[112] The 3-letter code for amino acids as defined herein is in conformity with the IUPAC- IUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[113] The phrase“conservative substitution” with regard to an amino acid residue of a peptide, peptide region, polypeptide region, protein, or molecule refers to a change in the amino acid composition of the peptide, peptide region, polypeptide region, protein, or molecule that does not substantially alter the function and structure of the overall peptide, peptide region, polypeptide region, protein, or molecule (see Creighton T, Proteins:

Structures and Molecular Properties (W. H. Freeman and Company, 2^nd ed., New York (1992))).

[114] As used herein, the term“conservative substitution” denotes that one or more amino acids are replaced by another, biologically similar amino acid residue. Examples include substitution of amino acid residues with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids (see, for example, Table B, infra). An example of a conservative substitution with a residue normally not found in endogenous, mammalian peptides and proteins is the conservative substitution of an arginine or lysine residue with, for example, ornithine, canavanine, aminoethylcysteine, or another basic amino acid. For further information concerning phenotypically silent substitutions in peptides and proteins see, e.g., Bowie J et al., Science 247: 1306-10 (1990).

TABLE B. Examples of Conservative Amino Acid Substitutions

[115] In the conservative substitution scheme in Table B above, exemplary conservative substitutions of amino acids are grouped by physicochemical properties - I: neutral, hydrophilic; II: acids and amides; III: basic; IV: hydrophobic; V: aromatic, bulky amino acids, VI hydrophilic uncharged, VII aliphatic uncharged, VIII non-polar uncharged, IX cycloalkenyl-associated, X hydrophobic, XI polar, XII small, XIII turn-permitting, and XIV flexible. For example, conservative amino acid substitutions include the following: 1) S may be substituted for C; 2) M or L may be substituted for F; 3) Y may be substituted for M; 4) Q or E may be substituted for K; 5) N or Q may be substituted for H; and 6) H may be substituted for N.

[116] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, such as, e.g, software and/or algorithms described herein (see section I-B. Variants of the Antibodies, and Fragments and Derivatives Thereof, of the Present Invention).

[117] As used herein, the term“antibody” refers to various immunoglobulin proteins having a structural domain known as an Ig domain which is involved in antigen binding, and thus, the term antibody encompasses the broadest of antibody formats having antigen binding capability. The term“antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, antibodies derived from any biological species, synthetic antibodies, chimeric antibodies, humanized antibodies, full-length antibodies, multiple-chain or single-chain antibodies, multispecific antibodies (e.g. bispecific antibodies and/or dual variable domain antibodies), antibody fragments (e.g. Fv, Fab, Fab’, Fab’-SH, F(ab’)2), antibody derivatives (e.g. single chain Fv (scFv), Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, surrobodies

(including a surrogate light chain construct)), and single domain antibodies (including camelized antibodies and/or heavy-chain only antibodies) as long as they exhibit the desired antigen-binding activity, such as, e.g. , binding to PD-L1 with an affinity characterized by a KD of < 100 nM, 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nanomolar (nM) (e.g. 10⁸ M or less, e.g. from 10⁸ M to 10¹³ M, e.g., from 10⁹ M to 10¹³ M). Unless dictated otherwise by contextual constraints the term“antibody” further comprises all classes of antibodies (e.g. IgA, IgD, IgE, IgG, and IgM) and all subclasses (e.g. IgGl, IgG2, IgG3,

IgG4, IgAl, and IgA2). If desired, the class of an antibody may be“switched” using methods known to the skilled worker. For example, an antibody that was originally produced as an IgM molecule may be class switched to an IgG antibody. Class switching techniques also may be used to convert one IgG subclass to another, for example: from IgGl to IgG2 or IgG4; from IgG2 to IgGl or IgG4; or from IgG4 to IgGl or IgG2. Engineering of antibodies to generate constant region chimeric molecules, such as by combining regions from different IgG subclasses, can also be performed by the skilled worker using known methods.

[118] As used herein, the phrase“antibody derivative” or derivative of an antibody fragment refers to a recombinant molecule that comprises at least one antibody binding domain (e.g. a CDR, HVR, VH, and/or VL) and that binds with requisite specificity to the same antigen to which the antibody comprising the same antibody binding domain(s) binds. For example, an antigen binding fragment of a parental antibody includes a polypeptide fragment or recombinant derivative of the parental antibody that immunospecifically binds or reacts with the target protein or antigen or competes with the parental antibody from which the fragment(s) or derivative was derived for specific binding to the same target protein or antigen.

[119] An“antibody fragment” refers to a molecule other than an intact antibody that comprises a portion(s) of an intact antibody that binds the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments, antigen binding fragments, and antibody derivatives include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2), linear antibodies, single-chain antibody molecules ( e.g . scFv’s), multispecific antibodies formed from antibody fragments, and numerous additional antigen-binding antibody derivatives described below and/or known to the skilled person. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody (e.g. a full-length antibody) as well as production by recombinant host cells (e.g. using E. coli and/or phage). For example, papain digestion of antibodies produces two identical antigen binding fragments, called“Fab” fragments, each with a single antigen binding site. Pepsin treatment yields an F(ab’) 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigens as exhibited by the intact antibody from which it was derived. Antibody fragments can be made by various techniques, including but not limited to recombinant engineering of heavy and/or light chains into a single-chain polypeptide, e.g. to form a scFv.

[120] An“isolated” antibody is one which has been separated and/or recovered from (an)other component(s) in the environment in which it was produced and/or that it has been purified from a mixture of two or more components present in the environment in which it was produced, such as the antibody’s natural environment. In certain embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, using an electrophoretic (e.g. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), isoelectric focusing (IEF), capillary electrophoresis (CE)) or chromatographic (e.g. ion exchange or reverse phase HPLC) method (see e.g. Flatman S et ak, J Chromatogr B Analyt Technol Biomed Life Sci 848: 79-87 (2007)).

[121] As used herein, the term“affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. an antibody or its paratope) and its binding partner (e.g. an antigen or epitope). ETnless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. an antibody and the antibody’s specific target). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by methods known to the skilled worker, including those described herein. Specific illustrative and exemplary embodiments of methods of measuring binding affinity are described herein. The term“epitope” may be used herein to refer the portion(s) of the PD-L1 protein or antigen to which the antibody or antigen-binding fragment specifically binds. A“linear epitope” is formed by contiguous amino acids in the antigen that allow for binding of the antibody. The ability to preferentially bind linear epitopes is typically maintained even when the antigen is denatured. Conversely, a“conformational epitope” usually comprises non-contiguous amino acid residues in the antigen’s amino acid sequence that, in the context of the antigen’s secondary, tertiary or quaternary structure, are sufficiently proximate to be bound concomitantly by a single antibody when the target protein is in its native folded state. When antigens with

conformational epitopes are denatured, the antibody will typically no longer recognize the antigen. An epitope (contiguous or non-contiguous) typically includes at least three and at least five or more amino acid residues in a unique spatial conformation, such as, e.g ., 8-10 or 12-20 amino acid residues.

[122] The terms“anti-PD-Ll antibody”,“anti-PD-Ll”,“PD-L1 antibody” or“an antibody that binds to PD-L1” refers to an antibody that is capable of binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-L1. In one embodiment, the extent of binding of an anti-PD-Ll antibody to an unrelated, non-PD-Ll protein is less than about 10% of the binding of the antibody to PD-L1 as measured, e.g. , by a radioimmunoassay. An“anti-PD-Ll antibody”, as used herein, is one that specifically binds to a PD-L1 molecule with sufficient specificity and affinity to be useful in targeting a therapeutic, targeting diagnostic, or method of detecting PD-L1 in a biological sample from a subject. In certain embodiments, an antibody that binds to PD-L1 has a dissociation constant (KD) of < 100 nM, 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10⁸ moles/liter (M) or less, e.g. from 10⁸ M to 10¹³ M, e.g., from 10⁹ M to 10¹³ M). In certain embodiments, an anti-PD-Ll antibody binds to an epitope of PD-L1 that is conserved among PD-L1 from different species.

[123] An“antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

[124] As used herein, the phrase“native antibodies” refer to naturally occurring

immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide bonded. From amino- to carboxy- terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from amino- to carboxy-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to certain types, e.g. kappa (K) and lambda (l), based on the amino acid sequence of its constant domain.

[125] As used herein, the term“variable region” or“variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH or VH and VL or VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see e.g. Kindt T et ak, Kuby Immunology, at pg. 91 (W.H. Freeman and Co., 6th ed., (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see e.g. Portolano S et ak, J Immunol 150: 880-7 (1993); Clarkson T et ak, Nature 352: 624-8 (1991)).

[126] The term“hypervariable region” or“HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (Ll, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (Ll), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96- 101 (H3) (Chothia C, Lesk A, J Mol Biol 196: 901-17 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3 (Rabat E, Sequences of Proteins of Immunological Interest, Public Health Service, National Institutes of Health (U.S.), Bethesda, MD, 5th ed. (1991)). With the exception of CDR1 in VH, CDRS generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise“specificity determining residues”, or“SDRs”, which are residues that contact antigen during binding. SDRs are contained within regions of the CDRs called abbreviated- CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a- CDR-H2, and a-CDR-EB) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3 ( see Almagro J, Fransson J, Front Biosci 13 : 1619-33 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain ( e.g . FR residues) are numbered herein according to Kabat E (1991), supra , referred to herein as“Kabat”. Certain sequence identifiers (SEQ ID NO:X) are herein followed by a parenetical indicating an alternative numbering scheme used instead of Kabat, such as, e.g.,“Chothia” (see Chothia C (1987), supra).

[127] As used herein, the term“heavy chain variable (VH) domain” or“light chain variable (VL) domain” respectively refer to any antibody VH or VL domain (e.g. a human VH or VL domain) as well as any derivative thereof that retains the antigen binding ability of the corresponding native antibody (e.g. a humanized VH or VL domain derived from a native murine VH or VL domain). A VH or VL domain consists of a“framework” region interrupted by the three CDRs or ABRs. As used herein, the term“framework” or“FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4.

Accordingly, the HVR and FR sequences generally appear in the following sequence in a VH (or VL): FRl-Hl(Ll)-FR2-H2(L2)-FR3-H3(L3)-FR4. The framework regions serve to align the CDRs or ABRs for specific binding to an epitope of an antigen. From amino-terminus to carboxy-terminus, both VH and VL domains comprise the following framework (FR) and CDR regions or ABR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; or, similarly, FR1, ABR1, FR2, ABR2, FR3, ABR3, and FR4. As used herein, the terms“HCDR1”, “HCDR2”, or“HCDR3” are used to refer to CDRs 1, 2, or 3, respectively, in a VH domain, and the terms“LCDR1”,“LCDR2”, and“LCDR3” are used to refer to CDRs 1, 2, or 3, respectively, in a VL domain. As used herein, the terms“HABRl”,“HABR2”, or“HABR3” are used to refer to ABRs 1, 2, or 3, respectively, in a VH domain, and the terms“LABR1”, “LABR2”, or“LABR3” are used to refer to CDRs 1, 2, or 3, respectively, in a VL domain.

For camelid VHH fragments, IgNARs of cartilaginous fish, VNAR fragments, certain single domain antibodies, and derivatives thereof, there is a single, heavy chain variable domain comprising the same basic arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

As used herein, the terms“HCDRl”,“HCDR2”, or“HCDR3” may be used to refer to CDRs 1, 2, or 3, respectively, in a single domain antibody, autonomous heavy chain antibody, heavy chain only antibody, VHH fragment, VH domain fragment, heavy-chain antibody domain derived from a camelid, heavy-chain antibody domain derived from a cartilaginous fish, immunoglobulin new antigen receptor (IgNAR), or VNAR fragment. A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[128] As used herein, the term“monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations ( e.g ., naturally occurring and/or spontaneous mutations), that may be present or arise in minor amounts. For example, monoclonal antibodies are typically derived from a single-cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone and may containing mutations arising during production of a monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of methods known to the skilled worker, including but not limited to the hybridoma methods, recombinant DNA methods, phage- display methods, and methods utilizing transgenic animals containing all or part of an immunoglobulin genetic locus from a different species.

[129] As used herein, the term“human antibody” refers to an antibody which possesses an amino acid sequence that those of skill in the art would recognize corresponds to that of an antibody produced by a human or human cell, derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, and/or has been made using any of the techniques for making human antibodies described herein. For example, the term“human antibody” is intended to include antibodies having variable regions in which at least a portion of a framework region and/or at least a portion of a CDR region are derived from a human germline immunoglobulin sequence. A human antibody may have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. This definition of a human antibody specifically excludes a humanized antibody comprising nonhuman antigen-binding residues ( e.g . CDRs). A human single-domain antibody is one comprising only a human heavy chain or human light chain; however, the CDR sequence may be naturally occurring or synthetic (see e.g. U.S. 6,248,516).

[130] As used herein, the term“chimeric antibody” refers to a recombinant protein that is derived from protein sequences from other than a single biological species or single class/subclass of antibody, such as, e.g. , wherein protein sequences from two biological species, classes of antibody, or isotypes of antibody have been covalently joined together.

For example, a chimeric antibody may comprise a portion of the heavy and/or light chain derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from one or more different sources or species. For example, in one embodiment, the chimeric antibody may contain the variable domains and complementarity determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody. In certain embodiments, chimeric antibodies of the invention are“CDR-grafted” antibodies, where the CDRs alone (as defined using Rabat, Chothia, Kabat/Chothia, McCallum/Contact, IMGT, Gelfand, Honneger, Martin, North, or AbM) are derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody is largely derived from an antibody from another species or belonging to another antibody class or subclass. For example, one or more selected rodent CDRs (e.g, mouse CDRs) may be grafted into a human recipient antibody, replacing one or more of the naturally occurring CDRs of the human antibody. Such antibodies may optionally possess certain advantages, such as, e.g, exhibiting less immunogenicity in human recipients while also providing the possibility of maximal levels of human antibody effector functions, e.g, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[131] As used herein, the term“humanized antibody” refers to a recombinant protein in which complementarity determining regions of a non-human species donor antibody have been transferred from heavy and light variable chains of the non-human species donor antibody into a recipient human variable domain. For example, a humanized antibody includes an antibody which possesses an amino acid sequence and/or residues involved in antigen-binding (e.g. a CDR) that are derived from a non-human source and wherein one or more other amino acid sequences is derived from a human source (e.g. a framework sequence). For example, humanized antibodies may be produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse antibody into a human variable domain. For example, a humanized antibody includes a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs ( e.g ., CDRs) correspond to those of a nonhuman antibody, and all or substantially all of the FRs correspond to those of a human antibody. In certain embodiments,“back mutations” can be introduced into the humanized antibody, in which residues in one or more framework domains (FRs) of the variable region of the recipient human antibody are replaced by corresponding residues from the non-human species donor antibody. Such back mutations may help maintain the appropriate three-dimensional configuration of the grafted CDR(s) and thereby improve antibody affinity and/or stability. Antibodies from various donor species may be used including, without limitation, mouse, rat, rabbit, or non-human primates. Furthermore, humanized antibodies may comprise new residues that are not found in the recipient antibody or in the donor antibody to, for example, to further refine antibody performance. In general, a humanized antibody will comprise at least one - typically two - variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human species donor antibody and in which all or substantially all of the FR residues are those of a recipient human variable domain. The humanized antibody may optionally comprise an Fc or at least a portion of an Fc region, corresponding to a human antibody Fc sequence. A“humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[132] As used herein, a“camelized antibody” is one which possesses an amino acid sequence derived from a non-camelid source and comprises two heavy chains and no light chains and further comprises a hinge region derived from a camelid source or species.

[133] As used herein, the term“anti-idiotype antibody” refers to an antibody that binds with the variable region domain of another antibody. For example, an anti-idiotype antibody may bind with the variable region of an anti-target antibody, and thus, an anti-idiotype antibody comprises a paratope which mimics an epitope of that target.

[134] As used herein, the symbol“a” is shorthand for an immunoglobulin-type binding region capable of binding to the biomolecule following the symbol. The symbol“a” is used to refer to the functional characteristic of an immunoglobulin-type binding region based on its ability to bind to the biomolecule following the symbol with a binding affinity described by a dissociation constant (KD) of 10⁵ M or less. [135] Throughout this specification, the term“bispecific” will be understood to include molecules which bind different extracellular target biomolecules or which bind the same extracellular target biomolecule at two or more different epitopes, whether non-overlapping or overlapping epitopes ( e.g . a bivalent biparatopic molecule).

[136] As used herein, the term“Fc region” refers to the portion of an antibody that is responsible for binding to either antibody receptors on cells and/or the Clq component of complement. The Fc region may consist of the disulfide-linked heavy chain hinge regions, CH2, and CH3 domains; or alternatively this term may be applied to a single chain consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-linked dimer with a second such chain (see e.g. Putnam F, The Plasma Proteins, vol. V (Academic Press, Inc., New York (1987)); Padlan E, Mol Immunol 31 : 169-217 (1994)).

[137] As used herein, the term“Fc region having ADCC activity” refers to an Fc region capable of antibody-dependent cell-mediated cytotoxicity (ADCC) through binding of a cytolytic Fc receptor (e.g. FcyRIIIa) on a cytolytic immune effector cell expressing the Fc receptor (e.g. an NK cell or CD8+ T cell).

[138] As used herein, the term“Fc region having CDC activity” refers to an Fc region capable of mediating complement-dependent cytotoxicity (CDC) through binding of Clq complement protein and activation of the classical complement system.

[139] As used herein, the term“complement” refers collectively to those components in normal serum that, together with antigen-bound antibodies, exhibit the ability to lyse cells. Complement consists of a group of serum proteins that act in concert and in an orderly sequence to exert their effect. The phrase“classical complement system”, as used herein, refers to a specific pathway for the activation of complement requiring the ordered function of nine major protein components designated Cl through C9 and providing amplification of large amounts of complement from a relatively small initial signal. For several steps in the activation process, the product is an enzyme that catalyzes the subsequent step.

[140] For purposes of the present invention, the term“effector” means providing a biological activity, such as cytotoxicity, biological signaling, enzymatic catalysis, subcellular routing, and/or intermolecular binding resulting in an allosteric effect(s) and/or the recruitment of one or more factors.

[141] As used herein, the phrase“antibody effector function” refer to those biological activities attributable to a Fc region of an antibody or derivative thereof, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and CDC; Fc receptor binding (including the neonatal Fc receptor (FcRn) or Brambell receptor), ADCC; phagocytosis; down-regulation of cell surface receptors (e.g. PD-L1, PD-l, or B7-1); T-cell activation, and B-cell activation.

[142] For purposes of the present invention, the phrase“derived from” when referring to a polypeptide or polypeptide region means that the polypeptide or polypeptide region comprises highly similar amino acid sequences originally found in a“parental” protein or molecule and which may now comprise certain amino acid residue additions, deletions, truncations, rearrangements, or other alterations relative to the original polypeptide or polypeptide region as long as a certain function(s) (e.g. antigen binding affinity) and a structure(s) of the“parental” molecule are substantially conserved. The skilled worker will be able to identify a parental molecule (e.g. an antibody sequence) from which a polypeptide or polypeptide region (e.g. a CDR, HVR, VH, and/or VL) was derived using techniques known in the art, e.g. , protein sequence alignment software.

[143] The terms“associated”,“associating”,“linked”, or“linking” with regard to the claimed invention refers to the state of two or more components of a molecule being joined, attached, connected, or otherwise coupled to form a single molecule or the act of making two molecules associated with each other to form a single molecule by creating an association, linkage, attachment, and/or any other connection between the two molecules. For example, the term“linked” may refer to two or more components associated by one or more atomic interactions such that a single molecule is formed and wherein the atomic interactions may be covalent and/or non-covalent. Non-limiting examples of covalent associations between two components include peptide bonds and cysteine-cysteine disulfide bonds. Non-limiting examples of non-covalent associations between two molecular components include ionic bonds.

[144] For purposes of the present invention, the term“linked” refer to two or more molecular components associated by one or more atomic interactions such that a single molecule is formed and wherein the atomic interactions includes at least one covalent bond. For purposes of the present invention, the term“linking” refers to the act of creating a linked molecule as described herein.

[145] For purposes of the present invention, the term“fused” refers to two or more proteinaceous components associated by at least one covalent bond which is a peptide bond, regardless of whether the peptide bond involves the participation of a carbon atom of a carboxyl acid group or involves another carbon atom, such as, e.g. , the a-carbon, b-carbon, g- carbon, s-carbon. Non-limiting examples of two proteinaceous components fused together include, e.g. , an amino acid, peptide, or polypeptide fused to a polypeptide via a peptide bond such that the resulting molecule is a single, continuous polypeptide. For purposes of the present invention, the term“fusing” refers to the act of creating a fused molecule as described herein, such as, e.g ., a fusion protein generated from the recombinant fusion of genetic regions which when translated produces a single proteinaceous molecule.

[146] The symbol means the polypeptide regions before and after it are physically linked together to form a continuous polypeptide.

[147] As used herein, the term“immunoconjugate” or“conjugate” is used broadly and includes the covalent or non-covalent association of any material with an antibody, regardless of the method of association. An“immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent or detection- promoting agent. As used herein, the term“heterologous” refers to a molecule, moiety or agent that is not natively found in association with the antibody, antigen-binding fragment or derivative thereof of the present invention.

[148] The terms“toxin”,“toxin agent”,“toxin component”, or“cytotoxin” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction, including tissue damage. The toxin component of an antibody, antibody drug conjugate, or antibody toxin immunoconjugate of the present invention may include, but is not limited to, natural toxins, biotoxins, proteinaceous toxins, venom, cytotoxins, small molecule toxins, and synthetic toxicants derived from any of the aforementioned, such as, e.g. any one of various toxin agents described herein or known to the skilled worker (see e.g. Table C).

[149] The term“cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g. At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹², and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various cytotoxic agents disclosed and described below.

[150] A“naked antibody” refers to an antibody that is not associated (e.g. linked, conjugated, or fused) with a heterologous moiety), such as, e.g. , a detection-promoting agent or a cytotoxic agent. The naked antibody may be present in a pharmaceutical formulation. [151] As used herein, the terms“expressed”,“expressing”, or“expresses”, and grammatical variants thereof, refer to translation of a polynucleotide or nucleic acid into a protein. The expressed protein may remain intracellular, become a component of the cell surface membrane, be secreted into an extracellular space, or alternatively expression may occur in a cell-free composition.

[152] As used herein, cells which express a significant amount of an extracellular target biomolecule at least one cellular surface are“target positive cells”,“target+ cells”, or“+ve cells” and are cells physically coupled to the specified, extracellular target biomolecule.

[153] The term“selective cytotoxicity” with regard to the cytotoxic activity of a molecule refers to the relative level of cytotoxicity between a biomolecule target positive cell population ( e.g . a targeted cell-type) and a non-targeted bystander cell population (e.g. a biomolecule target negative cell-type), which can be expressed as a ratio of the half-maximal cytotoxic concentration (CDso) for a targeted cell type over the CDso for an untargeted cell type to provide a metric of cytotoxic selectivity or indication of the preferentiality of killing of a targeted cell versus an untargeted cell.

[154] The cell surface representation and/or density of a given extracellular target biomolecule (or extracellular epitope of a given target biomolecule) may influence the applications for which certain anti-PD-Ll antibodies of the present invention may be most suitably used. Differences in cell surface representation and/or density of a given target biomolecule (e.g. PD-L1) between cells may alter, both quantitatively and qualitatively, the efficiency of cellular internalization and/or cytotoxicity potency of a given anti-PD-Ll antibody of the present invention. The cell surface representation and/or density of a given target biomolecule can vary greatly among target biomolecule positive cells or even on the same cell at different points in the cell cycle or cell differentiation. The total cell surface representation of a given target biomolecule and/or of certain extracellular epitopes of a given target biomolecule on a particular cell or population of cells may be determined using methods known to the skilled worker, such as methods involving fluorescence-activated cell sorting (FACS) flow cytometry.

[155] As used herein,“de-immunized” means reduced antigenic and/or immunogenic potential after administration to a chordate as compared to a reference molecule, such as, e.g. , a parental molecule, wild-type peptide region, polypeptide region, or protein. This includes a reduction in overall antigenic and/or immunogenic potential despite the introduction of one or more, de novo, antigenic and/or immunogenic epitopes as compared to a reference molecule. For certain embodiments,“de-immunized” means a molecule exhibited reduced antigenicity and/or immunogenicity after administration to a mammal as compared to a“parental” molecule from which it was derived under the same conditions measured by the same assay, such as, e.g ., an assay known to the skilled worker and/or described herein, e.g. , a

quantitative enzyme linked immunosorbent assay (ELISA), Western blot analysis, and/or measurement of anti-molecule antibodies produced in a mammal(s) after receiving administration of the molecule at a given time-point. The relative immunogenicity of a given molecule may be determined using an assay for mammalian in vivo antibody responses to the test molecule after repeat administrations over periods of time.

[156] As used herein, the terms“patient” and“subject” are used interchangeably to refer to any organism, commonly vertebrates such as humans and animals, which presents symptoms, signs, and/or indications of at least one disease, disorder, or condition. These terms include mammals, such as the non-limiting examples of primates (e.g. humans and nonhuman primates such as chimpanzees), domesticated/livestock animals/companion animals (e.g, cattle, cats, dogs, horses, pigs, and sheep), and laboratory animals (e.g. mice, macaques, rabbits, rats).

[157] The term“pharmaceutical composition” or“pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[158] A“pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A

pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[159] As used herein,“treat,”“treating,” or“treatment”, and grammatical variants thereof, refer to an approach for obtaining beneficial or desired clinical results. The terms may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, inhibition or reduction of an increase in severity of a pathological state, diminishment of extent of disease, stabilization (e.g. not worsening) of state of disease, delay or slowing of disease progression such as reduction in the increase in severity of symptoms, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment may refer to a clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Beneficial or desirable results of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. “Treat,”“treating,” or “treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject ( e.g . a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. With regard to tumors and/or cancers, treatment includes reduction in overall tumor burden and/or individual tumor size. For certain embodiments, the antibody of the present invention is used to delay development of a disease or to slow the progression of a disease.

[160] As used herein, the terms“prevent”,“preventing”,“prevention” and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition, disease, or disorder. Accordingly,“prevention” may refer to prophylactic or preventive measures. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g. a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term“prevention” includes slowing the onset of disease relative to the absence of treatment and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus“preventing” or“prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition or preventing or delaying the development of symptoms associated with the condition.

[161] As used herein, an“effective amount” or“therapeutically effective amount” is an amount, dose, or dosage of a composition of matter (e.g. a therapeutic composition, antibody, or theragnostic) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition. An“effective amount” includes an amount or dosage administrated for periods of time necessary to achieve the desired therapeutic or prophylactic result. The most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof.

This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including weight, age, sex, disease type, disease stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject’s response to administration of a composition and adjusting the dosage accordingly (see e.g. Remington: The Science and Practice of Pharmacy (Gennaro A, ed., Mack

Publishing Co., Easton, PA, U.S., l9th ed., 1995)). A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects.

[162] The term“detecting” is used in the broadest sense to include both qualitative and quantitative measurements of a target molecule. In one aspect, the detecting method as described herein is used to identify the mere presence of the PD-L1 of interest in a biological sample. In another aspect, the method is used to test whether the PD-L1 of interest in a sample is present at a detectable level. In yet another aspect, the method can be used to quantify the amount of the PD-L1 of interest in a sample and further to compare the antibody levels from different samples.

[163] The term“biological sample” refers to any biological substance that contains or is suspected to contain a PD-L1 of interest. A sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, and other constituents of the body that may contain the PD-L1 of interest. In various

embodiments, the sample is a body sample isolated or obtained from any animal. For certain embodiments, the sample is isolated or obtained from a mammal. In certain embodiments, the sample is isolated or obtained from a human subject. For certain embodiments, the biological sample is serum from a human subject. For certain embodiments the biological sample is biopsy material. For certain embodiments, the biological sample is biopsy material from a human subject. For certain embodiments, the biological sample is biopsy material from a tumor. For certain embodiments, the biological sample is biopsy material comprising tumor cells or cancer cells. For certain embodiments, the biological sample is plasma isolated or obtained from a human subject. For certain embodiments the biological sample is primary cell culture material. For certain embodiments, the biological sample is primary cell culture material isolated from a human subject. In certain embodiments, the biological sample is isolated or obtained from human patients or patients treated with a therapeutic anti- PD-L1 antibody or antibodies or some other therapeutic directed to an immune checkpoint, such as, e.g. one or more of an anti -PD- 1 antibody, anti-B7-l antibody, anti-PD-L2 antibody, and anti-CTLA-4 antibody.

[164] The term“capture reagent” refers to a reagent (e.g, an antibody) or mixture of such reagent that bind to a target (e.g. PD-L1) of interest and are capable of binding and capturing the target (e.g. PD-L1) of interest in a biological sample such that under suitable conditions, the complex of capture reagent and target (e.g. PD-L1) of interest can be separated from the rest of the sample. For example, a capture reagent can be an anti-PD-Ll antibody or mixture of such antibodies that bind an epitope of the PD-L1 of interest and are capable of binding and capturing the PD-L1 of interest in a biological sample such that under suitable conditions, the complex of capture reagent and PD-L1 of interest can be separated from the rest of the sample. In certain embodiments, the capture reagent is immobilized or immobilizable.

[165] The term“detectable antibody” refers to an antibody that binds the PD-L1 of interest and is capable of being detected either directly through a label amplified by a detection means, or indirectly through, e.g, another antibody that is labeled. In certain embodiments, the detectable antibody is an antibody from a non-human species that binds to human antibodies. In certain embodiments, the detectable antibody is an anti-PD-Ll antibody or mixture of such antibodies that bind an epitope of the PD-L1 of interest. For direct labeling, the antibody is typically conjugated to a moiety that is detectable by some means. In certain embodiments, the detectable antibody is conjugated to horseradish peroxidase (HRP).

[166] The term“detection means” refers to a moiety or technique used to detect the presence of the detectable antibody through signal reporting that is then read out in the assay herein. It includes reagents that amplify the immobilized label such as the label captured onto a microtiter plate.

[167] An“isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present at extrachromosomal location or at a chromosomal location that is different from its natural chromosomal location.

[168] “Isolated nucleic acid encoding an anti-PD-Ll antibody” refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an anti-PD-Ll antibody, antigen binding fragment or derivative thereof, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[169] The term“polynucleotide” is equivalent to the term“nucleic acid”, each of which includes one or more of: polymers of deoxyribonucleic acids (DNAs), polymers of ribonucleic acids (RNAs), analogs of these DNAs or RNAs generated using nucleotide analogs, and derivatives, fragments and homologs thereof.

[170] The term“vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing or controlling the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as“expression vectors”.

[171] The term“expression vector”, as used herein, refers to a polynucleotide or nucleic acid, linear or circular, comprising one or more expression units. The term“expression unit” denotes a polynucleotide segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell. An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in operable configuration. An expression vector contains one or more expression units.

[172] The term“host cell” refers to a cell which can support the replication or expression of the expression vector. The terms“host cell”,“host cell line”, and“host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include“transformants” and“transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but rather may contain non-identical mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included within the scope of the terms host cell”,“host cell line”, and “host cell culture”.

[173] The term“package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products. [174] The present invention is described more fully hereinafter using illustrative, non limiting embodiments, and references to the accompanying figures. This invention may, however, be embodied in many different forms and should not be construed as to be limited to the embodiments set forth below. Rather, these embodiments are provided so that this disclosure is thorough and conveys the scope of the invention to those skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

[175] Figure 1 depicts exemplary anti-PD-Ll antibodies, and derivatives thereof, comprising one or more antigen binding domain. The depictions of exemplary molecules in Figure 1 are for illustrative purposes of a limited set of embodiments of the present invention. It is to be understood that these exemplary molecules do not intend, nor should any be construed, to be wholly definitive as to the arrangement of any structural features and/or components of a molecule of the present invention. The relative size, location, or number of features shown in the schematics of Figure 1 have been simplified. The schematics in Figure 1 are not intended to accurately portray any information regarding the relative sizes of molecular structures in any embodiment of the present invention.

[176] Figure 2 graphically shows the results of a human membrane proteome array assay used to test the specificity and selectivity of PD-L1 binding by the exemplary anti-PD-Ll single-chain variable fragment (scFv) (SEQ ID NO: 13). The results shown in Figure 2 show that only PD-L1 (CD274) was identified and validated as being bound by anti-PD-Ll scFv (SEQ ID NO: 13) from among about 5,300 different proteins. Flow cytometry was used to identify the binning signal for each individual protein, and data was normalized to

background signal.

Introduction

[177] The present invention provides anti-PD-Ll antibodies, and derivatives thereof, such as immunoconjugates, and methods of using the same. The anti-PD-Ll antibodies of the present invention, and derivatives thereof, are useful, for e.g ., for the targeting of PD-L1 in vivo ; detecting, quantifying, or purifying PD-L1; delivery of cargos to PD-L1 -expressing cells; selective killing of specific cell-types in the presence of other cells; the stimulation of beneficial immune responses in vivo such as, e.g. , by modifying PD-L1 signaling pathways and/or killing PD-L1 -expressing cells; and diagnosing, preventing, and treating a variety of diseases, disorders, and conditions, such as, e.g. , cancers, tumors, other growth abnormalities, immune disorders, and microbial infections involving PD-L1 expression. Examples of beneficial immune responses which may be stimulated by an anti-PD-Ll antibody of the present invention include increased immunosurveillance; reductions in T-cell dysfunction; reversal of anergy conditions; increased T-cell activation, expansion, and/or survival; either increased or reduced inflammation; increasing epitope spreading; altering the

immunodominance of antigen-specific T-cells; reduced auto-immunity; reduced graft-versus- host response; and reduced transplant rejection.

[178] PD-L1, also recognized in the art as PDL1, programmed cell death 1 ligand 1,

PDCD1 Ligand 1, PDCD1L1, PDCD1LG1, B7 homolog 1 (B7-H1), and CD274, is a ligand for the programmed cell death-l receptor of T- and B-cells (Dong H et ah, Nat Med 5: 1365-9 (1999); Freeman G et ah, J Exp Med 192: 1027-34 (2000); Latchman Y et ah, Nat Immunol 2: 261-8 (2001)) and the B7-1 receptor and CD80 receptor found on T-cells (Butte M et ah, Immunity 27: 111-22 (2007); Park J et ah, Blood 116: 1291-8 (2010)). While the name PD-L1 might refer to multiple proteins with related structures and polypeptide sequences from various species, for the purposes of the structural examples of this section, the term“PD-L1” refers to the PD-l ligands present in humans whose exact sequence might vary slightly based on the isoform and from individual to individual (see e.g. SEQ ID NO:20). With regard to humans, PD-L1 refers to the protein represented by the predominant polypeptide sequence UniProt Q9NZQ7 and NCBI accession AAI13735.1; however, different isoforms and variants exist due to splicing, polymorphisms and/or mutations (see e.g. Abelson A et ah, Genes Immun 8: 69-74 (2007); Wang S et ah, J Clin Immunol 27: 563-7 (2007); Hayashi M et ah, Eur J Endocrinol 158: 817-22 (2008); Mitchell A et a\., J Clin Endocrinol Metab 94: 5139-45 (2009); Y ang Q et al . , Clin Exp Rheumatol 29 : 13-8 (2011); Ma Y et al . , Int J Clin Exp Med 15: 16585-91 (2015)). A skilled worker will be able to identify other PD-L1 proteins in humans, even if they differ from the referenced sequences ( see e.g. SEQ ID NOs: 20-23).

[179] PD-L1 may be absent from most healthy tissues under normal conditions; however, PD-L1 expression can be induced by exposure of most nucleated mammalian cells to an interferon(s) ( see e.g ., Dong H et al., Nat Med 8: 793-800 (2002); Chen L, Nat Rev Immunol 4: 336-47 (2004); Hirano F et al., Cancer Res 65: 1089-96 (2005); Zou W, Chen L, Nat Rev Immunol 8: 467-7 (2008); Flies D et al., Yale J Biol Med 84: 409-21 (2011); Chen J et al., Immunobiology 217: 385-93 (2012); Spranger S et al. Sci Transl Med 5: 200ral l6 (2013)). During certain malignancies, PD-L1 upregulation in tumor microenvironments might lead to over-suppression of immune responses to tumor cells, which fits common notions about PD- Ll’s involvement in adaptive immune resistance of tumor cells to a host’s immune system (see e.g., Zou W, Chen L, Nat Rev Immunol 8: 467-7 (2008); Zheng P, Zho Z, Biomark Cancer 7: 15-8 (2015)).

[180] PD-L1 is an attractive target for therapies because PD-L1 is strongly expressed by certain tumor cells and tumor-infiltrating lymphocytes whereas healthy, human tissues and cells seldom express high levels of PD-L1 at a cellular surface (see e.g. Dong H et al., Nat Med 8: 793-800 (2002); Chen L, Nat Rev Immunol 4: 336-47 (2004); Hirano F et al., Cancer Res 65: 1089-96 (2005); Chen L, Han X, J Clin Invest 125: 3384-91 (2015)). In humans, expression of cell-surface PD-L1 by tumor cells has been observed in a number of primary tumor biopsies and tumor-cell cultured cells assessed by immunohistochemistry, including cells and tissues related to, e.g, carcinomas, gliomas, B-cell lymphomas, adult T-cell leukemia/lymphoma (ATLL), angioimmunoblastic T-cell lymphomas (AITLs), bladder cancers, chronic lymphocytic leukemias (CLLs), epithelial malignancies, oral squamous cell carcinomas, esophageal squamous cell carcinomas (ESCCs), lung cancers, non-Hodgkin lymphomas (NHLs), pancreatic cancers, renal cell carcinomas (RCCs), small lymphocytic lymphomas (SLLs), squamous cell carcinomas of the head and neck (SCCHN), and virus- associated malignancies (see e.g., Brown J et al., Immunol 170: 1257-66 (2003); Strome S et al., Cancer Res 63 : 6501-5 (2003); Wintterle et al., Cancer Res 63 : 7462-7 (2003); Thompson R et al., Cancer Res 66: 3381-5 (2006); Nomi T et al., Clin Cancer Res 13 : 2151-7 (2007); Thompson et al., Clin Cancer Res 13 : 1757-61 (2007); Andorsky D et al., Clin Cancer Res 17: 4232-44 (2011); Chen B et al., Clin Cancer Res 19: 3462-73 (2013); Chen M et al., Oncotarget 7: 7913-24 (2016); Wu C et al., Sci Rep 6: 19740 (2016)).

[181] It may be possible to harness the power of the immune system using an anti-PD-Ll antibody of the present invention by altering immune checkpoint pathways involving PD-L1 expression as it relates to T-cell dysfunction and general anergy in order to increase the immune system’s surveillance and killing of malignant cells, e.g. tumor and/or infected cells, and at localized tissue sites, such as, e.g. , by targeting the tumor or infection

microenvironment. It may be possible to modulate the immune system using therapies directed against PD-L1 by altering immune checkpoint pathways involving PD-L1 as it relates to autoimmunity, immunodominance, and self-tolerance. Furthermore, it may be possible to modulate the immune system using therapies directed against PD-L1 by altering PD-L1 functions unrelated to immune checkpoint pathways, such as, e.g. PD-L1 functions in pre-activation events in the development of immune cells, including in central tolerance and allergies.

I. Anti-PD-Ll Antibodies, Antigen Binding Fragments, and Derivatives Thereof

[182] In one aspect, the present invention provides anti-PD-Ll antibodies that each specifically bind to a PD-L1 molecule. Antibodies of the present invention are useful, e.g. , for targeting PD-L1, such as for targeting therapeutics, diagnostics, and/or theragnostic in vivo. In addition, antibodies of the present invention are useful, e.g, for the detection, quantification, or purification of PD-L1 and PD-L1 epitopes in biological samples, including in vitro or ex vivo.

A. Anti-PD-Ll Antibodies, and Derivatives Thereof, which Specifically Bind PD-L1

[183] In one aspect, the invention provides anti-PD-Ll antibodies that binds to a PD-L1 antigen, such as, e.g. , a human PD-L1 protein described herein (e.g. SEQ ID NO:20), with an dissociation constant (KD) of less than 1 O⁴ moles/liter (M), less than 10⁵ M, or less than 10⁶ M. In certain further embodiments, the anti-PD-Ll antibody binds specifically and/or with high affinity to PD-L1 (e.g. SEQ ID NO:20). In certain further embodiments, the anti-PD-Ll antibody binds selectively and/or with high affinity to PD-L1 (e.g. SEQ ID NO:20). In certain embodiments of any of the aforementioned, the PD-L1 antigen is an extracellular epitope of the PD-L1 molecule. In certain further embodiments, the epitope comprises the natively positioned amino acid residue F42. In certain embodiments of any of the aforementioned, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, is isolated.

[184] The antibody of the invention may be defined by reference to one or more CDRs, such as those defined in SEQ ID NOs: 1-6 (Kabat), 18-19 (Chothia), and 24-29

(MacCallum). [185] The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 1-6. The antibody of the invention may comprise 1, 2, 3,

4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 1-3, 5-6, and 18 (Chothia). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 1-3, 6, and 18-19 (Chothia). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 24-29 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 18 (Chothia), 24-26 (MacCallum), and 28-29 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 18-19 (Chothia), 24-26 (MacCallum), and 29 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 1 and 25-29 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 2-6 and 24 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 3- 6 and 24-25 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 4-6 and 24-26 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 5-6 and 24-27 (MacCallum). The antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of the CDR sequences shown in SEQ ID NOs: 6 and 24-28 (MacCallum).

[186] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: an immunoglobulin region comprising three CDRs

comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO: 19 (Chothia), and SEQ ID NO:6.

In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:5, and SEQ ID NO:6. In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:6.

In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO:28 (MacCallum), and SEQ ID NO:29 (MacCallum). In certain embodiments, the anti- PD-L1 antibody, or antigen-binding fragment or derivative thereof, comprises an

immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:28 (MacCallum), and SEQ ID NO:29 (MacCallum). In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum). In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum).

[187] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3. In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum). In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum). In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum). In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises an immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3. [188] In certain embodiments, the anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, comprises three, four, five, six, or more CDRs and/or HVRs.

[189] In certain embodiments, the anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, comprises: (a) a light chain variable region (HVR-L) comprising three CDRs wherein one or more comprises, consists essentially of, or consists of an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum); and/or (b) a heavy chain variable region (HVR-H) comprising three CDRs wherein one or more comprises, consists essentially of, or consists of an amino acid sequence show in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), SEQ ID NO:27 (MacCallum), SEQ ID NO:28, and SEQ ID NO:29 (MacCallum). In certain further embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a light chain variable region (HVR-L) comprising three CDRs, each comprising, consisting of, or consisting essentially of an amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:24

(MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum); and/or (b) a heavy chain variable region (HVR-H) comprising three CDRs, each comprising, consisting of, or consisting essentially of an amino acid sequence show in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), SEQ ID NO:27 (MacCallum), SEQ ID NO:28, and SEQ ID NO:29 (MacCallum).

[190] In certain embodiments, the anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, comprises a heavy variable chain or VH as in any of the embodiments provided herein, and a light variable chain or VL as in any of the embodiments provided herein, optionally including post-translational modifications of those polypeptides.

[191] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[192] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[193] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) an

immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[194] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[195] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO:28 (MacCallum), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[196] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:28 (MacCallum), and SEQ ID NO:29

(MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3, respectively

[197] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[198] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[199] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[200] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum),

SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[201] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) an

immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively. [202] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[203] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO:28 (MacCallum), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[204] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:28 (MacCallum), and SEQ ID NO:29

(MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26

(MacCallum), respectively.

[205] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24

(MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[206] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[207] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[208] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[209] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) an

immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[210] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 1, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[211] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO:28 (MacCallum), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[212] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:28 (MacCallum), and SEQ ID NO:29

(MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[213] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 1, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[214] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: l, SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum), respectively.

[215] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[216] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[217] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) an

immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[218] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[219] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO:28 (MacCallum), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[220] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:28 (MacCallum), and SEQ ID NO:29

(MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[221] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[222] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24, SEQ ID NO:2, and SEQ ID NO:26 (MacCallum), respectively.

[223] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[224] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:4, SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum),

SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[225] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) an

immunoglobulin region comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:5, and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[226] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:6, respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[227] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO:28 (MacCallum), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[228] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO:28 (MacCallum), and SEQ ID NO:29

(MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[229] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:27, SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24

(MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[230] In certain embodiments, the anti-PD-Ll antibody, or antigen-binding fragment or derivative thereof, comprises: (a) a heavy chain variable region (HVR-H) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO: 18 (Chothia), SEQ ID NO: 19 (Chothia), and SEQ ID NO:29 (MacCallum), respectively; and (b) a light chain variable region (HVR-L) comprising three CDRs comprising, consisting of, or consisting essentially of the amino acid sequences show in SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:3, respectively.

[231] In certain embodiments, the antibody comprises the VH polypeptide having the sequence of SEQ ID NO: 8 and the VL polypeptide having the sequence of SEQ ID NO: 7 or 9, optionally including post-translational modifications of those polypeptides.

[232] In certain embodiments, the anti-PD-Ll antibody comprises a light chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:7, and/or a heavy chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:8, optionally including post- translational modifications of those polypeptides.

[233] In certain embodiments, the anti-PD-Ll antibody is a an antibody fragment and/or synthetically engineered antibody derivative, such as, e.g. an autonomous VH domain, single domain antibody domain (sdAb), camelized scaffold comprising a VH domain, VNAR fragment, single-chain variable (scFv) fragment, nanobody, Fd fragment consisting of the heavy chain and CH I domains, single chain FV-CH3 minibody, Fc antigen binding domain (Fcabs), scFv-Fc fusion, multimerizing scFv fragment (diabody, triabody, tetrabody), disulfide-stabilized antibody variable (Fv) fragment (dsFv), disulfide-stabilized antigen binding (Fab) fragment consisting of the VL, VH, CL and CH I domains, single-chain variable- region fragments comprising a disulfide-stabilized heavy and light chain (sc-dsFv), bivalent nanobody, bivalent minibody, bivalent F(ab’)2 fragment (Fab dimer), bispecific tandem VHH fragment, bispecific tandem scFv fragment, bispecific nanobody, bispecific minibody, and any genetically manipulated counterparts of the foregoing that retain paratope and binding function, such as, e.g. , wherein the relative orientation or order of the heavy and light chains is reversed or“flipped”.

[234] In certain embodiments, the anti-PD-Ll antibody is monoclonal and/or humanized.

[235] In certain embodiments, the anti-PD-Ll antibody is a single-chain variable fragment, such as, e.g. , consisting of, comprising, or consisting essentially of the polypeptide of any one of SEQ ID NOs: 10-17, optionally including post-translational modifications of those polypeptides. [236] In certain embodiments, the anti-PD-Ll antibody is a full-length antibody, e.g, an intact IgG2 or IgG4 antibody or other antibody class or isotype as defined herein. In certain further embodiments, the anti-PD-Ll antibody is an intact IgGl antibody or other antibody class or isotype as defined herein.

[237] In certain embodiments, the anti-PD-Ll antibody can be of, or derived from, any antibody class and/or isotype, including for example, IgA (e.g. IgAi, IgA_2. or slgA), IgD, IgE, IgG (e.g. IgGi, IgG₂, IgG3, or IgG₄), or IgM. The“class” of an antibody may refer to the type of constant domain or constant region possessed by its heavy chain. There are at least five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. , IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

The heavy chain constant domains that correspond to the different classes of

immunoglobulins are called a, d, e, g, and m, respectively.

[238] In certain embodiments, the anti-PD-Ll antibody can be of, or derived from, any antibody of any species, including but not limited to species of mammalian origin (e.g.

simians, rodents, goats, and rabbits).

[239] In certain embodiments, the PD-L1 -targeting molecule of the present invention comprises a binding region comprising an immunoglobulin-type polypeptide capable of exhibiting specific and/or high-affinity binding to human PD-L1 and/or PD-L1 present on a cellular surface of a cell, such as, e.g. , PD-L1 expressing cell or PD-L1 positive cell. In certain embodiments, the anti-PD-Ll antibody, antigen-binding fragment or derivative thereof binds to a human PD-L1 antigen (e.g, SEQ ID NO: 20), e.g. present or expressed on a surface of a cell. In certain embodiments, the antibody, antigen-binding fragment or derivative thereof binds to a primate PD-L1 antigen (e.g, SEQ ID NO: 21), e.g. present or expressed on a surface of a cell. In certain embodiments, the antibody, antigen-binding fragment or derivative thereof binds to a murine PD-L1 antigen (e.g, SEQ ID NOs: 22-23), e.g. present or expressed on a surface of a cell.

[240] In certain embodiments, a binding region of an anti-PD-Ll antibody of the present invention is a cell-targeting component, such as, e.g, a domain, molecular moiety, or agent, capable of binding specifically to an extracellular part of a PD-L1 target biomolecule on a cell surface (i.e. an extracellular target biomolecule) with high affinity. For purposes of the present invention, the term“PD-L1 binding region” refers to a molecular moiety (e.g. a proteinaceous molecule) or agent capable of specifically binding an extracellular part of a PD-L1 molecule with high affinity, such as, e.g, having a dissociation constant with regard to PD-L1 of 10⁵ to 10¹² M. As used herein, PD-L1 binding refers to the ability to bind to an extracellular part of PD-L1, including an isoform or variant of human PD-L1.

[241] An extracellular part of a target biomolecule refers to a portion of its structure exposed to the extracellular environment when the molecule is physically coupled to a cell, such as, e.g ., when the target biomolecule is expressed at a cellular surface by the cell. In this context, exposed to the extracellular environment means that part of the target biomolecule is accessible by, e.g. , an antibody or at least a binding moiety smaller than an IgG such as a single-domain antibody domain, a nanobody, a heavy-chain antibody domain derived from camelids or cartilaginous fishes, a single-chain variable fragment. The exposure to the extracellular environment of or accessibility to a part of target biomolecule physically coupled to a cell may be empirically determined by the skilled worker using methods well known in the art.

[242] In certain embodiments, an anti-PD-Ll antibody of the present invention comprises a binding region comprising one or more polypeptides capable of selectively and/or

specifically binding an extracellular part of PD-L1.

[243] Binding regions may be functionally defined by their ability to bind to target molecules. Immunoglobulin-type binding regions are commonly derived from antibody or antibody-like structures. Immunoglobulin (Ig) proteins (members of the Ig family) have a structural domain known as an Ig domain. Ig domains range in length from about 70-110 amino acid residues and possess a characteristic Ig-fold, in which typically 7 to 9 antiparallel beta strands arrange into two beta sheets which form a sandwich-like structure. The Ig fold is stabilized by hydrophobic amino acid interactions on inner surfaces of the sandwich and highly conserved disulfide bonds between cysteine residues in the strands. Ig domains may be variable (IgV or V-set), constant (IgC or C-set) or intermediate (Igl or I-set). Some Ig domains may be associated with a complementarity determining region (CDR), also called a “complementary determining region”, which is important for the specificity of antibodies binding to their epitopes. Ig-like domains are also found in non-immunoglobulin proteins and are classified on that basis as members of the Ig superfamily of proteins. The HUGO Gene Nomenclature Committee (HGNC) provides a list of members of the Ig-like domain containing family.

[244] An immunoglobulin-type binding region may be a polypeptide sequence of an antibody or antigen-binding fragment thereof wherein the amino acid sequence has been varied from that of a native antibody or an Ig-like domain of a non-immunoglobulin protein, for example by molecular engineering or selection by library screening. Because of the relevance of recombinant DNA techniques and in vitro library screening in the generation of immunoglobulin-type binding regions, antibodies can be redesigned to obtain desired characteristics, such as smaller size, cell entry, or other improvements for in vivo and/or therapeutic applications. The possible variations are many and may range from the changing of just one amino acid to the complete redesign of, for example, a variable region. Typically, changes in the variable region will be made in order to improve the antigen-binding characteristics, improve variable region stability, or reduce the potential for immunogenic responses.

[245] There are numerous immunoglobulin-type binding regions contemplated as components of the present invention. In certain embodiments, the immunoglobulin-type binding region is derived from an immunoglobulin binding region, such as an antibody paratope capable of binding an extracellular part of PD-L1. In certain other embodiments, the immunoglobulin-type binding region comprises an engineered polypeptide not derived from any immunoglobulin domain but which functions like an immunoglobulin binding region by providing high-affinity binding to an extracellular part of PD-L1. This engineered polypeptide may optionally include polypeptide scaffolds comprising or consisting essentially of complementary determining regions from immunoglobulins as described herein.

[246] There are also numerous binding regions that are useful for targeting polypeptides to specific cell-types via their high-affinity binding characteristics. In certain embodiments, the binding region of the anti-PD-Ll antibody of the present invention is selected from the group which includes autonomous VH domains, single-domain antibody domains (sdAbs), heavy- chain antibody domains derived from camelids (VHH fragments or VH domain fragments), heavy-chain antibody domains derived from camelid VHH fragments or VH domain fragments, heavy-chain antibody domains derived from cartilaginous fishes, immunoglobulin new antigen receptors (IgNARs), VNAR fragments, single-chain variable (scFv) fragments, nanobodies, Fd fragments consisting of the heavy chain and Gil domains, single chain Fv- CH3 minibodies, dimeric CH2 domain fragments (CH2D), FC antigen binding domains (Fcabs), isolated complementary determining region 3 (CDR3) fragments, constrained framework region 3, CDR3, framework region 4 (FR3-CDR3-FR4) polypeptides, small modular immunopharmaceutical (SMTP) domains, scFv-Fc fusions, multimerizing scFv fragments (diabodies, triabodies, tetrabodies), disulfide stabilized antibody variable (Fv) fragments, disulfide stabilized antigen-binding (Fab) fragments consisting of the VL, VH, CL and CH! domains, bivalent nanobodies, bivalent minibodies, bivalent F(ab’)2 fragments (Fab dimers), bispecific tandem VHH fragments, bispecific tandem scFv fragments, bispecific nanobodies, bispecific minibodies, and any genetically manipulated counterparts of the foregoing that retain its paratope and binding function, such as, e.g ., wherein the relative orientation or order of the heavy and light chains is reversed or flipped (see Ward E et al., Nature 341 : 544-6 (1989); Davies J, Riechmann L, Biotechnology (NY) 13 : 475-9 (1995); Reiter Y et al., Mol Biol 290: 685-98 (1999); Riechmann L, Muyldermans S, J Immunol Methods 231 : 25-38 (1999); Tanha J et al., J Immunol Methods 263 : 97-109 (2002); Vranken W et al., Biochemistry 41 : 8570-9 (2002); Jespers L et al., J Mol Biol 337: 893-903 (2004); Jespers L et al., Nat Biotechnol 22: 1161-5 (2004); To R et al., J Biol Chem 280: 41395-403 (2005); Saerens D et al., Curr Opin Pharmacol 8: 600-8 (2008); Dimitrov D, MAbs 1 : 26-8 (2009); Weiner L, Cell 148: 1081-4 (2012); Ahmad Z et al., Clin Dev Immunol 2012: 980250 (2012)).

[247] There are a variety of binding regions comprising polypeptides derived from the constant regions of immunoglobulins, such as, e.g. , engineered dimeric Fc domains, monomeric Fes (mFcs), scFv-Fcs, VHH-FCS, CH2 domains, monomeric CH3 S domains

(IUCH3 S), synthetically reprogrammed immunoglobulin domains, and/or hybrid fusions of immunoglobulin domains with ligands (Hofer T et al., Proc Natl Acad Sci U. S. A. 105: 12451-6 (2008); Xiao J et al., J Am Chem Soc 131 : 13616-13618 (2009); Xiao X et al., Biochem Biophys Res Commun 387: 387-92 (2009); Wozniak-Knopp G et al., Protein Eng Des Sel 23 289-97 (2010); Gong R et al., PLoS ONE 7: e42288 (2012); Wozniak-Knopp G et al., PLoS ONE 7: e30083 (2012); Ying T et al., J Biol Chem 287: 19399-408 (2012); Ying T et al., J Biol Chem 288: 25154-64 (2013); Chiang M et al., J Am Chem Soc 136: 3370-3 (2014); Rader C, Trends Biotechnol 32: 186-97 (2014); Ying T et al., Biochimica Biophys Acta 1844: 1977-82 (2014)).

[248] In certain embodiments, the binding region of the anti-PD-Ll antibody of the present invention is an intact antibody and/or comprises an Fc region. The term“Fc region” refers to part of the fragment crystallizable region, a C-terminal proximal region of certain heavy chains of native immunoglobulins that contains at least a portion of the constant region, such as, e.g. , at least the second and third constant (CH) domains and a glycosylation site.

However, as used herein, the term“Fc region” includes native sequence Fc regions and variant or mutated Fc regions or fragments thereof. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Rabat E (1991), supra. [249] In certain embodiments, the anti-PD-Ll antibody of the present invention includes polyclonal antibodies; monoclonal antibodies; multiclonal antibodies; chimeric antibodies; humanized antibodies; primatized antibodies; CDR grafted antibodies; human antibodies (including recombinantly produced human antibodies); recombinantly produced antibodies; intrabodies; multispecific antibodies; bispecific antibodies; monovalent antibodies;

multivalent antibodies; anti -idiotypic antibodies; synthetic antibodies, including muteins and variants thereof; immunoglobulin new antigen receptor (IgNAR); antigen binding fragments and derivatives thereof such as an antibody variable fragment (Fv), single-chain variable fragment (scFv), scFv-Fc fusion, single chain Fv-CFB minibody, bispecific tandem scFv fragment, multimerizing scFv fragment (diabody, triabody, tetrabody), Fd fragment, Fab fragment, F(ab') fragment, bivalent F(ab’)2 fragment, single-domain antibody fragment (sdAb) or nanobody®, bivalent nanobody®, bispecific nanobody®, bivalent minibody, bispecific minibody, autonomous VH domain, VHH, bispecific tandem VHH fragment, VNAR fragment, Fc antigen binding domain (Fcab), isolated complementary determining region 3 (CDR3) fragment, constrained framework region 3, framework region 4 (FR3-CDR3-FR4) polypeptide, dimeric CH2 domain fragment (CH2D), heavy-chain antibody domain derived from a camelid VHH fragment or VH domain fragment, heavy-chain antibody domain derived from a cartilaginous fish, and small modular immunopharmaceutical (SMIP) domain; and any other immunoreactive molecule or genetically manipulated counterparts of any of the foregoing which retain binding functionality (see Ward E et ak, Nature 341 : 544-6 (1989); Davies J, Riechmann L, Biotechnology (NY) 13 : 475-9 (1995); Reiter Y et ak, Mol Biol 290: 685-98 (1999); Riechmann L, Muyldermans S, J Immunol Methods 231 : 25-38 (1999);

Tanha J et ak, J Immunol Methods 263 : 97-109 (2002); Vranken W et ak, Biochemistry 41 : 8570-9 (2002); Jespers L et ak, J Mol Biol 337: 893-903 (2004); Jespers L et ak, Nat Biotechnol 22: 1161-5 (2004); To R et ak, J Biol Chem 280: 41395-403 (2005); Saerens D et ak, Curr Opin Pharmacol 8: 600-8 (2008); Dimitrov D, MAbs 1 : 26-8 (2009); Weiner L, Cell 148: 1081-4 (2012); Ahmad Z et ak, Clin Dev Immunol 2012: 980250 (2012)).

[250] Any of the aforementioned PD-L1 binding proteins may be suitable for use as a PD- Ll binding region or modified to create one or more PD-L1 binding regions for use in an anti-PD-Ll antibody of the present invention. Any of the above antibody structures may be used as a component of a molecule of the present invention as long as the binding region component has a dissociation constant of 10⁴ to 10¹³ M or less, preferably less than 200 nanomolar (nM), towards a PD-L1 molecule. B. Variants of the PD-L1 Antibodies, and Fragments and Derivatives Thereof

[251] In certain embodiments, amino acid sequence variants of the antibodies, antigen binding fragments, and antibody variable region domains provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody of the present invention and/or a fragment or derivatives thereof. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g ., a certain binding affinity level of antigen binding, a certain level of KD, and/or a certain level of Koff

[252] In certain embodiments, the anti-PD-Ll antibody can comprise modifications and/or mutations that alter the properties of the antibodies and/or antigen-binding fragments, such as those that increase half-life, decrease immunogenicity, increase or decrease cytotoxicity, alter glycosylation, and/or increase or decrease ADCC, CDC, antibody-dependent cell-mediated phagocytosis (ADCP), antigen cross-linking, PD-l antagonism, PD-L1 antagonism, PD-L1 agonist activity, and/or cellular internalization, as is known in the art. In certain

embodiments, the anti-PD-Ll antibody sequence is modified to provide for non-natural amino acid or radioisotope incorporation. In certain embodiments, the anti-PD-Ll antibody sequence is modified to provide for conjugation or fusion to a cargo or other moiety, e.g. site- specific attachment. In certain embodiments, the anti-PD-Ll antibody is monoclonal, camelized, chimeric, or humanized. In a further aspect of the invention, an anti-PD-Ll antibody according to any of the above embodiments is a monoclonal antibody, including a camelized, chimeric, humanized or human antibody.

1. Defining the Sequence and Sequence Variants of the Antibody of the Present Invention

[253] In certain embodiments, variants of the antibody of the present invention, or fragments or derivatives thereof, having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g. , retained/improved antigen binding, decreased immunogenicity, improved ADCC, and/or improved CDC, improved PD-L1 inhibition (e.g. inhibition of PD- Ll interaction with cognate receptors thereby inhibiting or otherwise altering downstream signal transduction mediated by PD-L1 receptor binding), improved apoptosis via PD-L1 binding, and/or improved ADCP.

[254] As used herein, the assignment of amino acids to each domain, framework region and CDR may be in accordance with one of the schemes provided by KabatE (1991), supra ; Chothia C, Lesk A, JMol Biol 196: 901-17 (1987); Chothia C et al. , Nature 342: 877-83 (1989); MacCallum R et al. , JMol Biol 262: 732-45 (1996); Handbook of Therapeutic Antibodies 3^rd Ed. (Dubel S, ed., Wily-VCH Verlag GmbH and Co (2007); or AbM (Oxford Molecular/MSI Pharmacopeia) unless otherwise noted. As is well known in the art variable region residue numbering is typically as set forth in Chothia or Rabat. Amino acid residues which comprise CDRs as defined by Rabat, Chothia, MacCallum (also known as Contact) and AbM are set out below in Table I. Note that MacCallum uses the Chothia numbering system.

Table I. CDR Identification and Demarcation

[255] Variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art, such as, as set out above, for example using the Rabat nomenclature system, or by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Antibody

Engineering (Rontermann R, Diibel S, eds., Springer, New York, NY (2001)) and Current Protocols in Immunology (Coligan J et al. (eds), John Wiley and Sons Inc., Hoboken, NJ, (2000)). Exemplary databases of antibody sequences are described in, and can be accessed through, the“Abysis” website and/or the VBASE2 website (see Retter et al., Nucl Acids Res 33(Database issue): D671-4 (2005)). Preferably the sequences are analyzed using the Abysis database, which integrates sequence data from Rabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB (Antibody Engineering (Rontermann R, Diibel S (eds.), Springer-Verlag, Heidelberg, (2010)). The Abysis database website further includes general rules that have been developed for identifying CDRs which can be used in accordance with the teachings herein.

[256] For heavy chain constant region amino acid positions, numbering is according to the Eu index described in Edelman G et al., Proc Natl Acad Sci U.S.A. 63 : 78-85 (1969), describing the amino acid sequence of the myeloma protein Eu, which reportedly was the first human IgGl sequenced. The Eu index of Edelman is also set forth in Kabat E ( 1991 ), supra. Thus, the terms“Eu index as set forth in Kabat” or“Eu index of Kabat” or“Eu index” or“Eu numbering” in the context of the heavy chain refers to the residue numbering system based on the human IgGl Eu antibody of Edelman et al, supra , as set forth in Kabat E

( 1991 ), supra. The numbering system used for the light chain constant region amino acid sequence is similarly set forth in Kabat et al., {supra).

[257] The present invention also provides‘variants’ of the antibody polypeptide sequence and‘variants’ of the antibody polynucleotide sequence that exhibit“sequence identity”, sequence similarity” or“sequence homology” to the reference polypeptide sequence of the antibody and the reference polynucleotide sequence of the antibody. A“homologous” polypeptide or polynucleotide may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the reference

polypeptide or polynucleotide. As used herein, the percent homology between two amino acid sequences or two polynucleotide sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences {e.g., % homology= # of identical positions/total # of positionsx 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as those as described herein.

[258] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g, segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g. CLETSTAL W and iterative refinement (see e.g. Thompson J et al., Nucleic Acids Res 22: 4673-80 (1994); Gotoh O, J Mol Biol 264: 823-38 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences, such as, e.g. Match-box, Gibbs sampling, or Align-M (see e.g. Depiereux E, Feytmans E, Comput Appl Biosci 8: 501-9 (1992); Lawrence C et la., Science 262: 208-14 (1993); Van Walle I et al., Bioinformatics 20: 1428-35 (2004)). Thus, percent sequence identity is determined by conventional methods, such as, as described in Altschul S, Erickson B, Bull Math Biol 48: 603-16 (1986); Henikoff S, Henikoff J, Proc Natl Acad Sci U.S.A. 89: 10915- 19 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).

Table II. Alignment Scores for Determining Sequence Identity

[259] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20 to 25 amino acid residues, or an affinity tag.

[260] In certain embodiments, the variant polypeptide sequence of the antibody may have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a polypeptide sequence recited herein (and which retain at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to the polypeptide sequences recited herein), as long as it retains antigen binding function.

[261] In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where there is a substitution with a non

conservative amino acid, the embodiments exhibiting the requisite sequence identity will retain the desired function or activity of the antibody of the invention.

[262] In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for clostridial polypeptide amino acid residues. The polypeptide sequence of the antibody can also comprise non-naturally occurring amino acid residues.

[263] Non-naturally occurring amino acids include, without limitation, trans-3- methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N- methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo- cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2- azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. In a second method, translation is accomplished in vivo using Xenopus oocytes by

microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti G et ah, J Biol Chem 271 : 19991-8 (1996)). Within a third method, transcription and translation is carried out in vivo using bacterial cells cultured in the absence of a natural amino acid that is to be replaced ( e.g . phenylalanine) and in the presence of the desired non- naturally occurring amino acid(s) (e.g. 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) ( see e.g. Koide H et al., Biochemistry 33 : 7470-6 (1994)). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site- directed mutagenesis to further expand the range of substitutions (Wynn R, Richards F, Protein Sci 2: 395-403 (1993)).

[264] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptide sequence of the antibody.

[265] Critical amino acid residues in the polypeptide sequence of the antibody can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham B, Wells J, Science 244: 1081-5, (1989)). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffmity labeling, in conjunction with mutation of putative contact site amino acids (see e.g. de Vos A et al., Science 255: 306-12 (1992); Smith L et al., JMol Biol 224: 899-904 (1992); Wlodaver A et al., FEBS Lett 309: 59-64 (1992)).

[266] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson J, Sauer R, Science 241 : 53-7 (1988); Bowie J, Sauer R, Proc Natl Acad Sci U.S.A. 86: 2152-6 (1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display and region-directed mutagenesis (e.g.

Derbyshire K et al., Gene 46: 145-52 (1986); Ner S et al., DNA 7: 127-34 (1988); Lowman H et al., Biochemistry 30: 10832-7 (1991)).

[267] In certain embodiments of the PD-L1 antibodies described herein, variants in the amino acid residue sequence of a component of the anti-PD-Ll antibody (e.g. an antibody- toxin conjugate) of the present invention herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody- toxin conjugate. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody component, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired

characteristics, e.g ., antigen-binding and/or toxin delivery.

[268] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g, improvements) in certain biological properties (e.g, increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g, using phage display -based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

[269] Alterations (e.g, substitutions) may be made in HVRs, e.g, to improve antibody affinity. Such alterations may be made in HVR“hotspots”, i.e.., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see e.g. Chowdhury, Methods Mol Biol 207: 179-96 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g, in Methods in Molecular Biology 178: 1-37 (O’Brien P et al., eds., Human Press, Totowa, NJ, (2001)). In certain embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g, error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g. 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[270] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs as long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g. conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR“hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided herein, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[271] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by

Cunningham, B. and Wells, J., Science , 244: 1081-5 (1989). In this method, a residue or group of target residues ( e.g . charged residues such as arginine, aspartate, histidine, lysing, and glutamine) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.

[272] Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[273] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

2. Structural/Functional Variants of the Antibodies of the Present Invention

[274] In certain embodiments, amino acid sequence variants of the antibodies, antigen binding fragments, and antibody variable region domains provided herein are contemplated. For example, the heavy chain of an antibody of the invention may comprise a HCDR1 sequence comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO: 18 (Chothia), or SEQ ID NO:27 (MacCallum), wherein one, two or three of these amino acid residues may be substituted by a different amino acid. The heavy chain of an antibody according to the invention may comprise a HCDR1 sequence comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO: 18 (Chothia), or SEQ ID NO:27 (MacCallum). In certain further embodiments, the antibody further comprises a light chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:7 or 9, optionally including post-translational modifications of those polypeptides.

[275] The heavy chain of an antibody of the invention may comprise a HCDR2 sequence comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO: 19 (Chothia), or SEQ ID NO:28, wherein one, two or three of these amino acids may be substituted by a different amino acid. The heavy chain of an antibody according to the invention may comprise a HCDR2 sequence comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO: 19 (Chothia), SEQ ID NO:28. In certain further embodiments, the antibody further comprises a light chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 7 or 9, optionally including post-translational modifications of those polypeptides.

[276] The heavy chain of an antibody of the invention may comprise a HCDR3 sequence comprising the amino acid sequence of SEQ ID NO:6, wherein one, two or three of these amino acids may be substituted by a different amino acid. The heavy chain of an antibody according to the invention may comprise a HCDR3 sequence comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:29 (MacCallum). In certain further embodiments, the antibody further comprises a light chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:7 or 9, optionally including post- translational modifications of those polypeptides.

[277] The light chain of an antibody of the invention may comprise a LCDR1 sequence comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:24 (MacCallum), wherein one, two or three of these amino acids may be substituted with a different amino acid. The light chain of an antibody according to the invention may comprise a LCDR1 sequence comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:24

(MacCallum). In certain further embodiments, the antibody further comprises a heavy chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 8, optionally including post-translational modifications of those polypeptides.

[278] The light chain of an antibody of the invention may comprise a LCDR2 sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:25 (MacCallum), wherein one, two or three of these amino acids may be substituted with a different amino acid. The light chain of an antibody according to the invention may comprise a LCDR2 sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:25

[279] The light chain of an antibody of the invention may comprise a LCDR3 sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:26 (MacCallum), wherein one, two or three of these amino acids may be substituted with a different amino acid. The light chain of an antibody according to the invention may comprise a LCDR3 sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:26

[280] The present invention provides anti-PD-Ll antibodies, and fragments and derivatives thereof, which comprise a polypeptide having a region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 1-17 and 24- 29 (MacCallum).

[281] In certain embodiments, the anti-PD-Ll antibody comprises a heavy chain variable domain (VH) sequence having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. In certain embodiments, a VH sequence having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8 contains substitutions (e.g. conservative substitutions), insertions, or deletions relative to the reference sequence (e.g. SEQ ID NO: 8) wherein the anti-PD-Ll antibody comprising that VH sequence retains the ability to bind to the same PD-L1 molecule as the anti-PD-Ll antibody comprising the reference sequence. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted relative to SEQ ID NO:8. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (e.g. in the FRs). Optionally, the anti-PD-Ll antibody comprises the VH polypeptide having the sequence of SEQ ID NO: 8, optionally including post-translational modifications. In certain embodiments, the VH comprises one, two or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6.

[282] In another aspect, an anti-PD-Ll antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9 contains substitutions (e.g. , conservative substitutions), insertions, or deletions relative to the reference sequence (e.g. SEQ ID NO:7 or SEQ ID NO:9), wherein the anti-PD-Ll antibody comprising that VL sequence retains the ability to bind to the same PD-L1 as the anti-PD-Ll antibody comprising the reference sequence. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted relative to SEQ ID NO:7 or SEQ ID NO:9. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (e.g. in the FRs). In certain embodiments, the anti-PD-Ll antibody comprises the VL sequence of SEQ ID NO:7 or SEQ ID NO:9. In certain embodiments, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: l or SEQ ID NO:24 (MacCallum); (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:25 (MacCallum); and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:26 (MacCallum).

[283] The antibody of the invention may be defined by reference to particular CDR sequences of its heavy chain variable domain and/or light chain variable domain.

[284] In certain embodiments, the anti-PD-Ll antibody comprises a heavy chain variable region (HVR-H) comprising three CDRs, each having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 4-6, 18-19

(Chothia), and 27-29 (MacCallum); or consisting essentially of an amino acid sequence show in any one of 4-6, 18-19 (Chothia), and 27-29 (MacCallum). In certain further

embodiments, the binding region further comprises: (a) a light chain variable region (HVR- L) comprising three CDRs, each having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3; or consisting essentially of an amino acid sequence shown in any one of SEQ ID

NO: 1, SEQ ID NO:2, and SEQ ID NO:3. In certain further embodiments, the binding region comprises: (a) a light chain variable region (HVR-L) comprising three CDRs, each comprising or consisting essentially of an amino acid sequence shown in any one of SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3; and (b) a heavy chain variable region (HVR-H) comprising three CDRs, each comprising or consisting essentially of an amino acid sequence show in any one of SEQ ID NOs: 4-6, 18-19 (Chothia), and 27-29 (MacCallum). [285] In certain embodiments, the anti-PD-Ll antibody comprises a heavy chain variable region (HVR-H) comprising three CDRs, each having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 4-6, 18-19

96%, 97%, 98%, or 99% identity to any one of SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum); or consisting essentially of an amino acid sequence shown in any one of SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum). In certain further embodiments, the binding region comprises: (a) a light chain variable region (HVR-L) comprising three CDRs, each comprising or consisting essentially of an amino acid sequence shown in any one of SEQ ID NO:24 (MacCallum), SEQ ID NO:25 (MacCallum), and SEQ ID NO:26 (MacCallum); and (b) a heavy chain variable region (HVR-H) comprising three CDRs, each comprising or consisting essentially of an amino acid sequence show in any one of SEQ ID NOs: 4-6, 18- 19 (Chothia), and 27-29 (MacCallum).

[286] The antibody of the invention may be defined by reference to the sequence of its heavy chain variable domain and/or light chain variable domain.

[287] In certain embodiments, the antibody comprises at least one heavy chain variable (VH) domain comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence shown in SEQ ID NO: 8. In certain further embodiments, the antibody comprises at least one heavy chain variable (VH) domain comprising, consisting essentially of, or consisting of SEQ ID NO:8, optionally including post-translational modifications of those polypeptides.

[288] In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment or derivative thereof, comprises: (a) a light chain region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identity to SEQ ID NO:7 or SEQ ID NO:9 or consisting essentially of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9; and/or (b) a heavy chain region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:8 or consisting essentially of the amino acid sequence of SEQ ID NO: 8, optionally including post-translational modifications of those polypeptides.

[289] In certain embodiments, the antibody comprises at least one light chain variable (VL) domain comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO:9. In certain further embodiments, the antibody comprises at least one light chain variable (VL) domain comprising, consisting essentially of, or consisting of SEQ ID NO:7 or SEQ ID NO:9, optionally including post-translational modifications of those polypeptides.

[290] Any of heavy chain variable domain polypeptides described herein may be used in combination with any of the light chain variable domain polypeptides described herein.

[291] In certain embodiments, the anti-PD-Ll antibody comprises a heavy chain variable domain (VH) sequence having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. In certain embodiments, a VH sequence having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8 contains substitutions (e.g. conservative substitutions), insertions, or deletions relative to the reference sequence (e.g. SEQ ID NO: 8) wherein the anti-PD-Ll antibody comprising that VH sequence retains the ability to bind to the same PD-L1 molecule as the anti-PD-Ll antibody comprising the reference sequence. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted relative to SEQ ID NO:8. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (e.g. in the FRs). Optionally, the anti-PD-Ll antibody comprises the VH polypeptide having the sequence of SEQ ID NO: 8, optionally including post-translational modifications. In certain embodiments, the VH comprises one, two or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6.

[292] In certain embodiments, the antibody may comprise (a) at least one heavy chain variable (VH) domain comprising, consisting essentially of, or consisting of SEQ ID NO:8; and (b) at least one light chain variable (VL) domain comprising, consisting essentially of, or consisting of SEQ ID NO:7 or SEQ ID NO:9, optionally including post-translational modifications of those polypeptides.

[293] In certain embodiments, the anti-PD-Ll antibody comprises a heavy chain variable domain (VH) sequence having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. In certain embodiments, a VH sequence having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8 contains substitutions (e.g. conservative substitutions), insertions, or deletions relative to the reference sequence (e.g. SEQ ID NO: 8) wherein the anti-PD-Ll antibody comprising that VH sequence retains the ability to bind to the same PD-L1 molecule as the anti-PD-Ll antibody comprising the reference sequence. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted relative to SEQ ID NO:8. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (e.g. in the FRs). Optionally, the anti-PD-Ll antibody comprises the VH polypeptide having the sequence of SEQ ID NO: 8, optionally including post-translational modifications. In certain embodiments, the VH comprises one, two or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6.

[294] In certain embodiments, the anti-PD-Ll antibody comprises a light chain variable region comprising a polypeptide having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO:7 and/or a heavy chain variable region comprising a polypeptide having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 8.

[295] In certain embodiments, the anti-PD-Ll antibody comprises a light chain variable region comprising a polypeptide having at least 85%, 87%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO:9 and/or a heavy chain variable region comprising a polypeptide having 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 8.

[296] In certain embodiments, the antibody comprises, consists essentially of, or consists of an amino acid sequence that is at least 85% (such as at least 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence of any one of SEQ ID NOs: 10-17. In certain further embodiments, the antibody comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 10-17.

[297] In certain of the above embodiments, the polypeptide sequence of the anti-PD-Ll antibodies of the present invention is varied by one or more conservative amino acid substitutions introduced into the polypeptide region(s) as long the anti-PD-Ll antibody retains the required PD-L1 binding activity and/or other functions discussed herein. In certain of the above embodiments, the polypeptide sequence of the anti-PD-Ll antibodies of the present invention are varied by one or more conservative amino acid substitutions introduced into the polypeptide region(s) as long as all required structural features are still present and the anti-PD-Ll antibody is capable of exhibiting any required function(s), either alone or, e.g ., as a component of a conjugate or ADC.

[298] It is within the scope of the present invention to use fragments, variants, and/or derivatives of the antibody of the present invention which retain binding functionality. The skilled worker will recognize that variations may be made to the antibody of the present invention, and polynucleotides encoding said antibody, without diminishing their biological activities (e.g. antigen binding function). For example, any antibody variant, or derivative thereof, of the present invention which binds PD-L1 with a dissociation constant (KD) of 10⁴ to 10¹² M, preferably less than 200 nM, may be an antibody of the present invention. For example, some modifications may facilitate expression, facilitate purification, improve pharmacokinetic properties, and/or improve immunogenicity. Such modifications are well known to the skilled worker and include, for example, a methionine added at the amino- terminus to provide an initiation site, additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons, and biochemical affinity tags fused to either terminus to provide for convenient detection and/or purification. A common modification to improve the immunogenicity of a polypeptide produced using a non-chordate system (e.g. a prokaryotic cell) is to remove, after the production of the polypeptide, the starting methionine residue, which may be formylated during production, such as, e.g. , in a bacterial host system, because, e.g. , the presence of N-formylmethionine (fMet) might induce undesirable immune responses in chordates.

[299] Also contemplated herein is the inclusion of additional amino acid residues at the amino and/or carboxy termini of the antibody of the present invention, such as sequences for labels, epitope tags, or other moieties. The additional amino acid residues may be used for various purposes including, e.g. , facilitating cloning, facilitating expression, post-translational modification, facilitating synthesis, purification, facilitating detection, and administration. Non-limiting examples of epitope tags and moieties are chitin binding protein domains, enteropeptidase cleavage sites, Factor Xa cleavage sites, FIAsH tags, FLAG tags, green fluorescent proteins (GFP), glutathione-S-transferase moieties, HA tags, maltose binding protein domains, myc tags, polyhistidine tags (e.g. His-tag), ReAsH tags, strep-tags, strep-tag II, TEY protease sites, thioredoxin domains, thrombin cleavage site, and V5 epitope tags. a) Substitution, Insertion, and Deletion Variants [300] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g. , retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[301] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. to create a humanized antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g. improvements) in certain biological properties (e.g. increased affinity or reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g. , using phage display -based affinity maturation techniques. Briefly, one or more HVR residues are mutated and the variant antibodies displayed and screened for a particular biological activity (e.g. binding affinity) (see e.g. WO 2015/120058).

[302] Alterations (e.g. substitutions, insertions, or deletions) may be made in HVRs, e.g. , to improve antibody affinity using methods known to the skilled worker. For example, alterations may be made in HVR“hotspots” or residues encoded by codons that undergo mutation at high frequency during the somatic maturation process and/or SDRs (a-CDRs), with the resulting variant heavy and/or light chains being tested for binding affinity (see e.g. Chowdhury P, Methods Mol Biol 207: 179-196 (2008)). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs as long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations that do not substantially reduce binding affinity may be made in HVRs, including outside of HVR“hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided herein, each HVR either is unaltered, or contains no more than one, two, or three amino acid substitutions.

[303] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an amino-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the amino- and/or carboxyl-terminus of the antibody to an enzyme (e.g. for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody. b) De-Immunized, Humanized, and/or Chimeric Variants

[304] In certain embodiments, the antibody component of the anti-PD-Ll antibody (e.g. an antibody-toxin conjugate) of the present invention is chimeric. For example, the chimeric antibody comprises a non-human variable region (e.g, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate) and a human constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or isotype has been changed from that of the parent antibody from which it was derived. In certain embodiments, the chimeric antibody is a humanized antibody. Chimeric antibodies include antigen-binding fragments thereof.

[305] In certain embodiments, the antibody component of the anti-PD-Ll antibody (e.g. an antibody-toxin conjugate) of the present invention is humanized. Typically, a non-human antibody is humanized to reduce immunogenicity in humans, while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which HVRs, e.g, CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a constant region from a human antibody. In certain embodiments, some FR residues in a humanized antibody have been substituted with corresponding residues from a non-human antibody (e.g. the antibody from which the HVR residues are derived), e.g, to restore or improve antibody specificity and/or affinity. c) Glycosylation Variants

[306] In certain embodiments, an antibody of the present invention is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. For example, an antibody component of the invention comprising a glycosylated Fc region may be altered such that the carbohydrate attached thereto is altered. In another example, the carbohydrate attached to an antibody component of the invention may be altered using methods known to the skilled worker.

[307] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see e.g. Wright A, Morrison S, TIBTECH 15: 26-32 (1997)). The oligosaccharide may include various carbohydrates, e.g. mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the“stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

[308] In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e.., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function (see e.g. WO 2003/035835; WO 2003/055993). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: WO 2002/031140; WO 2003/035835; WO 2000/61739; WO 2001/29246; US 2002/0164328; WO 2003/055993; WO 2003/084569; WO 2003/084570; WO 2003/085102; WO 2003/085107; WO 2003/085118; WO 2003/085119; US 2003/115614; WO 2005/035586; WO 2005/053742; WO 2005/035778; Okazaki A et al. JMol Biol 336: 1239-49 (2004); Yamane-Ohnuki N et al. Biotechnol Bioeng 87: 614-22 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka J et al. Arch Biochem Biophys 249: 533-45 (1986); WO 2003/035835; WO 2004/056312, see Example 11), and knockout cell lines, such as alpha- l,6-fucosyltransferase gene, FUT8, knockout CHO cells (see e.g. Yamane-Ohnuki N et al. Biotechnol Bioeng 87: 614-22 (2004); Kanda Y et al., Biotechnol Bioeng 94: 680-8 (2006); WO 2003/085107)).

[309] Antibodies variants are further provided with bisected oligosaccharides, e.g, in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by

GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g, in WO 1999/054342; WO 2003/011878; WO 2005/044859. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/030087; WO 1998/058964; and WO 1999/022764. d) Fc Region Variants

[310] In certain embodiments, the anti-PD-Ll antibody of the present invention may comprise an Fc region which retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as, e.g. FcRn binding, antibody half-life modulation, ADCC function, and complement binding.

[311] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.

For example, the Fc region variant may comprise a human Fc region sequence (e.g, a Fc region from a human IgGl, IgG2, IgG3, or IgG4) and may optionally comprise one or more amino acid alterations (e.g. a substitution at one or more amino acid positions). In certain embodiments, the antibody component of the invention comprises an Fc region that has ADCC and/or CDC activity. Such antibodies are particularly useful for mediating killing of target expressing cells. Antibodies with improved Fc effector functions can be generated, for example, through changes in amino acid residues involved in the interaction between the Fc domain and an Fc receptor (FcR) (e.g. FcyRI, FcyRIIA, FcyRIIB, or FcyRI 11 with FcRn), which may lead to increased cytotoxicity and/or altered pharmacokinetics, such as increased serum half-life. Certain antibody variants with improved or diminished binding to FcRs are known to skilled worker and/or described in Shields R et al., J Biol Chem 9: 6591-6604 (2001).

[312] Certain antibody variants with improved or diminished binding to FcRs are described (see e.g. US 6,737,056; WO 2004/056312; Shields R et al., J Biol Chem 276: 6591-604 (2001)). In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g, substitutions at positions 298,

333, and/or 334 of the Fc region (EU numbering of residues). In some embodiments, alterations are made in the Fc region that result in altered (e.g. improved or diminished) Clq binding and/or CDC, e.g, as described in US 6, 194,551; WO 1999/051642; Idusogie E et al. J Immunol 164: 4178-4184 (2000).

[313] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Kim J et al., Eur J Immunol 24: 2429-34 (1994)) are described in US2005/0014934. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, or 434, e.g ., substitution of Fc region residue 434 (US 7,371,826). Other exemplary Fc variants are described in Duncan A, Winter G, Nature 322: 738-40 (1988); WO 1988/007089; and WO 1994/029351 concerning other examples of Fc region variants.

[314] In certain embodiments, the antibody component of the invention comprises an Fc region that lacks one or more effector functions (e.g. lacks ADCC and/or CDC activity). Fc regions lacking or having substantially reduced effector function may be obtained, for example, by introducing one or more amino acid substitutions into a native Fc region sequence, such that the Fc region does not bind, or has substantially reduced binding, to cytolytic Fc receptors (e.g. DANA mutant) and/or the Clq complement protein (see e.g. Wilson N et ak, Cancer Cell 19: 101-113 (2011); Idusogie E et al. J Immunol 164: 4178-4184 (2000)). In certain embodiments, the antibody component is varied in that it possesses some but not all antibody effector functions, which make it a desirable candidate for applications in which the half-life of the anti-PD-Ll antibody in vivo is important yet certain effector functions (e.g. CDC or ADCC) are undesirable or deleterious.

[315] In certain embodiments, the antibody component of the invention comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g. , substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

[316] In certain embodiments, the antibody component of the invention comprises an Fc region with one or more amino acid substitutions resulting in altered Clq binding and/or CDC effector function (e.g. either improved or diminished) (see e.g. WO 1999/051642; U.S. 6,194,551).

[317] In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is described in Ravetch J, Kinet J, Annu Rev Immunol 9: 457-92 (1991). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art ( see e.g. Petkova S et ah, Int Immunol 18: 1759-69 (2006)). Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US 7,332,581). e) Cysteine-Engineered Variants

[318] In certain embodiments of the present invention, the antibody component of the anti- PD-L1 antibody (e.g. an antibody -toxin conjugate) of the present invention possesses one or more engineered cysteine residues. In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g. a ThioFab or THIOMAB™, in which one or more residues of an antibody are substituted with cysteine residues. In certain further

embodiments, the substituted residues occur at sites of the antibody that are readily available for conjugation (see e.g. Junutula J et ak, Nature Biotech 26: 925-32 (2008); Dornan D et al, Blood 114: 2721-29 (2009)). By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments of the antibody, it may be desirable to create cysteine engineered antibodies via one or more cysteine residue substitutions that do not significantly perturb antibody folding and assembly nor significantly alter antigen binding and/or antibody effector functions. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Rabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g. , in WO 2006/034488.

C. Antibody Cargos. Payloads and Additional Exogenous Materials

[319] The present invention provides various embodiments of anti-PD-Ll antibodies, or an antigen binding fragment or derivative thereof, which is associated, linked, fused, or conjugated to a cargo, such as, e.g. , a payload, drug, toxin, detection-promoting agent, radionucleide, and/or heterologous moiety, or any additional material. In certain embodiments of the anti-PD-Ll antibodies of the present invention described herein, the anti- PD-L1 antibody of the present invention further comprises an additional material, which may be present for delivery to a target cell. For example, the antibody of the invention may be conjugated, linked or fused to or otherwise associated with a pharmaceutically active moiety which is a therapeutic moiety or a drug, including, but not limited to, cytotoxic agents (or cytotoxins) such as a small molecule chemotherapeutic agent, anti -neoplastic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor.

1. Immunoconjugates/ Antibody Drug Conjugates

[320] In one embodiment, the antibody of the invention may be conjugated with

pharmaceutically active or diagnostic moieties to form an“antibody drug conjugate” (ADC) or immunoconjugate. The term“conjugate” is used broadly and means the covalent or non- covalent association of any pharmaceutically active or diagnostic moiety with an antibody of the invention regardless of the method of association. The disclosed ADCs or

immunoconjugates may be used for therapeutic and diagnostic purposes.

[321] The invention provides ADCs/immunoconjugates comprising any antibody provided herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins ( e.g ., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes (also known as a radioconjugate).

[322] The antibody of the invention may be conjugated, linked or fused to or otherwise associated with a diagnostic or detectable agent ( i.e . a detection-promoting agent), such as a tag, marker, or reporter (e.g. an enzyme), including a fluorophore, radioisotope, small molecule, and/or biological molecule (e.g, a peptide, protein, or nucleotide). Labeled antibodies can be useful for monitoring the development or progression of a target-associated disorder or as part of a clinical testing procedure to determine the efficacy of a particular therapy, including the disclosed antibodies or to determine a future course of treatment. Such markers or reporters may also be useful in purifying the selected antibody, for use in antibody analytics (e.g, epitope binding or antibody binning).

[323] In a further aspect of the invention, an anti-PD-Ll antibody according to any of the above embodiments or described herein is conjugated to a heterologous moiety or agent, such as, e.g. as described below and including any additional exogenous material as described herein. [324] The invention also provides immunoconjugates comprising an anti-PD-Ll antibody of the present invention conjugated to one or more other therapeutic agents or radioactive isotopes.

[325] In certain embodiments, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates, such as, e.g .,²¹¹At,¹³¹I,¹²⁵1,⁹⁰Y,^luIn,¹⁸⁶Re,¹⁸⁸Re,¹⁵³Sm,²¹²Bi,³²P,²¹²Pb,⁶⁰C, and/or radioactive isotopes of Lu. When the radioconjugate is used for PD-L1 detection, the antibody may comprise a radioactive atom for scintigraphic studies, e.g. tc99m or¹²³I, or a spin label for nuclear magnetic resonance (MR) imaging (e.g. nuclear magnetic resonance spectroscopy or MRI), such as¹²³I, ,¹³¹I,^luIn,¹⁹F,¹³C,¹⁵N,¹⁷0, gadolinium, manganese, or iron.

[326] The present invention provides various embodiments of anti-PD-Ll antibodies, wherein the antibody further comprises one or more heterologous molecule(s), meaning heterologous to native antibodies, including but not limited to a cytotoxic agent. An “immunoconjugate” is an antibody (including an antigen-binding antibody fragment or antibody derivative) conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent. Immunoconjugates, including antibody drug conjugates (ADCs), allow for the targeted delivery of a drug moiety to a tumor, and, in certain embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells (Polakis P, Curr Opin Pharmacol 5: 382-387 (2005); Carter P, Senter P, Cancer J 14: 154-69 (2008); Chari R, Acc Chem Res 41 :98-l07 (2008); Teicher B, Curr Cancer Drug Targets 9: 982- 1004 (2009)). Nonlimiting examples of such immunoconjugates are discussed in further detail herein.

[327] The ADC compounds of the invention include those with anticancer activity. In certain embodiments, the ADC compounds include an antibody conjugated, e.g. covalently attached, to the drug. In certain embodiments, the antibody is covalently attached to the drug via a linker. The drug of the antibody-drug conjugates of the present invention may include any compound, moiety or group that has a cytotoxic or cytostatic effect. Drug agents may impart their cytotoxic and cytostatic effects by mechanisms including but not limited to tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and/or topoisomerase. Exemplary drugs include, but are not limited to, a maytansinoid, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU- 159682, anthracy cline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[328] In certain embodiments of the anti-PD-Ll antibody of the present invention, the immunoconjugate is an antibody-toxin conjugate, which is an antibody conjugated to a toxin, such as, e.g., diphtheria A chain, exotoxin A chain (from P. aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (e.g. PAPI , PAPII, or PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, Shiga toxin A Subunit, and tricothecenes. Biological immunoconjugates comprising a toxin (e.g. a Shiga toxin A subunit fragment) linked to a PD-L1 binding region (e.g. an antibody or antibody fragment) are useful as therapeutic or diagnostic biological molecules. In addition, such therapeutic or diagnostic molecules may be improved by having a Shiga toxin effector polypeptide conjugated to an additional agent such as, e.g. , a detection- promoting agent, solubility-altering agent, pharmacokinetic-altering agent, immunogenicity- altering agent, and/or a pharmacodynamic-altering agent (see e.g. WO 2018/106895).

Typically, biopharmaceutical immunoconjugates are created by conjugating an antibody to other agents or cargos using chemical reactions involving a functional group(s) of the biological molecule and a functional group of the agent or cargo, or alternatively of a linker designed to bridge between the biological molecule and the agent or cargo (see section I.-D. Linkages Connecting Components of the Invention and/or Their Subcomponents, supra).

[329] In certain embodiments, the anti-PD-Ll antibody of the present invention is an immunoconjugate utilizing a cysteine engineered into the PD-L1 binding region, such as, e.g. , wherein the anti-PD-Ll antibody comprises a cysteine engineered antibody. In certain further embodiments, the anti-PD-Ll antibody of the present invention is an

immunoconjugate utilizing a cysteine engineered into the framework region (e.g. FR1) of an immunoglobulin variable region for conjugation (see e.g. WO 2011/000054).

[330] In certain embodiments, the anti-PD-Ll antibody of the present invention is an immunoconjugate utilizing a carbohydrate moiety attached to a Fc region, such as, e.g. , wherein the anti-PD-Ll antibody comprises a glycosylated antibody or antibody fragment.

[331] In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l, 3- dioxolane, poly-l, 3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols ( e.g . glycerol), polyvinyl alcohol, and mixtures thereof. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules (i.e. homo- or hetero-polymers). In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used, e.g., in a therapy or for diagnosis under defined conditions.

[332] In certain embodiments, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In certain embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam N et ah, Proc Natl Acad Sci U.S.A. 102: 11600-5 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

2. Antibody Fusions

[333] In certain embodiments, the anti-PD-Ll antibody of the present invention (or antigen binding fragment or derivative thereof) is linked to a heterologous moiety, such as a cargo or detection-promoting agent described herein and/or known to the skilled worker. In certain further embodiments, the linkage comprises a proteinaceous fusion. In certain further embodiments, the heterologous moiety is a cytotoxic agent. In certain further embodiments, the cytotoxic agent is a proteinaceous toxin. In certain further embodiments, the

proteinaceous toxin is a ribotoxic polypeptide, such as, e.g, a toxin polypeptide described herein and/or known to the skilled worker.

3. Anti-PD-Ll Antibodies Comprising a Heterologous and/or Additional Material for Delivery

[334] In addition to direct cell killing of cells with cell-surface PD-Ll-bound anti-PD-Ll antibodies, the anti-PD-Ll antibodies of the present invention optionally may be used for delivery of additional exogenous materials into the interiors of target cells. The delivery of additional exogenous materials may be used, e.g, for cytotoxic, cytostatic, immune system stimulation, immune cell targeting, information gathering, and/or diagnostic functions. Non- cytotoxic variants of the cytotoxic, anti-PD-Ll antibodies of the invention, or optionally toxic variants, may be used to deliver additional exogenous materials to and/or label the interiors of cells physically coupled with an extracellular target biomolecule of the anti-PD-Ll antibody. Various types of cells and/or cell populations which express target biomolecules (e.g. PD-L1) to at least one cellular surface may be targeted by the anti-PD-Ll antibodies of the invention for receiving exogenous materials.

[335] The heterologous moiety of an anti-PD-Ll antibody of the present invention may be any number of additional materials for delivery to a target cell, e.g. a PD-L1 expressing cell. “Additional exogenous material” as used herein refers to one or more molecules, often not generally present within a native target cell and/or present at undesirably low levels, where the proteins of the present invention can be used to specifically transport such material to the interior of a cell. Non-limiting examples of additional exogenous materials are cytotoxic agents, peptides, polypeptides, proteins, polynucleotides, detection-promoting agents, small molecule chemotherapeutic agents, and any cargo known to the skilled worker and/or described herein.

[336] In certain embodiments of the proteins of the present invention for delivery of additional exogenous material, the additional exogenous material is a cytotoxic agent, such as, e.g. , a small molecule chemotherapeutic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor. In certain further embodiments of the anti-PD-Ll antibodies of the present invention, the additional exogenous material is a cytotoxic agent, such as, e.g. , a small molecule chemotherapeutic agent, anti neoplastic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor. Non-limiting examples of cytotoxic agents suitable for use with the present invention include aziridines, cisplatins, tetrazines, procarbazine,

hexamethylmelamine, vinca alkaloids, taxanes, camptothecins, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin, amanitin, amatoxins, bleomycin, centanamycin (indolecarboxamide), plicamycin, mitomycin, daunorubicin, epirubicin, idarubicins, dolastatins, maytansines, maytansionoids, duromycin, docetaxel, duocarmycins, adriamycin, calicheamicin, auristatins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers (PBDs), carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C, paclitaxel, l,3-Bis(2-chloroethyl)-l-nitrosourea (BCNU), rifampicin, cisplatin, methotrexate, gemcitabine, aceglatone, acetogenins (e.g. bullatacin and bullatacinone), aclacinomysins, AG1478, AG1571, aldophosphamide glycoside, alkyl sulfonates (e.g, busulfan, improsulfan, and piposulfan), alkylating agents (e.g. thiotepa and

cyclosphosphamide), aminolevulinic acid, aminopterin, amsacrine, ancitabine, anthramycin, arabinoside, azacitidine, azaserine, aziri dines (e.g, benzodopa, carboquone, meturedopa, and uredopa), azauridine, bestrabucil, bisantrene, bisphosphonates (e.g. clodronate), bleomycins, bortezomib, bryostatin, cactinomycin, callystatin, carabicin, carminomycin, carmofur, carmustine, carzinophilin, CC-1065, chlorambucil, chloranbucil, chlornaphazine,

chlorozotocin, chromomycinis, chromoprotein enediyne antibiotic chromophores, CPT-l l, cryptophycins (e.g. cryptophycin 1 and cryptophycin 8), cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunomycin, defofamine, demecolcine, detorubicin, diaziquone, 6-diazo-5-oxo-L-norleucine, dideoxyuridine, difluoromethylomithine (DMFO), doxifluridine, doxorubicins (e.g, morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolinodoxorubicin, and deoxydoxorubicin), dynemicins, edatraxate, edatrexate, eleutherobins, elformithine, elliptinium acetate, enediyne antibiotics (e.g. calicheamicins), eniluracil, enocitabine, epirubicins, epothilone, esorubicins, esperamicins, estramustine, ethylenimines, 2-ethylhydrazide, etoglucid, fludarabine, folic acid analogues (e.g, denopterin, methotrexate, pteropterin, and trimetrexate), folic acid replenishers (e.g. frolinic acid), fotemustine, fulvestrant, gacytosine, gallium nitrate, gefitinib, gemcitabine,

hydroxyurea, ibandronate, ifosfamide, imatinib mesylate, erlotinib, fulvestrant, letrozole, PTK787/ZK 222584 (Novartis, Basel, CH), oxaliplatin, leucovorin, rapamycin, lapatinib, lonafamib, sorafenib, methylamelamines (e.g, altretamine, triethy lenemelamine, triethy lenephosphoramide, triethylenethiophosphoramide and trimethylomelamine), pancrati statins, sarcodictyins, spongi statins, nitrogen mustards (e.g, chlorambucil, chlornaphazine, cyclophosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard), nitrosureas (e.g, carmustine, fotemustine, lomustine, nimustine, and ranimnustine), dynemicins, neocarzinostatin chromophores, anthramycin, detorubicin, epirubicins, marcellomycins, mitomycins (e.g. mitomycin C), mycophenolic acid, nogalamycins, olivomycins,

peplomycins, potfiromycins, puromycins, quelamycins, rodorubicins, ubenimex, zinostatins, zorubicins, purine analogs (e.g, fludarabine, 6-mercaptopurine, thiamiprine, and

thioguanine), pyrimidine analogs (e.g, ancitabine, azacitidine, 6-azauridine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine), aceglatone, lentinan, lonidainine, maytansinoids (e.g. maytansins and ansamitocins), mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"trichlorotriethylamine, trichothecenes (e.g, T-2 toxin, verracurin A, roridin A, and anguidine), urethan, vindesine, mannomustine, mitobronitol, mitolactol, pipobroman, arabinoside, cyclophosphamide, toxoids (e.g. paclitaxel and doxetaxel), 6-thioguanine, mercaptopurine, platinum, platinum analogs (e.g. cisplatin and carboplatin), etoposide (VP- 16), mitoxantrone, vinorelbine, novantrone, daunomycin, xeloda, topoisomerase inhibitor RFS 2000, retinoids (e.g. retinoic acid), capecitabine, lomustine, losoxantrone, mercaptopurines, nimustine, nitraerine, rapamycin, razoxane, roridin A, spongi statins, streptonigrins, streptozocins, sutent, T-2 toxin, thiamiprine, thiotepa, toxoids (e.g. paclitaxel and doxetaxel), tubercidins, verracurin A, vinblastine, vincristine, and structural analogs of any of the aforementioned (e.g. synthetic analogs), and/or derivatives of any of the aforementioned (see e.g ., Lindell T et al., Science 170: 447-9 (1970); Remillard S et al., Science 189: 1002-5 (1975); Ravry M et al., Am ./ Clin Oncol 8: 148-50 (1985); Ravry M et al., Cancer Treat Rep 69: 1457-8 (1985); Sternberg C et al., Cancer 64: 2448-58 (1989); Bai R et al., Biochem Pharmacol 39: 1941-9 (1990); Boger D, Johnson D, Proc Natl Acad Sci USA 92: 3642-9 (1995); Beck J et al., Leuk Lymphoma 41 : 117-24 (2001); Cassady J et al., Chem Pharm Bull (Tokyo) 52: 1-26 (2004); Sapra P et al., Clin Cancer Res 11 : 5257-64 (2005); Okeley N et al., Cline Cancer Res 16: 888-97 (2010); Oroudjev E et al., Mol Cancer Ther 9: 2700-13 (2010); Ellestad G, Chirality 23 : 660-71 (2011); Kantarjian H et al., Lancet Oncol 13 : 403-11 (2012); Moldenhauer G et al., JNatl Cancer Inst 104: 622-34 (2012);

Meulendijks D et al., Invest New Drugs 34: 119-28 (2016)).

[337] In certain embodiments, the cargo, payload, heterologous moiety, or additional exogenous material of the anti-PD-Ll antibody of the present invention is a toxin. There are numerous proteinaceous toxins suitable for use as a toxin component of the present invention (see e.g. Table C, supra). For example, a ribotoxic protein for use in the present invention may be derived from the catalytic domains of members of the RIP Superfamily of protein ribotoxins. For example, argonaute enzymatic domains or hybrid enzymatic domains composed of fungal ribotoxins and argonaute sequences may be engineered for ribosome inactivation (see Pichinuk E, Wreschner D, Protein Sci 19: 1272-8 (2010)). Examples of RNases with enzymatic domains useful as ribotoxic components of the antibody of the present invention include bacterial RNases, such as, e.g. , binase, amphibian RNases, such as e.g. , ranpimase and Onconase®, and mammalian RNases, such as, e.g. , bovine semen RNase and the human RNases: RNase2, RNase3, and RNase5 (Newton D et al., J Biol Chem 269: 739-45 (1994); Netwon D et al., J ImmunolMeth 231 : 159-67 (1999); Yoon J et al., Life Sci 64: 1435-45 (1999); Hugh M et al., Cancer Res 61 : 8737-42 (2001); Makarov A, Ilinskaya N, FEBSLett 540: 15-20 (2003)).

Table C. Exemplary Protein Toxins and Sources of Toxin Component Polypeptides

[338] The toxin component of an anti-PD-Ll antibody, ADC, or antibody toxin conjugate (immunoconjugate) of the present invention may include, but is not limited to, natural toxins, biotoxins, proteinaceous toxins, venom, cytotoxins, small molecule toxins, and synthetic toxicants derived from any of the aforementioned, such as, e.g ., aconitine, adriamycin, amanitin, amatoxin, anthracycline, aroin, apitoxin, atropine, bufotoxin, cardiac glycoside, calicheamicin, celandine, cicutoxin, colchicine, coniine, convallatoxin, crotamine, curare, curcin, dauricine, digitalis, dolastatin, duocarmycin, evomonoside, grayanotoxin, gelsemine, gelseminine, hellebrin, helleborin, hyoscyamine, ligatoxin, ligustrin, maytansine, mitomycin C, muscarine, phallotoxin, phoratoxin, phytotoxin, picrotoxin, sea nettle toxin, taxine alkaloid, thionin, vinca alkaloid, viscotoxin, and various toxin agents described herein.

Pharmaceutically active cytotoxins suitable for use as a toxin component of the present invention also include, but are not limited to ABx toxins, ribosome inactivating protein toxin, anthrax toxin, cholix toxin, claudin, diphtheria toxin, heat-labile enterotoxin, pertussis toxin, Pseudomonas exotoxin A, ricin, Shiga toxin, and subtilase cytotoxin; alkylating agents (such as, e.g. bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, etramustine, ifosfamide, lomustine, mechlorethamine, melphalan, mustine, thiotepa, and treosulfan), antibiotics (such as, e.g. anthracyclines), anti-microtubule agens (such as, e.g. vinca alkaloids like vincristine, vinblastine, and etoposide or toxoids like paclitaxel and docetaxel), intercalating agents (such as, e.g. daunorubicin, bleomycin, dactinomycin, doxorubicin, epirubicin, mitoxatrone, idarubicin, plicamycin, mitomycin, and steptozotocin), anti metabolites (such as, e.g. methotrexate, pyrimidine antagonists, and purine antagonists), growth inhibitory agents (such as topoisomerase inhibitors and spindle poisons like camptothecin, colchicine, daunorubicin, fisetin, genistein, irinotecan, lamellarins, myricetin, paclitaxel, thaspine, tricitrinol B, topotecan, vinca alkaloids); enzymes and fragments thereof such as nucleolytic enzymes like asparaginase and certain RNAses such as, e.g. , bacterial RNases, fungal ribotoxins, argonaute polypeptides, binase, amphibian RNases, ranpirnase, Onconase®, and mammalian RNases, such as, e.g. , bovine semen RNase and the human RNases; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, such as, e.g. , abrins, agrostin, amarandins, amaranthin, Amaranthus antiviral/RIP, angiogenin, A. patens RIPs, Articulatin D, asparins, aspergillin, Aspfl, balsamin, B. hispida RIP, bouganin, Bougainvillea x buttiana antiviral protein 1, benincasins, bouganin, B. rubra RIPs, bryodins (e.g. bryodin 1, bryodin 2), B. spectabilis RIPs, B. vulgaris RIPs, C. album RIPs, camphorin, C. aculeatum- systemic resistance inducing protein, C. cristata RIPs, C.figarei RIPs, charantin, charybdin, cinnamomin, clavin, C. moschata RIP, cochinin B, colocins, crotins, cucurmosin, curcins, Dianthus spp. RIPs, Corynebacterium spp. diphtheria toxins (diphtheria toxins in C. ulcerans , C. omega , C. pseudotuberculosis ), dodecandrins, ebulins, ebulitins, E. hyemalis RIPs, euserratins, eutirucallin, flammin, flammulin, foetidissimin, gelonin, gigantin, gypsophilin, H. crepitans RIPs, Heterotepalin, hispin, hirsutellin A, H. orientalis RIPs, H. vulgare RIPs, hypsin, insularin, I. hollandica RIPs, lagenin, lamjapin, lanceolin, L.

cylindrical RIPs, luffacylin, luffaculin, luffagulin, luffms, L. usitatissimum RIPs, lychnin, lyophyllin, manutins, marmorin, mapalmin, M. charantia lectin, M. crystallinum RIPs, melonin, mexm, Mirabilis spp. RIPs, mitogillin, modeccins, MORs, Mormordica spp. RIPs, momorsgrovin, moschatin, musarmins, N tabacum RIPs, nigrins, nigritins, ocymoidin, pachyerosin, P. californicum lectin, pepocin, petroglaucin, petrograndin, Phytolacca spp. RIPs, pisavin, pleuturegin, Pluturegin, A. thaliana pectin methyl transferase (PME), P.

multiforum RIPs, pokeweed antiviral protein (PAP), porrectin, Aeromonas spp. Pseudomonas toxins (A. hydrophila pseudomonas-like toxin), pulchellin, quinqueginsin, R. communis agglutinins, restrictocin, ricins, riproximin, saporins, sarcins, sativin, S. cereale RIPs, sechiumin, Shiga toxin, Shiga-like toxins, sieboldin b, S. nigra RIPs (e.g. S. nigra agglutinins I-V), S. ocymoides RIPs, Spinacia oleracea protein, stellarin, stenodactylin, texanin, tricholin, Trichosanthes spp. RIPs (e.g. karasurins, kirilowins, trichoanguin, trichokirins,

trichosanthins, TYchi), Triticum spp. RIPs, V. album RIPs, velin, velutin, verotoxins, V.

hispanica RIPs, vircumin, volkensin, V. volvacea RIPs, Volvarin, Yucca leaf protein, Z diploperennis RIPs, Z. mays RIPs, and any ribotoxic fragment of any of the foregoing; and the various antitumor or anticancer agents described herein.

[339] With regard to the claimed invention, the phrases“ribotoxic polypeptide” refer to a polypeptide derived from proteins, including naturally occurring toxins and synthetic toxins, that is capable of effectuating ribosome inactivation in vitro , protein synthesis inhibition in vitro and/or in vivo , cytotoxicity, and/or cytostasis. Commonly, ribotoxic polypeptides are enzymatically active domains derived from naturally occurring protein toxins or toxin-like structures which are altered or engineered by human intervention (see e.g. Newton D et ah, Blood 97: 528-35 (2001); De Lorenzo C et ah, FEBS Lett 581 : 296-300 (2007); De Lorenzo C, D’ Alessio G, Curr Pharm Biotechnol 9: 210-4 (2008); Menzel C et ah, Blood 111 : 3830-7 (2008)). Thus, ribotoxic polypeptides may be derived from synthetic or engineered protein constructs with increased or decreased ribotoxicity, and/or naturally occurring proteins that have been otherwise altered to have a non-native characteristic, such as, e.g. increased stability, optimized expression in a laboratory species or cell line, improved solubility, improved pharmacokinetic properties, improved pharmacodynamic properties, and/or reduced antigenicity and/or immunogenicity.

[340] The ribotoxic polypeptides of the present invention may be derived from ribotoxic domains of proteins from diverse phyla, such as, e.g. , algae, bacteria, fungi, plants, and animals. For example, polypeptides derived from various toxins have been linked or fused to immunoglobulin domains, receptor ligands, or randomized peptides through chemical conjugation or recombinant protein engineering with the hope of creating cell-type-specific cytotoxic therapeutics ( see e.g. Pastan I et al., Annu Rev Biochem 61 : 331-54 (1992); Foss F et al., Curr Top Microbiol Immunol 234: 63-81 (1998); Olsnes S, Toxicon 44: 361-70 (2004); Pastan I, et al., Nat Rev Cancer 6: 559-65 (2006); Lacadena J et al., FEMS Microbiol Rev 31 : 212-37 (2007); de Virgilio M et al., Toxins 2: 2699-737 (2011); Walsh M, Virulence 4: 774- 84 (2013); Weidle U et al., Cancer Genomics Proteomics 11 : 25-38 (2014)).

[341] Ribotoxic polypeptide components of the invention may be derived from the catalytic domains of members of the Ribosome Inactivating Protein (RIP) Superfamily of protein ribotoxins (de Virgilio M et al., Toxins 2: 2699-737 (2011); Lapadula W et al., PLoS ONE 8: e72825 (2013); Walsh M, Virulence 4: 774-84 (2013)). RIPs are ribotoxic proteins expressed in algae, bacteria, fungi, and plants which are often potent inhibitors of eukaryotic and prokaryotic protein synthesis at sub-stoichiometric concentrations (see Stirpe, F, Biochem J 202: 279-80 (1982)). Various RIPs are considered promising sources for toxin effector polypeptide sequences for use in therapeutics for treating cancers (see Pastan I, et al., Nat Rev Cancer 6: 559-65 (2006); Fracasso G et al., Ribosome-inactivating protein-containing conjugates for therapeutic use , Toxic Plant Proteins 18, pp. 225-63 (Eds. Lord J, Hartley, M. Berlin, Heidelberg: Springer-Verlag, 2010); de Virgilio M et al., Toxins 2: 2699-737 (2011); Puri M et al., Drug Discov Today 17: 774-83 (2012); Walsh M, Virulence 4: 774-84 (2013)).

[342] The most commonly used ribotoxins in recombinant cytotoxic polypeptides include DT, PE, ricin, a-sarcin, saporin, and gelonin (see Shapira A, Benhar I, Toxins 2: 2519-83 (2010); Yu C et al., Cancer Res 69: 8987-95 (2009); Fuenmayor J, Montano R, Cancers 3 : 3370-93 (2011); Weldon, FEBS J 278: 4683-700 (2011); Carreras-Sangra N et al., Protein Eng Des Sel 25: 425-35 (2012); Lyu M at al., Methods Enzymol 502: 167-214 (2012);

Antignani, Toxins 5: 1486-502 (2013); Lin H et al., Anticancer Agents Med Chem 13 : 1259- 66 (2013); Polito L et al., Toxins 5: 1698-722 (2013); Walsh M, Virulence 4: 774-84 (2013)).

[343] Many different toxins are contemplated within the scope of the present invention to be utilized for sources of heterologous moieties of conjugates and fusions, such as, e.g. , to function as ribotoxic polypeptides. In certain embodiments of the present invention, the ribotoxic component of the present invention is derived from a member of the RIP

Superfamily which includes RIPs, fungal ribotoxins, and bacterial ribotoxins, such as, e.g. , cholix toxin, DT, and PE. In certain embodiments of the present invention, the ribotoxic component is derived from a nontoxic RNase. In certain embodiments of the present invention, the ribotoxic component is derived from a toxin selected from the group consisting of: abrins, agrostin, amarandins, amaranthin, Amaranthus antiviral/RIP, angiogenin, A.

patens RIPs, Articulatin D, asparins, aspergillin, Aspfl, balsamin, B. hispida RIP, bouganin, Bougainvillea x buttiana antiviral protein 1, benincasins, bouganin, B. rubra RIPs, bryodins (e.g. bryodin 1, bryodin 2), B. spectabilis RIPs, B. vulgaris RIPs, C. album RIPs, camphorin, C. aculeatum- systemic resistance inducing protein, C. cristata RIPs, C.figarei RIPs, charantin, charybdin, cinnamomin, clavin, C. moschata RIP, cochinin B, colocins, crotins, cucurmosin, curcins, Dianthus spp. RIPs, Corynebacterium spp. diphtheria toxins (diphtheria toxins in C. ulcerans , C. omega , C. pseudotuberculosis ), dodecandrins, ebulins, ebulitins, E. hyemalis RIPs, euserratins, eutirucallin, flammin, flammulin, foetidissimin, gelonin, gigantin, gypsophilin, H. crepitans RIPs, Heterotepalin, hispin, hirsutellin A, H. orientalis RIPs, H. vulgare RIPs, hypsin, insularin, I. hollandica RIPs, lagenin, lamjapin, lanceolin, L.

cylindrical RIPs, luffacylin, luffaculin, luffagulin, luffins, L. usitatissimum RIPs, lychnin, lyophyllin, manutins, marmorin, mapalmin, M. charantia lectin, M. crystallinum RIPs, melonin, mexm, Mirabilis spp. RIPs, mitogillin, modeccins, MORs, Mormordica spp. RIPs, momorsgrovin, moschatin, musarmins, N tabacum RIPs, nigrins, nigritins, ocymoidin, pachyerosin, P. californicum lectin, pepocin, petroglaucin, petrograndin, Phytolacca spp.

RIPs (e.g P. dioica RIPs PD-L1, PD-L2, PD-L3, PD-L4), pisavin, pleuturegin, Pluturegin, A. thaliana pectin methyl transferase (PME), P. multiforum RIPs, pokeweed antiviral protein (PAP), porrectin, Aeromonas spp. Pseudomonas toxins (A. hydrophila pseudomonas-like toxin), pulchellin, quinqueginsin, R. communis agglutinins, restrictocin, ricins, riproximin, saporins, sarcins, sativin, S. cereale RIPs, sechiumin, Shiga toxin, Shiga-like toxins, sieboldin b, S. nigra RIPs (e.g. S. nigra agglutinins I-V), S. ocymoides RIPs, Spinacia oleracea protein, stellarin, stenodactylin, texanin, tricholin, Trichosanthes spp. RIPs (e.g. karasurins, kirilowins, trichoanguin, trichokirins, trichosanthins, TYchi), Triticum spp. RIPs, V album RIPs, velin, velutin, verotoxins, V hispanica RIPs, vircumin, volkensin, V. volvacea RIPs, Volvarin, Yucca leaf protein, Z. diploperennis RIPs, Z. mays RIPs, and any functional fragment of any of the foregoing.

[344] In addition, the enzymatic domains of various RNases may be engineered as components of cell killing anti-PD-Ll antibodies. For example, nontoxic RNases when fused to targeting domains are capable of 1) cleaving rRNAs, mRNAs, tRNAs; 2) inhibiting cell growth and/or 3) killing cells (see Newton D et ak, J Biol Chem 269: 739-45 (1994); Netwon D et ak, J Immunol Meth 231 : 159-67 (1999); Yoon I et ak, Life Sci 64: 1435-45 (1999);

Hugh M et ak, Cancer Res 61 : 8737-42 (2001); Newton D et ak, Blood 97: 528-35 (2001); Krauss J et al., Biochem Biophys Res Commun 331 : 595-602 (2005); De Lorenzo C et al., FEBS Lett 581 : 296-300 (2007); Lacadena J et al., FEMS Microbiol Rev 31 : 212-37 (2007); De Lorenzo C, D’Alessio G, Curr Pharm Biotechnol 9: 210-4 (2008); Menzel C et al., Blood 111 : 3830-7 (2008); Riccio G et al., J Immunother 31 : 440-5 (2008)). Argonaute enzymatic domains or hybrid enzymatic domains composed of fungal ribotoxins and argonaute sequences may be engineered for ribosome inactivation (see Pichinuk E, Wreschner D, Protein Sci 19: 1272-8 (2010)). Examples of RNases with enzymatic domains useful as ribotoxic components include bacterial RNases, such as, e.g., binase, amphibian RNases, such as e.g, ranpirnase and Onconase®, and mammalian RNases, such as, e.g, bovine semen RNase and the human RNases: RNase2, RNase3, and RNase5 (Newton D et al., J Biol Chem 269: 739-45 (1994); Netwon D et al., J Immunol Meth 231 : 159-67 (1999); Yoon J et al., Life Sci 64: 1435-45 (1999); Hugh M et al., Cancer Res 61 : 8737-42 (2001); Makarov A,

Ilinskaya N, FEBS Lett 540: 15-20 (2003)).

[345] In certain embodiments, the additional material comprises a proapoptotic peptide, polypeptide, or protein, such as, e.g, BCL-2, caspases (e.g. fragments of caspase-3 or caspase-6), cytochromes, granzyme B, apoptosis-inducing factor (AIF), BAX, tBid (truncated Bid), and proapoptotic fragments or derivatives thereof (see e.g, Ellerby H et al., Nat Med 5: 1032-8 (1999); Mai J et al., Cancer Res 61 : 7709-12 (2001); Jia L et al., Cancer Res 63 : 3257-62 (2003); Liu Y et al , Mol Cancer Ther 2: 1341-50 (2003); Perea S et al., Cancer Res 64: 7127-9 (2004); Xu Y et al., J Immunol 173 : 61-7 (2004); Dalken B et al., Cell Death Differ 13 : 576-85 (2006); Wang T et al., Cancer Res 67: 11830-9 (2007); Kwon M et al., Mol Cancer Ther 7: 1514-22 (2008); Qiu X et al., Mol Cancer Ther 7: 1890-9 (2008); Shan L et al., Cancer Biol Ther 11 : 1717-22 (2008); Wang F et al., Clin Cancer Res 16: 2284-94 (2010); Kim J et al., 7 Virol 85: 1507-16 (2011)).

[346] In certain embodiments, the additional material comprises a protein or polypeptide comprising an enzyme. In certain other embodiments, the additional material is a nucleic acid, such as, e.g. a ribonucleic acid that functions as a small inhibiting RNA (siRNA) or microRNA (miRNA). In certain embodiments, the additional material is an antigen, such as antigens derived from pathogens, bacterial proteins, viral proteins, proteins mutated in cancer, proteins aberrantly expressed in cancer, or T-cell complementary determining regions. For example, additional materials include antigens, such as those characteristic of antigen-presenting cells infected by bacteria, and T-cell complementary determining regions capable of functioning as exogenous antigens. Additional materials comprising polypeptides or proteins may optionally comprise one or more antigens whether known or unknown to the skilled worker. Additional examples of suitable materials include polypeptides and proteins larger than an antigenic peptide, such as enzymes.

D. Linkages Connecting Components of the Invention and/or Their Subcomponents

[347] Individual PD-L1 binding regions, toxin components, and/or other components of the anti-PD-Ll antibodies present invention may be suitably linked to each, such as, e.g, fused directly or indirectly linked to each other via one or more linkers well known in the art and/or described herein. Individual polypeptide subcomponents of the binding regions, e.g. heavy chain variable regions (VH), light chain variable regions (VL), CDR, and/or ABR regions, may be suitably linked to each other via one or more linkers well known in the art and/or described herein, including via chemical conjugation. Proteinaceous components of the invention, e.g. , multi-chain binding regions, may be suitably linked to each other or other polypeptide components of the invention directly via peptide bonds and/or indirectly via one or more linkers well known in the art. Peptide components of the invention, e.g.,

endoplasmic reticulum retention/retrieval signal motifs, may be suitably linked to another component of the invention directly via peptide bonds or indirectly via one or more linkers, such as a proteinaceous linker, which are well known in the art.

[348] Suitable linkers are generally those which allow each polypeptide component of the present invention to fold with a three-dimensional structure very similar to the polypeptide components produced individually without any linker or other component. Suitable linkers include single amino acids, peptides, polypeptides, and linkers lacking any of the

aforementioned, such as various non-proteinaceous carbon chains, whether branched or cyclic.

[349] Suitable linkers may be proteinaceous and comprise one or more amino acids, peptides, and/or polypeptides. Proteinaceous linkers are suitable for both recombinant fusion proteins and chemically linked conjugates. A proteinaceous linker typically has from about 2 to about 50 amino acid residues, such as, e.g, from about 5 to about 30 or from about 6 to about 25 amino acid residues. The length of the linker selected will depend upon a variety of factors, such as, e.g, the desired property or properties for which the linker is being selected. In certain embodiments, the linker is proteinaceous and is linked near the terminus of a protein component of the present invention, typically within about 20 amino acids of the terminus.

[350] Suitable linkers may be non-proteinaceous, such as, e.g. chemical linkers. Various non-proteinaceous linkers known in the art may be used to link anti-PD-Ll antibodies to cargos and additional materials to form the anti-PD-Ll antibodies of the present invention, such as linkers commonly used to conjugate immunoglobulin polypeptides to heterologous polypeptides. For example, polypeptide regions may be linked using the functional side chains of their amino acid residues and carbohydrate moieties such as, e.g ., a carboxy, amine, sulfhydryl, carboxylic acid, carbonyl, hydroxyl, and/or cyclic ring group. For example, disulfide bonds and thioether bonds may be used to link two or more polypeptides. In addition, non-natural amino acid residues may be used with other functional side chains, such as ketone groups (see e.g. Axup J et ak, Proc Natl Acad Sci U.S.A. 109: 16101-6 (2012); Sun S et ak, Chembiochem Jul 18 (2014); Tian F et ak, Proc Natl Acad Sci USA 111 : 1766-71 (2014)). In addition, non-natural amino acid residues may be used with other functional side chains, such as ketone groups, alkyne groups, or azides (see e.g. WO 2013185115; W02 014176284). Examples of non-proteinaceous chemical linkers include but are not limited to N-hydroxysuccinimide esters (NHS esters) such as sulfo-NHS esters, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Further examples of non-proteinaceous chemical linkers include but are not limited to N- succinimidyl (4-iodoacetyl)-aminobenzoate, ri-(/V-succinimidyl) thioacetate (SATA), N- succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridyldithio) toluene (SMPT), N-succinimidyl 4- (2-pyridyldithio)-pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane carboxylate (SMCC or MCC), sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate, 4- succinimidyl-oxycarbonyl-a-(2-pyridyldithio) toluene, sulfosuccinimidyl-6-(a-methyl-a- (pyridyldithiol)-toluamido) hexanoate, N-succinimidyl-3-(-2-pyridyldithio)-proprionate (SPDP), succinimidyl 6(3(-(-2-pyridyldithio)-proprionamido) hexanoate, sulfosuccinimidyl 6(3(-(-2-pyridyldithio)-propionamido) hexanoate, maleimidocaproyl (MC),

maleimidocaproyl-valine-citrulline-p-aminobenzyloxy carbonyl (MC-vc-PAB), 3- maleimidobenzoic acid A-hydroxysuccinimide ester (MBS), alpha-alkyl derivatives, sulfoNHS-ATMBA (sulfosuccinimidyl N-[3-(acetylthio)-3-methylbutyryl-beta-alanine]), sulfodichlorophenol, 2-iminothiolane, 3-(2-pyridyldithio)-propionyl hydrazide, Ellman’s reagent, dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine.

[351] The immunoconjugates or ADCs provided herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC,

SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo- SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), any of which are commercially available.

[352] Conjugates of an antibody may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),

succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate H ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (pdiazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta E et al., Science 238: 1098-104 (1987). Carbon- l4-labeled l-isothiocyanatobenzyl-3-methyldi ethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (see e.g. WO 1994/011026).

[353] Suitable linkers, whether proteinaceous or non-proteinaceous, may include, e.g. , protease sensitive, environmental redox potential sensitive, pH sensitive, acid cleavable, photocleavable, and/or heat sensitive linkers.

[354] Proteinaceous linkers may be chosen for incorporation into recombinant fusion anti- PD-L1 antibodies of the present invention. For recombinant fusion cell-targeting proteins of the invention, linkers typically comprise about 2 to 50 amino acid residues, preferably about 5 to 30 amino acid residues. Commonly, proteinaceous linkers comprise a majority of amino acid residues with polar, uncharged, and/or charged residues, such as, e.g. , threonine, proline, glutamine, glycine, and alanine.

[355] Proteinaceous linkers may be selected based upon the properties desired.

Proteinaceous linkers may be chosen by the skilled worker with specific features in mind, such as to optimize one or more of the fusion molecule’s folding, stability, expression, solubility, pharmacokinetic properties, pharmacodynamic properties, and/or the activity of the fused domains in the context of a fusion construct as compared to the activity of the same domain by itself. For example, proteinaceous linkers may be selected based on flexibility, rigidity, and/or cleavability. The skilled worker may use databases and linker design software tools when choosing linkers. In certain linkers may be chosen to optimize expression. In certain linkers may be chosen to promote intermolecular interactions between identical polypeptides or proteins to form homomultimers or different polypeptides or proteins to form heteromultimers. For example, proteinaceous linkers may be selected which allow for desired non-covalent interactions between polypeptide components of the anti-PD- Ll antibodies of the invention, such as, e.g ., interactions related to the formation dimers and other higher order multimers.

[356] Flexible proteinaceous linkers are often greater than 12 amino acid residues long and rich in small, non-polar amino acid residues, polar amino acid residues, and/or hydrophilic amino acid residues, such as, e.g. , glycines, serines, and threonines. Flexible proteinaceous linkers may be chosen to increase the spatial separation between components and/or to allow for intramolecular interactions between components. For example, various“GS” linkers are known to the skilled worker and are composed of multiple glycines and/or one or more serines, sometimes in repeating units.

[357] Rigid proteinaceous linkers are often stiff alpha-helical structures and rich in proline residues and/or one or more strategically placed prolines. Rigid linkers may be chosen to prevent intramolecular interactions between linked components.

[358] Suitable linkers may be chosen to allow for in vivo separation of components, such as, e.g. , due to cleavage and/or environment-specific instability. In vivo cleavable proteinaceous linkers are capable of unlinking by proteolytic processing and/or reducing environments often at a specific site within an organism or inside a certain cell type. In vivo cleavable proteinaceous linkers often comprise protease sensitive motifs and/or disulfide bonds formed by one or more cysteine pairs. In vivo cleavable proteinaceous linkers may be designed to be sensitive to proteases that exist only at certain locations in an organism, compartments within a cell, and/or become active only under certain physiological or pathological conditions (such as, e.g. , involving proteases with abnormally high levels, proteases overexpressed at certain disease sites, and proteases specifically expressed by a pathogenic microorganism). For example, there are proteinaceous linkers known in the art which are cleaved by proteases present only intracellularly, proteases present only within specific cell types, and proteases present only under pathological conditions, e.g. , cancer or inflammation.

[359] In certain embodiments of the anti-PD-Ll antibodies of the present invention, a linker may be used which comprises one or more protease sensitive sites to provide for cleavage by a protease present within a target cell. In certain embodiments of the anti-PD-Ll antibodies of the invention, a linker may be used which is not cleavable to reduce unwanted toxicity after administration to a vertebrate organism.

[360] Suitable linkers may include, e.g. , protease sensitive, environmental redox potential sensitive, pH sensitive, acid cleavable, photocleavable, and/or heat sensitive linkers, whether proteinaceous or non-proteinaceous ( see e.g., Doronina S et ak, Bioconjug Chem 17: 114-24 (2003); Saito G et al., Adv Drug Deliv Rev 55: 199-215 (2003); Jeffrey S et al., J Med Chem 48: 1344-58 (2005); Sanderson R et al., Clin Cancer Res 11 : 843-52 (2005); Erickson H et al., Cancer Res 66: 4426-33 (2006); Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013)). Suitable cleavable linkers may include linkers comprising cleavable groups which are known in the art.

[361] Suitable linkers may include pH sensitive linkers. For example, certain suitable linkers may be chosen for their instability in lower pH environments to provide for dissociation inside a subcellular compartment of a target cell (see e.g. , van Der Velden V et al., Blood 97: 3197-204 (2001); Ulbrich K, Subr V, Adv Drug Deliv Rev 56: 1023-50 (2004)). For example, linkers that comprise one or more trityl groups, derivatized trityl groups, bismaleimideothoxy propane groups, adipic acid dihydrazide groups, and/or acid labile transferrin groups, may provide for release of components of the anti-PD-Ll antibodies of the invention, e.g. a polypeptide component, in environments with specific pH ranges. In certain linkers may be chosen which are cleaved in pH ranges corresponding to physiological pH differences between tissues, such as, e.g, the pH of tumor tissue is lower than in healthy tissues.

[362] Photocleavable linkers are linkers that are cleaved upon exposure to electromagnetic radiation of certain wavelength ranges, such as light in the visible range. Photocleavable linkers may be used to release a component of an anti-PD-Ll antibody of the invention, e.g. a polypeptide component, upon exposure to light of certain wavelengths. Non-limiting examples of photocleavable linkers include a nitrobenzyl group as a photocleavable protective group for cysteine, nitrobenzyloxycarbonyl chloride cross-linkers,

hydroxypropylmethacrylamide copolymer, glycine copolymer, fluorescein copolymer, and methylrhodamine copolymer. Photocleavable linkers may have particular uses in linking components to form anti-PD-Ll antibodies of the invention designed for treating diseases, disorders, and conditions that can be exposed to light using fiber optics.

[363] In certain embodiments of the anti-PD-Ll antibodies of the present invention, the molecule comprises a binding region which is a scFv with a linker connecting a heavy chain variable (VH) domain and a light chain variable (VL) domain. There are numerous linkers known in the art suitable for this purpose.

E. Methods of Characterizing Anti-PD-Ll Antibodies of the Present Invention

[364] Anti-PD-Ll antibodies provided herein may be identified, screened for, or

characterized for their physicochemical properties and/or biological activities by various assays known in the art and/or described herein. The anti-PD-Ll antibody of the present invention may be defined by reference to one or more characteristics, including but not limited to its ability to act as an antagonist or agonist, its ability to internalize into a target cell, its ability to cause death of the target cell, and its binding specificity. Non-limiting examples of target cells for use in studying the characteristics of an anti-PD-Ll antibody of the present invention, such as in vitro or ex vivo , primary cell or cell derived from primary tissue, organ or tumor samples from a mammalian subject; immortalized or transformed mammalian cells grown in culture or on surfaces such as on plates or dishes and in arrays.

[365] For certain methods of anti-PD-Ll antibody characterization, the cells used are mammalian cells that express PD-L1, such as, e.g ., MC38 or B16F10 mouse cells. For certain methods of anti-PD-Ll antibody characterization, the cells used are immortalized human cells that express PD-L1, such as, e.g., HCC1954, HCC827, L1236, MDA-MB-231, JIMT-l, NCI-H226, MALME-3M, A375, and/or SK-BR-3 cells. In addition, immortalized human cells such as human embryonic kidney 293 (HEK293) and non-human mammalian Chinese hamster Ovarian (CHO) cell lines have be modified to overexpress a species-specific PD-L1 molecule for purposes of detection of antibody affinity specificity.

1. Binding Affinity, Epitope Mapping, and Binding Competition

[366] An antibody’s binding affinity for its antigen can be measured by common methods known in the art. For example, an antibody of the present invention may be tested for PD-L1 antigen binding activity using methods known to skilled worker such as, e.g, enzyme-linked immunosorbent assay (ELISA), isothermal titration calorimetry, Western blot, surface- plasmon resonance, protein display, fluorescent polarization, alanine scanning mutants, and/or other methods described herein.

[367] For certain embodiments, the KD of an antibody is measured by a radiolabeled antigen binding assay performed with the Fab version of the antibody and antigen molecule as described by the following assay that measures solution binding affinity of Fabs for antigen by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate ( see e.g. Chen Y et ak, JMol Biol 293 : 865-81 (1999)). To establish conditions for the assay, microtiter plates are coated overnight with 5 pg/mL of a capturing anti-Fab antibody in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w/v) bovine serum albumin in phosphate-buffered saline (PBS) for two to five hours at room temperature (approximately 23 °C). In a non-adsorbant plate, about 100 pM or 26 pM [¹²⁵I]- antigen are mixed with serial dilutions of a Fab of interest. The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period ( e.g . 2 to 3 days) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature for one hour. The solution is then removed, and the plate washed eight times with 0.1% TWEEN®-20 in PBS. When the plates have dried, 150 pL of scintillant is added, and the plates are counted for radioactive counts using standard techniques known to the skilled worker. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

[368] For certain embodiments, the KD is measured by using surface-plasmon resonance assays using a Biacore™ instrument (GE Healthcare, Chicago, IL, ET.S.A.) at 25 °C with immobilized antigen using standard techniques known to the skilled worker. Briefly, carboxy methylated dextran biosensor chips are activated with N-ethyl-N’-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/mL (0.2 pM) before injection at a flow rate of 5 pL/minute to achieve

approximately 10 response units (RU) of the coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.50 nM to 500 nM) are injected in PBS with 0.05%

TWEEN®-20 (PBST) at 25 °C at a flow rate of approximately 25 pL/min. Association rates (Kon) and dissociation rates (Koff) are calculated using a simple one-to-one Langmuir binding model by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio Koff/Kon (see e.g. Chen Y et ak, JMol Biol 293 : 865-81 (1999)). If the on-rate exceeds 10⁶ M V¹ by the surface-plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence-emission intensity

(excitation = 295 nanometer (nm); emission = 340 nm, 16 nanometer (nm) band-pass) at 25° C of anti-antigen antibody (Fab form) at 20 nM in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow-equipped spectrophotometer or a spectrophotometer with a stirred cuvette.

[369] For certain embodiments, competition assays may be used to identify another anti- PD-L1 antibody that competes for binding of an anti-PD-Ll antibody with any of anti-PD-Ll antibodies described herein. In certain embodiments, such a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) of an anti-PD-Ll antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Epitope Mapping Protocols: Methods in Molecular Biology, vol. 66 (Morris G, ed., Humana Press, Totowa, NJ (1996)).

[370] In an exemplary competition assay, immobilized anti-PD-Ll antibody is incubated in a solution comprising a first labeled antibody ( e.g. , a first labeled“anti- anti-PD-Ll antibody”) that binds to the anti-PD-Ll antibody, and a second unlabeled antibody (e.g, a second unlabeled anti-PD-Ll antibody), respectively, that is being tested for its ability to compete with the first anti-PD-Ll antibody for binding to PD-L1. The second antibody may be present in a hybridoma supernatant. As a control, immobilized anti-PD-Ll antibody is incubated in a solution comprising the first labeled antibody but not the second unlabeled anti-PD-Ll antibody. After incubation under conditions permissive for binding of the first labeled antibody to the anti-PD-Ll antibody, excess unbound antibody is removed, and the amount of label associated with immobilized anti-PD-Ll antibody is measured. If the amount of label associated with immobilized anti-PD-Ll antibody is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to PD-L1 (see e.g. Antibodies: A Laboratory Manual (Harlow E, Lane D, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988)). Competition assays can also be performed in a manner as described herein with FACS using cells transfected with anti-PD-Ll antibody and expressed on the cell surface. Additionally, ELISA with anti-PD-Ll antibody can also be used in a competition assay.

[371] The antibody of the invention may“bind specifically” to the PD-L1 protein. An antibody is considered to be specifically binding if the antibody binds to the PD-L1 with a threshold level of binding activity. Binding affinity of an antibody can be characterized using the dissociation constant“KD” or the association constant“Ka”. Binding affinity may be determined using various techniques known in the art, for example, surface plasmon resonance, bio-layer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical

ultracentrifugation, and flow cytometry.

[372] In one embodiment, the antibody specifically binds if it binds to a target PD-L1 polypeptide, peptide, or epitope with an dissociation constant (KD) of less than 10⁴ M, less than 10⁵ M, less than 10⁶ M, less than 10⁷ M, less than 10⁸ M, less than 10⁹ M, less than 10¹⁰ M, less than l0^u M, less than 10¹² M, less than 10¹³ M, M, less than 10¹⁴ M, or less than 10¹⁵ M. In one embodiment, the antibody specifically binds if it binds to a PD-L1 polypeptide, peptide, or epitope with an association constant (Ka) of at least l0⁵ M¹s¹, at least l0⁶ M¹s¹, at least l0⁷ M¹s¹, at least l0⁸ M¹s¹ or at least l0⁹ M¹s¹. [373] An antibody of the present invention may specifically bind the extracellular domain of PD-L1. An antibody of the present invention may specifically bind the extracellular domain of a PD-L1 variant comprising the amino acid residue phenylalanine at position 42. An antibody of the present invention may specifically bind human PD-L1. An antibody of the present invention may specifically bind the human PD-L1 variant comprising the amino acid residue phenylalanine at position 42. An antibody of the present invention may specifically bind a polypeptide comprising, consisting essentially of, or consisting of amino acids 1 to 239 of SEQ ID NO:20.

[374] An antibody of the present invention may specifically bind a polypeptide comprising, consisting essentially of, or consisting of amino acids 1 to 239 of any one of SEQ ID NOs: 20-23. An antibody of the invention may specifically bind a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 75% ( e.g . at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to amino acids 1 to 239 of any one of SEQ ID NOs: 20-23. An antibody of the invention may specifically bind a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 75% (e.g. at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to amino acids 1 to 239 of any one of SEQ ID NOs: 20-23 as long as the sequence comprises a phenylalanine at position 42. An antibody of the invention may specifically bind a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 75% (e.g. at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to amino acids 1 to 239 of any one of SEQ ID NOs: 20-23 as long as the sequence comprises a phenylalanine at position 42, aspartate at position 122, and/or tyrosine at position 123.

[375] Competitive binding assays known in the art may be used to assess the binding and specificity of an antibody for a target. The antagonistic antibody of the invention may be considered to inhibit or reduce binding of the target to a binding partner or substrate when an excess of antibody reduces the quantity of binding partner bound to the target by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more as measured, for example, in an in vitro competitive binding assay. The agonistic antibody of the invention may be considered to stimulate or increase binding of the target to a binding partner or substrate when an excess of antibody increases the quantity of binding partner bound to the target by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,

95%, 97%, 99% or more as measured, for example, in an in vitro competitive binding assay.

In one embodiment, the antibody of the invention alters (e.g. increases or decreases) the biological activity of the target by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,

90%, 95%, 97%, 99% or more. The modified activity of the target may be measured directly using art-recognized techniques or may be measured by the impact that the altered activity has downstream ( e.g ., PD-L1/PD-1 signal interference as measured using the PD-1/PD-L1 Blockade Bioassay described herein).

[376] It is also possible to characterize the antibody of the invention in terms of the group or “bin” to which it belongs. “Binning” refers to the use of competitive antibody binding assays to identify pairs of antibodies that are incapable of simultaneously binding to an antigen, thereby identifying antibodies that“compete” for binding. Competing antibodies may be determined by an assay in which the antibody or antigen binding fragment being tested prevents or inhibits specific binding of a reference antibody to a common antigen. Typically, such an assay involves the use of purified antigen (e.g., target protein or a domain or fragment thereof) bound to a solid surface or cells, an unlabeled test antibody and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more. Conversely, when the reference antibody is bound it will preferably inhibit binding of a subsequently added test antibody (e.g. an anti-PD-Ll antibody) by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding of the test antibody is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

[377] Generally binning or competitive binding may be determined using various art- recognized techniques, such as, for example, immunoassays such as western blots, radioimmunoassays, ELISA,“sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays. Such immunoassays are routine and well known in the art (see, Current Protocols in Molecular Biology Vol. 1 (Ausubel F et ah, eds, John Wiley & Sons, Inc., New York). Additionally, cross-blocking assays may be used, such as described in Antibodies: A Laboratory Manual (Harlow E, Lane D, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988). Other technologies known to the skilled worker for determining competitive inhibition (and hence“bins”), include: surface plasmon resonance using, for example, a Biacore™ system; bio-layer interferometry using, for example, a ForteBio® Octet RED (ForteBio, Fremont CA, U.S.A.); or flow cytometry bead arrays, e.g, using a BD Canto system (BD Biosciences) or a multiplex LUMINEX® detection assay (Luminex, Austin, TX, U.S.A.).

[378] Once a bin, encompassing a group of competing antibodies, has been defined further characterization can be carried out to determine the specific domain or epitope on the antigen to which that group of antibodies binds ( see e.g. Cochran J et al., J Immunol Methods 287: 147-58 (2004)). Fine epitope mapping is the process of determining the specific amino acids on the antigen that comprise the epitope of a target to which the antibody binds.

[379] In certain embodiments fine epitope mapping can be performed using phage or yeast display. Other compatible epitope mapping techniques include alanine scanning mutants, peptide blots, or peptide cleavage analysis (see e.g. Reineke U, Methods Mol Biol 248: 443- 63 (2004)). In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed using enzymes such as proteolytic enzymes (e.g. trypsin, endoproteinase Glu-C, endoproteinase Asp-N, chymotrypsin); chemical agents such as, e.g. , succinimidyl esters and their derivatives, primary amine-containing compounds, hydrazines and carbohydrazines, and free amino acids (see e.g. Hochleitner E et al., Protein Sci 9: 487-96 (2000)). In another embodiment, Modification-Assisted Profiling, also known as Antigen Structure-based Antibody Profiling (ASAP) can be used to categorize large numbers of monoclonal antibodies directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see e.g. ETS 2004/0101920).

[380] As well as competition binding methods, other methods known in the art for characterizing the epitope for a given antibody include mutagenesis, X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and Hydrogen deuterium exchange Mass Spectrometry (HX-MS).

2. Antibody Functional Activity Assays

[381] Antibody activity can be determined by common methods known in the art. For example, an antibody of the present invention may be tested for various biological activities besides binding affinity using methods known in the art. In one aspect, assays are provided herein for identifying anti-PD-Ll antibodies having useful biological activity. Biological activity may include, e.g. , binding of such antibodies to different PD-L1 molecules or epitopes, targeting cell-surface PD-L1 in vitro or in vivo , and detecting PD-L1 levels, stimulating ADCC, stimulating CDC, stimulating programmed cell death (e.g. apoptosis) via PD-L1 binding, stimulating ADCP, and modulating PD-L1 signaling (e.g. inhibition of signal transduction). Antibodies having such biological activity in vivo and/or in vitro are also provided.

[382] For certain embodiments, an antibody of the present invention is tested for such biological activity. For example, numerous routine assays exist to investigate ADCC, CDC, target cross-linking (e.g. Fc-dependent cross-linking), and apoptosis induced by antibodies of the present invention. a). Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) Activity Assays

[383] The Fc region of an antibody of the present invention can signal while bound to the surface of a target cell through the FcyRIIIa (CD 16) receptor on natural killer (NK) cells and other myeloid cells, inducing these cells to release cytokines such as interferon-gamma (IFNy) and cytotoxic granules containing perforin and granzymes that leads to DNA degradation, induces apoptosis and results in cell death, i.e.“ADCC”. Over the years, numerous versions and variations of the ADCC assay have been developed based on the use of peripheral blood mononuclear cells (PBMC) from donated blood as a source of the induced effector cells (NK cells). In some cases, NK cells have been further isolated from leukopheresis products of normal donors using MACS NK cell isolation kits. The ADCC activity of an anti-PD-Ll antibody of the present invention may be assessed using routine ADCC activity assays by performing routine and conventional methods. It is important to note that the successful ADCC assay is dependent on the optimization of the ratio of target to effector cells in the assay because density of receptors (FcyRIIIa) will likely differ from donor-to-donor cell populations.

[384] Non-limiting examples of in vitro assays to assess ADCC activity of an antibody of interest are described in WO 1988/004936; Hellstrom I el al ., Proc Natl Acad Sci U.S.A. 82: 1499-1502 (1985); Hellstrom I et al, Proc Natl Acad Sci U.S.A. 83 : 7059-63 (1986); WO 1999/022653; Briiggemann M et al., J Exp Med 166: 1351-61 (1987). Alternatively, non radioactive assays methods may be employed, such as non-radioactive cytotoxicity assays, see e.g. aCella-TOX (Cell Technology, Mountain View, CA, U.S.A.), and CytoTox 96® (Promega, Madison, WI, U.S.A.). Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, ADCC activity of the antibody of interest may be assessed in vivo, e.g, in an animal model such as that disclosed in Clynes R et al. Proc Natl Acad Sci U.S.A. 95: 652-6 (1998). ADCC may be monitored in such assays by measuring the specific markers, such as, e.g. , using FACS, fluorescent dye, a radioisotope (e.g.⁵¹Cr), and/or routine methods used to detect cell death and proliferation known to skilled worker (e.g. lactate dehydrogenase (LDH) release). b). Complement-Dependent Cytotoxicity (CDC) Activity Assays

[385] Similar to ADCC activities, the Fc region of an antibody of the present invention can elicit CDC killing effects via binding to serum complement components, particularly Clq. Following complicated enzyme activations and cleavage events, involving over 20 highly regulated elements, CDC can lead to the formation of membrane attacking complex (MAC), resulting in target cell death. Classical CDC manifests as one vital mechanism of action of most therapeutic antibodies; meanwhile, it can collaborate with ADCC as an enhancer. The Fc portion of antibodies bound to the surface of a target cell can bind to Clq and cause the deposition of complement and the activation of a cascade resulting in CDC. The CDC activity of an anti-PD-Ll antibody of the present invention may be assessed using routine CDC activity assays by performing routine and conventional methods known to the skilled worker. There are various CDC assays known in the art, such as, e.g. , using serum from a healthy human as a source of complement or using purified complement components. To assess complement activation, a CDC assay may be performed as described in Gazzano- Santoro et al., J Immunol Methods 202: 163-71 (1997); Cragg M el al., Blood 101 : 1045-52 (2003); Cragg M, Glennie M, Blood 103 : 2738-43 (2004). Clq binding assays may also be carried out to determine whether that the antibody is able to bind Clq or not, and hence lacks CDC activity (see e.g. Clq and C3c binding ELISA in WO 2006/029879 and WO

2005/100402). c). ADCP Activity Assays

[386] An antibody’s antibody-dependent cellular phagocytosis (ADCP) activity can be determined by common methods known in the art, such as, e.g. cell reporter assays based on FcyRIIa, FcyRI, and FcyRIIIa signaling to a luciferase reporter. The amount of ADCP activity of an anti-PD-Ll antibody of the present invention may be assessed using routine ADCP activity assays by performing routine and conventional methods. d). Internalizing Activity Assays

[387] An antibody’s cellular internalization activity can be determined by common methods known in the art. For certain embodiments, the antibody of the present invention may have an internalizing function such that when the antibody binds to a target protein (e.g. PD-L1), the antibody will be internalized (along with any conjugated pharmaceutically active moiety) into a selected target cell. The number of antibody molecules internalized may be sufficient to kill an antigen-expressing cell. Depending on the potency of the antibody, the uptake of a single antibody molecule into the cell may be sufficient to kill the target cell to which the antibody binds. Whether an antibody internalizes upon binding to a mammalian cell can be determined by various art-recognized assays known to the skilled worker. e). PD-L1 Signaling Modulation Assay

[388] The PD-L1 signaling modulation activity of an anti-PD-Ll antibody of the present invention may be assessed using PD-L1 signaling modulation assays by performing routine and conventional methods. Various PD-L1 signaling assays are known in the art. For example, there are PD-L1 inhibition assays involving purified PD-L1 and PD-l used to detect inhibition of receptor ligand binding, e.g. a biochemical interaction assay. i) PD-L1 Antagonistic Antibodies

[389] In one embodiment, the antibody of the invention may act as an antagonist that blocks, reduces or inhibits the biological activity of PD-L 1. The antagonistic antibody of the invention may bind to or interact with PD-L1 or its receptor or ligand and prevent binding or association of the PD-L1 to a binding partner such as a receptor (e.g. PD-l and/or B7-1) or substrate, thereby interrupting a biological response that otherwise would result from the interaction of the molecules. For example, cell-based reporter assays for immune checkpoint activation known in the art may be used by the skilled worker to characterize a PD-L1 antibody of the present invention. One example is a coculture cell assay using PD-l effector cells (like Jurkat cells expressing PD-l) and PD-L1 expressing cells (like CHO-K1 cells expressing PD-L1 and a cell surface protein designed to activate cognate T-cell receptors (TCRs) in an antigen dependent manner) (see e.g. Promega, Madison, WI: “PD-l /PD-L 1 Blockade Bioassay Protocol”). ii) PD-L1 Agonist Antibodies

[390] In another embodiment, the antibody of the invention may act as an agonist that stimulates or increases the biological activity of the PD-L1. The agonistic antibody of the invention may bind to or interact with a PD-L1 molecule and facilitate binding or association of PD-L 1 to a binding partner such as a receptor or substrate, thereby promoting a biological response (e.g. PD-L1 reverse signaling) that results from the interaction of the molecules. For example, cell-based reporter assays for immune checkpoint signaling known in the art may be used by the skilled worker to characterize agonist action of a PD-L1 antibody of the present invention (see e.g. Kehry M et ah, Discovery of Checkpoint Agonist Antibodies for Autoimmune/Inflammatory Disease, W. 73 FOCIS Annual Meeting (2019)). f). Depleting Antibodies

[391] In one embodiment, the antibody of the invention is a depleting antibody. The term “depleting” antibody refers to an antibody that binds to a target protein on or near the cell surface and induces, promotes or causes the death of the cell (e.g. by CDC, ADCC or introduction of a cytotoxic agent). In certain embodiments, the depleting antibody is conjugated to a cytotoxin or radioisotope.

[392] Any embodiment of the anti-PD-Ll antibody of the present invention may be used with each individual embodiment of the methods of the present invention.

II. Methods of Using and Uses of Anti-PD-Ll Antibodies and Antigen Binding Fragments and Derivatives Thereof and Compositions Comprising the Aforementioned

[393] The anti-PD-Ll antibodies of the present invention are useful in diverse applications involving, e.g. , cell-killing; tumor growth inhibition; cargo delivery; biological information gathering; immune response stimulation, and/or remediation of a health condition. The anti- PD-Ll antibodies of the present invention are useful as therapeutic and/or diagnostic molecules, such as, e.g. , as cell-targeting, cytotoxic, therapeutic molecules; cell-targeting, nontoxic, delivery vehicles; and/or cell-targeting, diagnostic molecules; for examples in applications involving the in vivo targeting of specific cell types for the diagnosis or treatment of a variety of diseases, including cancers, immune disorders, and microbial infections involving PD-L1 -expressing cells. In certain embodiments, the anti-PD-Ll antibodies of the present invention are capable of binding an extracellular part of PD-L1 molecules associated with cell surfaces of particular cell types and entering those cells or delivering a cargo to the interior of those cells.

[394] The binding of PD-l to PD-L1 expressed on a cell can transmit survival, growth, and/or proliferation signals to PD-L 1 -expressing cells (“reverse signaling”) (see e.g. Lecis D et ak, Cancers 11 : E624 (2019)). For example, the presence of PD-l, whether in a membrane bound or soluble form, can function to activate PD-L1 signaling within PD-L 1 -expressing tumor cells to protect from IFNy-induced cell death of colon tumor cells or support survival and proliferation of lymphoma cells (Lau J et ak, Nat Commun 8: 14572 (2017); Jalali S et al., Blood Cancer J 9 : 22 (2019)). The inhibition of PD-L1 signaling or antagonistic binding to PD-L1 may be used to block survival, growth, and/or proliferation signals in PD-L1- expressing tumor cells (Gato-Canas M et al., Cell Rep 20: 1818-29 (2017)).

[395] For example, the binding of PD-l to PD-L1 expressed on a tumor-associated macrophage can transmit an activation signal by down-regulating constitutively active, inhibitory signaling of PD-L1 in the absence of PD-l ( see e.g. Lecis D et al., Cancers 11 : E624 (2019)). The treatment of macrophages with anti-PD-Ll antibodies increased macrophage activation, survival, and proliferation, thereby releasing the macrophages from a constitutive immuno-suppressive state, which was not affected by anti -PD-l treatment (Hartley G et al., Cancer Immunol Res 6: 1260-73 (2018)). Thus, PD-L1 is a putative target for immunomodulatory agents for the activation of tumor-associated PD-L 1 -expressing immune cells via directly antagonizing intrinsic PD-L1 signaling within a PD-L 1 -expressing cell.

[396] Blockade of the PD-L1/PD-1 signaling axis by antibodies may promote epitope spreading by reducing CD8+ T-cell fratricidal lysis of neighboring CD8+ T-cells restricted to less immunodominant antigens (Haeryfar S and Schell, Cancer Immunol Immunother 67: 1669-72 (2018)), and T-cell effector cells specific to certain less dominant neoantigens may be important for therapeutic effectiveness (see e.g. Braunlein E, Krackhardt A, Front Immunol 8: 1702 (2017)). The reduction in fratricide of CD8+T-cells restricted to less immunodominant antigens reinvigorates subdominant CD8+ T-cells responses that might contribute to antitumor immunity (Memamejadian A et al., J Immunol 199: 3348-59 (2017); Haeryfar S and Schell, Cancer Immunol Immunother 67: 1669-72 (2018)). Thus, PD-L1 is a putative target for immunomodulatory agents for increasing immune responses to less dominant antigens, such as, e.g. locally in the tumor microenvironment.

A PD-L1/PD-1 Signaling Interference

[397] In addition to cytotoxic, cytostatic, and immune stimulation applications, anti-PD-Ll antibodies of the present invention optionally may be used for inhibiting PD-l signaling, such as, e.g. , in applications involving immune checkpoint inhibition and anti-cancer

immunotherapy. Certain embodiments of the PD-L 1 -targeting molecules of the present invention when exogenously administered to cells can interfere with the PD-1/PD-L1 interaction. Although certain embodiments of the anti-PD-Ll antibodies of the present invention exhibit half-maximal inhibitory concentrations (EC so) for PD-L1 signaling inhibition that are much less potent (e.g. greater than 500 nM or 1 mM) than their cytotoxic CD50 (e.g. 0.1 to 50 nM), for a given target cell type, this is not always the case. Certain embodiments of the anti-PD-Ll antibodies of the present invention can exhibit EC50 values equivalent to their CD50 values, indicating potent levels of both PD-l signaling inhibition and cytotoxicity could occur concurrently. For certain embodiments, the anti-PD-Ll antibodies of the present invention exhibit EC50 values (e.g. 1 to 200 nM) that are greater than their cytotoxic CD50 values (e.g. greater than 1,000 or 10,000 nM). Certain anti-PD-Ll antibodies of the present invention exhibiting EC50 values greater than their cytotoxic CD50 value may be used at certain concentrations for effectuating PD-l signaling inhibition in the absence of any significant cytotoxic activity.

B. Killing PD-L 1 -Expressing Cells

[398] The present invention provides the use of the anti-PD-Ll antibody of the present invention (such as an antigen-binding fragment or derivative thereof) for killing PD-L1 expressing cells. For certain embodiments, the anti-PD-Ll antibody of the present invention is capable, upon contacting a PD-L 1 -expressing cancer or immune cell, such as, e.g. , in vivo.

[399] For certain embodiments, the anti-PD-Ll antibody of the present invention is capable, upon contacting a PD-L1 positive peripheral blood mononuclear cell of causing the death of the PD-L1 positive peripheral blood mononuclear cell, such as, e.g. , in vivo.

C. Methods for Detecting PD-L1

[400] In certain embodiments, the anti-PD-Ll antibodies of the present invention have uses in the in vitro and/or in vivo detection of PD-L1 and specific cells, cell types, and/or cell populations. The present invention provides the use of the anti-PD-Ll antibody, antigen binding fragment or derivative thereof, for detecting PD-L1 in specific cells, cell types, and/or cell populations, as well as specific subcellular compartments of any of the

aforementioned. Anti-PD-Ll antibodies of the invention that are conjugated to detection- promoting agents optionally may be used for diagnostic functions, such as for companion diagnostics used in conjunction with a therapeutic regimen comprising the same or a related binding region, such as, e.g. , a binding region with high-affinity binding to the same target biomolecule, an overlapping epitope, and/or the same epitope.

[401] In certain embodiments, the anti-PD-Ll antibodies of the present invention, including antigen binding fragments and derivates thereof, or compositions comprising the

aforementioned and/or provided herein are useful for detecting the presence of PD-L1, e.g. in vivo or in a biological sample. The anti-PD-Ll antibodies, or compositions comprising such antibodies, can be used in a variety of different detection assays, including but not limited to ELISA, immunoprecipitation, bead-based immunoassays, mass spectrometry, and

immunofixation electrophoresis (IFE). In certain embodiments, the anti-PD-Ll antibody is immobilized to a solid support or conjugated to biotin and bound to a streptavidin coated microtiter plate. For certain embodiments, the antibody is directly detectable, such as, e.g ., due to the presence of a label including but not limited to an enzyme (e.g. horseradish peroxidase), a fluorescent agent, or a colorimetric reagent.

[402] PD-L1 expression may serve as a diagnostic marker for the characterization of a cell- type, tissue, disease, disorder, or condition. PD-L1 expression may serve as a diagnostic marker for the selection or stratification of patients most likely to respond to certain therapies or therapeutic approaches or to monitor changes in patients during or after receipt of a therapeutic regimen or other intervention. Thus, PD-L1 is a target for diagnostic detection and characterization, such as, e.g. , to detect, quantify, or characterize cells capable of internalizing an anti-PD-Ll antibody for information-gathering regarding the status of certain diseases, disorders, and conditions, including the progression and effects of treatments thereof (see e.g. Ma W et ak, J Hematol Oncol 9: 47 (2016)).

[403] For certain embodiments, the anti-PD-Ll antibody or compositions comprising such antibodies as provided herein are useful to quantitate PD-L1 amounts in a sample, e.g. a biological sample as described herein. The present invention provides the use of the anti-PD- Ll antibody, antigen-binding fragment or derivative thereof for detection and/or

quantification of PD-L1 in a sample, e.g. , a biological sample. In certain embodiments, a biological sample is a biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, urine and other constituents of the body that may contain the PD-L1 of interest. In various embodiments, the sample is a body sample isolated or obtained from any vertebrate. In certain embodiments, the sample is isolated or obtained from a mammal. In certain embodiments, the sample is isolated or obtained from a human subject. In certain embodiments, the biological sample is serum from a human subject. In certain embodiments the biological sample is biopsy material. In certain embodiments, the biological sample is biopsy material from a human subject. In certain embodiments the biological sample is biopsy material from a tumor. In certain embodiments the biological sample is biopsy material comprising tumor cells or cancer cells. In certain embodiments the biological sample is primary cell culture material. In certain embodiments, the biological sample is primary cell culture material from a human subject. In certain embodiments, the biological sample is from a human subject or subjects treated with a therapeutic anti-PD-Ll antibody or antibodies or some other therapeutic directed to an immune checkpoint, such as, e.g. one or more of an anti -PD- 1 antibody, anti-B7-l antibody, anti-PD-L2 antibody, and anti-CTLA-4 antibody.

[404] In certain embodiments any of the anti-PD-Ll antibodies, or compositions comprising such antibodies, as provided herein, are useful for detecting the presence of PD-L1 in an immunoassay without affecting PD-Ll’s ability to bind to another molecule (e.g. PD-l or B7- 1). The present invention provides the use of the anti-PD-Ll antibody, antigen-binding fragment or derivative thereof for detecting the presence of PD-L 1 in an immunoassay without affecting PD-Ll’s ability to bind to another molecule, such as a natural ligand or receptor, e.g. , PD-l or B7-1.

[405] In certain embodiments any of the anti-PD-Ll antibodies that bind to an anti-PD-Ll antibody, or compositions comprising such antibodies, as provided herein, are useful for depleting, detecting, or differentiating PD-L1 from a patient sample. The present invention provides the use of the anti-PD-Ll antibody, antigen-binding fragment or derivative thereof for depleting, detecting, or differentiating PD-L1 from a patient sample.

[406] In certain embodiments, the sample is isolated or obtained from a mammal. In certain embodiments, the sample is isolated or obtained from a human subject, e.g. , when measuring PD-L1 expression in a clinical sample. In certain embodiments, the biological sample is isolated or obtained from a human subject or a patient treated with a therapy/therapeutic (e.g. atezolizumab, avelumab, and/or durvalumab), such as, e.g. an anti-PD-Ll antibody, anti-PD- 1 antibody, anti-B7-l antibody, anti-PD-L2 antibody, and/or anti-CTLA-4 antibody. In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a human subject. In certain embodiments the biological sample is biopsy material. In certain embodiments, the biological sample is biopsy material from a human subject. In certain embodiments, the biological sample is biopsy material from a tumor. In certain embodiments the biological sample is biopsy material comprising tumor cells or cancer cells. In certain embodiments the biological sample is primary cell culture material. In certain embodiments, the biological sample is primary cell culture material from a human subject.

[407] In certain embodiments of the methods and uses of detecting and/or quantifying the presence of PD-L 1 described herein, the method or use comprises the detection of a“label” or“detecting promoting agent”. In certain further embodiments, the detection-promoting agent is a fluorescent, chromophoric, electron-dense, chemiluminescent, radioactive, and/or other label, which may be detected directly using routine methods known to the skilled worker. In certain further embodiments, the detection-promoting agent is an enzyme or ligand, which may be detected indirectly using routine methods known to the skilled worker. Exemplary labels include, but are not limited to, the radioisotopes³²P,¹⁴C,¹²⁵1,³H, and¹³¹I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferin, luciferases ( e.g ., firefly luciferase and bacterial luciferase), 2,3 -dihydrophthalazinedi ones, biotin/avidin, horseradish peroxidase (HRP), alkaline phosphatase, J3-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g. glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g. uricase or xanthine oxidase) coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, spin labels, bacteriophage labels, and relatively stable radicals (also known as stable free radicals).

[408] In certain embodiments, the anti-PD-Ll antibodies described herein are used for both diagnosis and treatment, or for diagnosis alone. When the same cytotoxic anti-PD-Ll antibody is used for both diagnosis and treatment, for certain embodiments of the present invention the anti-PD-Ll antibody variant which incorporates a detection-promoting agent for diagnosis may have its cytotoxicity reduced or may be rendered nontoxic via one or more amino acid substitutions, including exemplary substitutions described herein. For example, certain nontoxic variants of the anti-PD-Ll antibodies of the present invention exhibit less than 5%, 4%, 3%, 2%, or 1% death of target cells after administration of a dose less than 1 mg/kg. Reduced-cytotoxicity variants may still be cytotoxic at certain concentrations or dosages but exhibit reduced cytotoxicity.

[409] The ability to conjugate detection-promoting agents known in the art to various anti- PD-Ll antibodies of the present invention provides useful compositions for the detection of certain cells, such as, e.g. , cancer, tumor, immune, and/or infected cells. These diagnostic embodiments of the anti-PD-Ll antibodies of the invention may be used for information gathering via various imaging techniques and assays known in the art. For example, diagnostic embodiments of the anti-PD-Ll antibodies of the invention may be used for information gathering via imaging of intracellular organelles (e.g. endocytotic, Golgi, endoplasmic reticulum, and cytosolic compartments) of individual cancer cells, immune cells, and/or infected cells in a patient or biopsy sample.

[410] Various types of information may be gathered using the diagnostic embodiments of the anti-PD-Ll antibodies of the invention whether for diagnostic uses or other uses. This information may be useful, for example, in diagnosing neoplastic cell types, determining therapeutic susceptibilities of a patient’s disease, determining the progression of anti neoplastic therapies over time, determining the progression of immunomodulatory therapies over time, determining the progression of antimicrobial therapies over time, evaluating the presence of infected cells in transplantation materials, evaluating the presence of unwanted cell types in transplantation materials, and/or evaluating the presence of residual tumor cells after surgical excision of a tumor mass.

[411] For example, subpopulations of patients might be ascertained using information gathered using the diagnostic variants of the anti-PD-Ll antibodies of the invention, and then individual patients could be further categorized into subpopulations based on their unique characteristic(s) revealed using those diagnostic embodiments. For example, the

effectiveness of specific pharmaceuticals or therapies might be a criterion used to define a patient subpopulation. For example, a nontoxic diagnostic variant of a particular cytotoxic, anti-PD-Ll antibody of the invention may be used to differentiate which patients are in a class or subpopulation of patients predicted to respond positively to a cytotoxic variant of that anti-PD-Ll antibody of the invention. Accordingly, associated methods for patient identification, patient stratification, and diagnosis using anti-PD-Ll antibodies of the present invention, including non-toxic variants of cytotoxic, anti-PD-Ll antibodies of the present invention, are considered to be within the scope of the present invention.

[412] The expression of the target biomolecule by a cell need not be native in order for cell targeting by an anti-PD-Ll antibody of the present invention, such as, e.g ., for direct cell-kill, indirect cell-kill (including ADCC and/or CDC), delivery of exogenous materials, e.g. , a T- cell epitope, and/or information gathering. Cell surface expression of the target biomolecule could be the result of an infection, the presence of a pathogen, and/ or the presence of an intracellular microbial pathogen. Expression of a target biomolecule could be artificial such as, for example, by forced or induced expression after infection with a viral expression vector, see e.g. adenoviral, adeno-associated viral, and retroviral systems. Expression of PD- Ll can be induced by exposing a cell to interferon-g (see e.g. Chen L, Nat Rev Immunol 4: 336-47 (2004); Hirano F et ak, Cancer Res 65: 1089-96 (2005); Zou W, Chen L, Nat Rev Immunol 8: 467-7 (2008); Flies D et ak, Yale J Biol Med 84: 409-21 (2011); Chen J et ak, Immunobiology 217: 385-93 (2012); Spranger S et ak Sci Transl Med 5: 200ral l6 (2013)).

D. Delivery of Cargo into the Interior of Targeted Cells [413] Certain anti-PD-Ll antibodies of the present invention optionally may be used for targeted delivery functions. Cargos include toxic payloads (including indirectly cytotoxic antigens) and non-toxic cargos, such as, e.g. certain detection-promoting agents, enzymes, or polynucleotides.

[414] Anti-PD-Ll antibodies of the present invention which are not capable of killing a target cell, such as at certain concentration ranges, certain cell-types, or specific situations (e.g. for in vitro ), may still be useful for delivering exogenous materials into cells, such as, e.g. , drugs or detection-promoting agents, at such concentrations, to such cell-types, and/or in such situations. The present invention therefore provides the use of the anti-PD-Ll antibody, antigen-binding fragment or derivative thereof, for delivering exogenous materials into cells.

[415] Certain anti-PD-Ll antibodies of the invention may be rendered non-cytotoxic, such as, e.g. , via the addition of one or more amino acid substitutions known to the skilled worker (e.g. to a Fc region) to render the antibody more suitable in certain situations for delivery of a cargo to target cell. Certain reduced-activity ribotoxic polypeptides may be particularly useful for delivering an additional exogenous material to certain intracellular locations or subcellular compartments of target cells.

[416] Various types of cells and/or cell populations which express PD-L1 to at least one cellular surface may be targeted by the anti-PD-Ll antibodies of the invention for receiving cargos such as an exogenous material or material heterologous to the anti-PD-Ll antibody.

[417] The cargo delivery function of certain anti-PD-Ll antibodies of the present invention may be accomplished under varied conditions and in the presence of non-targeted bystander cells, such as, e.g. , an ex vivo manipulated target cell, a target cell cultured in vitro , a target cell within a tissue sample cultured in vitro , or a target cell in an in vivo setting, such as within a multicellular organism. Furthermore, the selective delivery of exogenous material to certain cells by certain anti-PD-Ll antibodies of the present invention may be accomplished under varied conditions and in the presence of non-targeted bystander cells, such as ex vivo manipulated mixtures of cell types, in vitro cultured tissues with mixtures of cell types, or in vivo in the presence of multiple cell types (e.g, in situ or in a native location within a multicellular organism).

III. Production, Manufacture, and Purification of PD-L1 Binding Proteins

[418] A description follows as to exemplary techniques for the production of the anti-PD-Ll antibodies of the present invention for use in accordance with the methods of the present invention. The anti-PD-Ll antibody of the present invention can be produced using a variety of methods known in the art, which are discussed in detail below.

[419] The anti-PD-Ll antibodies and antigen binding fragments or derivatives thereof of the present invention may be produced using routine techniques known to those of skill in the art. For example, anti-PD-Ll antibodies of the invention may be manufactured by standard synthetic methods, by use of recombinant expression systems, or by any other suitable method. Thus, anti-PD-Ll antibodies of the invention may be synthesized in a number of ways, including, e.g. methods comprising: (1) synthesizing a polypeptide or polypeptide component of an anti-PD-Ll antibody using standard solid-phase or liquid-phase

methodology, either stepwise or by fragment assembly, and isolating and purifying the final polypeptide compound product; (2) expressing a polynucleotide that encodes a protein or protein component of an anti-PD-Ll antibody of the invention in a host cell and recovering the expression product from the host cell or host cell culture; or (3) cell-free, in vitro expression of a polynucleotide encoding a polypeptide or polypeptide component of an anti- PD-Ll antibody of the invention, and recovering the expression product; or by any combination of the methods of (1), (2) or (3) to obtain fragments of the protein component, subsequently joining (e.g. ligating) the peptide or polypeptide fragments to obtain a polypeptide component, and recovering the polypeptide component.

[420] It may be desirable to synthesize an anti-PD-Ll antibody of the present invention, or a protein component of an anti-PD-Ll antibody of the invention, by means of solid-phase or liquid-phase peptide synthesis. Anti-PD-Ll antibodies of the present invention may suitably be manufactured by standard synthetic methods. Thus, peptides may be synthesized by, e.g. methods comprising synthesizing the peptide by standard solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the final peptide product. In this context, reference may be made to WO 1998/011125 or, inter alia , Fields G et ak, Principles and Practice of Solid-Phase Peptide Synthesis (Synthetic Peptides, Grant G, ed., Oxford University Press, U.K., 2nd ed., 2002) and the synthesis examples therein.

[421] Anti-PD-Ll antibodies of the present invention may be prepared (produced and purified) using recombinant techniques well known in the art. In general, methods for preparing proteins by culturing host cells transformed or transfected with a vector comprising the encoding polynucleotide and purifying or recovering a substantially pure protein from cell culture are described in, e.g., Sambrook J et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY, U.S., 1989); Dieffenbach C et ak, PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, N.Y., U.S., 1995). Any suitable host cell may be used to produce an anti-PD-Ll antibody of the invention. Host cells may be cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors which drive expression of a polypeptide of the invention. In addition, an anti-PD-Ll antibody may be produced by modifying the polynucleotide encoding an anti-PD-Ll antibody of the invention that result in altering one or more amino acids or deleting or inserting one or more amino acids in order to achieve desired properties, such as changed immunogenicity, off-target toxicity, side effects, on-target cytotoxicity, changed cytostatic effects, and/or changed serum half-life.

[422] There are a wide variety of expression systems which may be chosen to produce an anti-PD-Ll antibody of the present invention. For example, host organisms for expression of certain anti-PD-Ll antibodies of the invention include prokaryotes, such as E. coli and B. subtilis , eukaryotic cells, such as yeast and filamentous fungi (like S. cerevisiae , P. pastoris , A. awamori , and A. lactis ), algae (like C. reinhardtii ), insect cell lines, mammalian cells (like simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein), plant cell lines, and eukaryotic organisms such as transgenic plants (like A. thaliana and N. benthamiana).

[423] For certain embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell or lymphoid cell ( e.g ., YO, NSO, Sp20 cell). In one embodiment, a method of making an anti- PD-Ll antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[424] Accordingly, the present invention also provides methods for producing an anti-PD- Ll antibody of the present invention according to above recited methods and using a polynucleotide encoding part or all of a polypeptide of the invention or a protein component of an anti-PD-Ll antibody of the invention, an expression vector comprising at least one polynucleotide of the invention capable of encoding part or all of an anti-PD-Ll antibody of the invention when introduced into a host cell, and/or a host cell comprising a polynucleotide or expression vector of the invention.

[425] When a protein is expressed using recombinant techniques in a host cell or cell-free system, it is advantageous to separate (or purify) the desired protein away from other components, such as host cell factors, in order to obtain preparations that are of high purity or are substantially homogeneous. Purification can be accomplished by methods well known in the art, such as centrifugation techniques, extraction techniques, chromatographic and fractionation techniques ( e.g . size separation by gel filtration, charge separation by ion- exchange column, hydrophobic interaction chromatography, reverse phase chromatography, chromatography on silica or cation-exchange resins such as diethylaminoethyl (DEAE), chromatofocusing, and protein A sepharose chromatography to remove contaminants), and precipitation techniques (e.g. ethanol precipitation or ammonium sulfate precipitation). Any number of biochemical purification techniques may be used to increase the purity of an anti- PD-L1 antibody of the present invention. In certain embodiments, the anti-PD-Ll antibodies of the invention may optionally be purified in homo-multimeric forms (e.g. a molecular complex comprising two or more PD-L1 binding antibody-derivatives of the present invention).

[426] Antibodies may be produced using recombinant methods and compositions (see e.g. ET.S. 4,816,567). In certain embodiments, isolated nucleic acid encoding an antibody or antibody fragment described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g. a light and/or heavy chain of an antibody). A method of making an antibody of the present invention comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of an antibody, nucleic acid encoding an antibody, e.g. as described herein, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using routine methods known to the skilled worker.

[427] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein and/or known to the skilled worker. For example, antibodies may be produced in bacteria, in particular when glycosylation and/or Fc effector function are not required (see e.g. ET.S. 5,648,237, U.S. 5,789, 199, and ET.S.

5,840,523). After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[428] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of an antibody with a partially or fully human glycosylation pattern (see e.g. Gemgross T, Nat Biotech 22: 1409-14 (2004); Li H et ak, Nat Biotech 24: 210-15 (2006)). [429] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Plant cells may be utilized as hosts (see e.g. U.S. 5,959,177, U.S. 6,040,498, U.S. 6,420,548, U.S. 7,125,978, and U.S. 6,417,429). Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of S. frugiperda cells. Vertebrate cells may be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line 293 cells; baby hamster kidney cells (BHK); mouse sertoli cells (e.g. TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells (Graham F et ak, J Gen Virol 36: 59-74 (1977); Mather J et ak, Biol Reprod 23 : 243-52 (1980); Mather J et ak, Ann NY Acad Sci 383 : 44-68 (1992)). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO, and myeloma cell lines such as Y0, NS0 and Sp2/0 cells (see e.g. Urlaub G et ak, Proc Natl Acad Sci U.S.A. 77: 4216-20 (1980)). For a review of certain mammalian host cell lines suitable for antibody production, see Yazaki P, Wu A, Methods in Molecular Biology , vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, New Jersey (2003)).

[430] Methods of immunoconjugation include but are not limited to reactive thiols, aldehyde-tagged, sortase-mediated conjugation, MTGase-mediated conjugation,

transglutaminase conjugation, bis-linkages, and using a spacer or multifunctional linker (see e.g WO 2009/052249, WO 2012/097333, WO 2013/173391, WO 2014/140317, WO

2014/159835, WO 2015/155753, WO 2015/191883, WO 2016/102632, WO 2018/185526).

[431] An antibody -toxin conjugate or immunoconjugate of the present invention may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including reaction of a nucleophilic group of an antibody with a bivalent linker reagent to form a covalent bond between the linker and the antibody, followed by reaction with a toxin component; and reaction of a nucleophilic group of a toxin component with a bivalent linker reagent, to form a covalent bond between the linker and the toxin, followed by reaction with a nucleophilic group of an antibody.

[432] Nucleophilic groups on antibodies include but are not limited to: (i) amino-terminal amine groups, (ii) side chain amine groups, e.g. of a lysine residue, (iii) side chain thiol groups, e.g. of a cysteine residue, and (iv) sugar hydroxyl or amino groups of a carbohydrate moiety when the antibody is glycosylated. Amine, hydroxyl, and thiol groups are

nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, e.g. cysteine disulfide bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), such that the antibody is fully or partially reduced (see e.g. WO 2013/173391, WO 2013/173392, WO 2013/173393, WO 2013/190272, WO 2014/064424, WO 2014/114207, WO 2015/155753, WO 2018/185526). Additional nucleophilic groups can be introduced into antibodies through modification of lysine residues, e.g. , by reacting lysine residues with 2-iminothiolane (Traut’s reagent), resulting in conversion of an amine into a thiol. Reactive thiol groups may also be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.g. by preparing variant antibodies comprising one or more non-native cysteine amino acid residues).

[433] Antibody-toxin conjugates or immunoconjugates of the present invention may also be produced by reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or toxin component. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In one embodiment, an antibody is modified to introduce electrophilic moieties that are capable of reacting with nucleophilic substituents on the linker reagent or toxin component. In another embodiment, the sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or toxin components. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the toxin component. In another embodiment, antibodies containing amino-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (see e.g. U.S. 5,362,852). Such an aldehyde can be reacted with a toxin component or linker nucleophile.

[434] Carbohydrate(s) on the Fc region of an antibody is a natural site for attaching compounds. Generally, the carbohydrate is modified by periodate oxidation to generate reactive aldehydes, which can then be used to attach reactive amine containing compounds by Schiff base formation. As the aldehydes can react with amine groups, reactions are carried out at low pH so that lysine residues in the antibody or antigen binding domain are protonated and unreactive. Hydrazide groups are most suitable for attachment to the aldehydes generated since they are reactive at low pH to form a hydrazone linkage. The linkage can then be further stabilized by reduction with sodium cyanoborohydride to form a hydrazine linkage.

[435] Exemplary nucleophilic groups on a toxin component include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[436] The loading of an ADC (e.g. drug/antibody ratio) may be controlled in different ways, such as by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.

[437] Conjugate loading may be expressed as an average number of conjugate moieties per antibody (x). Conjugate loading may range from 1 to 20 conjugate moieties per antibody.

The average number of conjugate moieties per antibody in preparations of antibody-drug conjugates or immunoconjugates from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA, and high-performance liquid chromatography (HPLC). The quantitative distribution of immunoconjugate in terms of x may also be determined, such as, e.g. , by separation, purification, and characterization of homogeneous immunoconjugate where p is a certain value from immunoconjugate with other conjugate loadings may be achieved by means such as reverse phase HPLC or

electrophoresis.

[438] It is to be understood that where more than one nucleophilic group reacts with a drug- linker intermediate or linker reagent, then the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody.

Individual ADC molecular species may be identified in the mixture by using mass spectroscopic methods and may be separated by using chromatographic methods, e.g.

hydrophobic interaction chromatography ( see e.g. Chari R, Acc Chem Res 41 : 98-107 (2008); McDonagh C et al., Protein EngDes Sel 19: 299-307 (2006); Hamblett K et al., Clin Cancer Res 10: 7063-70 (2004)). In certain embodiments, a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis,

chromatography, and/or other techniques known to the skilled worker.

[439] Any form of antigen, cells (e.g. a PD-L1 -expressing cell), or preparations containing the antigen, can be used to generate an antibody that is specific for the target protein. The term“antigen” may comprise any antigenic fragment of the selected target protein including a single epitope, multiple epitopes, single or multiple domains or the entire extracellular domain (ECD). The antigen may be an isolated full-length protein, a cell surface protein, or a soluble protein. The target antigen may include a fusion polypeptide, such as fusions of the target protein or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The antigen may be produced in a genetically modified cell. The DNA encoding the antigen may be genomic or non-genomic (e.g. an extrachromosomal DNA molecule) and may encode at least a portion of the ECD, sufficient to elicit an antigenic response. Any vector may be employed to transform the cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors, such as cationic lipids.

[440] In certain embodiments, the anti-PD-Ll antibody fragments of the present invention are isolated by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter G et al., Ann Rev Immunol 12: 433-55 (1994). Such phage libraries are screened for binding to the desired PD-L1 molecule, PD-L1 antigen, and/or PD-L1 epitope. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption/elution.

[441] In certain embodiments, the anti-PD-Ll antibody, or antibody fragments thereof, are isolated from antibody phage libraries generated using the techniques described in Clackson T et al. , Nature 352: 624-8 (1991); Marks J et al. , J Mol Biol, 222: 581-97 (1991);

McCafferty J et al., Nature 348: 552-4 (1990). [442] Screening of the libraries can be accomplished by various techniques known in the art. For example, PD-L1 can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries.

[443] In certain embodiments, the anti-PD-Ll antibody of the present invention is a humanized antibody. Humanization of an antibody may be carried out using recombinant techniques known to the person skilled in the art (see e.g. Antibody Engineering Methods in Molecular Biology, vol. 248 (Lo B, ed., Totowa, NJ (2004)). Various art-recognized techniques can be used to determine which human sequences to use as recipient antibodies to provide humanized constructs in accordance with the present invention. A suitable human recipient framework for both the light and heavy chain variable domain may be identified by, for example, sequence or structural homology. Alternatively, fixed human recipient frameworks may be used, e.g. , based on knowledge of structure, biophysical and biochemical properties. The recipient frameworks can be germline derived or derived from a mature antibody sequence. Non-limiting examples of compatible human germline sequences and methods of determining their suitability as recipient sequences are described in Tomlinson I et al. , J Mol Biol 221: 776-98 (1992); Chothia C et al. , J Mol Biol 227: 799-817 (1992); Cook G, Tomlinson I, Immunol Today 16: 237-42 (1995); Tomlinson I et al., EMBO J 14: 4628-38 (1995); Handbook of Therapeutic Antibodies, 2nd Ed. (Wiley -Blackwell GmbH, Dubel S, Reichert J, eds., (2014)). The V-BASE directory (VBASE2: Retter I et al., Nucleic Acid Res 33 : 671-4, (2005)), which provides a comprehensive directory of human immunoglobulin variable region sequences, may also be used to identify compatible recipient sequences. In general, human framework recipient sequences are selected based on homology with the donor framework sequences along with an analysis of the CDR canonical structures of the donor and recipient antibodies. The derived sequences of the heavy and light chain variable regions of the derived antibody may then be synthesized using art recognized techniques. CDR regions from the donor sequence or antibody can be transferred by CDR grafting. The CDR grafted humanized antibody can be further optimized, such as, e.g, for affinity, functionality and biophysical properties by identification of critical framework positions where re-introduction (back-mutation) of the amino acid residue from the donor sequence or antibody has beneficial impact on the properties of the humanized antibody. In addition to donor sequence or antibody derived back-mutations, the humanized antibody can be engineered, e.g, by introduction of germline residues in the CDR or framework regions, elimination of immunogenic epitopes, site-directed mutagenesis, and/or affinity maturation. [444] The anti-PD-Ll antibody of the present invention may be an isolated antibody, meaning that the antibody has been recovered its natural environment and/or separated from contaminants that would interfere with uses of the antibody, such as, e.g. diagnostic or therapeutic uses. Isolated antibodies include antibodies produced in situ within recombinant cells. These isolated preparations may be purified using various art-recognized techniques known to the skilled worker, such as, for example, ion exchange and size exclusion chromatography, dialysis, diafiltration, and affinity chromatography, particularly Protein A or Protein G affinity chromatography.

IV. Pharmaceutical and Diagnostic Compositions Comprising Anti-PD-Ll Antibodies

[445] The present invention provides anti-PD-Ll antibodies for use, alone or in combination with one or more additional therapeutic agents, in a pharmaceutical composition, for treatment or prophylaxis of conditions, diseases, disorders, or symptoms described in further detail below (e.g. cancers, malignant tumors, non-malignant tumors, growth abnormalities, immune disorders, and microbial infections). The present invention further provides pharmaceutical compositions comprising an anti-PD-Ll antibody of the present invention (such as the antigen-binding fragments and derivatives thereof, including any ADC or immunoconjugate thereof), or a pharmaceutically acceptable salt or solvate thereof, according to the invention, together with at least one pharmaceutically acceptable carrier, excipient, or vehicle. The pharmaceutical compositions of the invention are useful in methods of treating, ameliorating, or preventing a disease, condition, disorder, or symptom described in further detail below. Each such disease, condition, disorder, or symptom is envisioned to be a separate embodiment with respect to uses of a pharmaceutical composition according to the invention. The invention further provides pharmaceutical compositions for use in at least one method of treatment according to the invention, as described in more detail below. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject’s response to administration of a composition and adjusting the dosage accordingly (see e.g. Remington: The Science and Practice of Pharmacy (Gennaro A, ed., Mack Publishing Co., Easton, PA, U.S., 19th ed., 1995)).

[446] An effective amount of an agent, e.g. , a pharmaceutical composition or formulation of an anti-PD-Ll antibody of the present invention, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The effective amount of the drug for treating cancer may reduce the number of cancer cells; reduce the tumor size; inhibit (e.g. slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (e.g. slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. The effective amount may extend progression free survival (e.g. as measured by Response Evaluation Criteria for Solid Tumors, RECIST, or CA-125 changes), result in an objective response (including a partial response, PR, or complete response, CR), increase overall survival time, and/or improve one or more symptoms of cancer (e.g. as assessed by FOSI).

[447] Pharmaceutical compositions (e.g. pharmaceutical formulations) of an anti-PD-Ll antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, diluents, and/or excipients (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers, diluents, and excipients are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: sterile water, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;

benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as ethylenediamine tetraacetic acid (EDTA); sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases. [448] Exemplary lyophilized antibody formulations are described in US 6,267,958.

Aqueous antibody formulations include those described in US 6,171,586 and WO

2006/044908, such as, e.g ., a histidine-acetate buffer.

[449] The formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in

combination in amounts that are effective for the purpose intended.

[450] In certain embodiments, the pharmaceutical composition of the present invention may comprise two or more anti-PD-Ll antibodies of the present invention.

[451] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example,

hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)

microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in

macroemulsions. Such techniques are known to skilled worker, see e.g. in Remington's Pharmaceutical Sciences 16^th edition (Osol, A. ed. (1980)).

[452] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g, by filtration through sterile filtration membranes.

[453] Diagnostic compositions of the present invention comprise an anti-PD-Ll antibody of the present invention comprising one or more detection-promoting agents. When producing or manufacturing a diagnostic composition of the present invention, an anti-PD-Ll antibody of the present invention may be directly or indirectly linked to one or more detection- promoting agents. There are numerous standard techniques known to the skilled worker for incorporating, affixing, and/or conjugating various detection-promoting agents to proteins or proteinaceous components of molecules, especially to immunoglobulins and

immunoglobulin-derived domains.

[454] There are numerous detection-promoting agents known to the skilled worker, such as isotopes, dyes, colorimetric agents, contrast enhancing agents, fluorescent agents,

bioluminescent agents, and magnetic agents, which can be operably linked to the anti-PD-Ll antibodies of the present invention for information gathering methods, such as for diagnostic and/or prognostic applications to diseases, disorders, or conditions of an organism (see e.g. Cai W et al., J Nucl Med 48: 304-10 (2007); Nayak T, Brechbiel M, Bioconjug Chem 20: 825-41 (2009); Paudyal P et al., Oncol Rep 22: 115-9 (2009); Qiao J et al., PLoS ONE 6: el8l03 (2011); Sano K et al., Breast Cancer Res 14: R61 (2012)). These agents may be associated with, linked to, and/or incorporated within an anti-PD-Ll antibody of the present invention at any suitable position. For example, the linkage or incorporation of the detection- promoting agent may be via an amino acid residue(s) of a molecule of the present invention or via some type of linkage known in the art, including via linkers and/or chelators. The incorporation of the agent is in such a way to enable the detection of the presence of the diagnostic composition in a screen, assay, diagnostic procedure, and/or imaging technique.

[455] Similarly, there are numerous imaging approaches known to the skilled worker, such as non-invasive in vivo imaging techniques commonly used in the medical arena, for example: computed tomography imaging (CT scanning), optical imaging (including direct, fluorescent, and bioluminescent imaging), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound, and x-ray computed tomography imaging.

V. Production or Manufacture of Pharmaceutical and/or Diagnostic Compositions

Comprising an Anti-PD-Ll Antibody of the Present Invention

[456] Pharmaceutically acceptable salts or solvates of any of anti-PD-Ll antibodies, or an antigen binding fragment or derivative thereof, of the present invention are within the scope of the present invention.

[457] The term“solvate” in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu , a proteinaceous compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent. The solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate.

[458] Polypeptides and proteins of the present invention, or salts thereof, may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of a molecule of the present invention, or a salt thereof, in a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic molecule use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences (Mack Publishing Co. (A. Gennaro, ed., 1985). As used herein,“pharmaceutically acceptable carrier” includes any and all physiologically acceptable (i.e. compatible) solvents, dispersion media, coatings, antimicrobial agents, isotonic, and absorption delaying agents. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. Exemplary pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, or PEG), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyloleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration ( e.g . by injection or infusion). Depending on selected route of administration, the protein or other pharmaceutical component may be coated in a material intended to protect the compound from the action of low pH and other natural inactivating conditions to which the active protein may encounter when administered to a patient by a particular route of administration.

[459] The formulations of the pharmaceutical compositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In such form, the composition is divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen. Compositions may be formulated for any suitable route and means of administration. Subcutaneous or transdermal modes of administration may be particularly suitable for therapeutic proteins described herein.

[460] The pharmaceutical compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Preventing the presence of microorganisms may be ensured both by sterilization procedures and the inclusion of various antibacterial and antifungal agents, such as, e.g ., paraben, chlorobutanol, and/or phenol sorbic acid. The inclusion in the compositions, of isotonic agents, such as sugars and/or sodium chloride, may be desirable. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin.

[461] A pharmaceutical composition of the present invention also optionally includes a pharmaceutically acceptable antioxidant. Exemplary pharmaceutically acceptable antioxidants are water soluble antioxidants such as, e.g. , ascorbic acid, cysteine

hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite; oil-soluble

antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, and alpha-tocopherol; and metal chelating agents, such as citric acid, EDTA, sorbitol, tartaric acid, and phosphoric acid.

[462] In another aspect, the present invention provides pharmaceutical compositions comprising one or a combination of different anti-PD-Ll antibodies of the present invention, or an ester, salt or amide of any of the foregoing, and at least one pharmaceutically acceptable carrier.

[463] Therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g, glycerol, propylene glycol, and liquid PEG), or any suitable mixtures. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art. In certain embodiments, isotonic agents, e.g, sugars and polyalcohols such as mannitol, sorbitol, or sodium chloride, may be desirable in the composition. Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.

[464] Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, PEGs, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as, e.g, sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, and/or acetate. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[465] Sterile injectable solutions may be prepared by incorporating anti-PD-Ll antibodies of the present invention in the required amount in an appropriate solvent with one or a combination of ingredients described herein, as required, followed by sterilization microfiltration. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and other ingredients, such as those described herein. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.

[466] When a therapeutically effective amount of an anti-PD-Ll antibody of the present invention is designed to be administered by, e.g. intravenous, cutaneous or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable protein solutions, taking into consideration appropriate pH, isotonicity, stability, and other parameters, are within the skill of the skilled worker. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to binding agents, an isotonic vehicle such as sodium chloride injection, Ringer’s injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. A pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.

[467] As described elsewhere herein, an anti-PD-Ll antibody of the present invention may be prepared with carriers that will protect the active therapeutic agent against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art ( see e.g. Sustained and Controlled Release Drug Delivery Systems (Robinson J, ed., Marcel Dekker, Inc., NY, U.S., 1978)).

[468] In certain embodiments, the composition of the present invention (e.g. a

pharmaceutical and/or diagnostic composition) may be formulated to ensure a desired in vivo distribution of an anti-PD-Ll antibody of the present invention. For example, the blood-brain barrier excludes many large and/or hydrophilic compounds. To target a therapeutic molecule, diagnostic molecule, or composition of the present invention to a particular in vivo location, the aforementioned can be formulated, for example, in liposomes which may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery. Exemplary targeting moieties include folate or biotin, mannosides, immunoglobulin domains, surfactant protein A receptor, and pl20 catenin.

[469] Pharmaceutical compositions include parenteral formulations designed to be used as implants or particulate systems. Examples of implants are depot formulations composed of polymeric or hydrophobic components such as emulsions, ion exchange resins, and soluble salt solutions. Examples of particulate systems are microspheres, microparticles,

nanocapsules, nanospheres, and nanoparticles ( see e.g. Honda M et al., Int J Nanomedicine 8: 495-503 (2013); Sharma A et al., Biomed Res Int 2013 : 960821 (2013); Ramishetti S, Huang L, Ther Deliv 3 : 1429-45 (2012)). Controlled release formulations may be prepared using polymers sensitive to ions, such as, e.g. liposomes, polaxamer 407, and hydroxyapatite.

VI. Polynucleotides, Expression Vectors, and Host Cells of the Present Invention

[470] Beyond the anti-PD-Ll antibodies of the present invention, the polynucleotides that encode the anti-PD-Ll antibodies of the invention, or antigen binding portions thereof, are also encompassed within the scope of the present invention. The term“polynucleotide” is equivalent to the term“nucleic acid”, each of which includes one or more of: polymers of deoxyribonucleic acids (DNAs), polymers of ribonucleic acids (RNAs), analogs of these DNAs or RNAs generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The polynucleotide of the present invention may be single-, double-, or triple-stranded. Such polynucleotides are specifically disclosed to include all polynucleotides capable of encoding an exemplary protein, for example, taking into account the wobble known to be tolerated in the third position of RNA codons, yet encoding for the same amino acid as a different RNA codon (see Stothard P, Biotechniques 28: 1102-4 (2000)).

[471] Anti-PD-Ll antibodies, or antigen binding derivatives of the aforementioned, within the scope of the present invention may be variants or derivatives of the polypeptides and molecules described herein that are produced by modifying the polynucleotide encoding a polypeptide and/or proteinaceous component of a molecule of the invention by altering one or more amino acids or deleting or inserting one or more amino acids that may render it more suitable to achieve desired properties, such as more optimal expression by a host cell. [472] In one aspect, the present invention provides polynucleotides which encode an anti- PD-L1 antibody of the present invention, or antigen binding fragment or derivative thereof. The polynucleotides may include, e.g ., a nucleic acid sequence encoding a polypeptide at least 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, identical to a polypeptide comprising one of the amino acid sequences of an anti-PD-Ll antibody of the present invention. The invention also includes polynucleotides comprising nucleotide sequences that hybridize under stringent conditions to a polynucleotide which encodes an anti-PD-Ll antibody of the invention, or a fragment or derivative thereof, or the antisense or complement of any such sequence.

[473] Derivatives or analogs of the molecules of the present invention (e.g, a nucleic acid encoding an anti-PD-Ll antibody) include, inter alia , polynucleotide (or polypeptide) molecules having regions that are substantially homologous to the polynucleotides (or anti- PD-Ll antibodies of the present invention), e.g. by at least about 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a polynucleotide (or polypeptide) sequence of the same size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art. An exemplary program is the GAP program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI, U.S.) using the default settings, which uses the algorithm of Smith T, Waterman M, Adv ApplMath 2: 482-9 (1981). Also included are polynucleotides capable of hybridizing to the complement of a sequence encoding an anti-PD-Ll antibody of the invention under stringent conditions (see e.g.

Ausubel F et ah, Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, U.S., 1993)), and below. Stringent conditions are known to those skilled in the art and may be found, e.g., in Current Protocols in Molecular Biology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6 (1989)).

[474] Nucleic acids (DNA fragments) comprising sequences encoding VH and/or VL segments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein or protein fragment, such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, means that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame. [475] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3 in the case of IgGl). The sequences of human heavy chain constant region genes are known in the art (KabatE (1991), supra ) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[476] Isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g. KabatE (1991), supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[477] The present invention further provides expression vectors that comprise the polynucleotides within the scope of the present invention. The polynucleotides capable of encoding the anti-PD-Ll antibodies of the present invention, or an antigen binding fragment or derivative thereof, may be inserted into known vectors, including bacterial plasmids, viral vectors and phage vectors, using material and methods well known in the art to produce expression vectors. Such expression vectors will include the polynucleotides necessary to support production of contemplated an anti-PD-Ll antibody of the invention within any host cell of choice or cell-free expression systems. The specific polynucleotides comprising expression vectors for use with specific types of host cells or cell-free expression systems are well known to one of ordinary skill in the art, can be determined using routine

experimentation, and/or may be purchased.

[478] An expression vector contains one or more expression units. Thus, in the context of the present invention, an expression vector encoding a single-chain anti-PD-Ll antibody includes at least an expression unit for the single polypeptide chain, whereas an anti-PD-Ll antibodies comprising, e.g. two or more polypeptide chains (e.g. one chain comprising a VL domain and a second chain comprising a VH domain linked to a toxin effector polypeptide) includes at least two expression units, one for each of the two polypeptide chains of the protein. For expression of multi-chain anti-PD-Ll antibodies of the invention, an expression unit for each polypeptide chain may also be separately contained on different expression vectors ( e.g . expression may be achieved with a single host cell into which expression vectors for each polypeptide chain has been introduced).

[479] Expression vectors capable of directing transient or stable expression of polypeptides and proteins are well known in the art. The expression vectors generally include, but are not limited to, one or more of the following: a heterologous signal sequence or peptide, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a

transcription termination sequence, each of which is well known in the art. Optional regulatory control sequences, integration sequences, and useful markers that can be employed are known in the art.

[480] In certain embodiments, a host cell of the present invention comprises (e.g., has been transfected or transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of an anti-PD-Ll antibody of the invention and an amino acid sequence comprising the VH of an anti-PD-Ll antibody of the invention, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of an anti-PD-Ll antibody of the invention and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of an anti-PD-Ll antibody of the invention. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g. Y0, NS0, or Sp20 cells).

VII. Molecules of the Present Invention Immobilized on a Solid Substrate

[481] Certain embodiments of the present invention include a molecule of the present invention (e.g. an anti-PD-Ll antibody, fusion protein, or polynucleotide of the present invention) immobilized on a solid substrate. Solid substrates contemplated herein include, but are not limited to, microbeads, nanoparticles, polymers, matrix materials, microarrays, microtiter plates, or any solid surface known in the art (see e.g. US 7,771,955). In accordance with these embodiments, a molecule of the present invention may be covalently or non-covalently linked to a solid substrate, such as, e.g, a bead, particle, or plate, using techniques known to the skilled worker (see e.g. Jung Y et ah, Analyst 133 : 697-701 (2008)). Immobilized molecules of the invention may be used for screening applications using techniques known in the art (see e.g. Bradbury A et ah, Nat Biotechnol 29: 245-54 (2011); Sutton C, Br J Pharmacol 166: 457-75 (2012); Diamante L et ah, Protein Eng Des Sel 26: 713-24 (2013); Houlihan G et ah, J Immunol Methods 405: 47-56 (2014)).

[482] Non-limiting examples of solid substrates to which a molecule of the invention may be immobilized on include: microbeads, nanoparticles, polymers, nanopolymers, nanotubes, magnetic beads, paramagnetic beads, superparamagnetic beads, streptavidin coated beads, reverse-phase magnetic beads, carboxy terminated beads, hydrazine terminated beads, silica (sodium silica) beads and iminodiacetic acid (IDA) -modified beads, aldehyde-modified beads, epoxy-activated beads, diaminodipropylamine (DADPA) -modified beads (beads with primary amine surface group), biodegradable polymeric beads, polystyrene substrates, amino- polystyrene particles, carboxyl-polystyrene particles, epoxy-polystyrene particles, dimethylamino-polystyrene particles, hydroxy-polystyrene particles, colored particles, flow cytometry particles, sulfonate-polystyrene particles, nitrocellulose surfaces, reinforced nitrocellulose membranes, nylon membranes, glass surfaces, activated glass surfaces, activated quartz surfaces, polyvinylidene difluoride (PVDF) membranes, polyacrylamide- based substrates, poly-vinyl chloride substrates, poly-methyl methacrylate substrates, poly(dimethyl siloxane) substrates, and photopolymers which contain photoreactive species (such as nitrenes, carbenes, and ketyl radicals) capable of forming covalent linkages. Other examples of solid substrates to which a molecule of the invention may be immobilized on are commonly used in molecular display systems, such as, e.g ., cellular surfaces, phages, and virus particles.

VIII. Delivery Devices and Kits of the Present Invention

[483] In a further aspect of the invention, is a delivery device or kit involving a composition of matter of the present invention.

[484] In certain embodiments, the invention relates to a device comprising one or more compositions of matter of the present invention, such as a pharmaceutical composition or diagnostic composition, for delivery to a subject in need thereof. Thus, a delivery device comprising one or more compositions of the present invention can be used to administer to a patient a composition of matter of the present invention by various delivery methods, including: intravenous, subcutaneous, intramuscular or intraperitoneal injection; oral administration; transdermal administration; pulmonary or transmucosal administration;

administration by implant, osmotic pump, cartridge or micro pump; or by other means recognized by a person of skill in the art.

[485] In certain other embodiments, the invention relates to kits comprising at least one composition of matter of the invention, and optionally, packaging and instructions for use. Kits may be useful for drug administration and/or diagnostic information gathering. A kit of the invention may optionally comprise at least one additional reagent (e.g, a standard or control, a blocking agent, a marker, a secondary antibody, or a detection agent/substrate for a colorimetric, fluorometric, or radiometric assay). Kits typically include a label or package insert indicating the intended use of the contents of the kit. The kit may further comprise reagents and other tools for detecting a cell type ( e.g . a PD-L1 -expressing tumor cell or immune cell) in a sample or in a subject, or for diagnosing whether a patient belongs to a group that responds to a therapeutic strategy which makes use of a compound, composition, or related method of the present invention, e.g., such as a method described herein.

[486] In various embodiments of the kits described herein, the anti-PD-Ll antibody is one or more of any of the anti-PD-Ll antibodies, or an antigen binding derivative thereof, disclosed herein.

[487] The PD-L1 detection and/or quantification methods of the present invention can be provided in the form of a kit. In certain embodiments, such a kit comprises an anti-PD-Ll antibody, antigen-binding fragment or derivative thereof of the present invention or a composition comprising the aforementioned, such as an antibody or composition described herein. In certain embodiments, such a kit is a packaged combination including the basic elements of: an anti-PD-Ll antibody of the present invention which binds to a PD-L1 molecule of interest and comprises a detectable moiety, and instructions on how to perform an assay method using the aforementioned reagents. In certain embodiments, such a kit is a packaged combination including the basic elements of: an anti-PD-Ll antibody of the present invention which binds to a PD-L1 molecule of interest, a detectable (labeled or unlabeled) antibody that binds to PD-L1 or to the anti-PD-Ll antibody of the present invention, and instructions on how to perform an assay method using the aforementioned reagents. In certain embodiments, such a kit is a packaged combination including the basic elements of: a capture reagent comprising an anti-PD-Ll antibody of the present invention which binds to a PD-L1 molecule of interest, a detectable (labeled or unlabeled) antibody that binds to PD-L1 or to the anti-PD-Ll antibody of the present invention, and instructions on how to perform an assay method using the aforementioned reagents.

[488] The kit may further comprise a solid support for the capture reagents, which may be provided as a separate element or on which the capture reagents are already immobilized.

[489] Hence, the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit. In certain embodiments, the capture reagents are coated on or attached to a solid material (e.g. to beads, a microtiter plate, or a comb). The detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies, such as, e.g. antibodies raised in a different species. Where the label is an enzyme, the kit will ordinarily include substrates and cofactors required by the enzyme; where the label is a fluorophore, a dye precursor that provides the detectable chromophore; and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to HRP or b-galactosidase for use in a fluorescent b- galactosidase assay.

[490] The kit also typically contains additives such as stabilizers, washing and incubation buffers for performing the assay method(s). The components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay(s). Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.

[491] In various embodiments, a kit comprising an anti-PD-Ll antibody as described herein is for use in a method as described herein ( e.g ., in a method of detecting PD-L1 in a biological sample from a subject). In certain embodiments, the kit further comprises an anti- PD-Ll antibody coated or attached to a surface.

A. Enzyme-Linked Immuno-Sorbent Assay (ELISA)

[492] In certain embodiments, the anti-PD-Ll antibodies are used in an ELISA assay. The assay described herein is an ELISA that utilizes anti-PD-Ll antibody as a capture reagent for a PD-L1 molecule of interest. In the first step of the assay the biological sample suspected of containing or containing the PD-L1 of interest is contacted and incubated with the capture (or coat) antibodies so that the capture antibodies capture or bind to the PD-L1 molecule of interest so that it can be detected in a detection step. The detection step involves use of a detectable antibody, which, when contacted with any of the bound PD-L1 of interest, binds to the PD-L1 of interest, if present. A detection means may be used to detect the label on the detecting antibody and hence the presence or amount of PD-L1 of interest present.

Alternatively, a secondary antibody may be used to detect the PD-L1 binding antibody bound to the PD-L1, if any.

[493] In certain embodiments, the assay utilizes the following steps. In the first step of the assay herein, the biological sample suspected of containing or containing the PD-L1 molecule of interest as defined herein is contacted and incubated with the immobilized capture (or coat) reagents, which are anti-PD-Ll antibodies which specifically bind the PD-L1 molecule of interest. In certain embodiments, the anti-PD-Ll antibody is a monoclonal antibody, and may be from any species, or hybridizations thereof. In certain embodiments, these anti-PD- Ll antibodies are murine or rodent antibodies, in further embodiments mouse or rat.

[494] Immobilization conventionally is accomplished by insolubilizing the capture reagents either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (US 3,720,760) or non-covalent or covalent coupling (for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g ., nitric acid and a reducing agent (see e.g. US 3,645,852; Rotmans J, Delwel H et al.; J Immunol Methods 57: 87-98 (1983)), or afterward, e.g., by immunoprecipitation. In certain

embodiments, the capture antibody is conjugated to biotin and is bound to a streptavidin coated surface. In other embodiments, the capture antibody is conjugated to a protein tag, such as a His-tag or GST, and is bound to a suitable surface, e.g. , a nickel or copper coated surface, or a glutathione coated surface.

[495] The solid phase is coated with the capture reagents as defined herein, which may be linked by a non-covalent or covalent interaction or physical linkage as desired. Techniques for attachment include those described in US 4,376,110 and the references cited therein. If covalent, the plate or other solid phase is incubated with a cross-linking agent together with the capture reagent under conditions well known to the skilled worker, such as for one hour at room temperature.

[496] The solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, e.g. , surfaces, particles, porous matrices. Examples of commonly used supports include small sheets, SEPHADEX® gels, polyvinyl chloride, plastic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, or polystyrene, including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water-insoluble matrices such as cyanogen-bromide-activated carbohydrates and the reactive substrates described in US 3,691,016; US 3,969,287; US 4,195, 128; US 4,229,537; US 4,247,642; and US 4,330,440 are suitably employed for capture-reagent immobilization. In certain embodiments, the immobilized capture reagents are coated on a microtiter plate. In certain embodiments, the solid phase used is a multi- well microtiter plate that can be used to analyze several samples at one time.

[497] Commonly used cross-linking agents for attaching the capture reagents to the solid phase substrate include, e.g. , l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3’-dithiobis(succinimidylpropionate), and bifunctional maleimides, such as bis-N-maleimido-l, 8-octane. Derivatizing agents, e.g. methyl-3 -((p-azidophenyl)-dithio)propioimidate, yield photoactivatable intermediates capable of forming cross-links in the presence of light.

[498] If 96-well plates are utilized, they may be coated with the mixture of capture reagents typically diluted in a buffer such as 0.05 M sodium carbonate by incubation for at least about ten hours. In certain embodiments, incubation is at least overnight, at temperatures of about 4 to 20 °C or about 4 to 8 °C, and at a pH of about 8-12, about 9-10, or about 9.6. For shorter coating times (1-2 hours), the skilled worker can use 96-well plates with nitrocellulose filter bottoms or coat at 37°C with routine methods. The plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.

[499] The coated plates are then typically treated with a blocking agent that binds nonspecifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate. Examples of appropriate blocking agents for this purpose include, e.g. , gelatin, bovine serum albumin, egg albumin, casein, and non fat milk. The blocking treatment typically takes place under conditions of ambient temperatures for about 1 to 4 hours, or about 1.5 to 3 hours.

[500] After coating and blocking, the biological sample to be analyzed, appropriately diluted, is added to the immobilized phase. In certain embodiments the dilution rate is about 5-15%, or about 10%, by volume. Buffers that may be used for dilution for this purpose include (a) PBS containing 0.5% BSA, 0.05% TWEEN®-20 detergent (P20), 0.05%

PROCLIN™ 300 antibiotic, 5 mM EDTA, 0.25% 3-((3-cholamidopropyl)

dimethylammonio)-l-propanesulphonate (CHAPS) surfactant, 0.2% beta-gamma globulin, and 0.35M NaCl; (b) PBS containing 0.5% bovine serum albumin (BSA), 0.05% P20, and 0.05% PROCLIN™ 300, pH 7; (c) PBS containing 0.5% BSA, 0.05% P20, 0.05%

PROCLIN™ 300, 5 mM EDTA, and 0.35 M NaCl, pH 6.35; (d) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mM EDTA, 0.2% beta-gamma globulin, and 0.35 M NaCl; and (e) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mM EDTA, 0.25% CHAPS, and 0.35 M NaCl.

[501] The amount of capture reagents employed is sufficiently large to give a signal of a strength comparable with the standards, but not in molar excess compared to the maximum expected level of antibody of interest in the sample. In certain embodiments, the amount of biological sample added is such that the immobilized capture reagents are in molar excess of the maximum molar concentration of free antibody of interest anticipated in the biological sample after appropriate dilution of the sample. This anticipated level depends mainly on any known correlation between the concentration levels of the PD-L1 of interest in the particular biological sample being analyzed with the clinical condition of the patient.

[502] The concentration of the capture reagents may be determined by the concentration range of interest of the PD-L1 of interest, taking any necessary dilution of the biological sample into account. The final concentration of the capture reagents may also be determined empirically to maximize the sensitivity of the assay over the range of interest. Generally, a molar excess is suitably less than about ten-fold of the maximum expected molar

concentration of PD-L1 of interest in the biological sample after any appropriate dilution of the sample.

[503] The conditions for incubation of sample and immobilized capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation, and to ensure that any antibody of interest present in the sample binds to the immobilized capture reagent. The incubation is accomplished at fairly constant temperatures, ranging from about 0 °C to about 40 °C, for example at or about room temperature. The time for incubation is generally no greater than about 10 hours. In various embodiments, the incubation time is from about 0.5 to 3 hours, or from about 1.5 to 3 hours at or about room temperature to maximize binding of the antibody of interest to the capture reagents. The duration of incubation may be longer if a protease inhibitor is added to prevent proteases in the biological fluid from degrading the antibody of interest.

[504] At this stage, the pH of the incubation mixture will ordinarily be in the range of about 4-9.5, or in the range of about 6-9, or about 7 to 8. The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagents to the PD-L1 of interest being captured. Various buffers may be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, TRIS-HC1 or TRIS- phosphate, acetate, or barbital. The particular buffer employed is not critical to the invention, but in individual assays one buffer may be desirable over another.

[505] In an optional second step of the assay method of the present invention, the biological sample is separated (for example by washing) from the immobilized capture reagents to remove uncaptured PD-L1 of interest. The solution used for washing is generally a buffer (“washing buffer”) with a pH determined using the considerations and buffers described herein for the incubation step, with a pH range of about 6-9. The washing may be done three or more times. The temperature of washing is generally from refrigerator to moderate temperatures, with a constant temperature maintained during the assay period, typically from about 0-40°C, or about 4-30°C. For example, the wash buffer can be placed in ice at 4°C in a reservoir before the washing, and a plate washer can be utilized for this step. A cross- linking agent or other suitable agent may also be added at this stage to allow the now-bound PD-L1 of interest to be covalently attached to the capture reagents if there is any concern that the captured antibody of interest may dissociate to some extent in the subsequent steps.

[506] For certain embodiments, the next step involves contacting the immobilized capture reagents bound to any PD-L1 of interest with detectable antibody at a temperature of about 20-40°C, or about 36-38°C, with the exact temperature and time for contacting the two being dependent primarily on the detection means employed. For example, when 4- methylumbelliferyl -P-galactoside (MUG), streptavidin-HRP, or streptavidin -P-galactosidase is used as the means for detection, the contacting may be carried out overnight ( e.g ., about 15-17 hours or more) to amplify the signal to the maximum. While the detectable antibody may be a polyclonal or monoclonal antibody, preferably it is a monoclonal antibody, to reduce background noise. For certain embodiments, the same anti-PD-Ll antibody is used for coat and detection in the assay. In other embodiments, different anti-PD-Ll antibodies can be used for coat and detection which are selected so that the background noise is minimized.

[507] In certain embodiments, the detectable antibody is an antibody from a non-human species that binds to human antibodies. In certain embodiments, the detectable antibody is an anti-huIgG Fc antibody. In certain embodiments, the detectable antibody is a mouse anti- huIgG Fc antibody. In certain embodiments, the detectable antibody is directly detectable. In certain embodiments, the detectable antibody is biotinylated. In such cases, the detection means for the biotinylated label may be avidin or streptavidin-HRP, and the readout of the detection means may be fluorometric or colorimetric. In certain embodiments, the antibody is conjugated to HRP, and the detection means is colorimetric.

[508] An excess of detectable antibody with respect to the maximum concentration of free PD-L1 of interest expected (as described herein) is added to the plate after it is washed. The affinity of the detectable antibody must be sufficiently high that small amounts of the free antibody of interest can be detected, but not so high that it causes the antibody of interest to be pulled from the capture reagents.

[509] In certain embodiments, the last step of the assay method of the present invention involves measuring the level of any PD-L1 of interest from the sample that is now bound to the capture reagents using a detection means for the detectable antibody. If the biological sample is from a human subject, the measuring step comprises comparing the reaction that occurs as a result of the above three steps with a standard curve to determine the level of the PD-L1 epitope of interest compared to the known amount. The antibody added to the immobilized capture reagents will be either directly labeled, or detected indirectly by addition, after washing off of excess first antibody, of a molar excess of a second, labeled antibody directed against the species first antibody, including against a non-variable part of the first antibody, such as a species-antibody class/isotype. In the latter, indirect assay, labeled antisera against the first antibody are added to the sample so as to produce the labeled antibody in situ.

[510] The label used for either the first or second antibody is any detectable functionality that does not interfere with the binding of unbound PD-L1 to the anti-PD-Ll antibody of the present invention.

[511] Examples of suitable labels are those numerous labels known for use in immunoassay, including moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes³²P,¹⁴C,¹²⁵I,

³H, and^{13 1}1, fluorophores such as rare-earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g ., firefly luciferase and bacterial luciferase, luciferin, 2,3 -dihydrophthalazinedi ones, HRP, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g. , glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin (detectable by, e.g. , avidin, streptavidin, streptavidin-HRP, and streptavidin -P-galactosidase with MUG), spin labels, bacteriophage labels, and stable free radicals.

[512] Conventional methods are available to bind these labels covalently to proteins or polypeptides. For instance, coupling agents such as dialdehydes, carbodiimides,

dimaleimides, bis-imidates, and/or bis-diazotized benzidine may be used to label or tag the antibodies with a fluorescent, chemiluminescent, and enzymatic label, such as one described herein (see e.g. US 3,940,475; Hunter et ah, Nature , 144: 945 (1962); David G, Reisfeld R, Biochemistry 13 : 1014-21 (1974); Hashida S et ah, J Immunoassay 6: 111-23 (1985); Jeanson A et ah, Anal Biochem 172: 392-6 (1988)).

[513] The conjugation of such label, including the enzymes, to the antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques (see e.g. Methods in Enzymology vol. 73 (J. Langone and H. Van Vunakis, eds., Academic Press, New York (1981)). Suitable commercially available labeled antibodies may also be used.

[514] Following the addition of last labeled antibody, the amount of bound antibody is determined by removing excess unbound labeled antibody through washing and then measuring the amount of the attached label using a detection method appropriate to the label, and correlating the measured amount with the amount of the antibody of interest in the biological sample. For example, in the case of enzymes, the amount of color developed and measured will be a direct measurement of the amount of the antibody of interest present. Specifically, if HRP is the label, the color may be detected using the substrate 3,3^s, 5, 5'- tetramethylbenzidine (TMB), using a 450 nm read wavelength and a 620 or 630 nm reference wavelength.

[515] In one example, after an enzyme-labeled second antibody directed against the first unlabeled antibody is washed from the immobilized phase, color or chemiluminescence is developed and measured by incubating the immobilized capture reagent with a substrate of the enzyme. Then the concentration of the antibody of interest is calculated by comparing with the color or chemiluminescence generated by the standard antibody of interest run in parallel.

B. Mass Spectrometry

[516] In certain embodiments, the anti-PD-Ll antibodies are used in a mass spectrometry assay for PD-L1 target molecules in a sample. The assays described herein utilize anti-PD- Ll antibodies for immunoaffmity capture of PD-L1 molecules of interest from a biological sample. The sample may be further processed using a separation technique, such as chromatography, prior to quantification of the PD-L1 by mass spectroscopy. In certain embodiments, characteristic peptide fragments are produced by proteolysis and are measured as surrogate analytes for the PD-L1 of interest. In certain embodiments, the surrogate peptides are quantified using HPLC with detection by tandem mass spectroscopy (MS/MS).

C. Processing Biological Samples

[517] The biological sample may be tissue, cells, blood or blood products such as serum or plasma, or another body fluid containing the PD-L1 of interest. The biological samples are processed to form analysis samples by conventional procedures including: formulating, immobilizing, centrifugation, isolating, digesting, inducing or preventing blood cell clotting, hydrolyzing, or purifying. Processing biological samples serves to remove impurities and reduce sample heterogeneity which may hinder separation of the sample constituents, or obscure data collection or analysis. Alternatively, or in addition to, processing simplifies sample handling, preserves from degradation, minimizes sample volume, or selects for the sample constituents (analytes) of interest in the mass spectrometric analysis. Alternatively, or in addition to, processing converts biological samples into metabolites, fragments, or derivatives which are of interest in determining PD-L1 degradation, PD-L1 consumption, or PD-L1 metabolism.

Capturing PD-L1 Molecules (and optionally associated molecules) from Processed Samples

[518] The PD-L 1 of interest is captured on immune-affinity beads where the beads have an immobilized anti-PD-Ll antibody. In various embodiments, the anti-PD-Ll is any anti-PD- Ll antibody disclosed herein. In certain embodiments, the immobilized anti-PD-Ll antibody is specific for the PD-L1 epitope bound by an anti-PD-Ll antibody previously administered to the subject. The anti-PD-Ll antibody may be conjugated to the immunoaffmity beads using any suitable method known in the art.

[519] For certain embodiments, the anti-PD-Ll specific for the PD-L1 of interest is biotinylated and bound to streptavidin coated paramagnetic beads through strong biotin- streptavidin interaction ( e.g . having a KD about 10¹⁵ M).

[520] For certain embodiments, bound antibodies may be deglycosylated on the beads, e.g. with PNGaseF. The bound sample constituents may be eluted into a sample plate, with segregated receiving vessels or wells. The eluted samples may then be addressed by manual pipetting or by robotic transfer and separated by reverse phase chromatography and the separated sample constituents are analyzed by mass spectrometry.

[521] For certain embodiments, the biological sample may be digested with a protease. Characteristic peptide fragments are produced by proteolysis and are measured as surrogate analytes for the PD-L1 of interest. In an exemplary embodiment, the biological sample may be digested with trypsin digestion. For trypsin digestion, samples may be reduced with DTT, S-carboxymethylated with sodium iodoacetate, and then digested with trypsin. Digested samples may be analyzed by a separation method, for example, reverse phase HPLC, size- exclusion chromatography (SEC), or boronate affinity chromatography.

D. Separation of Sample Constituents

[522] To form the analysis sample, the biological sample may be applied to a separation media to effect separation of more than one sample constituent. Separation methods include affinity, chromatography, and electrophoresis methods. Affinity methods include affinity chromatography, adsorption, and immobilized affinity matrices. Chromatography methods include HPLC, hydrophobic interaction (HIC), anion exchange, cation exchange, reverse- phase, normal phase, ion-pair reverse-phase, thin-layer, capillary flow, and size-exclusion. Electrophoretic methods include single dimensional, slab gel, capillary (CE), polyacrylamide, denaturing, native, free solution, paper, 2-dimensional, isoelectric focusing (IEF), and gradient voltage. Other suitable separation methods may include: dialysis, centrifugation, sedimentation, floatation, precipitation, immunoprecipitation, and gel filtration.

[523] Separation methods may effect separation of the constituents of the biological sample by one or more physicochemical properties including, but not limited to, elution time, hydrophobicity, hydrophilicity, migration time, rate, velocity, chromatographic retention time, solubility, molecular volume or size, net charge, charge state, ionic charge, isoelectric point, dissociation constant (pKa), antibody affinity, electrophoretic mobility, ionization potential, dipole moment, hydrogen-bonding capability, and ion mobility in gas phase.

[524] In certain embodiments, a method of the present invention is used to detect PD-L1 in a biological sample. In certain embodiments, the method comprises (a) contacting the biological sample with an anti-PD-Ll antibody or composition as described herein under a condition to allow binding of the anti-PD-Ll antibody to any PD-L1 present to form a complex; (b) comparing the biological sample that has been contacted with the anti-PD-Ll antibody to the biological sample that has not been contacted with the anti-PD-Ll antibody by some assay or measurement, such as, e.g ., immunofixation electrophoresis. Several commercially available systems provide automated options that consolidate one or more of these steps.

[525] The methods of the present invention for detecting/measuring PD-L1 are appropriate for the analysis of biomolecular mixtures derived from biological samples where different chemical constituents of the mixture are first isolated, separated, or partially separated by one or more processes including affinity or chromatography which cause the constituents to elute sequentially or in a batch wise manner, or to be directly detected by mass spectrometry. For example, a variety of mass spectrometry systems capable of high mass accuracy, high sensitivity, and high resolution are known in the art and can be employed in the methods of the invention. The mass analyzers of such mass spectrometers include, but are not limited to, quadrupole (Q), time of flight (TOF), ion trap, magnetic sector or Fourier transform ion cyclotron resonance (FT-ICR) or combinations thereof. The ion source of the mass spectrometer should yield mainly sample molecular ions, or pseudo- molecular ions, and certain characterizable fragment ions. Examples of such ion sources include atmospheric pressure ionization sources, e.g. electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and Matrix Assisted Laser Desorption Ionization (MALDI). ESI and MALDI are the two most commonly employed methods to ionize proteins for mass spectrometric analysis of small molecules, such as, e.g. , by liquid chromatography mass spectrometry (LC/MS) (Lee M, LC/MS Applications in Drug Development (J. Wiley & Sons, New York (2002)). Another example is surface enhanced laser desorption ionization

(SELDI). SELDI is a surface-based ionization technique that allows for high-throughput mass spectrometry. Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface such as a“protein chip” to interact with analytes, e.g. , proteins, in solution. Such surfaces selectively interact with analytes and immobilize them thereon. Thus, the analytes of the invention can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing multiple reactive moieties at different sites on a substrate surface, throughput may be increased.

IX. Methods for Usinu PD-L1 Binding Proteins of the Present Invention and/or

Pharmaceutical and/or Diagnostic Compositions Thereof

[526] Generally, it is an object of the present invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the prevention and/or treatment of diseases, disorders, and conditions, such as certain cancers, tumors, growth abnormalities, immune disorders, or further pathological conditions mentioned herein. Accordingly, the present invention provides methods of using the anti-PD-Ll antibodies (such as the antigen-binding fragments and derivatives thereof, including any ADC or immunoconjugate thereof) and pharmaceutical compositions of the invention for the targeted killing of cells, for delivering additional exogenous materials into targeted cells, for labeling of the interiors of targeted cells, for collecting diagnostic information, for the delivering of T- cell epitopes, and for treating diseases, disorders, and conditions as described herein. For example, the methods of the present invention may be used to prevent or treat cancers, cancer initiation, tumor initiation, metastasis, and/or disease reoccurrence.

[527] In particular, it is an object of the present invention to provide such pharmacologically active agents, compositions, and/or methods that have certain advantages compared to the agents, compositions, and/or methods that are currently known in the art. Accordingly, the present invention provides methods of using anti-PD-Ll antibodies with specified protein sequences and pharmaceutical compositions thereof. For example, any of the amino acid sequences described herein may be specifically utilized as a component of the anti-PD-Ll antibody used in the following methods or any method for using an anti-PD-Ll antibody known to the skilled worker.

[528] The present invention provides methods of killing a cell comprising the step of contacting the cell, either in vitro or in vivo , with an anti-PD-Ll antibody, or pharmaceutical composition of the present invention. The anti-PD-Ll antibodies and pharmaceutical compositions of the present invention can be used to kill a specific cell type upon contacting a cell or cells with one of the claimed compositions of matter. For certain embodiments, an anti-PD-Ll antibody or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of different cell types, such as mixtures comprising cancer cells, infected cells, immune cells, and/or hematological cells. For certain embodiments, an anti-PD-Ll antibody or pharmaceutical composition of the present invention can be used to kill cancer cells in a mixture of different cell types. For certain embodiments, an anti-PD-Ll antibody or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of different cell types, such as pre-transplantation tissues. For certain embodiments, an anti-PD-Ll antibody or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of cell types, such as pre administration tissue material for therapeutic purposes. In certain embodiments, an anti-PD- Ll antibody or pharmaceutical composition of the present invention can be used to selectively kill cells infected by viruses or microorganisms, or otherwise selectively kill cells expressing a particular extracellular target biomolecule, such as a cell surface PD-L1 molecule. The anti-PD-Ll antibodies and pharmaceutical compositions of the present invention have varied applications, including, e.g ., uses in depleting unwanted cell types from tissues either in vitro or in vivo , uses in modulating immune responses to treat graft versus host or tissue rejection, uses as antiviral agents, uses as anti -parasitic agents, and uses in purging transplantation tissues of unwanted cell types.

[529] In certain embodiments, certain anti-PD-Ll antibodies and pharmaceutical compositions of the present invention, alone or in combination with other compounds or pharmaceutical compositions, can show potent cell-kill activity when administered to a population of cells, in vitro or in vivo in a subject such as in a patient in need of treatment.

By targeting the delivery of a drug using high-affinity immunoglobulin binding regions to specific cell types, cell-kill activities can be restricted to specifically and selectively killing certain cell types within an organism, such as certain cancer cells, neoplastic cells, malignant cells, non-malignant tumor cells, immune cells, and/or infected cells.

[530] The present invention provides a method of killing a cell in a patient in need thereof, the method comprising the step of administering to the patient at least one anti-PD-Ll antibody of the present invention or a pharmaceutical composition thereof.

[531] In certain embodiments, the anti-PD-Ll antibody of the present invention or pharmaceutical compositions thereof can be used to kill a cancer cell in a patient by targeting an extracellular PD-L1 found physically coupled with a cancer or tumor cell. The terms “cancer cell” or“cancerous cell” refers to various neoplastic cells which grow and divide in an abnormally accelerated and/or unregulated fashion and will be clear to the skilled person. The term“tumor cell” includes both malignant and non-malignant cells. Generally, cancers and/or tumors can be defined as diseases, disorders, or conditions that are amenable to treatment and/or prevention. The cancers and tumors (either malignant or non-malignant) which are comprised of cancer cells and/or tumor cells which may benefit from methods and compositions of the invention will be clear to the skilled person. Neoplastic cells are often associated with one or more of the following: unregulated growth, lack of differentiation, local tissue invasion, angiogenesis, and metastasis. The diseases, disorders, and conditions resulting from cancers and/or tumors (either malignant or non-malignant) which may benefit from the methods and compositions of the present invention targeting certain cancer cells and/or tumor cells will be clear to the skilled person.

[532] Certain embodiments of the anti-PD-Ll antibodies and compositions of the present invention may be used to kill cancer stem cells, tumor stem cells, pre-malignant cancer- initiating cells, and tumor-initiating cells, which commonly are slow dividing and resistant to cancer therapies, such as chemotherapy and radiation.

[533] Compositions of matter of the present invention may be more effectively used in methods involving in combination with other therapies, such as, e.g ., chemotherapies, immunotherapies, radiation, stem cell transplantation, and immune checkpoint inhibitors, including PD-L1 -targeted therapies targeting other than the same epitope on, non

overlapping, or different targets for the same disease disorder or condition.

[534] Certain embodiments of the anti-PD-Ll antibodies of the present invention, or pharmaceutical compositions thereof, can be used to kill an immune cell (whether healthy or malignant) in a patient by targeting an extracellular PD-L1 found physically coupled with an immune cell. [535] It is within the scope of the present invention to utilize an anti-PD-Ll antibody of the present invention, or pharmaceutical composition thereof, for the purposes of purging patient cell populations ( e.g . bone marrow) of malignant, neoplastic, or otherwise unwanted immune cells, e.g. T-cells, B-cells, and/or macrophages, and then reinfusing the T-cell and/or B-cells depleted material into the patient ( see e.g. van Heeckeren W et al., Br J Haematol 132: 42-55 (2006); ( see e.g. Alpdogan O, van den Brink M, Semin Oncol 39: 629-42 (2012)).

[536] It is within the scope of the present invention to utilize the anti-PD-Ll antibody of the present invention, or pharmaceutical composition thereof, for the purposes of ex vivo depletion of T cells and/or B-cells from isolated cell populations removed from a patient. In one non-limiting example, the anti-PD-Ll antibody of the invention can be used in a method for prophylaxis of organ and/or tissue transplant rejection wherein the donor organ or tissue is perfused prior to transplant with a cytotoxic, anti-PD-Ll antibody of the invention or a pharmaceutical composition thereof in order to purge the organ of donor T-cells and/or B- cells ( see e.g. Alpdogan O, van den Brink M, Semin Oncol 39: 629-42 (2012)).

[537] It is also within the scope of the present invention to utilize the anti-PD-Ll antibody of the invention, or pharmaceutical composition thereof, for the purposes of depleting T-cells and/or B-cells from a donor cell population as a prophylaxis against graft-versus-host disease, and induction of tolerance, in a patient to undergo a bone marrow and or stem cell transplant

( see e.g. van Heeckeren W et al., Br J Haematol 132: 42-55 (2006); ( see e.g. Alpdogan O, van den Brink M, Semin Oncol 39: 629-42 (2012)).

[538] In certain embodiments of the anti-PD-Ll antibody of the present invention, or pharmaceutical compositions thereof, can be used to kill an infected cell in a patient by targeting an extracellular PD-L1 found physically coupled with an infected cell.

[539] In certain embodiments of the anti-PD-Ll antibodies of the present invention, or pharmaceutical compositions thereof, can be used to“seed” a locus within a chordate with non-self, T-cell epitope-peptide presenting cells in order to activate the immune system to enhance policing of the locus. In certain further embodiments of this“seeding” method of the present invention, the locus is a tumor mass or infected tissue site. In preferred embodiments of this“seeding” method of the present invention, the non-self, T-cell epitope- peptide is selected from the group consisting of: peptides not already presented by the target cells of the anti-PD-Ll antibody, peptides not present within any protein expressed by the target cell, peptides not present within the proteome or transcriptome of the target cell, peptides not present in the extracellular microenvironment of the site to be seeded, and peptides not present in the tumor mass or infect tissue site to be targeting. This“seeding” method of using an anti-PD-Ll antibody of the present invention can provide a temporary vaccination-effect by inducing adaptive immune responses to attack the cells within the seeded microenvironment, such as, e.g. a tumor mass or infected tissue site, whether presenting an anti-PD-Ll antibody-delivered T-cell epitope(s) or not. This“seeding” method may also induce the breaking of immuno-tolerance to a target cell population, a tumor mass, and/or infected tissue site within a chordate.

[540] Certain methods of the present invention involving the seeding of a locus within a chordate with one or more antigenic and/or immunogenic epitopes may be combined with the administration of immunologic adjuvants, whether administered locally or systemically, to stimulate the immune response to certain antigens, such as, e.g. , the co-administration of a composition of the present invention with one or more immunologic adjuvants, such as a cytokine, bacterial product, or plant saponin. Other examples of immunologic adjuvants which may be suitable for use in the methods of the present invention include aluminum salts and oils, such as, e.g. , alums, aluminum hydroxide, mineral oils, squalene, paraffin oils, peanut oils, and thimerosal.

[541] Additionally, the present invention provides a method of treating a disease, disorder, or condition in a patient, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of at least one of the anti-PD-Ll antibodies of the present invention, or a pharmaceutical composition thereof. Contemplated diseases, disorders, and conditions that can be treated using this method include cancers, malignant tumors, non-malignant tumors, growth abnormalities, immune disorders, and microbial infections, such as, e.g., involving a PD-L1 -expressing cell. Administration of a

“therapeutically effective dosage” of a composition of the present invention can result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

[542] The therapeutically effective amount of a composition of the present invention will depend on the route of administration, the type of organism being treated, and the physical characteristics of the specific patient under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention and may be confirmed in properly designed clinical trials. An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person.

[543] An acceptable route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal ( e.g . topical administration of a cream, gel or ointment, or by means of a transdermal patch). “Parenteral administration” is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.

[544] For administration of a pharmaceutical composition of the present invention, the dosage range will generally be from about 0.001 to 10 milligrams per kilogram (mg/kg), and more, usually 0.001 to 0.5 mg/kg, of the subject’s body weight. Exemplary dosages may be 0.01 mg/kg body weight, 0.03 mg/kg body weight, 0.07 mg/kg body weight, 0.9 mg/kg body weight or 0.1 mg/kg body weight or within the range of 0.01 to 0.1 mg/kg. An exemplary treatment regime is a once or twice daily administration, or a once or twice weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every two or three months or once every three to 6 months. Dosages may be selected and readjusted by the skilled health care professional as required to maximize therapeutic benefit for a particular patient.

[545] Pharmaceutical compositions of the present invention will typically be administered to the same patient on multiple occasions. Intervals between single dosages can be, for example, two to five days, weekly, monthly, every two or three months, every six months, or yearly. Intervals between administrations can also be irregular, based on regulating blood levels or other markers in the subject or patient. Dosage regimens for a composition of the present invention include intravenous administration of 1 mg/kg body weight or 3 mg/kg body weight with the composition administered every two to four weeks for six dosages, then every three months at 3 mg/kg body weight or 1 mg/kg body weight.

[546] A pharmaceutical composition of the present invention may be administered via one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by the skilled worker, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for anti-PD-Ll antibodies and pharmaceutical compositions of the present invention include, e.g. intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example by injection or infusion. For other embodiments, an anti-PD-Ll antibody or pharmaceutical composition of the invention may be administered by a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[547] Therapeutic anti-PD-Ll antibodies or pharmaceutical compositions of the present invention may be administered with one or more of a variety of medical devices known in the art. For example, in one embodiment, a pharmaceutical composition of the invention may be administered with a needleless hypodermic injection device. Examples of well-known implants and modules useful in the present invention are in the art, including e.g. , implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; variable flow implantable infusion devices for continuous drug delivery; and osmotic drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art.

[548] The anti-PD-Ll antibody or pharmaceutical composition of the present invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents. A combination therapy may include an anti-PD-Ll antibody of the present invention, or pharmaceutical composition thereof, combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated. Examples of other such agents include, inter alia , a cytotoxic, anti-cancer or chemotherapeutic agent, an anti inflammatory or anti-proliferative agent, an antimicrobial or antiviral agent, growth factors, cytokines, an analgesic, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates one or more signaling pathways, and similar modulating therapeutic molecules which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.

[549] Treatment of a patient with anti-PD-Ll antibody or pharmaceutical composition of the present invention may lead to cell death of targeted cells and/or the inhibition of growth of targeted cells. As such, cytotoxic, anti-PD-Ll antibodies of the present invention, and pharmaceutical compositions comprising them, will be useful in methods for treating a variety of pathological disorders in which killing or depleting target cells may be beneficial, such as, inter alia , cancer, tumors, other growth abnormalities, immune disorders, and infected cells. The present invention provides methods for suppressing cell proliferation, and treating cell disorders, including neoplasia, overactive B-cells, and overactive T-cells.

[550] In certain embodiments, the anti-PD-Ll antibodies and pharmaceutical compositions of the present invention can be used to treat or prevent cancers, tumors (malignant and non- malignant), growth abnormalities, immune disorders, and microbial infections. In a further aspect, the above ex vivo method can be combined with the above in vivo method to provide methods of treating or preventing rejection in bone marrow transplant recipients, and for achieving immunological tolerance.

[551] In certain embodiments, the present invention provides methods for treating malignancies or neoplasms and other blood cell associated cancers in a mammalian subject, such as a human, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a cytotoxic anti-PD-Ll antibody or pharmaceutical composition of the present invention.

[552] The anti-PD-Ll antibodies and pharmaceutical compositions of the present invention have varied applications, including, e.g ., uses in removing unwanted T-cells, uses in modulating immune responses to treat graft versus host, uses as antiviral agents, uses as antimicrobial agents, and uses in purging transplantation tissues of unwanted cell types. The anti-PD-Ll antibodies and pharmaceutical compositions of the present invention are commonly anti-neoplastic agents - meaning they are capable of treating and/or preventing the development, maturation, or spread of neoplastic or malignant cells by inhibiting the growth and/or causing the death of cancer or tumor cells.

[553] In certain embodiments, the anti-PD-Ll antibody or pharmaceutical composition of the present invention is used to treat a B-cell-, plasma cell- or antibody- mediated disease or disorder, such as for example leukemia, lymphoma, myeloma, rheumatic disease, spondylitis, Human Immunodeficiency Virus-related diseases, amyloidosis, hemolytic uremic syndrome, polyarteritis, septic shock, Crohn’s Disease, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, ulcerative colitis, psoriasis, asthma, allergic asthma, Sjogren’s syndrome, graft-versus-host disease, graft rejection, diabetes, vasculitis, scleroderma, and systemic lupus erythematosus.

[554] In another aspect, certain embodiments of the anti-PD-Ll antibodies and

pharmaceutical compositions of the present invention are antimicrobial agents - meaning they are capable of treating and/or preventing the acquisition, development, or consequences of microbiological pathogenic infections, such as caused by viruses, bacteria, fungi, prions, or protozoans.

[555] It is within the scope of the present invention to provide a prophylaxis or treatment for diseases or conditions mediated by T-cells or B-cells by administering the anti-PD-Ll antibody the present invention, or a pharmaceutical composition thereof, to a patient for the purpose of killing T-cells or B-cells in the patient. This usage is compatible with preparing or conditioning a patient for bone marrow transplantation, stem cell transplantation, tissue transplantation, or organ transplantation, regardless of the source of the transplanted material, e.g. human or non-human sources.

[556] It is within the scope of the present invention to provide a bone marrow recipient for prophylaxis or treatment of host-versus-graft disease via the targeted cell-killing of host T- cells using a cytotoxic anti-PD-Ll antibody or pharmaceutical composition of the present invention.

[557] Certain embodiments of the anti-PD-Ll antibodies and pharmaceutical compositions of the present invention can be utilized in a method of treating cancer comprising

administering to a patient, in need thereof, a therapeutically effective amount of an anti-PD- Ll antibody and/or pharmaceutical composition of the present invention. In certain embodiments of the methods of the present invention, the cancer being treated is selected from the group consisting of: bone cancer (such as multiple myeloma or Ewing’s sarcoma), breast cancer, central/peripheral nervous system cancer (such as glioma, brain cancer, neurofibromatosis, or glioblastoma), gastrointestinal cancer (such as stomach cancer or colorectal cancer), germ cell cancer (such as ovarian cancers and testicular cancers, glandular cancer (such as pancreatic cancer, parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroid cancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer, or pharyngeal cancer), hematological cancers (such as leukemia, lymphoma, or myeloma), kidney -urinary tract cancer (such as renal cancer, bladder cancer, or urothelial carcinoma), liver cancer, lung/pleura cancer (such as mesothelioma, small cell lung carcinoma, or non small cell lung carcinoma, and/or triple negative for HER2, estrogen receptor, and

progesterone receptor), prostate cancer, sarcoma (such as angiosarcoma, fibrosarcoma, Kaposi’s sarcoma, or synovial sarcoma), skin cancer (such as Merkel cell cancer, basal cell carcinoma, squamous cell carcinoma, or melanoma), uterine cancer, acute lymphoblastic leukemia (ALL), T acute lymphocytic leukemia/lymphoma (ALL), acute myelogenous leukemia, acute myeloid leukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL), B- cell prolymphocytic lymphoma, Burkitt’s lymphoma (BL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML-BP), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia (HCL), Hodgkin’s

Lymphoma (HL), intravascular large B-cell lymphoma, lymphomatoid granulomatosis, lymphoplasmacytic lymphoma, MALT lymphoma, mantle cell lymphoma, multiple myeloma (MM), natural killer cell leukemia, nodal marginal B-cell lymphoma, Non-Hodgkin’s lymphoma (NHL), plasma cell leukemia, plasmacytoma, primary effusion lymphoma, pro- lymphocytic leukemia, promyelocytic leukemia, small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma, T-cell lymphoma (TCL), B-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL), angioimmunoblastic T-cell lymphoma (AITL), epithelial malignancies, oral squamous cell carcinoma, esophageal squamous cell carcinoma (ESCC), squamous cell carcinoma of the head and neck (SCCHN), and virus-associated neoplastic malignancies.

[558] Certain embodiments of the anti-PD-Ll antibodies and pharmaceutical compositions of the present invention can be utilized in a method of treating an immune disorder comprising administering to a patient, in need thereof, a therapeutically effective amount of the anti-PD-Ll antibodies and/or pharmaceutical composition of the present invention. In certain embodiments of the methods of the present invention, the immune disorder is associated with a disease selected from the group consisting of: AIDS, rheumatic disease, spondylitis, amyloidosis, ankylosing spondylitis, asthma, allergic asthma, autism,

cardiorheumatic disease, Crohn’s disease, diabetes, erythematosus, gastritis, graft rejection, graft-versus-host disease, Grave’s disease, Hashimoto’s thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, lymphoproliferative disorders (including post-transplant lymphoproliferative disorders), multiple sclerosis, myasthenia gravis, neuroinflammation, polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjogren’s syndrome, systemic lupus erythematosus, ulcerative colitis, vasculitis, cell proliferation, inflammation, leukocyte activation, leukocyte adhesion, leukocyte chemotaxis, leukocyte maturation, leukocyte migration, neuronal differentiation, acute lymphoblastic leukemia (ALL), T acute lymphocytic leukemia/lymphoma (ALL), acute myelogenous leukemia, acute myeloid leukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL), B-cell prolymphocytic lymphoma, Burkitt’s lymphoma (BL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML-BP), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia (HCL), Hodgkin’s Lymphoma (HL), intravascular large B-cell lymphoma, lymphomatoid granulomatosis, lymphoplasmacytic lymphoma, MALT lymphoma, mantle cell lymphoma, multiple myeloma (MM), natural killer cell leukemia, nodal marginal B-cell lymphoma, Non- Hodgkin’s lymphoma (NHL), plasma cell leukemia, plasmacytoma, primary effusion lymphoma, pro-lymphocytic leukemia, promyelocytic leukemia, small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma, T-cell lymphoma (TCL), heavy chain disease, monoclonal gammopathy, monoclonal immunoglobulin deposition disease, myelodusplastic syndromes (MDS), smoldering multiple myeloma, and Waldenstrom macroglobulinemia.

[559] Among certain embodiments of the present invention is using the anti-PD-Ll antibody of the present invention as a component of a pharmaceutical composition or medicament for the treatment or prevention of a cancer, tumor, other growth abnormality, immune disorder, and/or microbial infection. For example, immune disorders presenting on the skin of a patient may be treated with such a medicament in efforts to reduce

inflammation. In another example, skin tumors may be treated with such a medicament in efforts to reduce tumor size or eliminate the tumor completely.

[560] Among certain embodiment of the present invention is a method of using an anti-PD- Ll antibody, pharmaceutical composition, and/or diagnostic composition of the present invention to label or detect the interiors of neoplastic cells and/or immune cell types. This method may be based on the ability of certain anti-PD-Ll antibodies of the present invention to enter specific cell types and delivery a cargo which routes within cells via retrograde intracellular transport, to the interior compartments of specific cell types are labeled for detection. This can be performed on cells in situ within a patient or on cells and tissues removed from an organism, e.g. biopsy material.

[561] Among certain embodiment of the present invention is a method of using an anti-PD- Ll antibody, pharmaceutical composition, and/or diagnostic composition of the present invention to detect the presence of a cell type for the purpose of information gathering regarding diseases, conditions and/or disorders. The method comprises contacting a cell with a diagnostically sufficient amount of an anti-PD-Ll antibody of the present invention in order to detect the molecule by an assay or diagnostic technique. The phrase“diagnostically sufficient amount” refers to an amount that provides adequate detection and accurate measurement for information gathering purposes by the particular assay or diagnostic technique utilized. Generally, the diagnostically sufficient amount for whole organism in vivo diagnostic use will be a non-cumulative dose of between 0.001 to 10 milligrams of the detection-promoting agent linked anti-PD-Ll antibody of the invention per kg of subject per subject. Typically, the amount of anti-PD-Ll antibody of the invention used in these information gathering methods will be as low as possible provided that it is still a diagnostically sufficient amount. For example, for in vivo detection in an organism, the amount of anti-PD-Ll antibody or pharmaceutical composition of the invention administered to a subject will be as low as feasibly possible.

[562] The cell-type specific targeting of anti-PD-Ll antibodies of the present invention combined with detection-promoting agents provides a way to detect and image cells physically coupled with an extracellular PD-L1 bound by the binding region of the molecule of the invention. Imaging of cells using the anti-PD-Ll antibodies of the present invention may be performed in vitro or in vivo by any suitable technique known in the art. Diagnostic information may be collected using various methods known in the art, including whole body imaging of an organism or using ex vivo samples taken from an organism. The term “sample” used herein refers to any number of things, but not limited to, fluids such as blood, urine, serum, lymph, saliva, anal secretions, vaginal secretions, and semen, and tissues obtained by biopsy procedures. For example, various detection-promoting agents may be utilized for non-invasive in vivo tumor imaging by techniques such as magnetic resonance imaging (MRI), optical methods (such as direct, fluorescent, and bioluminescent imaging), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound, x-ray computed tomography, and combinations of the aforementioned (see, Kaur S et ak, Cancer Lett 315: 97-111 (2012), / r review).

[563] Among certain embodiment of the present invention is a method of using an anti-PD- Ll antibody, or pharmaceutical composition of the present invention in a diagnostic composition to label or detect the interiors of a hematologic cell, cancer cell, tumor cell, infected cell, and/or immune cell (see e.g., Koyama Y et ak, Clin Cancer Res 13 : 2936-45 (2007); Ogawa M et ak, Cancer Res 69: 1268-72 (2009); Yang L et ak, Small 5: 235-43 (2009)). Based on the ability of certain anti-PD-Ll antibodies of the invention to enter specific cell types (and for certain embodiments, to deliver a cargo to the interior of a target cell) allows for the interior of a cell being labeled for detection. This can be performed on cells in situ within a patient or on cells and tissues removed from an organism, e.g. biopsy material.

[564] Diagnostic compositions of the present invention may be used to characterize a disease, disorder, or condition as potentially treatable by a related pharmaceutical composition of the present invention. In certain compositions of matter of the present invention may be used to determine whether a patient belongs to a group that responds to a therapeutic strategy which makes use of a compound, composition or related method of the present invention as described herein or is well suited for using a delivery device of the invention.

[565] Diagnostic compositions of the present invention may be used after a disease, e.g. a cancer, is detected in order to better characterize it, such as to monitor distant metastases, heterogeneity, and stage of cancer progression. The phenotypic assessment of disease disorder or infection can help prognostic and prediction during therapeutic decision making. In disease reoccurrence, certain methods of the invention may be used to determine if local or systemic problem.

[566] Diagnostic compositions of the present invention may be used to assess responses to therapies regardless of the type of the type of therapy, e.g. small molecule drug, biological drug, or cell-based therapy, such as a therapy involving an immune checkpoint inhibitor. For example, certain embodiments of the diagnostics of the invention may be used to measure changes in tumor size, changes in antigen positive cell populations including number and distribution, or monitoring a different marker than the antigen targeted by a therapy already being administered to a patient ( see Smith-Jones P et ah, Nat Biotechnol 22: 701-6 (2004); Evans M et ah, Proc Natl Acad Sci U.S.A. 108: 9578-82 (2011)).

[567] In certain embodiments of the method used to detect the presence of a cell type may be used to gather information regarding diseases, disorders, and conditions, such as, for example bone cancer (such as multiple myeloma or Ewing’s sarcoma), breast cancer, central/peripheral nervous system cancer (such as glioma, brain cancer, neurofibromatosis, or glioblastoma), gastrointestinal cancer (such as stomach cancer or colorectal cancer), germ cell cancer (such as ovarian cancers and testicular cancers, glandular cancer (such as pancreatic cancer, parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroid cancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer, or pharyngeal cancer), hematological cancers (such as leukemia, lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancer, bladder cancer, or urothelial carcinoma), liver cancer, lung/pleura cancer (such as mesothelioma, small cell lung carcinoma, or non-small cell lung carcinoma, and/or triple negative for HER2, estrogen receptor, and progesterone receptor), prostate cancer, sarcoma (such as angiosarcoma, fibrosarcoma, Kaposi’s sarcoma, or synovial sarcoma), skin cancer (such as Merkel cell cancer, basal cell carcinoma, squamous cell carcinoma, or melanoma), uterine cancer, B-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL), angioimmunoblastic T-cell lymphoma (AITL), epithelial malignancies, oral squamous cell carcinomas, esophageal squamous cell carcinoma (ESCC), squamous cell carcinoma of the head and neck (SCCHN), virus-associated malignancies, AIDS, rheumatic disease, spondylitis, amyloidosis, ankylosing spondylitis, asthma, allergic asthma, autism, cardiorheumatic disease, Crohn’s disease, diabetes, erythematosus, gastritis, graft rejection, graft-versus-host disease, Grave’s disease, Hashimoto’s thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, lymphoproliferative disorders

(including post-transplant lymphoproliferative disorders), multiple sclerosis, myasthenia gravis, neuroinflammation, polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjogren’s syndrome, systemic lupus erythematosus, ulcerative colitis, vasculitis, cell proliferation, inflammation, leukocyte activation, leukocyte adhesion, leukocyte chemotaxis, leukocyte maturation, leukocyte migration, neuronal differentiation, acute lymphoblastic leukemia (ALL), T acute lymphocytic leukemia/lymphoma (ALL), acute myelogenous leukemia, acute myeloid leukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL), B-cell prolymphocytic lymphoma, Burkitt’s lymphoma (BL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML-BP), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia (HCL), Hodgkin’s Lymphoma (HL), intravascular large B-cell lymphoma, lymphomatoid granulomatosis, lymphoplasmacytic lymphoma, MALT lymphoma, mantle cell lymphoma, multiple myeloma (MM), natural killer cell leukemia, nodal marginal B-cell lymphoma, Non- Hodgkin’s lymphoma (NHL), plasma cell leukemia, plasmacytoma, primary effusion lymphoma, pro-lymphocytic leukemia, promyelocytic leukemia, small lymphocytic lymphoma, splenic marginal zone lymphoma, T-cell lymphoma (TCL), heavy chain disease, monoclonal gammopathy, monoclonal immunoglobulin deposition disease, myelodusplastic syndromes (MDS), smoldering multiple myeloma, and Waldenstrom macroglobulinemia.

[568] The present invention is further illustrated by the following non-limiting examples of anti-PD-Ll antibody derivatives comprising aforementioned structures and functions, in particular the function of specific and high affinity binding to PD-L1 with a KD of less than 10⁹ M.

EXAMPLES

[569] The following examples demonstrate certain embodiments of the present invention. However, it is to be understood that these examples are for illustration purposes only and do not intend, nor should any be construed, to be wholly definitive as to conditions and scope of this invention. The experiments in the following examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described. [570] The following examples describe an exemplary, anti-PD-Ll antibody of the present invention. The anti-PD-Ll antibodies of the present invention bind to various mammalian and primatial PD-L1 proteins, typically with high affinities. The exemplary, anti-PD-Ll antibodies, and proteins comprising the aforementioned, are useful for targeting and detecting PD-L1, such as, e.g., in vitro or in vivo. In the Examples, anti-PD-Ll antibodies of the present invention are used to quantify PD-L1 levels in a biological sample and to target a cargo to a PD-L1 expressing cell.

Example 1. Anti-PD-Ll Antibody Derivative - Single-Chain Variable Fragment

[571] A single-chain variable fragment polypeptide (SEQ ID NO: 13) was constructed comprising the CDRs shown in SEQ ID NOs: 1-6. This scFv was fused to a heterologous moiety consisting of a polypeptide. Then a PD-L1 binding assay was performed to analyze the dissociation constant (KD) of the anti-PD-Ll fusion protein to PD-L1. Binding to an extracellular portion of both a human PD-L1 (UniProt Q9NZQ7) and a PD-L1 (NCBI accession XP 005581836) from a cynomolgus monkey were tested. The results of the binding assay are shown in Table 1, below. The dissociation constants measured showed the scFv-fusion protein bound both mammalian PD-Lls with high affinity.

Table 1. Binding Affinity of anti-PD-Ll scFv to Primate PD-L1 Molecules

[572] The binding specificity and selectivity of the exemplary anti-PD-Ll scFv (SEQ ID NO: 13) was tested by analyzing binding of this purified fusion protein to a membrane proteome array, which comprised 5,300 different human proteins known to be expressed on the cell surface of HEK-293T cells (Integral Molecular, Inc., Philadelphia, PA, U.S.). The results shown in Figure 2, which shows that only PD-L1 (CD274) was identified and validated among the 5,300 proteins as a selective target of SEQ ID NO: 13.

[573] The epitope of human PD-L1 bound by SEQ ID NO: 13 was mapped to identify contact residues using a shotgun mutagenesis method by Integral Molecular (Philadelphia, PA, U.S.A.). An alanine scanning mutagenesis library based on a human PD-L1 protein was created and screened in duplicate by high-throughput flow cytometry for binding by three proteins comprising SEQ ID NO: 13. By this assay, a critical residue in the PD-L1 epitope for binding by SEQ ID NO: 13 included F42, thus the epitope in human PD-L1 bound by SEQ ID NO: 13 appeared to comprise F42. An analysis of the FDA approved anti-PD-Ll antibodies atezolizumab, durvalumab, and avelumab revealed that all three share an epitope comprising Y56, E58, Rl 13, Ml 15, and Y123. In particular, durvalumab binds an epitope in PD-L1 involving Rl 13, D122, Y123, and R125 (Lee H et al., Sci Rep 7: 5532 (2017)).

Example 2. PD-L1 Binding Proteins for Detecting PD-L1 in a Sample

[574] An exemplary anti-PD-Ll antibody of the present invention is used in an

immunoprecipitati on-mass-spectrometry assay for PD-L1 molecules in a sample. The anti- PD-Ll antibody is used for immunoaffmity capture of PD-L1 molecules of interest from a biological sample. The sample is first processed using a separation technique prior to quantification of the PD-L1 by mass spectroscopy.

[575] The biological sample is biopsy tissue comprising tumor cells which may or may not be expressing PD-L1. The biological samples are processed to form analysis samples by conventional procedures to purify the cell-surface proteins. To form the analysis sample, the biological sample is applied to chromatography.

[576] The sample is interrogated for PD-L1 of interest by capturing PD-L1 using immune- affinity beads where the beads have an immobilized anti-PD-Ll antibody of the present invention, prepared using routine methods wherein the anti-PD-Ll antibody is biotinylated and bound to streptavidin coated paramagnetic beads. Briefly, the analysis sample is contacted with the immobilized anti-PD-Ll antibody immune-affinity beads under a condition to allow binding of the anti-PD-Ll antibody to any PD-L1 present to form a complex. The beads are washed and then the bound PD-L1 is cleaved from the beads, eluted, and subjected to mass spectrometry.

Example 3. Anti-PD-Ll Antibodies Comprising a Cytotoxic Agent

[577] Shiga toxin A Subunit polypeptides are fused to anti-PD-Ll antibodies of the present invention, or antigen binding fragments or derivatives thereof, to create PD-L1 -targeted immunotoxins. Previously, Shiga toxin A Subunit derived proteins have been constructed and shown to promote cellular internalization, direct intracellular routing of their Shiga toxin components to the cytosol, and provide cytotoxicity ( see e.g. WO 2014/164680, WO

2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, WO 2015/191764, WO 2016/196344, WO 2017/019623, WO 2018/106895, WO

2018/140427, US 2007/298434, US 2009/156417, US 2013/196928, US 2016/177284, US 2017/143814, and US 2017/275382).

[578] Exemplary, PD-L1 binding proteins of the present invention are constructed comprising (1) an anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, (2) a de-immunized Shiga toxin A Subunit fragment, and optionally (3) a payload or cargo (such as, e.g. , a fused, heterologous, CD8+ T-cell epitope-peptide which is neither embedded nor inserted into a Shiga toxin effector polypeptide). These exemplary, PD-L1 binding proteins of the present invention specifically bind to PD-L1 biomolecules expressed by target cell- types and enter target cells. Then, the internalized exemplary PD-L1 binding protein effectively routes its Shiga toxin component to the cytosol of the target cell and optionally kills the target cell directly via ribosome inhibition or indirectly via intracellular delivery of a payload or cargo.

Example 4. PD-L1 Binding Proteins for Detecting PD-L1 Using Immunohistochemistry

[579] The PD-L1 binding proteins of the present invention may be used in PD-L1 detection methods to detect expression levels of PD-L1 polypeptides in a biological sample in vitro or in vivo. In vitro techniques for detection of PD-L1 polypeptides include ELIS As, Western blots, flow cytometry, immunoprecipitations, radioimmunoassay, and immunofluorescence (e.g. immunohistochemistry).

[580] For example, PD-L1 expression in cells or tissues can be detecting using classical immunohistochemical (IHC) staining methods employing an anti-PD-Ll antibody of the present invention. An exemplary anti-PD-Ll antibody of the present invention is used in IHC methods to detect PD-L1 in a biological sample. Various tissue samples from human subjects are tested with the exemplary anti-PD-Ll antibody, such as, e.g. , biopsy samples of cervical squamous cell carcinoma, Hodgkin’s lymphoma, placental, pancreas

adenocarcinoma, prostate adenocarcinoma, tonsil, or skin squamous cell carcinoma tissue. The samples are prepared using routine methods known to the skilled worker, such as, e.g. , by generating formalin-fixed paraffin embedded tissue samples. The anti-PD-Ll antibody exhibits detectable staining in the samples at various concentrations, e.g. 0.1 to 100 pg/mL, as visualized by a common technique in the art, such as, e.g, via indirect

immunofluorescence. These results demonstrate that exemplary anti-PD-Ll antibodies of the present invention detect PD-L1 in fixed tissue samples. [581] While some embodiments of the invention have been described by way of illustration, it will be apparent that the invention may be put into practice with many modifications, variations and adaptations, and with the use of numerous equivalents or alternative solutions that are within the scope of persons skilled in the art, without departing from the spirit of the invention or exceeding the scope of the claims.

[582] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. The international patent application publications WO 2014/164680, WO

2014/164693, WO 2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007, WO 2015/191764, WO 2016/196344, WO 2017/019623, WO 2018/106895, and WO

2018/140427 are each incorporated herein by reference in its entirety. The disclosures of U.S. patent applications US 2007/298434, US 2009/156417, US 2013/196928, US

2016/177284, US 2017/143814, US 2017/275382, and US 62/746153 are each incorporated herein by reference in its entirety. The complete disclosures of all electronically available biological sequence information from GenBank (National Center for Biotechnology

Information, U.S.) and The UniProt Consortium (Universal Protein Resource (UniProt) Consortium) for amino acid and nucleotide sequences cited herein are each incorporated herein by reference in their entirety.

Claims

CLAIMS What is claimed is:

1. An isolated anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, that is capable of specifically binding to PD-L1, wherein the anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, comprises:

(a) a light chain variable region comprising:

(i) a CDR1 comprising the amino acid sequence TGTSSDVGSYNRVS (SEQ ID NO: 1),

(ii) a CDR2 comprising the amino acid sequence EVSNRPS (SEQ ID NO: 2), and

(iii) a CDR3 comprising the amino acid sequence SSHTTSGTYV (SEQ ID NO:

3);

and

(b) a heavy chain variable region comprising:

(i) a CDR1 comprising the amino acid sequence SYAIS (SEQ ID NO: 4),

(ii) a CDR2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 5), and

(iii) a CDR3 comprising the amino acid sequence DQGYAHAFDI (SEQ ID NO: 6)·

2. The isolated anti-PD-Ll antibody, antigen binding fragment, or derivative thereof of claim 1, which comprises:

(a) a light chain region having at least 90% identity to SEQ ID NO:7; and/or

(b) a heavy chain region having at least 90% identity to SEQ ID NO: 8.

3. The isolated anti-PD-Ll antibody, antigen binding fragment, or derivative thereof of claim 1, which comprises:

(a) a light chain region having at least 90% identity to SEQ ID NO:9; and/or

(b) a heavy chain region having at least 90%identity to SEQ ID NO: 8.

4. The isolated anti-PD-Ll antibody, antigen binding fragment or derivative thereof of any one of claims 1-3, which comprises a polypeptide having at least 90% identity to any one of SEQ ID NOs: 10-17.

5. The isolated anti-PD-Ll antibody, antigen binding fragment, or derivative thereof of any one of claims 1-4, which is linked to a heterologous moiety or additional material, and which is optionally a cargo, therapeutic agent, or detection-promoting agent.

6. The isolated anti-PD-Ll antibody, antigen binding fragment, or derivative thereof of claim 5, wherein the heterologous moiety is a cytotoxic agent.

7. The isolated anti-PD-Ll antibody, antigen binding fragment, or derivative thereof of claim 5, wherein the heterologous moiety is a ribotoxic polypeptide.

8. The isolated anti-PD-Ll antibody, antigen binding fragment, or derivative thereof of claim 5, wherein the detectable moiety is a label, biotin, enzyme reporter, and/or fluorescent tag.

9. A composition comprising an anti-PD-Ll antibody, antigen binding fragment, or

derivative thereof according to any one of claims 1-8.

10. A pharmaceutical composition comprising

an isolated anti-PD-Ll antibody, antigen binding fragment, or derivative thereof according to any one of claims 1-8; and

at least one pharmaceutically acceptable excipient or carrier.

11. A diagnostic composition comprising

a detection promoting agent.

12. An isolated nucleic acid encoding an anti-PD-Ll antibody, antigen binding fragment, or derivative thereof according to any one of claims 1-8.

13. An expression vector comprising a nucleic acid according to claim 12.

14. A host cell comprising an expression vector according to claim 13.

15. A method of killing a PD-L1 expressing cell, the method comprising contacting the cell with an anti-PD-Ll antibody, antigen binding fragment, or derivative thereof according to any one of claims 1-8, a composition according to claim 9, or a pharmaceutical composition according to claim 10.

16. A method of treating a disease, disorder, or condition, the method comprising

administering to a patient in need thereof a pharmaceutically effective amount of an anti- PD-Ll antibody, antigen binding fragment, or derivative thereof according to any one of claims 1-8, a composition according to claim 9, or a pharmaceutical composition according to claim 10.

17. A process for making an anti-PD-Ll antibody, antigen binding fragment, or derivative thereof comprising culturing the host cell of claim 14 under conditions suitable for the expression of the expression vector encoding the anti-PD-Ll antibody, antigen binding fragment, or derivative thereof; and recovering the anti-PD-Ll antibody, antigen binding fragment, or derivative thereof.

18. A method for detecting PD-L1 in a biological sample, the method comprising:

(a) contacting the biological sample with an anti-PD-Ll antibody, antigen binding fragment, or derivative thereof according to any one of claims 1-8, a composition according to claim 9, or a pharmaceutical composition according to claim 10 under conditions that allow binding of the anti-PD-Ll antibody, antigen binding fragment, or derivative thereof to a PD-L1 molecule to form an immunocomplex;

(b) detecting the immunocomplex to detect the presence of PD-L1.

19. The method of claim 18, wherein the anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, is immobilized to a solid support and the method further comprises after step (b), the step of:

(c) separating an immunocomplex of immobilized anti-PD-Ll antibody, or antigen binding fragment or derivative thereof, bound to PD-L1 from the biological sample.

20. The method of claim 18 or 19, further comprising before step (b), the step of: (a)’ contacting the sample from (a) with a detectable antibody that is capable of binding to the immunocomplex.

21. The method of claim 20, wherein the detectable antibody, is directly detectable, or is conjugated to horseradish peroxidase, alkaline phosphatase, glucose oxidase, or b- galactosidase; or is detected by a fluorometric or colorimetric reagent.

22. The method of claim 20 or 21, wherein the detectable antibody is an antibody from a non human species that binds to human or humanized antibodies.

23. The method of any one of claims 18-22, wherein the biological sample is isolated from a human subj ect.

24. A kit comprising:

(i) an anti-PD-Ll antibody, antigen binding fragment, or derivative thereof according to any one of claims 1-8;

(ii) a composition according to claim 9;

(iii) a pharmaceutical composition according to claim 10;

(iv) a diagnostic composition according to claim 11;

(v) a nucleic acid according to claim 12;

(vi) an expression vector according to claim 13; and/or

(vii) a host cell according to claim 14; and

an additional reagent and/or pharmaceutical delivery device.

25. The kit of claim 24, wherein the kit is an immunoassay kit for specifically detecting PD- Ll in a biological sample, which comprises:

(a) an anti-PD-Ll antibody, antigen binding fragment, or derivative thereof according to any one of claims 1-8, a composition according to any one of claims 9-10;

(b) a detectable antibody that binds to the anti-PD-Ll antibody, antigen binding fragment, or derivative thereof; and

(c) instructions for detecting said detectable antibody.

26. The kit of claim 25, wherein the kit is for use in an immunoassay for detecting PD-L1.