOs: 58-95, 315-318, or 350-354, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 58-95, 315-318, or 350-354; and / or an immunoglobulin light chain polypeptide comprising:

[0102] an LCDR1 comprising or consisting of any one of SEQ ID NOs: 96-128, 319-323, 355, or 356, or a sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 96-128, 319-323, 355, or 356;

[0103] an LCDR2 comprising or consisting of any one of SEQ ID NOs: 129-151 or 357-359, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 129-151 or 357-359; and

[0104] An LCDR3 comprising or consisting of any one of SEQ ID NOs: 152-176 or 360, or a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 152-176 or 360.

[0105] In certain embodiments, the binding agent comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, wherein:

[0106] (1) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 1, an HCDR2 comprising or consisting of SEQ ID NO: 24, and an HCDR3 comprising or consisting of SEQ ID NO: 58; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 96, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 152;

[0107] (2) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:2, an HCDR2 comprising or consisting of SEQ ID NO:25, and an HCDR3 comprising or consisting of SEQ ID NO:59; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:97, an LCDR2 comprising or consisting of SEQ ID NO:129, and an LCDR3 comprising or consisting of SEQ ID NO:153;

[0108] (3) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 3, an HCDR2 comprising or consisting of SEQ ID NO: 26, and an HCDR3 comprising or consisting of SEQ ID NO: 60; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 154. Includes;

[0109] (4) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:4, an HCDR2 comprising or consisting of SEQ ID NO:27, and an HCDR3 comprising or consisting of SEQ ID NO:61; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:99, an LCDR2 comprising or consisting of SEQ ID NO:130, and an LCDR3 comprising or consisting of SEQ ID NO:155;

[0110] (5) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:5, an HCDR2 comprising or consisting of SEQ ID NO:28, and an HCDR3 comprising or consisting of SEQ ID NO:62; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:100, an LCDR2 comprising or consisting of SEQ ID NO:129, and an LCDR3 comprising or consisting of SEQ ID NO:153;

[0111] (6) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:6, an HCDR2 comprising or consisting of SEQ ID NO:29, and an HCDR3 comprising or consisting of SEQ ID NO:63; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:101, an LCDR2 comprising or consisting of SEQ ID NO:131, and an LCDR3 comprising or consisting of SEQ ID NO:156;

[0112] (7) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:7, an HCDR2 comprising or consisting of SEQ ID NO:30, and an HCDR3 comprising or consisting of SEQ ID NO:64; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:102, an LCDR2 comprising or consisting of SEQ ID NO:132, and an LCDR3 comprising or consisting of SEQ ID NO:157;

[0113] (8) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:2, an HCDR2 comprising or consisting of SEQ ID NO:31, and an HCDR3 comprising or consisting of SEQ ID NO:65; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:103, an LCDR2 comprising or consisting of SEQ ID NO:133, and an LCDR3 comprising or consisting of SEQ ID NO:155;

[0114] (9) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:8, an HCDR2 comprising or consisting of SEQ ID NO:32, and an HCDR3 comprising or consisting of SEQ ID NO:66; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:104, an LCDR2 comprising or consisting of SEQ ID NO:134, and an LCDR3 comprising or consisting of SEQ ID NO:158;

[0115] (10) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:9, an HCDR2 comprising or consisting of SEQ ID NO:33, and an HCDR3 comprising or consisting of SEQ ID NO:67; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:97, an LCDR2 comprising or consisting of SEQ ID NO:135, and an LCDR3 comprising or consisting of SEQ ID NO:159;

[0116] (11) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:7, an HCDR2 comprising or consisting of SEQ ID NO:34, and an HCDR3 comprising or consisting of SEQ ID NO:64; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:102, an LCDR2 comprising or consisting of SEQ ID NO:132, and an LCDR3 comprising or consisting of SEQ ID NO:160;

[0117] (12) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 10, an HCDR2 comprising or consisting of SEQ ID NO: 35, and an HCDR3 comprising or consisting of SEQ ID NO: 68; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 105, an LCDR2 comprising or consisting of SEQ ID NO: 136, and an LCDR3 comprising or consisting of SEQ ID NO: 161;

[0118] (13) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:2, an HCDR2 comprising or consisting of SEQ ID NO:25, and an HCDR3 comprising or consisting of SEQ ID NO:69; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:106, an LCDR2 comprising or consisting of SEQ ID NO:129, and an LCDR3 comprising or consisting of SEQ ID NO:162;

[0119] (14) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 11, an HCDR2 comprising or consisting of SEQ ID NO: 36, and an HCDR3 comprising or consisting of SEQ ID NO: 70; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 107, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 163;

[0120] (15) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 12, an HCDR2 comprising or consisting of SEQ ID NO: 37, and an HCDR3 comprising or consisting of SEQ ID NO: 71; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 108, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 164;

[0121] (16) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 1, an HCDR2 comprising or consisting of SEQ ID NO: 38, and an HCDR3 comprising or consisting of SEQ ID NO: 72; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 109, an LCDR2 comprising or consisting of SEQ ID NO: 138, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0122] (17) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 13, an HCDR2 comprising or consisting of SEQ ID NO: 39, and an HCDR3 comprising or consisting of SEQ ID NO: 73; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0123] (18) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 40, and an HCDR3 comprising or consisting of SEQ ID NO: 74; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 110, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 166;

[0124] (19) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 14, an HCDR2 comprising or consisting of SEQ ID NO: 41, and an HCDR3 comprising or consisting of SEQ ID NO: 75; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 111, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0125] (20) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 15, an HCDR2 comprising or consisting of SEQ ID NO: 42, and an HCDR3 comprising or consisting of SEQ ID NO: 74; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 139, and an LCDR3 comprising or consisting of SEQ ID NO: 152;

[0126] (21) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 14, an HCDR2 comprising or consisting of SEQ ID NO: 43, and an HCDR3 comprising or consisting of SEQ ID NO: 76; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 112, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0127] (22) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 16, an HCDR2 comprising or consisting of SEQ ID NO: 44, and an HCDR3 comprising or consisting of SEQ ID NO: 77; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 113, an LCDR2 comprising or consisting of SEQ ID NO: 140, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0128] (23) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 9, an HCDR2 comprising or consisting of SEQ ID NO: 45, and an HCDR3 comprising or consisting of SEQ ID NO: 78; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 114, an LCDR2 comprising or consisting of SEQ ID NO: 141, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0129] (24) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 46, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0130] (25) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 9, an HCDR2 comprising or consisting of SEQ ID NO: 25, and an HCDR3 comprising or consisting of SEQ ID NO: 80; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 115, an LCDR2 comprising or consisting of SEQ ID NO: 142, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0131] (26) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 41, and an HCDR3 comprising or consisting of SEQ ID NO: 81; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 116, an LCDR2 comprising or consisting of SEQ ID NO: 143, and an LCDR3 comprising or consisting of SEQ ID NO: 167;

[0132] (27) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:7, an HCDR2 comprising or consisting of SEQ ID NO:47, and an HCDR3 comprising or consisting of SEQ ID NO:82; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:117, an LCDR2 comprising or consisting of SEQ ID NO:144, and an LCDR3 comprising or consisting of SEQ ID NO:155;

[0133] (28) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO:2, an HCDR2 comprising or consisting of SEQ ID NO:41, and an HCDR3 comprising or consisting of SEQ ID NO:83; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:118, an LCDR2 comprising or consisting of SEQ ID NO:131, and an LCDR3 comprising or consisting of SEQ ID NO:168;

[0134] (29) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 18, an HCDR2 comprising or consisting of SEQ ID NO: 48, and an HCDR3 comprising or consisting of SEQ ID NO: 84; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 119, an LCDR2 comprising or consisting of SEQ ID NO: 145, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0135] (30) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 19, an HCDR2 comprising or consisting of SEQ ID NO: 49, and an HCDR3 comprising or consisting of SEQ ID NO: 85; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 120, an LCDR2 comprising or consisting of SEQ ID NO: 146, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0136] (31) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 50, and an HCDR3 comprising or consisting of SEQ ID NO: 86; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 121, an LCDR2 comprising or consisting of SEQ ID NO: 147, and an LCDR3 comprising or consisting of SEQ ID NO: 169;

[0137] (32) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 51, and an HCDR3 comprising or consisting of SEQ ID NO: 87; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 122, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0138] (33) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 20, an HCDR2 comprising or consisting of SEQ ID NO: 44, and an HCDR3 comprising or consisting of SEQ ID NO: 88; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 123, an LCDR2 comprising or consisting of SEQ ID NO: 148, and an LCDR3 comprising or consisting of SEQ ID NO: 170;

[0139] (34) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 3, an HCDR2 comprising or consisting of SEQ ID NO: 52, and an HCDR3 comprising or consisting of SEQ ID NO: 60; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 171;

[0140] (35) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 53, and an HCDR3 comprising or consisting of SEQ ID NO: 89; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 147, and an LCDR3 comprising or consisting of SEQ ID NO: 172;

[0141] (36) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 21, an HCDR2 comprising or consisting of SEQ ID NO: 38, and an HCDR3 comprising or consisting of SEQ ID NO: 90; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 109, an LCDR2 comprising or consisting of SEQ ID NO: 150, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0142] (37) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 22, an HCDR2 comprising or consisting of SEQ ID NO: 41, and an HCDR3 comprising or consisting of SEQ ID NO: 91; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 124, an LCDR2 comprising or consisting of SEQ ID NO: 151, and an LCDR3 comprising or consisting of SEQ ID NO: 173;

[0143] (38) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 54, and an HCDR3 comprising or consisting of SEQ ID NO: 92; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 126, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0144] (39) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 55, and an HCDR3 comprising or consisting of SEQ ID NO: 93; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 149, and an LCDR3 comprising or consisting of SEQ ID NO: 174;

[0145] (40) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 23, an HCDR2 comprising or consisting of SEQ ID NO: 56, and an HCDR3 comprising or consisting of SEQ ID NO: 94; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 125, an LCDR2 comprising or consisting of SEQ ID NO: 142, and an LCDR3 comprising or consisting of SEQ ID NO: 175;

[0146] (41) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 14, an HCDR2 comprising or consisting of SEQ ID NO: 43, and an HCDR3 comprising or consisting of SEQ ID NO: 76; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 127, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 176;

[0147] (42) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 3, an HCDR2 comprising or consisting of SEQ ID NO: 57, and an HCDR3 comprising or consisting of SEQ ID NO: 95; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 128, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0148] (43) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 46, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0149] (44) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 14, an HCDR2 comprising or consisting of SEQ ID NO: 310, and an HCDR3 comprising or consisting of SEQ ID NO: 315 (ERFLGGXMDV, where X can be any amino acid); and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0150] (45) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 311, and an HCDR3 comprising or consisting of SEQ ID NO: 316 (EAVAGPXMDV, where X can be any amino acid); and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0151] (46) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 310, and an HCDR3 comprising or consisting of SEQ ID NO: 317; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0152] (47) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0153] (48) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 46, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0154] (49) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 46, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0155] (50) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 51, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0156] (51) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 313, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0157] (52) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 51, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0158] (53) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 311, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0159] (54) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 314, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0160] (55) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 319, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0161] (56) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 320, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0162] (57) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 321, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0163] (58) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 322, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0164] (59) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 99, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0165] (60) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0166] (61) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 323, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0167] (62) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0168] (63) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0169] (64) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0170] (65) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 312, and an HCDR3 comprising or consisting of SEQ ID NO: 318; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155;

[0171] (66) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 346, and an HCDR3 comprising or consisting of SEQ ID NO: 350; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0172] (67) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 347, and an HCDR3 comprising or consisting of SEQ ID NO: 351; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 357, and an LCDR3 comprising or consisting of SEQ ID NO: 165;

[0173] (68) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 9, an HCDR2 comprising or consisting of SEQ ID NO: 348, and an HCDR3 comprising or consisting of SEQ ID NO: 352; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 358, and an LCDR3 comprising or consisting of SEQ ID NO: 360;

[0174] (69) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 310, and an HCDR3 comprising or consisting of SEQ ID NO: 353; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 355, an LCDR2 comprising or consisting of SEQ ID NO: 359, and an LCDR3 comprising or consisting of SEQ ID NO: 152;

[0175] (70) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 345, an HCDR2 comprising or consisting of SEQ ID NO: 349, and an HCDR3 comprising or consisting of SEQ ID NO: 354; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 356, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155; and / or

[0176] (71) The immunoglobulin heavy and light chain polypeptides comprise any combination of CDRs listed in Table 1.

[0177] In certain embodiments, the binding agent comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, wherein the immunoglobulin heavy chain polypeptide comprises a first framework region, a second framework region, a third framework region, and / or a fourth framework region; and / or the immunoglobulin light chain polypeptide comprises a first framework region, a second framework region, a third framework region, and / or a fourth framework region; and / or the immunoglobulin heavy chain polypeptide and light chain polypeptide comprise any combination of the framework regions listed in Tables 2 and 3.

[0178] [Table 1-1]

[0179] [Table 1-2]

[0180] [Table 1-3]

[0181] [Table 1-4]

[0182] [Table 1-5]

[0183] [Table 1-6]

[0184] [Table 1-7]

[0185] Table 2-1

[0186] Table 2-2

[0187] Table 2-3

[0188] Table 2-4

[0189] Table 2-5

[0190] Table 2-6

[0191] Table 2-7

[0192] Table 3-1

[0193] Table 3-2

[0194] Table 3-3

[0195] Table 3-4

[0196] Table 3-5

[0197] Table 3-6

[0198] Table 4-1

[0199] Table 4-2

[0200] Table 4-3

[0201] Table 4-4

[0202] Table 4-5

[0203] Table 4-6

[0204] Table 4-7

[0205] Table 4-8

[0206] [Table 4-9]

[0207] [Table 4-10]

[0208] [Table 4-11]

[0209] As mentioned above, a binding agent can comprise an Ig heavy and / or light chain variable region having at least about 90% (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identity to the specific heavy or light chain variable region sequences provided herein. Similarly, the CDRs of the Ig heavy and / or light chain variable regions can have at least about 90% (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identity to the specific CDR sequences provided herein. Thus, the Ig heavy and light chain variable region or CDR sequences can comprise at least one (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, etc., as appropriate based on the length of the sequence) amino acid modification (e.g., substitution, addition, or deletion) compared to the specific sequences provided herein, provided that the binding agent maintains the ability to specifically bind to PD-L1, and optionally, provided that the binding agent maintains the affinity of and / or competes with a binding agent having a particular sequence for binding to PD-L1.

[0210] Amino acids in the provided sequences can be substituted with any other amino acid. Amino acids include naturally occurring alpha amino acids and their stereoisomers, as well as non-naturally occurring amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to isomers that have the same molecular formula and intramolecular bond(s) but differ in the three-dimensional arrangement of bonds and atoms (e.g., L-amino acids and the corresponding D-amino acids). Amino acids can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glypiation) or deglycosylated.

[0211] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0212] Non-naturally occurring amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner similar to naturally occurring amino acids. For example, an "amino acid analog" may be an unnatural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon bonded to a hydrogen, a carboxyl group, and an amino group) but has modified side groups or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. An "amino acid mimetic" refers to a compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0213] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0214] Amino acid substitutions can be conservative, semi-conservative, or non-conservative with respect to the basic properties of the original amino acid residue. A "conservative" substitution refers to the replacement of one amino acid with another amino acid that shares common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). Such an analysis reveals that amino acids within a group preferentially substitute for each other, and thus allows the definition of groups of amino acids that are most similar to each other in their impact on the overall protein structure (Schulz and Schirmer, supra).

[0215] Amino acids are broadly grouped as "aromatic" or "aliphatic." Aromatic amino acids contain an aromatic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as "aliphatic." Examples of "aliphatic" amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine ​​(C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).

[0216] Aliphatic amino acids can be subdivided into four subgroups: the "large aliphatic nonpolar subgroup" consisting of valine, leucine, and isoleucine; the "aliphatic slightly polar subgroup" consisting of methionine, serine, threonine, and cysteine; the "aliphatic polar / charged subgroup" consisting of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine; and the "small residue subgroup" consisting of glycine and alanine. The group of charged / polar amino acids can be subdivided into three subgroups: the "positively charged subgroup" consisting of lysine and arginine, the "negatively charged subgroup" consisting of glutamic acid and aspartic acid, and the "polar subgroup" consisting of asparagine and glutamine.

[0217] The aromatic amino acids can be subdivided into two subgroups: the "nitrogen ring subgroup" consisting of histidine and tryptophan, and the "phenyl subgroup" consisting of phenylalanine and tyrosine.

[0218] Examples of conservative amino acid substitutions include substitutions of amino acids within the above subgroups, such as arginine for lysine and vice versa so that a positive charge can be maintained, aspartic acid for glutamic acid and vice versa so that a negative charge can be maintained, threonine for serine so that a free -OH can be maintained, and asparagine for glutamine so that a free -NH can be maintained.

[0219] "Semi-conservative mutations" include substitutions of amino acids within the same group as above, but do not include substitutions of amino acids within the same subgroup. For example, substitutions of aspartic acid with asparagine, or asparagine with lysine, involve amino acids within the same group but from different subgroups. "Non-conservative mutations" involve substitutions of amino acids between different groups, such as lysine for tryptophan or phenylalanine for serine.

[0220] Furthermore, one or more amino acids can be inserted into the aforementioned immunoglobulin heavy or light chain variable region polypeptides. Any number of any suitable amino acids can be inserted into the amino acid sequence of an immunoglobulin heavy or light chain variable region polypeptide. In this regard, at least one amino acid (e.g., two or more, five or more, or ten or more amino acids) and up to 20 amino acids (e.g., up to 18, 15 or 12 amino acids) can be inserted into the amino acid sequence of an immunoglobulin heavy or light chain variable region polypeptide. Preferably, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) are inserted into the amino acid sequence of an immunoglobulin heavy or light chain variable region polypeptide. In this regard, one or more amino acids can be inserted into any one of the aforementioned immunoglobulin heavy or light chain variable region polypeptides at any suitable position. In some embodiments, one or more amino acids are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of an immunoglobulin heavy or light chain variable region polypeptide; in other embodiments, the amino acids are inserted into a framework region.

[0221] Additionally provided are PD-L1-binding agents (e.g., antibodies or antibody fragments) that compete with a PD-L1-binding agent (e.g., an antibody or antibody fragment) having immunoglobulin heavy and light chain variable regions specifically provided herein (e.g., one of binding agents 1-42 provided herein).

[0222] The "biological activity" of a PD-L1-binding agent refers to, for example, binding affinity for PD-L1 or a particular PD-L1 epitope, neutralization or inhibition of PD-L1 protein binding to PD-L1, neutralization or inhibition of PD-L1 protein activity in vivo (e.g., IC 50 ), PD-L1 internalization, pharmacokinetics, and cross-reactivity (e.g., with non-human homologs or orthologs of the PD-L1 protein, or with other proteins or tissues). Other biological properties or characteristics of antigen-binding agents recognized in the art include, for example, avidity, selectivity, solubility, folding, immunotoxicity, expression, and formulation. The foregoing properties or characteristics can be observed, measured, and / or assessed using standard techniques, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™), or KINEXA™, in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and / or secretion assays, and signal transduction and immunohistochemistry assays.

[0223] The terms "inhibit" or "neutralize," as used herein with respect to the activity of a PD-L1-binding agent, refer to the ability to substantially antagonize, prohibit, prevent, suppress, slow, disrupt, alter, remove, halt, or reverse, for example, the biological activity of the PD-L1 protein, or the progression or severity of a disease or condition associated with the PD-L1 protein. The isolated PD-L1-binding agents of the invention inhibit or neutralize the activity of the PD-L1 protein by at least about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or a range defined by any two of the foregoing values. In other embodiments, the PD-L1-binding agent does not substantially inhibit the activity of the PD-L1 protein.

[0224] In some embodiments, the PD-L1-binding agent (e.g., an antibody or antibody fragment) exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a mechanism of cell-mediated immune defense in which effector cells of the immune system lyse target cells whose membrane surface antigens have been bound by specific antibodies. ADCC is independent of the immune complement system, which also lyses targets, but does not require any other cells, and is part of the adaptive immune response.

[0225] In some embodiments, the PD-L1-binding agent (e.g., an antibody or antibody fragment) promotes antibody-dependent cell-mediated phagocytosis (ADCP). ADCP is a cellular process by which phagocytic effector cells, such as monocytes and macrophages, can internalize target cells. Once engulfed, the target cell resides within a phagosome, which fuses with a lysosome, initiating target cell degradation through oxygen-dependent or -independent mechanisms. This function relies on opsonization, i.e., the identification of the target cell by the binding agent, which then acts as a bridge between the target cell and the phagocyte. The binding agent binds to its cognate antigen on the target cell through its antigen recognition domain and attracts phagocytes to the target via its Fc region. Once the target cell is bound to the Fc receptor on the phagocyte, it is ingested and degraded. This process also results in the production of soluble factors by effector cells that help initiate and drive the immune response.

[0226] In some embodiments, the PD-L1-binding agent (e.g., an antibody or antibody fragment) exhibits complement-dependent cytotoxicity (CDC). CDC is an effector function of IgG and IgM antibodies. Binding of the binding agent to a surface antigen triggers the classical complement pathway, leading to formation of the membrane attack complex (MAC) and lysis of the target cell.

[0227] In some embodiments, binding agents comprising an Fc region comprise one or more modifications in the Fc region (e.g., amino acid insertions, deletions, and / or substitutions) that result in modulated binding (e.g., increased or decreased binding to one or more Fc receptors (e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), and / or FcγRIIIB (CD16b)) compared to a binding agent or antibody having a native Fc region lacking the mutations). In some embodiments, the binding agents comprise one or more modifications in the Fc region (e.g., amino acid insertions, deletions, and / or substitutions) that decrease binding of the Fc region to FcγRIIB. In some embodiments, the binding agents comprise one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region of the antibody that reduce binding to FcγRIIB while having the same or increased binding to FcγRI (CD64), FcγRIIA (CD32A), and / or FcRγIIIA (CD16a) compared to a binding agent or antibody having a native Fc region lacking the mutations. In some embodiments, the binding agents comprise one or more modifications in the Fc region that increase binding of the Fc region to FcγRIIB. In some embodiments, the modifications substantially reduce or eliminate antibody effector function.

[0228] Mutations in the Fc region are H 2 domains, C HThe Fc region may be one of the three domains, or a combination thereof. A "native Fc region" is synonymous with a "wild-type Fc region" and includes an amino acid sequence identical to that of an Fc region found in nature or that found in a native antibody. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions, native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native-sequence Fc includes various Fc allotypes (see, e.g., Jefferis et al., mAbs, 1(4), 332-338 (2009)).

[0229] In some embodiments, the mutations in the Fc region that result in modulated binding to one or more Fc receptors are one or more of the following mutations: SD(S239D), SDIE(S239D / I332E), SE(S267E), SELF(S267E / L328F), SDIE(S239D / I332E), SDIEAL(S239D / I332E / A330L), GA(G236A), ALIE(A330L / I333E), GA(G236B), GA(G236C), GA(G236D), GA(G236E), GA(G236F ... 2E), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328, and A330. Additional Fc region modifications for modulating Fc receptor binding are described, for example, in U.S. Patent Application Publication No. 2016 / 0145350 and U.S. Patent Nos. 7,416,726 and 5,624,821, which are incorporated by reference in their entireties.

[0230] In some embodiments, the Fc region of the binding agent is modified to alter the glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0231] Human immunoglobulins are glycosylated at the Asn297 residue in the Cγ2 domain of each heavy chain. This N-linked oligosaccharide consists of a core heptasaccharide: N-acetylglucosamine 4 mannose 3 (GlcNAc4Man3). Removal of this heptasaccharide by endoglycosidases or PNGase F causes structural changes in the Fc region, significantly reducing binding affinity to activating FcγRs and potentially leading to impaired effector function. The core heptasaccharide is often decorated with galactose, bisecting GlcNAc, fucose, or sialic acid, which differentially influence Fc binding to activating and inhibitory FcγRs. Furthermore, α2,6-sialylation has been shown to enhance anti-inflammatory activity in vivo, while defucosylation improves FcγRIIIa binding and leads to a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Therefore, specific glycosylation patterns can be used to control inflammatory effector functions.

[0232] In some embodiments, the modification to alter the glycosylation pattern is a mutation, e.g., a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods of regulating immune responses using antibodies that modulate FcγR regulatory signaling are described, for example, in U.S. Pat. No. 7,416,726 and U.S. Patent Application Publication Nos. 2007 / 0014795 and 2008 / 0286819, which are incorporated by reference in their entireties.

[0233] In some embodiments, binding agents are modified to contain an engineered Fab region with a non-naturally occurring glycosylation pattern. For example, hybridomas can be engineered to secrete an afucosylated mAb, a desialylated mAb, or a deglycosylated Fc with specific mutations that allow for increased FcRγIIIa binding and effector function. In some embodiments, binding agents are engineered to be afucosylated.

[0234] In some embodiments, the entire Fc region is exchanged for a different Fc region, and the Fab region is conjugated to a non-native Fc region. For example, the Fab region of atezolizumab, which normally contains an IgG1 Fc region, can be conjugated to IgG2, IgG3, IgG4, or IgA, or the Fab region of nivolumab, which normally contains an IgG4 Fc region, can be conjugated to IgG1, IgG2, IgG3, IgA1, or IgG2. In some embodiments, Fc-modified binding agents with a non-native Fc domain contain one or more additional amino acid modifications that modulate the stability of the described Fc domain, such as the S228P mutation in the IgG4 Fc. In some embodiments, Fc-modified binding agents with a non-native Fc domain also contain one or more amino acid modifications described herein that modulate Fc binding to FcR.

[0235] In some embodiments, modifications that modulate binding of the Fc region to an FcR do not alter binding of the Fab region to its antigen compared to an unmodified Fab, while in other embodiments, modifications that modulate binding of the Fc region to an FcR also increase binding of the Fab region to its antigen compared to an unmodified Fab.

[0236] In some embodiments, the Fc region is modified by attaching or including a transforming growth factor beta 1 (TGFβ1) receptor, or a fragment thereof, capable of binding TGFβ1. For example, the receptor can be TGFβ receptor II (TGFβRII) (see U.S. Patent No. 9,676,863, incorporated herein in its entirety). In some embodiments, the TGFβ receptor is a human TGFβ receptor. In some embodiments, the Fc region (e.g., IgG) has a C-terminal fusion with the extracellular domain (ECD; e.g., amino acids 24-159 of SEQ ID NO: 9 in U.S. Patent No. 9,676,863) of a TGFβ receptor (e.g., TGFβRII). An "Fc linker," e.g., a G4S4G Fc linker, may be used to attach the IgG to the TGFβR extracellular domain. The Fc linker may be a short, flexible peptide that allows for proper three-dimensional folding of the molecule while maintaining binding specificity for the target. In some embodiments, the N-terminus of the TGFβ receptor is fused to an Fc region (with or without an Fc linker). In some embodiments, the C-terminus of an immunoglobulin heavy chain is fused to a TGFβ receptor (with or without an Fc linker). In some embodiments, the C-terminal lysine residue of the antibody heavy chain is mutated to alanine. In some embodiments, the antibody comprises SEQ ID NO: 308.

[0237] A PD-L1 binding agent can have any suitable affinity for the PD-L1 protein or its epitope. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents, and is also referred to as the dissociation constant (K D). The affinity of the binding agent for its ligand, like the affinity of an antibody for its epitope, can be, for example, about 1 picomolar (pM) to about 100 micromolar (uM) (e.g., about 1 picomolar (pM) to about 1 nanomolar (nM), about 1 nM to about 1 micromolar (uM), or about 1 uM to about 100 uM). In one embodiment, the PD-L1-binding agent can bind to PD-L1 protein with a KD of 1 nanomolar or less (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values). In another embodiment, the PD-L1-binding agent may bind to PD-L1 with a KD of 200 pM or less (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values). Immunoglobulin affinity for an antigen or epitope of interest can be measured using any art-recognized assay. Such methods include, for example, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface plasmon resonance (SPR), solution phase competition (KINEXA™), antigen panning, and / or ELISA (see Janeway et al. (eds.), Immunobiology, 9th Ed., Garland Publishing, New York, NY (2017)).

[0238] nucleic acid The invention also provides nucleic acids encoding the immunoglobulin heavy chain polypeptides and / or immunoglobulin light chain polypeptides of the PD-L1-binding agents.

[0239] The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which can be single- or double-stranded, and can include non-naturally occurring or altered nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymers of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms include, by way of equivalents, analogs of either RNA or DNA made from nucleotide analogs, and modified polynucleotides, such as methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate linkages to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (e.g., phosphorothioates, borane phosphate, etc.).

[0240] The nucleic acid may be part of a vector. Thus, vectors containing one or more nucleic acid sequences encoding the immunoglobulin heavy chain polypeptide, the immunoglobulin light chain polypeptide, or both of the PD-L1-binding agent are also provided. Any type of vector can be used, particularly an expression vector useful for expressing a polypeptide in a cell. The vector can be, for example, a plasmid, an episome, a cosmid, a viral vector (e.g., a retrovirus or adenovirus), or a phage. Suitable vectors and methods for vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0241] The vector preferably contains expression control sequences, such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc., that provide for expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0242] Numerous promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative sources of promoters include, for example, viral, mammalian, insect, plant, yeast, and bacterial, and suitable promoters from these sources are readily available or can be synthetically produced based on publicly available sequences, for example, from depositories such as the American Type Culture Collection (ATCC) and other commercial or private sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Examples of promoters include, but are not limited to, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93: 3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, CA), and the LACSWITC system (Invitrogen, Carlsbad, CA). H (trademark) system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Patent 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308: 123-144 (2005)).

[0243] The term "enhancer," as used herein, refers to a DNA sequence that, for example, increases the transcription of a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, changes in DNA methylation patterns, or changes in DNA structure. Many enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as the ATCC, as well as other commercial or private sources). Many polynucleotides that contain a promoter (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, internal, or downstream of a coding sequence.

[0244] A vector may also contain a "selectable marker gene." As used herein, the term "selectable marker gene" means a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected, either positively or negatively, in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publications WO 1992 / 008796 and WO 1994 / 028143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150: 1-14 (1981); Santerre et al., Gene, 30: 147-156 (1984); Kent et al., Science, 237: 901-903 (1987); Wigler et al., Cell, 11: 223-232 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026-2034 (1962); Lowy et al., Cell, 22: 817-823 (1980); and U.S. Patents 5,122,464 and 5,770,359.

[0245] In some embodiments, a vector is an "episomal expression vector" or "episome," which is capable of replicating in a host cell and persists as an extrachromosomal segment of DNA within the host cell under appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize the Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of episomal vectors that use T-antigen and the SV40 origin of replication in place of EBNA1 and orip.

[0246] Other suitable vectors include integrative expression vectors, which may integrate randomly into the DNA of a host cell or may contain recombination sites to allow specific recombination between the expression vector and the host cell chromosome. Such integrative expression vectors can utilize endogenous expression control sequences of the host cell chromosome to effect expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, components of the flp-in system (e.g., pcDNA™5 / FRT) from Invitrogen (Carlsbad, CA) or the cre-lox system, such as can be found in the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that integrate randomly into the host cell chromosome include, for example, pcDNA3.1 (when introduced in the absence of T-antigen) from Invitrogen (Carlsbad, CA), UCOE from Millipore (Billerica, MA), and pCI or pFN10A(ACT)FLEXI from Promega (Madison, WI). TM (trademark).

[0247] Viral vectors can also be used. Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentivirus-based pLP1 from Invitrogen (Carlsbad, CA), and the retroviral vectors pFB-RV plus pCFB-EGSH from Stratagene (La Jolla, CA). cell

[0248] Nucleic acid sequences encoding heavy and light chain immunoglobulin sequences can be provided to cells on the same vector (i.e., in cis). A unidirectional promoter can be used to control the expression of each nucleic acid sequence. In another embodiment, a combination of bidirectional and unidirectional promoters can be used to control the expression of multiple nucleic acid sequences. Nucleic acid sequences encoding the amino acid sequences of the invention can alternatively be provided to a population of cells on separate vectors (i.e., in trans). Each nucleic acid sequence on each separate vector can contain the same or different expression control sequences. The separate vectors can be provided to cells simultaneously or sequentially.

[0249] The vector(s) containing the nucleic acid(s) encoding the amino acid sequences of the invention can be introduced into a host cell capable of expressing the encoded polypeptides, including any suitable prokaryotic or eukaryotic cell. Thus, the invention provides in vitro (isolated) cells or cell lines containing the vectors of the invention that express immunoglobulin heavy and light chain polypeptides. Preferred host cells are those that can be grown easily and reliably, have reasonably fast growth rates, possess well-characterized expression systems, and can be easily and efficiently transformed or transfected.

[0250] Examples of suitable prokaryotic cells include, but are not limited to, cells of the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of E. coli (e.g., K12, HB101 (ATCC No. 33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102).

[0251] Preferably, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerevisiae and Pichia pastoris.

[0252] Suitable insect cells are described, for example, in Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67:4566-4579 (1993). Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, CA).

[0253] Preferably, mammalian cells are utilized in the present invention. Numerous suitable mammalian host cells are known in the art, and many are available from the ATCC. Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells, such as CHO-K1 cells (ATCC No. CCL61), CHODHFR- cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), JIMT-1, and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro culture of primary tissue, and primary culture explants are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from the American Type Culture Collection (ATCC). Methods for selecting appropriate mammalian host cells, and for transforming, culturing, amplifying, screening, and purifying the cells, are known in the art.

[0254] The mammalian cells may be human cells. For example, the mammalian cells may be lymphocyte-derived cell lines, such as cell lines derived from human lymphocytes or pre-B lymphocytes. Examples of human lymphoid cell lines include, but are not limited to, RAMOS (CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al., Proc. Natl. Acad. Sci. USA, 85: 1581-1585 (1988)), Raji cells (CCL-86), and derivatives thereof.

[0255] Nucleic acid sequences encoding the amino acid sequences of the present invention can be introduced into cells by "transfection," "transformation," or "transduction." As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Many transfection techniques are known in the art, including calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after propagation of infectious particles in suitable packaging cells, many of which are commercially available. composition

[0256] The present invention provides compositions comprising a PD-L1-binding agent or a nucleic acid(s) encoding same, optionally in a vector. Preferably, the compositions are pharmaceutically acceptable (e.g., physiologically acceptable) compositions comprising a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the PD-L1-binding agent or a nucleic acid(s) encoding same. Any suitable carrier can be used within the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. Optionally, the compositions can be sterile. The compositions can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be made according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0257] The compositions can be formulated for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ. Alternatively, the compositions can be injected intratumorally. Injectable compositions generally contain the active ingredient dissolved or suspended in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed include isotonic solutions of water and one or more salts, such as sodium chloride, e.g., Ringer's solution. Additionally, sterile, fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland, fixed oil, including synthetic monoglycerides or diglycerides, can be employed. Additionally, fatty acids, such as oleic acid, can also be used in the preparation of injectables. These compositions are desirably sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to achieve approximately physiological conditions, such as pH adjusting agents and buffers, and toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0258] The compositions can contain any suitable concentration of the PD-L1-binding agent or nucleic acid(s) encoding same, optionally in a vector, and in some embodiments, at a concentration effective to elicit a therapeutic response. The concentration can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the patient. In certain embodiments, the concentration of the immunoglobulin heavy chain polypeptides of the invention, immunoglobulin light chain polypeptides of the invention, PD-L1-binding agents of the invention, nucleic acid sequences of the invention encoding any of the foregoing, or vectors of the invention comprising nucleic acid sequences of the invention in an injectable solution formulation will range from about 0.1% (w / w) to about 10% (w / w). method

[0259] The PD-L1-binding agents provided herein can be used for any suitable purpose. For example, the PD-L1-binding agents can be used to target PD-L1-expressing cells and deliver a payload to such cells. The payload can be any agent (e.g., a therapeutic agent, a diagnostic / imaging agent, etc.) that is desired to be delivered to the cells. Accordingly, one aspect of the present disclosure provides a method of delivering a payload to cells that express PD-L1, the method comprising administering to the cells or comprises administering to a mammal containing the cells a PD-L1-binding agent provided herein conjugated to a payload.

[0260] Provided herein are methods of enhancing (e.g., increasing) the immune response of a mammal, comprising administering to the mammal a PD-L1-binding agent described herein alone or conjugated to a suitable agent or other agent suitable for enhancing an immune response, or a composition comprising same. The immune response can be an immune response to any type of antigen, for use in any application in which it is desirable to enhance the immune response to the antigen. In some embodiments, the antigen can be an infectious agent or pathogen (e.g., a virus or bacterium) or a cancer antigen (e.g., an endogenous cancer antigen within a cell, an administered antigen such as a peptide vaccine, or a neoantigen).

[0261] In another embodiment, there is provided a method of reducing (e.g., inhibiting) an immune response in a mammal comprising administering to the mammal a PD-L1-binding agent described herein alone or conjugated to a suitable agent or other agent suitable for reducing an immune response, or a composition comprising the same. The immune response can be, for example, an autoimmune response.

[0262] In this regard, the invention also provides methods of treating a disease, condition, or disorder responsive to PD-L1 inhibition in a mammal by administering to the mammal a PD-L1-binding agent as described herein, or a composition comprising same. In some embodiments, the disease or disorder is characterized by inappropriate expression (e.g., overexpression) or increased activity of PD-L1 protein, which causes or contributes to the pathological effects of the disease, and in which reduction of PD-L1 protein levels or activity has a therapeutic effect in a mammal, preferably a human.

[0263] As used herein, the terms "treat," "treatment," and "treating" refer to any indication of successful treatment or amelioration of an injury, pathology, condition (e.g., cancer, infection, or autoimmune disorder), or symptom (e.g., cognitive impairment), including any objective or subjective parameter, such as relief, remission, reduction of symptoms or making the symptom, injury, pathology, or condition more tolerable to the patient, reducing the rate of progression of a symptom, reducing the frequency or duration of a symptom or condition, or, in some circumstances, preventing the onset of a symptom. Treatment or amelioration of a symptom can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0264] The terms "cancer," "neoplasia," and "tumor" are used herein to refer to cells that exhibit autonomous, uncontrolled growth, such that the cells exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, and / or treatment in the context of the present invention include cancer cells (e.g., cancer cells from an individual with cancer), malignant cancer cells, premetastatic cancer cells, metastatic cancer cells, and nonmetastatic cancer cells. Cancer of almost any tissue is known. The term "cancer burden" refers to the amount of cancer cells or cancer volume in a subject. Reducing cancer burden therefore refers to reducing the number of cancer cells or the volume of cancer cells in a subject. As used herein, the term "cancer cell" refers to either a cancer cell (e.g., isolated from an individual with cancer, e.g., from any cancer for which an individual can be treated) or a cell derived from a cancer cell, e.g., a clone of a cancer cell. For example, a cancer cell can be derived from an established cancer cell line, a primary cell isolated from an individual with cancer, a progeny cell from a primary cell isolated from an individual with cancer, etc. In some embodiments, the term can also refer to a portion of a cancer cell, such as an intracellular portion of the cancer cell, a cell membrane portion, or a cell lysate of the cancer cell. Many types of cancer are known to those skilled in the art, and include solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, as well as circulating cancers such as leukemias.

[0265] As used herein, the term "cancer" includes any form of cancer, including, but not limited to, solid tumor cancers (e.g., skin, lung, prostate, breast, stomach, bladder, colon, ovarian, pancreatic, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinoma, melanoma, and neuroendocrine) and liquid cancers (e.g., blood cancers); carcinoma; soft tissue tumors; sarcoma; teratoma; melanoma; leukemia; lymphoma; and brain tumors, including minimal residual disease, and including both primary and metastatic tumors.

[0266] Any PD-L1 expressing or overexpressing cancer is a suitable cancer to be treated by the subject methods and compositions. As used herein, "PD-L1 expression" refers to cells that have PD-L1 receptors on their cell surface. As used herein, "PD-L1 overexpression" refers to cells that have more PD-L1 receptors than corresponding non-cancerous cells of the same type.

[0267] As used herein, an "internalizing binding agent" refers to a binding agent that, upon binding to a receptor or other ligand on the cell surface, is transported inside the cell (e.g., to a vacuole or other organelle, or into the cytoplasm of the cell). In some embodiments, the internalizing binding agent is an internalizing antibody.

[0268] Carcinomas are malignant tumors that originate in epithelial tissue. Epithelial cells cover the body's exterior surfaces, line internal cavities, and form the lining of glandular tissue. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancers derived from glandular (secretory) cells, such as breast cancer, pancreatic cancer, lung cancer, prostate cancer, stomach cancer, gastroesophageal junction cancer, and colon cancer), adrenocortical carcinoma, hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, carcinoma in situ, ductal carcinoma, breast cancer, basal cell carcinoma, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), transitional cell carcinoma, colon cancer, nasopharyngeal carcinoma, multinodular cystic renal cell carcinoma, oat cell carcinoma, large cell lung carcinoma, small cell lung carcinoma, and non-small cell lung carcinoma. Cancers can be found in the prostate, pancreas, colon, brain (usually as secondary metastases), lung, breast, and skin.

[0269] Soft tissue tumors are a highly diverse group of rare tumors derived from connective tissue. Examples of soft tissue tumors include alveolar soft part sarcoma, angiomatoid fibrous histiocytoma, chondroid fibroma, skeletal chondrosarcoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, desmoplastic small round cell tumor, dermatofibrosarcoma protuberans, endometrial stromal tumor, Ewing's sarcoma, fibromatosis (desmoid), infantile fibrosarcoma, gastrointestinal stromal tumor, giant cell tumor of bone, tenosynovial giant cell tumor, inflammatory myofibroblastoma, uterine leiomyoma, leiomyosarcoma, lipoblastoma, typical lipoma, spindle cell or pleomorphic lipoma, atypical lipoma, chondroid lipoma, well-differentiated liposarcoma, myxoid / round cell liposarcoma, and pleomorphic These include, but are not limited to, liposarcoma; myxoid malignant fibrous histiocytoma; high-grade malignant fibrous histiocytoma; myxofibrosarcoma, malignant peripheral nerve sheath tumor, mesothelioma, neuroblastoma, osteochondroma, osteosarcoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma; benign or malignant nerve sheath tumor; synovial sarcoma; Evan's tumor; nodular fasciitis; desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcoma protuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma; tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis (PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovial sarcoma; malignant peripheral nerve sheath tumor; neurofibroma; pleomorphic adenoma of soft tissue; and neoplasms derived from fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelial cells, and nerve sheath cells.

[0270] Sarcoma is a rare type of cancer that arises in the soft tissues of the body, including cells of mesenchymal origin, such as bone or cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissue. Different types of sarcoma are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examples of sarcomas include, but are not limited to, Askin tumor; botulinum sarcoma; chondrosarcoma; Ewing's sarcoma; malignant hemangioendothelioma; malignant sheath tumor; osteosarcoma; and soft tissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma; cystosarcoma phyllodes; dermatofibrosarcoma protuberans (DFSP); desmoid tumor; desmoplastic small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; hemangiosarcoma (more commonly called "angiosarcoma"); Kaposi's sarcoma; leiomyosarcoma; liposarcoma; lymphosarcoma; malignant peripheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma).

[0271] Teratomas are a type of germ cell tumor that can involve several different types of tissue (e.g., tissue derived from any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas most commonly occur in the ovaries of women, the testicles of men, and the coccyx of children.

[0272] Melanoma is a form of cancer that originates in melanocytes (cells that produce the pigment melanin). Melanoma can begin in a mole (cutaneous melanoma) or in other pigmented tissues, such as the eye or intestine.

[0273] Merkel cell carcinoma is a rare type of skin cancer that typically appears as a flesh-colored or bluish-red nodule. It commonly occurs on the face, head, and neck. Merkel cell carcinoma is also called neuroendocrine carcinoma of the skin. In some embodiments, the Merkel cell carcinoma has metastasized when administration begins.

[0274] Leukemia is a cancer that begins in blood-forming tissues, such as the bone marrow, and results in the production of large numbers of abnormal blood cells that enter the bloodstream. For example, leukemia can arise from bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named based on how quickly the disease develops and progresses (e.g., acute vs. chronic) and the type of white blood cell involved (e.g., myeloid vs. lymphocytic). Myeloid leukemia is also called myeloid leukemia or myeloblastic leukemia. Lymphoid leukemia is also called lymphoblastic leukemia or lymphocytic leukemia. Lymphoid leukemia cells can collect in lymph nodes, causing them to swell. Examples of leukemia include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0275] Lymphoma is a cancer that begins in cells of the immune system. For example, lymphoma can arise from cells derived from the bone marrow that normally mature in the lymphatic system. There are two basic categories of lymphoma. One category of lymphoma is Hodgkin lymphoma (HL), which is characterized by the presence of a type of cell called Reed-Sternberg cell. Currently, six types of HL are recognized. Examples of Hodgkin lymphoma include nodular sclerosing classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-depleted CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0276] Another category of lymphoma is non-Hodgkin's lymphoma (NHL), which includes a large and diverse group of cancers of immune system cells. Non-Hodgkin's lymphomas can be further divided into indolent (slow-growing) and aggressive (fast-growing) cancers. Currently, 61 types of non-Hodgkin's lymphoma are recognized. Examples of non-Hodgkin's lymphomas include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathic T-cell lymphoma, follicular lymphoma, hepatosplenic gamma delta T-cell lymphoma, T-cell leukemia, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, childhood lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, therapy-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0277] Brain tumors include any cancer of brain tissue, including, but not limited to, gliomas (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymoma, etc.), meningiomas, pituitary adenomas, vestibular schwannomas, and primitive neuroectodermal tumors (medulloblastomas).

[0278] The "pathology" of cancer includes all phenomena that impair the well-being of the patient, including, but not limited to, abnormal or uncontrolled cell proliferation, metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretions, suppression or exacerbation of inflammatory or immune responses, neoplasia, premalignancy, malignancy, and invasion of surrounding or distant tissues or organs such as lymph nodes.

[0279] As used herein, the terms "cancer recurrence" and "tumor recurrence," and grammatical variations thereof, refer to the further growth of neoplastic or cancerous cells after a cancer diagnosis. In particular, recurrence can occur when additional cancerous cell proliferation occurs in cancerous tissue. "Tumor spread" similarly occurs when tumor cells spread to local or distant tissues or organs, and thus includes tumor metastasis. "Tumor invasion" occurs when tumor growth spreads locally and impairs the function of the involved tissue by compressing, destroying, or preventing normal organ function.

[0280] As used herein, the term "metastasis" refers to the growth of a cancerous tumor in an organ or body part that is not directly connected to the organ of the original cancerous tumor. Metastasis is understood to include micrometastasis, which is the presence of undetectable amounts of cancer cells in an organ or body part that is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several stages of a process such as the detachment of cancer cells from the original tumor site and the migration and / or invasion of cancer cells to other parts of the body.

[0281] As used herein, the phrase "autoimmune disease" refers to a disease or disorder caused by a reaction produced by the body against its own tissues or organs, or the co-occurrence or manifestation of these disorders, or a condition related thereto. Examples of autoimmune diseases or disorders include arthritis (including acute arthritis, rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis, juvenile rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, primary chronic arthritis, reactive arthritis, and rheumatoid arthritis, such as ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis, such as plaque psoriasis, liquid psoriasis, pustular psoriasis, and nail psoriasis; contact dermatitis, chronic contact psoriasis, and the like. Dermatitis, including dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis (conjugated with X-linked hyper-IgM syndrome); urticaria, such as chronic allergic urticaria and chronic idiopathic urticaria (including chronic autoimmune urticaria); multiple sclerosis (MS), including primary progressive MS (APP), which involves destruction of the spinal cord and organs of vision; and) relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple sclerosis, ataxic sclerosis; inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune gastroenteropathy, ulcerative colitis, ulcerative colitis, microscopic colitis, collagen disease colitis, polypoid colitis, necrotic colitis, enteritis, and other colitis, and autoimmune inflammatory bowel disease);Pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), respiratory distress syndromes including adult respiratory distress syndrome (ARDS) or acute respiratory distress syndrome, meningitis, inflammation of all or part of the uveal tract, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis, allergic rhinitis, atopic rhinitis, encephalitis such as Rasmussen's encephalitis and lesional limb and / or brainstem encephalitis, anterior uveitis, acute anterior uveitis, granulocytic uveitis, granulomatous Chronic or acute glomerulonephritis (GN) with or without nephrotic syndrome, e.g., primary GN, immune-mediated GN, membranous GN (membranous nephropathy), glomerulonephrit type I and type II, and rapidly progressive GN, including idiopathic membranous GN or idiopathic membranous nephropathy, membranoproliferative or membranoproliferative GN (MPGN), allergic conditions, allergic reactions, eczema, including allergic or atopic eczema; asthma, e.g., bronchial asthma and autoimmune asthma; diseases caused by T-cell infiltration or chronic inflammatory responses; chronic inflammatory lung diseases, autoimmune myocarditis, and insufficient leukocyte adhesion; systemic lupus erythematosus (SLE), e.g., cutaneous SLE or subacute cutaneous SLE; erythematodes), lupus neonatal syndrome (HRV), disseminated lupus erythematosus, lupus (including nephritis, encephalitis, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus, and alopecia); juvenile diabetes mellitus (type 1) including childhood insulin-dependent diabetes mellitus (IDDM), adult diabetes mellitus (type 2 diabetes), autoimmune diabetes, idiopathic diabetes insipidus, cytokine- and T-lymphocyte-mediated acute and delayed hypersensitivity-related immune responses; tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosis, and granulomatous diseases including agranulocytosis;Vasculitis, including vasculitis, large vessel vasculitis (including polymyalgia rheumatica, giant cell arteritis (Takayasu's)), secondary vasculitis (including Kavazaki disease, polyarteritis nodosa), capillary polyarteritis, CNS necrotic vasculitis, cutaneous vasculitis, or vasculitis-associated hypersensitivity syndrome, necrotic systemic vasculitis and vasculitis, or Cherga-Strauss syndrome (SC; HANCA-associated vasculitis such as ANCA-associated vasculitis (ANCA-associated vasculitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, a-positive anemia, Coombs' disease, hemolytic anemia or immune hemolytic anemia including Diamond-Blakemann's disease, autoimmune hemolytic anemia (AIHA), pernicious anemia, Addison's disease, pure red cell anemia or aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases resulting in leukocyte detachment, central nervous system inflammatory diseases, multiple organ injury syndromes such as secondary syndromes, dissociated ass with sepsis, trauma, or hemorrhage; diseases mediated by complex formation "antigen-antibody" disease glomerular basement membrane disease membranes), antibody-antigen catalytic reactions, antiphospholipid syndrome, allergic neuritic disease / Behcet's syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigus such as bullous pemphigus and cutaneous pemphigus, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus, mucosal pemphigus, and pemphigus erythematous), utoimmunnaya poliendokrinopatiya disease or Reiter's syndrome, chronic neurological disorders such as immune complex-associated nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathy, IgM polyneuropathy, or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP) (including chrome-lytic or acute ITP), thrombocytopenia (e.g., occurring in patients with myocardial infarction);Autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis; primary hypothyroidism, thyroiditis such as gipoparathyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis; endocrine disorders, including autoimmune thyroid diseases; idiopathic hypothyroidism, Grave's disease, polyglandular syndromes, such as autoimmune polyglandular syndrome (or polyglandular endocrinopathy syndrome); myasthenia gravis syndrome, paraneoplastic symptoms including neurological paraneoplastic syndromes such as Lambert-Eaton syndrome or Eaton-Lambert syndrome, "stiff-man" syndrome, encephalomyelitis such as allergic encephalomyelitis (or allergic encephalomyelitis) and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as myasthenia gravis associated with thymoma, cerebellar degeneration, nebromyotonia, opsoclonus or syndrome (LNG) and neurosensory symptoms, multifocal motor neuropathy, Weiss syndrome system We), Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonia, obstructive bronchitis (non-communicable unlike NSIP); Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonia, autoimmune enteropathy syndrome, enteropathy or celiac disease, intestinal sprue (gluten enteropathy), unamenable sprue treatment, idiopathic sprue, cryoglobulinemia, Amylofiszewski's lateral sclerosis (ALS; Lou Gehrig's disease), coronary heart disease; autoimmune ear diseases such as autoimmune labyrinthitis (AZVU); autoimmune hearing loss; "Dancing Polychondritides, such as "LNG" syndrome (LNG), refractory or relapsing polychondritis; primary lymphoproliferations, such as pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, and monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of unknown etiology, MGUS); peripheral neuropathy, paraneoplastic syndromes;Epilepsy, migraine, cardiac arrhythmias, muscle disorders, hearing loss, blindness, periodic paralysis and "channelopathies" of the CNS, autism, inflammatory myopathy, "channelopathies" such as focal segmental glomerulosclerosis (OSGS), endocrine ophthalmopathy, uveitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine deficiency, Schmidt's syndrome, adrenal gland (adrenalitis), gastric atrophy, presenile dementia, autoimmune demyelinating diseases, diabetic nephrosis, Dressler's syndrome, alopecia areata, demyelinating diseases such as CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, ankylosis, telangiectasia), autoimmune infertility (male and female), mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent miscarriage, lung disease farmers), erythema multiforme, myocardial infarction syndrome, Cushing's syndrome, lung diseases, bird lover's disease, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion-related diseases, leprosy, malaria, leishmaniasis, kipanosomiasis, schistosomiasis, ascaridosis, aspergillosis, Semptera syndrome, Caplan syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, resistant erythema, fetal erythroblastosis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, fly apoplexy, chondromic cycle, chondromic cycle and iridotsyklit Fuchs' cyclitis, Henoch-Schonlein purpura, infections caused by human immunodeficiency virus (HIV), infections caused by echoviruses; cardiomyopathy, Alzheimer's disease, infections caused by parvoviruses; infections caused by rubella virus; syndromes that develop after vaccination; hereditary infections caused by rubella virus; infections caused by Epstein-Barr virus;Mumps, Evans syndrome, autoimmune hypogonadism, Sydenham tremor, poststreptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes, choroiditis, giant cell polymyalgia, endocrine ophthalmopathy, pneumonia associated with chronic hypersensitivity syndrome, idiopathic keratoconjunctivitis, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, nephropathy characterized by minimal changes in renal tissue, benign hereditary diseases due to ischemia and rheumatoid arthritis, autoimmune diseases of the retina, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthous stomatitis, atherosclerotic diseases, aspermia, autoimmune Hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, fakoanafilakticheskaya endophthalmitis, allergic enterocolitis, nodular lepromatous erythema, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Haman-Rich syndrome, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, focal ileitis, leukopenia, infectious mononucleosis, rhabdomyositis, primary idiopathic miksidema, nephrosis, sympathetic ophthalmopathy, granulomatous orchitis, pancreatitis, acute multiple sclerosis, pyoderma gangrenosum, Quervain's thyroiditis Acquired spinal cord atrophy, antisperm infertility, nonmalignant thymoma, vitiligo, severe combined immunodeficiency (SCID) and Epstein-Barra virus diseases; acquired immunodeficiency syndrome (AIDS), parasitic diseases such as leishmaniasis, toxic shock syndrome, food poisoning, immune responses associated with T cell infiltration, defective leukocyte adhesion, cytokine- and T lymphocyte-mediated acute and delayed hypersensitivity, diseases associated with leukocyte leakage, multiple organ injury syndrome, and diseases mediated by antigen-antibody complex formation Glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopatia, orbititis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmopathy, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine glandular insufficiency, peripheral organ neuropathy, autoimmune polyendocrine syndrome type 1, idiopathic adult gipoparatireoiditis (IIV), alopecia universalis, congestive cardiomyopathy, epidermolysis bullosa acquisita (PBE), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis; rheumatoid sinusitis, frontal sinusitis, maxillary sinusitis or sphenoiditis;Eosinophilic diseases such as eosinophilic diseases, pulmonary infiltrative eosinophilic disease, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, allergic pulmonary aspergillosis, aspergillomas or eosinophil-containing granulomas; anaphylaxis, seronegative spondylitis, polyendocrine autoimmune diseases, sclerosing cholangitis, scleritis, episcleritis, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia in children with Wiskott-Aldrich syndrome, ataxia-vasodilation syndrome, autoimmune diseases associated with collagen diseases, rheumatism, neurological diseases, ischemia-reperfusion injury, hypotensive reactions, vascular insufficiency, vascular dysplasia, tissue damage, vascular ischemia, hyperalgesia, cerebral ischemia, diseases associated with angiogenesis, allergic diseases associated with hypersensitivity; These include, but are not limited to, diseases, glomerulonephritis, reperfusion injury, reperfusion injury of the myocardium and other tissues, skin diseases with an acute inflammatory component, acute purulent meningitis and other inflammatory diseases of the central nervous system, inflammatory diseases of the eye and orbit; syndromes associated with granulocyte transfusions; cytokine-induced toxicity, acute serous inflammation, chronic refractory inflammation, pyelitis, diabetic retinopathy, diabetic lesions of the aorta, peripheral arterial hyperplasia, peptic ulcer disease, and endometriosis.

[0282] As used herein, the term "infection" means a condition caused by an infectious agent, such as a virus, bacterium, fungus, or parasite.

[0283] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the dosage of a substance, such as a binding agent, that produces the therapeutic effect for which it is administered. The exact dosage will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman, The Pharmacological Basis of Therapeutics, 11 th Edition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22 nd Edition, (Pharmaceutical Press, London, 2012).

[0284] As used herein, the terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., a human) for whom diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is a human.

[0285] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal, or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or implantation of a sustained-release device, e.g., a mini-osmotic pump, into a subject.

[0286] The methods of the invention can also be used to treat any type of infectious disease (i.e., a disease or disorder caused by bacteria, viruses, fungi, or parasites). Examples of infectious diseases that can be treated by the methods of the invention include, but are not limited to, diseases caused by human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), influenza virus, dengue virus, hepatitis B virus (HBV, or hepatitis C virus (HCV)). Administration of a composition comprising an immunoglobulin heavy chain polypeptide of the invention, an immunoglobulin light chain polypeptide of the invention, a PD-L1-binding agent of the invention, a nucleic acid sequence of the invention encoding any of the foregoing, or a vector of the invention comprising a nucleic acid sequence of the invention induces an immune response against cancer or an infectious disease in a mammal. An "immune response" can involve, for example, antibody production and / or activation of immune effector cells (e.g., T cells).

[0287] The methods of the invention involve administering a "therapeutically effective amount" of a binding agent. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's medical condition, age, sex, and weight, as well as the ability of the binding agent to elicit a desired response in the individual. For example, a therapeutically effective amount of a binding agent of the invention may be sufficient to reduce the biological activity of PD-L1 protein in humans and / or to prevent cancer or other diseases. is an amount that enhances the immune response to infection.

[0288] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the invention include administering a "prophylactically effective amount" of a binding agent. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

[0289] Typical dosages can range, for example, from 1 pg / kg to 20 mg / kg of animal or human body weight, although dosages below or above this exemplary range are within the scope of the present invention. Daily parenteral dosages range from about 0.00001 μg / kg to about 20 mg / kg (e.g., about 0.001 μg / kg, about 0.1 μg / kg) of total body weight. , about 1 ug / kg, about 5 ug / kg, about 10 ug / kg, about 100 ug / kg, about 500 ug / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, or a range defined by any two of the foregoing values), preferably about 0.1 ug / kg to about 10 mg / kg (e.g., about 0.5 ug / kg, about 1 ug / kg, about 50 ug / kg, about 150 ug / kg, about 300 ug / kg, about 750 ug / kg, about 1.5 mg / kg, about 5 mg / kg, or a range defined by any two of the foregoing values), more ... The therapeutic or prophylactic effect can be monitored by periodic evaluation of the treated patient. For repeated administration over several days or longer, treatment can be repeated until a desired suppression of disease symptoms occurs, depending on the condition. However, other administration regimens may be useful and are within the scope of the present invention. The desired dosage can be delivered by a single bolus of the composition, by multiple boluses of the composition, or by continuous infusion of the composition.

[0290] An effective amount of an immunoglobulin heavy chain polypeptide of the invention, an immunoglobulin light chain polypeptide of the invention, a binding agent of the invention, a composition comprising a nucleic acid sequence of the invention encoding any of the foregoing, or a vector of the invention comprising a nucleic acid sequence of the invention can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Preferably, the composition is suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0291] Once administered to a mammal (e.g., a human), the biological activity of a binding agent of the invention can be measured by any suitable method known in the art. For example, biological activity can be assessed by determining the stability of a particular binding agent. In one embodiment of the present invention, the binding agent (e.g., an antibody) has an in vivo half-life of between about 30 minutes and 45 days (e.g., about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values). In another embodiment, the PD-L1-binding agent has an in vivo half-life of between about 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or a range defined by any two of the foregoing values). In another embodiment, the binding agent has an in vivo half-life of between about 10 days and about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values).

[0292] The PD-L1-binding agents of the invention can be administered alone or in combination with other agents, either as separate moieties or conjugates with the PD-L1-binding agent. For example, the binding agents can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein. In this regard, the binding agents can be used in conjunction with at least one other anti-cancer agent, including any chemotherapeutic agent known in the art, ionizing radiation, small molecule anti-cancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy, and adoptive T-cell transfer, immunoconjugates, or cytotoxic antibody-drug conjugates), and / or surgery. When the method of the invention treats an infection, the binding agent can be administered in combination with at least one antibacterial agent or at least one antiviral agent. In this regard, the antibacterial agent can be any suitable antibiotic known in the art. The antiviral agent may be any suitable type of vaccine that specifically targets a particular virus, such as a live attenuated vaccine, a subunit vaccine, a recombinant vector vaccine, and a small molecule antiviral therapy (e.g., viral replication inhibitors and nucleoside analogues).

[0293] In another embodiment, the binding agents of the invention can be administered in combination with other agents that inhibit innate and adaptive immune checkpoint pathways, for example, the binding agents of the invention can be administered in combination with agents that inhibit or antagonize the CTLA-4, TIM-3, LAG-3, CD47, CD24, and SIRPA pathways.

[0294] In addition to therapeutic applications, the binding agents described herein can be used in diagnostic or research applications. In this regard, the binding agents can be used in methods for diagnosing cancer or infectious diseases. Similarly, the binding agents can be used in assays for monitoring PD-L1 protein levels in subjects being tested for diseases or disorders associated with aberrant PD-L1 expression. Research applications include, for example, methods utilizing the binding agent and a label to detect PD-L1 protein in a sample, for example, in a human body fluid or in a cell or tissue extract. The binding agents can be used with or without modification, such as labeling by covalent or non-covalent attachment to a detectable structural moiety. For example, the detectable structural moiety can be a radioisotope (e.g., 3 H, 14 C. 32 P, 35 S, or 125 The detectable moiety may be a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase), or a prosthetic group. Any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable moiety may be employed in the context of the present invention (see, e.g., Hunter et al., Nature, 194:495-496 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Meth., 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982)).

[0295] PD-L1 protein levels can be measured using the binding agents of the invention by any suitable method known in the art. Such methods include, for example, immunohistochemistry, immunofluorescence, radioimmunoassay (RIA), and FACS. A normal or standard expression value for PD-L1 protein can be established using any suitable technique, for example, by combining a sample containing, or suspected of containing, PD-L1 polypeptide with a PD-L1-specific antibody under conditions suitable for the formation of an antigen-antibody complex. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, radioactive materials, etc. (See, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)). The amount of PD-L1 polypeptide expressed in the sample is then compared with the standard value.

[0296] kit The binding agent can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts along with instructions for performing the diagnostic assay. When the binding agent is labeled with an enzyme, the kit desirably includes substrates and cofactors required by the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), and the like, may be included in the kit. The relative amounts of the various reagents can be varied to provide concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. The reagents can be provided as dry powders (typically lyophilized) that include excipients that, upon dissolution, provide a reagent solution having the appropriate concentration.

[0297] Examples of Non-Limiting Aspects of the Disclosure Aspects, including embodiments of the invention described herein, may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the disclosure, numbered (1)-(32), are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any preceding or subsequent individually numbered aspect. This is intended to provide support for all combinations of such aspects, and is not limited to the combinations of aspects explicitly provided below.

[0298] (1) A programmed death-ligand 1 (PD-L1) binding agent comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide, wherein: the immunoglobulin heavy chain variable region polypeptide comprises a complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 1-23, 309, or 345, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 24-57, 310-314, or 346-349, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 58-95, 315-318, or 350-354; or The immunoglobulin light chain variable region polypeptide comprises a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 96-128, 319-323, 355, or 356, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 129-151 or 357-359, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 152-176 or 360.

[0299] (2) a PD-L1-binding agent comprising an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 223-264, 324-334, or 361-365, or at least the CDRs thereof; and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 265-306, 335-344, or 366-370, or at least the CDRs thereof.

[0300] (3) a PD-L1-binding agent comprising an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 223-264, 324-334, or 361-365, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 265-306, 335-344, or 366-370.

[0301] (4) The PD-L1-binding agent of any one of Aspects 1-3, comprising a heavy chain and a light chain immunoglobulin polypeptide of a PD-L1-binding agent in Table 1, or at least the CDRs thereof.

[0302] (5) The PD-L1-binding agent of any one of Aspects 1-4, wherein the binding agent is an antibody, antibody conjugate, or antigen-binding fragment thereof.

[0303] (6) The PD-L1-binding agent of Aspect 5, wherein the binding agent is an antibody fragment selected from F(ab')2, Fab', Fab, Fv, scFv, dsFv, dAb, and single-chain binding polypeptides.

[0304] (7) The PD-L1-binding agent of Aspect 5, wherein the binding agent is an antibody.

[0305] (8) The PD-L1-binding agent of any of Aspects 1-7, further comprising an immunoglobulin Fc region.

[0306] (9) The PD-L1-binding agent of Aspect 8, further comprising a transforming growth factor beta 1 (TGFβ1) receptor, or a fragment thereof that binds TGFβ1, linked to an Fc region.

[0307] (10) The PD-L1-binding agent of any one of aspects 7-9, wherein the antibody is an IgG, IgM, IgA, IgD, or IgE antibody.

[0308] (11) The PD-L1-binding agent of any one of aspects 7-9, wherein the antibody is an IgG antibody.

[0309] (12) The PD-L1-binding agent of any one of aspects 7-11, wherein the antibody exhibits antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0310] (13) The PD-L1-binding agent of any of Aspects 1-12, wherein the binding agent is part of a bispecific antibody, a chimeric antigen receptor, a chimeric T cell receptor, or a bispecific T cell engager.

[0311] (14) The PD-L1-binding agent of any one of Aspects 1-12, wherein the binding agent is a cell-internalizing binding agent.

[0312] (15) A nucleic acid encoding a heavy chain immunoglobulin polypeptide of the anti-PD-L1 binding agent of any one of aspects 1-14.

[0313] (16) A nucleic acid encoding a light chain immunoglobulin polypeptide of the anti-PD-L1 binding agent of any one of aspects 1-14.

[0314] (17) A nucleic acid encoding the heavy and light chain immunoglobulin polypeptides of the PD-L1-binding agent of any one of aspects 1-14.

[0315] (18) A vector comprising the nucleic acid sequence of any one of aspects 15-17.

[0316] (19) An isolated cell comprising the nucleic acid of any one of aspects 15-17, optionally in a vector.

[0317] (20) A method of providing a PD-L1-binding agent according to any of Aspects 1-14, comprising expressing one or more nucleic acids encoding the immunoglobulin heavy and light chain polypeptides in a cell in vitro.

[0318] (21) A composition comprising the PD-L1-binding agent of any one of Aspects 1-14 or the nucleic acid of any one of Aspects 15-17 and a pharmaceutically acceptable carrier, optionally wherein the nucleic acid is in a vector.

[0319] (22) The PD-L1-binding agent of any one of Aspects 1-14 or the composition of Aspect 21 for use as a medicament for treating a disease, disorder, or condition in a mammal that is responsive to PD-L1 inhibition or binding.

[0320] (23) The PD-L1-binding agent or composition of Aspect 22, wherein the disease, disorder, or condition is cancer.

[0321] (24) The PD-L1-binding agent of any one of Aspects 1-14 or the composition of Aspect 19 for use as a medicament for enhancing, reducing, or inhibiting an immune response in a mammal.

[0322] (25) The PD-L1-binding agent for use according to Aspect 24, wherein the immune response is an anti-cancer immune response.

[0323] (26) A method for treating a disease, disorder, or condition in a mammal that is responsive to PD-L1 inhibition or binding, the method comprising administering to the mammal a PD-L1-binding agent of any one of Aspects 1-14 or a composition of Aspect 21.

[0324] (27) The method of Aspect 26, wherein the disease, disorder, or condition is cancer.

[0325] (28) The method of Aspect 26, wherein the disease, disorder, or condition is an autoimmune disorder.

[0326] (29) The method of Aspect 26, wherein the disease, disorder, or condition is an infectious disease.

[0327] (30) A method for enhancing, or reducing or suppressing, an immune response in a mammal, comprising administering to the mammal a PD-L1-binding agent of any one of Aspects 1-14, or a composition of Aspect 21.

[0328] (31) The method of Aspect 30, wherein the immune response is an anti-cancer immune response.

[0329] (32) A method of delivering a payload to a cell that expresses PD-L1, comprising administering to the cell, or to a mammal containing the cell, a conjugate comprising (1) the PD-L1-binding agent of any one of Aspects 1-14, and (2) a payload.

[0330] (33) A hybridoma or cell line expressing the PD-L1-binding agent of any of aspects 1-14.

[0331] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0332] Example [Example]

[0333] This example demonstrates that binding agents according to embodiments of the invention are effective at binding human PD-L1 (hPD-L1) on the cell surface.

[0334] The binding of binding agents of embodiments of the invention to human PD-L1 (hPD-L1) on the cell surface was assessed by incubating the binding agents with JIMT1 cells, which express endogenous hPD-L1. Briefly, binding agents were serially diluted in FACS buffer (PBS + 1% FBS + 5mM EDTA, pH 7.4) at 4°C. The diluted binding agents were then incubated with JIMT1 cells (~1x10 5 -2x10 5 / well) and incubated at 4℃. The cells were then washed twice to remove unbound binders, and then incubated with a fluorescent anti-human IgGFc antibody at 4℃ to detect cell-bound anti-PD-L1 binders. After washing away unbound secondary antibody, the cells were resuspended in FACS buffer + 2% formaldehyde and analyzed by flow cytometry. Fluorescence intensity, expressed as geometric mean fluorescence intensity, was used to determine binding of the anti-PDL1 binders to hPD-L1 on the cell surface.

[0335] As demonstrated by the data presented in Figures 1, 2, and 4, binding agents according to embodiments of the invention successfully bound to human PD-L1. The numbers along the x-axis correlate to the number of binding agents according to embodiments of the invention. "Ref Ab" in the figures refers to a reference binding agent that is not an embodiment of the invention. The binding agent concentrations are listed in the figure legends. [Example]

[0336] This example demonstrates that binding agents according to embodiments of the invention were internalized when exposed to cells bearing PD-L1 on the cell surface.

[0337] The internalization of anti-PDL1 binders was measured by estimating the degree of cell surface persistence upon incubation of cells with pre-bound antibodies at 37°C for extended periods. Briefly, to measure the surface persistence of anti-PDL1 antibodies, JIMT-1 cells were cultured at ~2x10 per well. 5 -4x10 5JIMT-1 cells were seeded with 100 μg / ml of anti-PDL1 antibody in cell culture medium. One set of samples (the "chase" set) was generated on day 1 by incubating JIMT-1 cells with 10 μg / ml of anti-PDL1 antibody in cell culture medium for 60 minutes at 4°C. After incubation, unbound binder was removed by serial washing with culture medium, and the cells were incubated in fresh culture medium at 37°C for 24 hours. On day 2, cells were harvested from the microtiter plate by a brief incubation with PBS + 10 mM EDTA. A second set of samples (the "no chase" set) was generated on day 2 by incubating JIMT-1 cells with the anti-PDL1 binder as described above, removing unbound antibody, and then immediately harvesting the cells with PBS + 10 mM EDTA.

[0338] Harvested JIMT-1 cells were transferred to a 96-well microtiter plate, pelleted, and incubated with fluorescent anti-human IgG Fc antibody in FACS buffer (PBS + 1% FBS + 5 mM EDTA, pH 7.4) at 4°C for at least 45 minutes to detect cell-bound anti-PDL1 antibody. Unbound binders were removed by washing the cells twice, and the cells were resuspended in FACS buffer + 2% formaldehyde and analyzed by flow cytometry.

[0339] The fluorescence intensity of the secondary anti-Fc channel, expressed as the geometric mean fluorescence intensity, was used to determine anti-PDL1 binding. After incubation with a given anti-PDL1 binder, the ratio of the fluorescence intensity of chased versus unchased cells for 24 hours is an indicator of the surface persistence of that binder. For example, a ratio of 1.0 indicates no loss of surface-bound binder during the 24-hour chase, while a ratio <1 indicates that some of the binder was internalized within the cells.

[0340] As shown by the data in Figure 3, binding agents according to embodiments of the invention were successfully internalized into cells. The numbers along the x-axis correlate to the number of binding agents according to embodiments of the invention. "Ref Ab" in the figure refers to a reference binding agent that is not an embodiment of the invention. The binding agent concentrations are listed in the figure legend. [Example]

[0341] This example demonstrates that binding agents according to embodiments of the invention inhibit PD-L1 / PD-1 binding.

[0342] The effects of the binders and two commercially available antibodies, avelumab and atezolizumab, on PD-L1 / PD-1 interaction were determined using a reporter assay (Promega). The assay was performed according to the manufacturer's instructions. Briefly, cells expressing both PD-L1 and T cell receptor (TCR)-activated αAPCs on their surface were incubated with cells expressing surface recombinant TCR and PD-1 and carrying a luciferase gene under the control of an NFAT (nuclear factor of activated T cells) response element.

[0343] Trans-PD-L1 / PD-1 interaction prevents trans-αAPC-TCR binding, TCR signaling, and the induction of luciferase expression, which occurs through activation of the NFAT response element. Anti-PDL1 antibodies block the interaction with PD-1, thereby allowing TCR signaling and luciferase expression. Luciferase expression is measured using a bioluminescent substrate and a plate reader set to luminescence detection mode.

[0344] Stimulator CHO-K1 / PD-L1 / αAPC cells were suspended in Ham's F-12 medium supplemented with 10% fetal bovine serum (FBS) and plated into six wells of a 96-well, clear-bottom, white-walled plate (Corning). The plate was placed in a 37°C incubator under a 5% CO2 atmosphere for 14 hours. The medium was removed, and test articles diluted to 2x the final concentration in RPMI-1640 medium supplemented with 1% FBS were added to the wells. Next, an equal volume of reporter Jurkat / PD-1 / luciferase cells suspended in RPMI-1640 + 1% FBS was added to each well. Final concentrations ranged from 4.12 to 27,000 ng / ml in 3-fold increments. Avelumab was included as a reference. After 8 hours in a 37°C incubator under a 5% CO2 atmosphere, the plate was removed and allowed to equilibrate to room temperature for 10 minutes. BIOGLOW cell permeable luciferase substrate (Promega) was then added to the wells for 10 minutes with gentle agitation.

[0345] Luminescence was measured using a Molecular Devices M3 plate reader. Luminescence data were analyzed using GraphPad PRISM v8 for four-parameter sigmoidal dose-response curve fitting and determination of EC50 values. Concentrations were converted to log10 values ​​for EC50 determination and graphing. "No test article" concentrations were converted to (lowest concentration tested) / 3 to allow for log10 transformation. Conversion of EC50 values ​​from ng / ml to nM was performed using IgG molecular weight data.

[0346] PD-L1 / PD-1 blockade was demonstrated by increasing luminescence (RLU = relative luminescence units) induced by increasing concentrations of anti-PDL1 antibody. The dose-response curve was closely fitted using a four-parameter sigmoidal model, consistent with saturation of antibody binding.

[0347] [Table 5]

[0348] In summary, binding agents according to embodiments of the present invention inhibited protein binding with EC50 values ​​ranging from 129.4-290.8 ng / ml (0.33-2.02 nM).

[0349] The terms "about" and "around" used herein to modify a numerical value indicate a close range surrounding that numerical value. Thus, when "X" is a numerical value, "about" or "around" refers to a numerical value of 0.9X to 1.1X, e.g., 0.95X to 1.05X or 0.99X to 1.01X. References to "about" or "around" specifically refer to at least the value X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, or 1.05X. Thus, "about" and "around" are intended to inform and provide descriptive support for claim limitations, e.g., "0.98X."

[0350] All references cited herein, including publications, patent applications, and patents, are incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0351] The use of the terms "a," "an," "the," "at least one," and similar referential terms in the context of describing the present invention (particularly in the context of the claims below) shall be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") shall be interpreted to mean one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" shall be interpreted as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referencing each separate value falling within the range, and each separate value is incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc."), are intended merely to better illustrate the invention and do not pose a limitation on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0352] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that skilled artisans will adopt such variations as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. 1. A programmed death-ligand 1 (PD-L1)-binding agent comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide, wherein: the immunoglobulin heavy chain variable region polypeptide comprises a complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 1-23, 309, or 345, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 24-57, 310-314, or 346-349, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 58-95, 315-318, or 350-354; or a PD-L1-binding agent, wherein the immunoglobulin light chain variable region polypeptide comprises a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 96-128, 319-323, 355, or 356, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 129-151, or 357-359, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 152-176, or 360.

2. A PD-L1-binding agent comprising an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 223-264, 324-334, or 361-365, or at least the CDRs thereof; and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 265-306, 335-344, or 366-370, or at least the CDRs thereof.

3. 1. A PD-L1-binding agent comprising an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 223-264, 324-334, or 361-365, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 265-306, 335-344, or 366-370.

4. The PD-L1-binding agent of any one of claims 1 to 3, comprising a heavy and light chain immunoglobulin polypeptide of a PD-L1-binding agent in Table 1, or at least the CDRs thereof.

5. 5. The PD-L1-binding agent of any one of claims 1 to 4, wherein the binding agent is an antibody, an antibody conjugate, or an antigen-binding fragment thereof.

6. The binding agent is F(ab') 2 6. The PD-L1-binding agent of claim 5, which is an antibody fragment selected from: , Fab', Fab, Fv, scFv, dsFv, dAb, and single-chain binding polypeptide.

7. 6. The PD-L1-binding agent of claim 5, wherein the binding agent is an antibody.

8. 8. The PD-L1-binding agent of any of claims 1-7, further comprising an immunoglobulin Fc region.

9. 9. The PD-L1-binding agent of claim 8, further comprising a transforming growth factor beta 1 (TGFβ1) receptor linked to an Fc region, or a fragment thereof that binds TGFβ1.

10. The PD-L1-binding agent of any one of claims 7 to 9, wherein the antibody is an IgG, IgM, IgA, IgD, or IgE antibody.

11. The PD-L1-binding agent of any one of claims 7 to 9, wherein the antibody is an IgG antibody.

12. The PD-L1-binding agent of any one of claims 7-11, wherein the antibody exhibits antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

13. 13. The PD-L1-binding agent of any of claims 1-12, wherein the binding agent is part of a bispecific antibody, a chimeric antigen receptor, a chimeric T cell receptor, or a bispecific T cell engager.

14. The PD-L1-binding agent of any one of claims 1 to 12, wherein the binding agent is a cell-internalizing binding agent.

15. A nucleic acid encoding the heavy chain immunoglobulin polypeptide of the anti-PD-L1 binding agent of any one of claims 1 to 14.

16. A nucleic acid encoding the light chain immunoglobulin polypeptide of the anti-PD-L1 binding agent of any one of claims 1 to 14.

17. A nucleic acid encoding the heavy and light chain immunoglobulin polypeptides of the PD-L1-binding agent of any one of claims 1 to 14.

18. A vector comprising the nucleic acid sequence according to any one of claims 15 to 17.

19. An isolated cell comprising a nucleic acid according to any one of claims 15 to 17, optionally in a vector.

20. 15. A method of providing a PD-L1-binding agent according to any one of claims 1 to 14, comprising expressing one or more nucleic acids encoding the immunoglobulin heavy and light chain polypeptides in a cell in vitro.

21. 18. A composition comprising the PD-L1-binding agent of any one of claims 1 to 14 or the nucleic acid of any one of claims 15 to 17, and a pharmaceutically acceptable carrier, optionally comprising the nucleic acid in a vector.

22. The PD-L1-binding agent of any one of claims 1 to 14, or a conjugate comprising same, or the composition of claim 21, for use as a medicament for treating a disease, disorder, or condition in a mammal that responds to PD-L1 inhibition or binding.

23. 23. The PD-L1-binding agent or composition of claim 22, wherein the disease, disorder, or condition is cancer.

24. The PD-L1-binding agent of any one of claims 1 to 14 or the composition of claim 19 for use as a medicament for modulating an immune response in a mammal.

25. 25. The PD-L1-binding agent for use according to claim 24, wherein the immune response is an anti-cancer immune response.

26. 22. A method for treating a disease, disorder, or condition in a mammal that responds to PD-L1 inhibition or binding, comprising administering to the mammal the PD-L1-binding agent of any one of claims 1 to 14 or a conjugate comprising same, or the composition of claim 21.

27. 27. The method of claim 26, wherein the disease, disorder, or condition is cancer.

28. 27. The method of claim 26, wherein the disease, disorder, or condition is an autoimmune disorder.

29. 27. The method of claim 26, wherein the disease, disorder, or condition is an infectious disease.

30. A method for enhancing or suppressing an immune response in a mammal, comprising administering to the mammal the PD-L1-binding agent of any one of claims 1 to 14 or a conjugate comprising the same, or the composition of claim 21.

31. 31. The method of claim 30, wherein the immune response is an anti-cancer immune response.

32. 15. A method for delivering a payload to a cell expressing PD-L1, comprising administering to the cell or to a mammal containing the cell a conjugate comprising (1) the PD-L1-binding agent of any one of claims 1 to 14 and (2) a payload.

33. A hybridoma or cell line expressing the PD-L1-binding agent of any one of claims 1 to 14.

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