Biopharmaceutical compositions and related methods

Abstract

Compositions comprising anti-PD-1 antibodies and related methods for treating cancer and other disorders responsive to PD-1 antagonism are provided.SOLUTION: A composition comprising an oxidized variant of an anti-PD-1 antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence comprising CDRH1-3 each consisting of a specific amino acid sequence and a light chain amino acid sequence comprising CDRL1-3 each consisting of a specific amino acid sequence; wherein the composition comprises no more than 65% oxidized variants.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to compositions comprising anti-PD-1 antibodies and related methods for treating cancer or infectious conditions and disorders. [Background technology]

[0003] Background of the Invention Programmed death 1 (PD-1), also known as programmed cell death 1, is a 268-amino acid type I transmembrane protein that was first identified by subtractive hybridization of murine T cell lines undergoing apoptosis. PD-1 is a member of the CD28 / CTLA-4 family of T cell regulators and is expressed on activated T cells, B cells, and myeloid cells.

[0004] Two ligands for PD-1, PD ligand 1 (PD-L1) and PD ligand 2 (PD-L2), have been identified, both of which belong to the B7 protein superfamily. PD-L1 is expressed on various cell types, including cells of the lung, heart, thymus, spleen, and kidney. PD-L1 expression is upregulated on macrophages and dendritic cells (DCs) in response to lipopolysaccharide (LPS) and GM-CSF treatment, and on T cells and B cells upon signaling through the T cell and B cell receptors. PD-L1 is also expressed on various murine tumor cell lines. In contrast, PD-L2 shows a more restricted expression pattern, primarily expressed by antigen-presenting cells (e.g., dendritic cells or macrophages) and some tumor cell lines. High PD-L1 expression in tumors, whether on tumor cells, stroma, or other cells within the tumor microenvironment, correlates with poor clinical prognosis, likely by inhibiting effector T cells and upregulating intratumoral regulatory T cells (Tregs).

[0005] PD-1 negatively regulates T cell activation, and this inhibitory function is linked to an immunoreceptor tyrosine-based switch motif (ITSM) in its cytoplasmic domain. PD-1 deficiency can also lead to autoimmunity. In humans, single nucleotide polymorphisms in the PD-1 gene are associated with a higher incidence of systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, and multiple sclerosis progression. Aberrant PD-1 expression has also been linked to impaired T cell function in several pathologies, such as tumor immune evasion and chronic viral infections.

[0006] Previous studies have shown that PD-1-induced T cell suppression also plays a role in suppressing antitumor immunity. For example, PD-L1 is expressed on a variety of human and mouse tumors, and PD-1 binding to PD-L1 on tumors results in T cell suppression and immune evasion and protection of the tumor. PD-L1 expression by tumor cells is directly correlated with resistance to lysis by antitumor T cells in vitro. PD-1 knockout mice are resistant to tumor challenge, and T cells from PD-1 knockout mice are highly effective in tumor rejection when adoptively transferred into tumor-bearing mice. Blocking PD-1 inhibitory signals using monoclonal antibodies can enhance host antitumor immunity in mice, and high levels of PD-L1 expression on tumors are associated with poor prognosis in many human cancer types.

[0007] In light of the above, strategies have been developed to inhibit PD-1 activity to treat various types of cancer and to enhance immunity (e.g., to treat infectious diseases). In this regard, monoclonal antibodies targeting PD-1 have been developed for cancer treatment. For example, nivolumab (also known as Opdivo) is a human IgG4 monoclonal antibody against PD-1 and is commercially available for the treatment of melanoma, non-small cell lung cancer, or kidney (renal cell) cancer. As another example, pembrolizumab (Keytruda) is a humanized IgG4 monoclonal antibody against PD-1 and is commercially available for the treatment of a range of cancers, including non-small cell lung cancer, head and neck cancer, melanoma, and Hodgkin's lymphoma. Furthermore, recent evidence suggests that therapies targeting PD-1 may enhance immune responses against pathogens such as HIV. Anti-PD-1 antibodies are described in WO2014 / 179664, WO2018 / 085468 and WO2018 / 129559. Summary of the Invention

[0008] Summary of the Invention According to one aspect of the invention, there is provided a composition comprising an oxidized variant of an anti-PD-1 antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 65% oxidized variant.

[0009] According to a further aspect of the invention, there is provided a composition comprising aggregation variants of an anti-PD-1 antibody, said aggregation variants comprising a heavy chain sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 36% aggregation variants.

[0010] According to a further aspect of the present invention, there is provided a composition comprising an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises (i) no more than 65% oxidation variants; and / or (ii) no more than 36% aggregation variants.

[0011] According to a further aspect of the invention, there is provided a composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 100% acidic variants; and / or no more than 35% basic variants; and / or 1% or more of the major isoform.

[0012] According to a further aspect of the present invention, there is provided a composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; the composition comprises 10 to 97% acidic variants; and / or 0.1 to 35% basic variants; and / or 2 to 80% of the major isoform.

[0013] According to a further aspect of the invention, there is provided a composition comprising an anti-PD-1 antibody charge variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises 35% or less acidic variants; and / or 5% or less basic variants; and / or 55% or more major isoforms.

[0014] According to a further aspect of the present invention, there is provided a composition comprising an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO: 13, and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises no more than 64% oxidation variants.

[0015] According to a further aspect of the present invention, there is provided a composition comprising an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO: 13, and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises no more than 36% aggregation variants.

[0016] According to a further aspect of the invention, there is provided a composition comprising a variant of an anti-PD-1 antibody, said variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition has at least 60% of the potency of a composition comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, 10 to 97% acidic variants, 0.1 to 35% basic variants, 2 to 80% major isoforms, 4.8% or less light chain W50 oxidation variants, 1% or less heavy chain M34 oxidation variants, 1.2% or less heavy chain M103 oxidation variants, 15.2% or less light chain W50 oxidation variants, 1% or less heavy chain M34 oxidation variants, 1.2% or less heavy chain M103 oxidation variants, 1.2% or less light chain M104 oxidation variants, 1.2% or less light chain M105 oxidation variants, 1.2% or less light chain M106 oxidation variants, 1.2% or less light chain M107 oxidation variants, 1.2% or less light chain M108 oxidation variants, 1.2% or less light chain M109 ... % or less aggregation mutants, 16.7% or less heavy chain M354 oxidation mutants, 29.0% or less heavy chain M424 oxidation mutants, 47.1% or less heavy chain M248 oxidation mutants, 20.8% or less heavy chain D147 isomerization mutants, 13.1% or less heavy chain D151 or D167 isomerization mutants, 3.1% or less heavy chain D261, D266 or D276 isomerization mutants, 4.6% or less fragmentation mutants, 27.8% or less heavy chain N380 deamidation mutants, 27.2% or less heavy chain N385 deamidation mutants, about 7.4% or less heavy chain N311 deamidation mutants, about 2.0% or less heavy chain N430 deamidation mutants, 90% or more heavy chain C-terminal lysine deletion mutants (ΔK443), and 1% or less heavy chain N-terminal pyroglutamic acid mutants.

[0017] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a composition described herein and at least one pharmaceutically acceptable excipient.

[0018] According to a further aspect of the present invention, there is provided a formulation comprising the pharmaceutical composition described herein, the formulation comprising about 20 mg / mL to about 125 mg / mL of an antibody and a buffer having a pH of about 5.5 to about 6.5.

[0019] According to a further aspect of the present invention, there is provided a formulation comprising the pharmaceutical composition described herein, comprising: (a) about 20 mg / mL to about 125 mg / mL of an antibody; (b) about 10 mM to about 40 mM citrate buffer or histidine buffer; (c) about 80 mM to about 120 mM arginine or about 2 to about 10% w / v trehalose; (d) about 20 mM to about 40 mM sodium chloride; and (e) about 0.01% to about 0.1% w / v polysorbate 80 at a pH of about 5.5 to about 6.5.

[0020] According to a further aspect of the present invention there is provided an injection device comprising a composition as described herein. According to a further aspect of the present invention there is provided an injection device comprising a pharmaceutical composition as described herein. According to a further aspect of the present invention there is provided an injection device comprising a formulation as described herein.

[0021] According to a further aspect of the present invention, there is provided a cell culture medium comprising the composition described herein.

[0022] According to a further aspect of the present invention, there is provided an eluate comprising the composition described herein.

[0023] According to a further aspect of the present invention, there is provided a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a composition described herein. According to a further aspect of the present invention, there is provided a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition described herein. According to a further aspect of the present invention, there is provided a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a formulation described herein.

[0024] According to a further aspect of the present invention there is provided a composition as described herein for use in therapy. According to a further aspect of the present invention there is provided a composition as described herein for use in therapy. According to a further aspect of the present invention there is provided a formulation as described herein for use in therapy.

[0025] According to a further aspect of the present invention there is provided a composition as described herein for use in the treatment of cancer. According to a further aspect of the present invention there is provided a pharmaceutical composition as described herein for use in the treatment of cancer. According to a further aspect of the present invention there is provided a formulation as described herein for use in the treatment of cancer.

[0026] According to a further aspect of the present invention there is provided the use of a composition as described herein in the manufacture of a medicament for use in the treatment of cancer. According to a further aspect of the present invention there is provided the use of a pharmaceutical composition as described herein in the manufacture of a medicament for use in the treatment of cancer. According to a further aspect of the present invention there is provided the use of a formulation as described herein in the manufacture of a medicament for use in the treatment of cancer. [Brief explanation of the drawings]

[0027] [Figure 1] Representative cIEF electropherograms of dostarlimab (A: full scale, and B: zoomed scale). DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description of the Invention General definition The invention described herein provides compositions comprising anti-programmed death 1 (PD-1) antibodies and related methods of treatment via inhibition of PD-1 activity. "Programmed death 1 (PD-1) antibody" refers to an antibody that specifically binds to the programmed death 1 protein (PD-1). A composition comprising an anti-PD-1 antibody as described herein may also refer to a population of anti-PD-1 antibodies as described herein, and it will be understood that such phrases are interchangeable. In one embodiment, the PD-1 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:9 and a light chain having the amino acid sequence set forth in SEQ ID NO:10.

[0029] The term "antibody," as used herein, broadly refers to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of this type.

[0030] The terms complete antibody, whole antibody, or intact antibody are used interchangeably herein to refer to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 daltons. Intact antibodies are composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains, including two antigen-binding fragments known as the "Fab" fragment and the "Fc" crystallizable fragment. The Fab fragment consists of an amino-terminal variable region, a variable heavy chain (VH) or variable light chain (VL), and a carboxyl-terminal constant region, CH1 (heavy chain) and CL (light chain). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. The Fc can trigger effector functions by binding to receptors on immune cells or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by different heavy chain amino acid sequences, called μ, α, γ, ε, and δ, respectively, and each heavy chain can pair with either κ or λ light chains. Most antibodies in serum belong to the IgG class, and human IgG has four isotypes: IgG1, IgG2, IgG3, and IgG4, which differ primarily in the hinge region.

[0031] Fully human antibodies can be obtained using a variety of methods, such as using yeast-based libraries or transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies. Yeast displaying human antibodies that bind to the antigen of interest can be selected using FACS (fluorescence-activated cell sorting)-based methods or by capturing the antigen on beads using labeled antigens. Transgenic animals engineered to express human immunoglobulin genes can be immunized with the antigen of interest, and antigen-specific human antibodies can be isolated using B cell sorting techniques. Human antibodies produced using these techniques can then be characterized for desired properties such as affinity, developability, and selectivity.

[0032] Monoclonal antibodies can be produced by eukaryotic or prokaryotic cell clones expressing a single antibody. Monoclonal antibodies can also be produced by eukaryotic cell lines that can recombinantly express antibody heavy and light chains by having nucleic acid sequences encoding them introduced into the cells. Exemplary methods for producing antibodies from different eukaryotic cell lines, such as Chinese hamster ovary cells, hybridomas, or immortalized antibody cells derived from animals (e.g., humans), are well known to those skilled in the art.

[0033] Antibodies may be derived from, for example, rat, mouse, primate (e.g., cynomolgus monkey, Old World monkey or great ape), human, or other sources, such as nucleic acids encoding antibody molecules generated using molecular biology techniques known to those skilled in the art.

[0034] The antibody may comprise a constant region, which may be of any isotype or subclass. The constant region may be of an IgG isotype, such as IgG1, IgG2, IgG3, IgG4, or a variant thereof. In one embodiment, the antibody comprises a constant region derived from IgG4. In one embodiment, a single serine-to-proline point mutation is present in the hinge region of the IgG4 heavy chain for the purpose of hinge stabilization. This mutation is at canonical position S228 (Kabat numbering), which corresponds to residue 224 of the full-length heavy chain sequence when numbered consecutively (SEQ ID NO: 9).

[0035] The antibody can be either fully human, humanized, or chimeric. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a monoclonal antibody.

[0036] The antibody may contain one or more modifications including, for example, a mutated constant domain so that the antibody has enhanced effector function / ADCC and / or complement activation.

[0037] An antibody may comprise two immunoglobulin (Ig) heavy chains ("HC") and two Ig light chains ("LC"). The basic antibody structural unit may comprise, for example, a tetramer of subunits. Each tetramer may comprise two pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain may comprise a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region may initially be expressed linked to a cleavable signal peptide. A variable region without a signal peptide is sometimes referred to as a mature variable region. Thus, in one example, a light chain mature variable region may comprise a light chain variable region without a light chain signal peptide. The carboxy-terminal portion of each chain may define a constant region. In one embodiment, the antibodies of the compositions described herein are full-length antibodies.

[0038] The terms "VH" and "VL" are used herein to refer to the heavy and light chain variable regions, respectively, of an antibody.

[0039] The mature variable regions of each light / heavy chain pair can form an antibody binding site (also called an antigen-binding site). An "antigen-binding site" refers to the site on an antibody that can specifically bind to an antigen, which can be a single variable domain or a paired VH / VL domain as found in a standard antibody. Thus, an intact antibody, for example, can have two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites can be the same. All chains can exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity-determining regions or "CDRs." The CDRs of the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. Thus, in one example, both the light and heavy chains comprise, from N- to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0040] Acceptable heavy chain variable region and light chain variable region framework 1, framework 2, and framework 3 regions are readily recognized by those skilled in the art. Acceptable heavy chain constant region (including hinge) regions and light chain constant region regions are also readily recognized by those skilled in the art. Acceptable antibody isotypes are also readily recognized by those skilled in the art.

[0041] "CDR" is defined as the amino acid sequence of an antibody's complementarity-determining region. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDR," as used herein, refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0042] Throughout this specification, the terms "CDR," "CDRL1," "CDRL2," "CDRL3," "CDRH1," "CDRH2," and "CDRH3" follow the Kabat numbering convention. Amino acid residues in variable region sequences and full-length antibody sequences are numbered consecutively to represent any antibody variant positions or post-translationally modified variant positions, such as oxidation variants (e.g., W50), deamidation variants (e.g., N380), or isomerization variants (e.g., D147).

[0043] Those skilled in the art will appreciate that there are alternative numbering conventions for amino acid residues in variable region sequences and full-length antibody sequences. CDR sequences also have alternative numbering conventions, such as those described by Chothia et al. (1989) Nature 342: 877-883. The structure and protein folding of antibodies may mean that other residues are considered part of the CDR sequence and are understood as such by those skilled in the art. Other numbering conventions for CDR sequences available to the skilled artisan include the "AbM" (University of Bath) and "contact" (University of London) systems. Table 1 below presents one definition using each numbering convention for each CDR or binding unit. Table 1 uses the Kabat numbering scheme to number the amino acid sequences of the variable region. It should be noted that some of the CDR definitions may vary depending on the individual publication used.

[0044] [Table 1]

[0045] The terms "variant," "antibody variant," "CDR variant," and "post-translationally modified variant" refer to variant antibody sequences in which at least one amino acid sequence has been altered relative to the antibody sequence, for example, by post-translational modification, chemical change, or sequence alteration by at least one deletion, substitution, or addition. Some post-translational modifications result in a chemical change that does not alter the sequence (e.g., Met to oxidized Met; or Asp to isomerization / isoAsp; or aggregation), while others result in a sequence change, such as converting one amino acid residue to another (e.g., converting Asn to Asp by deamidation, or deleting lysine). Additional post-translationally modified variants are described below. Variant antibody sequences comprising sequence changes can be the result of engineered sequence changes or post-translational modifications.

[0046] Amino acid substitutions or replacements can be conservative, semi-conservative, or non-conservative. Amino acids are broadly classified as "aromatic" or "aliphatic." Aromatic amino acids contain an aromatic ring (e.g., histidine, phenylalanine, tyrosine, and tryptophan). Non-aromatic amino acids are broadly classified as "aliphatic."

[0047] In one embodiment, the substitution is conservative. It is well recognized in the art that certain amino acid substitutions are considered "conservative." Amino acids can be further divided into groups based on common side chain properties, and substitutions within a group that maintain all or substantially all of the antibody's binding affinity are considered conservative substitutions. For example, amino acid groups include amino acid residues with hydrophobic side chains such as methionine, alanine, valine, leucine, and isoleucine; amino acids with neutral, hydrophilic side chains such as cysteine, serine, and threonine; amino acids with acidic side chains such as aspartic acid and glutamic acid; amino acids with basic side chains such as asparagine, glutamine, histidine, lysine, and arginine; amino acids with residues that affect chain orientation such as glycine and proline; and amino acids with aromatic side chains such as tryptophan, tyrosine, and phenylalanine. The antibodies disclosed herein may contain such "conservative" amino acid substitutions. In another embodiment, antibody variants contain at least one substitution while retaining the canonical identity of the antibody.

[0048] "Semi-conservative mutations" include amino acid substitutions of amino acids within a broad group (i.e., aromatic or aliphatic), but not within the same side chain subgroup. For example, substitution of aspartic acid with asparagine, or asparagine with lysine, involves amino acids within the same group (i.e., aliphatic), but different subgroups. "Non-conservative mutations" include amino acid substitutions between different groups, e.g., substitution of lysine with tryptophan, or phenylalanine with serine.

[0049] In one embodiment, the antibody variant is an antibody that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical (i.e., has sequence identity) to the antibody primary sequence. In another embodiment, the antibody variant comprises an antibody comprising a heavy chain amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:9 and / or a light chain amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:10.

[0050] "Percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is an "identity" value expressed as a percentage, calculated over the entire length of the query sequence after a pairwise global sequence alignment is performed using an appropriate algorithm or software, such as BLASTN, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign. Importantly, the query sequence may be described by a nucleic acid sequence identified in one or more claims herein.

[0051] "Percent identity" between a query amino acid sequence and a subject amino acid sequence is an "identity" value expressed as a percentage, calculated over the entire length of the query sequence after a pairwise global sequence alignment has been performed using an appropriate algorithm or software such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign. Importantly, the query sequence may be described by an amino acid sequence identified in one or more claims herein.

[0052] The query sequence may be 100% identical to the subject sequence, or may contain up to a certain integer number of amino acid or nucleotide changes compared to the subject sequence, such that the percent identity is less than 100%. For example, the query sequence may be at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. Such alterations include deletions, substitutions (including conservative and non-conservative substitutions), or insertions of at least one amino acid, which may be at the amino- or carboxy-terminal positions of the query sequence, or may be interspersed among amino acids or nucleotides within the query sequence anywhere between these terminal positions, or individually in one or more contiguous groups within the query sequence.

[0053] The % identity can be determined over the entire length of the query sequence, including the CDRs. Alternatively, the % identity may exclude one or more or all of the CDRs, e.g., all of the CDRs are 100% identical to the subject sequence, with the % identity varying in the remainder of the query sequence, e.g., the framework sequences, and thus the CDR sequences remain fixed and intact.

[0054] Amino acid sequences useful in and that may be included in the compositions and related methods of the present disclosure may have about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100% identity to an amino acid sequence identified in this disclosure (e.g., an antibody heavy chain or antibody light chain). In this disclosure, the percent identity between the recited amino acid sequences may include any discrete subrange of the above percent identity range (e.g., any range of integer values ​​within a particular range or discrete subvalues ​​within a particular range).

[0055] The antibody specifically binds to the target antigen, human PD-1. Exemplary anti-PD-1 antibodies and methods for making them are disclosed in International Publication No. WO2014 / 179664, which is incorporated herein by reference in its entirety. Further exemplary anti-PD-1 antibodies include those described in WO2018 / 085468 and WO2018 / 129559, each of which is incorporated herein by reference in its entirety.

[0056] The term "specifically binds," as used herein with respect to antibodies, means that the antibody binds to a target antigen as well as a distinct domain or distinct amino acid sequence within the target antigen, and has no or little binding to other (e.g., unrelated) proteins. However, this term does not exclude the fact that antibodies may also cross-react with closely related molecules (e.g., those with a high degree of sequence identity or from another genus or species). The antibodies described herein may bind to human PD-1 with at least 2-, 5-, 10-, 50-, 100-, or 1000-fold greater affinity than they bind to closely related molecules.

[0057] Affinity, also referred to as "binding affinity," is the strength of binding at a single interaction site, i.e., a single binding site, of one molecule, e.g., an antibody, to another molecule, e.g., its target antigen. The binding affinity of an antibody to its target can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., surface plasmon resonance analysis using a BIACORE or similar instrument).

[0058] The binding affinity (K D ) can be, for example, about 1 picomolar (pM) to about 100 micromolar (mM) (e.g., about 1 picomolar (pM) to about 1 nanomolar (nM)), about 1 nM to about 1 micromolar (μM), or about 1 μM to about 100 μM. In some embodiments, the anti-PD-1 antibody has a K of 1 nanomolar or less (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values). D In some embodiments, the anti-PD-1 antibody can bind to the PD-1 protein with a K of 200 pM or less (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values). D can bind to PD-1.

[0059] Or, K. D The binding affinity of an antibody can be expressed as a binding constant (K a ) and dissociation constant (K d ) is determined by (K D =K d / K a) Binding affinity can be measured by BIACORE (surface plasmon resonance), for example, by capturing the test antibody on a protein A-coated sensor surface and flowing the target antigen over this surface. Alternatively, binding affinity can be measured by FORTEBIO, for example, by capturing the test antibody receptor on a protein A-coated needle and flowing the target antigen over this surface.

[0060] K d is 1 x 10 -3 Ms -1 It can be: K d is 1 x 10 -5 Ms -1 ~1×10 -3 Ms -1 ; or 1 × 10 -4 Ms -1 ~1×10 -3 Ms -1 It can be. K d A slower β-glucanation rate may result in slower dissociation of the antibody-target antigen complex, improving neutralization of the target antigen.

[0061] The term "specific antigen binding activity" as used herein refers to antigen binding activity as measured by surface plasmon resonance (SPR). PD-1 specific binding activity can be determined, for example, by SPR using a BIACORE instrument running in binding mode. This is the binding activity divided by the total protein (e.g., dostarlimab) content in the sample. The term "FcRn binding activity" as used herein refers to neonatal Fc (FcRn) receptor binding activity as measured by surface plasmon resonance (SPR). FcRn binding can be determined using a BIACORE instrument. This is the binding activity to the FcRn receptor divided by the total protein concentration of the sample.

[0062] The SPR method for specific antigen binding and FcRn binding uses a dostarlimab reference standard. The dostarlimab reference standard can be used in the assay to obtain system suitability and sample comparability data to confirm that the method is performing properly. The reference standard allows for the generation of a calibration curve from which sample concentrations are interpolated.

[0063] Potency is defined herein as the inhibitory activity of an anti-PD-1 antibody, or a composition as described herein, in inhibiting the binding of a ligand (PD-L1) to PD-1. This can be measured by specific binding to the antigen PD-1, by a potency assay (e.g., an MSD assay), or by a potency bioassay (e.g., a cellular assay). The potency assay can be a cellular competitive binding assay that measures the dose-dependent ability of an antibody or composition to inhibit the binding of a PD-L1 ligand to PD-1 constitutively expressed by cells. The potency bioassay can be a cell-based intracellular signaling bioassay that measures the dose-dependent ability of an antibody or composition to inhibit the binding of a PD-L1 ligand to PD-1, resulting in T cell receptor (TCR) activation and nuclear factor of activated T cell response element (NFAT-RE) activation. Results can be reported as a percentage of potency relative to a reference substance (e.g., a control sample).

[0064] A Meso Scale Discovery (MSD) potency assay can be used, comprising engineered CHO K1 cells that constitutively express the PD-1 protein. The activity of an antibody or composition can be assessed using competitive binding, which measures the dose-dependent ability of an antibody to inhibit ligand binding to PD-1 on CHO K1 cells. A ligand (PD-L1) is used as the ligand in the assay (PD-L1-mFc). Readouts can be measured using a specific detection antibody mixture that emits a quantifiable electrochemiluminescence (ECL) signal. Results can be reported as a percentage of potency relative to a reference substance (e.g., a control sample).

[0065] A cellular potency bioassay developed using the Promega PD-1 / PD-L1 Blockade Bioassay (Cat. No. J1250 or J1255) can be used. Specifically, PD-1 effector cells (Jurkat T cells (Promega #J1155)) expressing human PD-1 and a luciferase reporter gene driven by the nuclear factor of activated T cell response element (NFAT-RE) can be co-cultured with PD-L1-expressing PD-L1 artificial antigen-presenting cells (aAPCs) (CHO-K1 cells (Promega #J1095)). When the two cell types are co-cultured, PD-1 / PD-L1 interaction inhibits TCR signaling and the resulting NFAT-RE luminescence. Addition of an antibody or composition releases an inhibitory signal, leading to TCR activation and NFAT-RE-mediated luminescence. Blockade of this inhibitory signal is dose-dependent, and the resulting luminescence can be quantified using a plate reader. From the signal response, four-parameter curves can be generated for both the reference and antibody / composition samples by plotting relative luciferase units (RLU) on the y-axis against the log2-transformed concentration on the x-axis. Median effective concentration (EC50) values ​​can be interpolated to calculate the potency of the antibody / composition sample relative to that of the reference (e.g., control sample).

[0066] The terms "peptide," "polypeptide," "protein," and "peptide chain" each refer to a molecule containing two or more amino acid residues. Peptides can be monomeric or polymeric.

[0067] As used herein, the term "about" when referring to measurable values ​​such as amounts, durations, etc., is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1%, from the particular value, where such variations are appropriate to perform the disclosed methods.

[0068] Anti-PD-1 antibody The composition may comprise an anti-PD-1 antibody comprising one or more CDRs according to the invention described herein, or one or both of the heavy or light chain variable regions according to the invention described herein, or one or both of the heavy or light chains according to the invention described herein.

[0069] In one aspect, the composition comprises an antibody having a heavy chain sequence comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain sequence comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:4, a CDRL2 comprising the amino acid sequence of SEQ ID NO:5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6.

[0070] In one embodiment, the anti-PD-1 antibody comprises a heavy chain variable region CDR1 ("CDRH1") comprising an amino acid sequence having one or two amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:1 ("CDR variant").

[0071] In one embodiment, the anti-PD-1 antibody comprises a heavy chain variable region CDR2 ("CDRH2") ("CDR variant") comprising an amino acid sequence that has five or fewer, e.g., four or fewer, three or fewer, two or fewer, or one amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 2. In a further embodiment, CDRH1 comprises an amino acid sequence that has one or two amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 2.

[0072] In one embodiment, the anti-PD-1 antibody comprises a heavy chain variable region CDR3 ("CDRH3") comprising an amino acid sequence having one or two amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:3 ("CDR variant").

[0073] In one embodiment, the anti-PD-1 antibody comprises a light chain variable region CDR1 ("CDRL1") ("CDR variant") comprising an amino acid sequence having three or fewer amino acid mutations, e.g., one or two, relative to the amino acid sequence set forth in SEQ ID NO:4.

[0074] In one embodiment, the anti-PD-1 antibody comprises a light chain variable region CDR2 ("CDRL2") comprising an amino acid sequence having one or two amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:5 ("CDR variant").

[0075] In one embodiment, the anti-PD-1 antibody comprises a light chain variable region CDR3 ("CDRL3") ("CDR variant") comprising an amino acid sequence having three or fewer amino acid mutations, e.g., one or two, relative to the amino acid sequence set forth in SEQ ID NO:6.

[0076] In one embodiment, the anti-PD-1 antibody comprises a CDRH1 comprising an amino acid sequence that has up to one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO:1; a CDRH2 comprising an amino acid sequence that has up to five amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 comprising an amino acid sequence that has up to one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO:3; a CDRL1 comprising an amino acid sequence that has up to three amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:4; a CDRL2 comprising an amino acid sequence that has up to one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO:5; and / or a CDRL3 comprising an amino acid sequence that has up to three amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:6.

[0077] In one embodiment, the anti-PD-1 antibody comprises a heavy chain variable region ("VH") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the VH comprises an amino acid sequence having at least one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO: 7, e.g., 1 to 5, e.g., 1 to 3, particularly up to 2 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 7.

[0078] In one embodiment, the anti-PD-1 antibody comprises a light chain variable region ("VL") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In one embodiment, the VL comprises an amino acid sequence having at least one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO: 8, e.g., 1 to 5, e.g., 1 to 3, particularly up to 2 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 8.

[0079] In one embodiment, the anti-PD-1 antibody comprises a VH having the amino acid sequence set forth in SEQ ID NO:7; and a VL having the amino acid sequence set forth in SEQ ID NO:8.

[0080] In one embodiment, the anti-PD-1 antibody comprises a VH comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and a VL comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0081] In one embodiment, the anti-PD-1 antibody comprises a heavy chain sequence ("HC") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In one embodiment, the HC comprises an amino acid sequence having at least one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO: 9, e.g., 1 to 10, e.g., 1 to 7, particularly up to 6 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 9. In a further embodiment, the HC comprises 1, 2, 3, 4, 5, 6, or 7 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 9.

[0082] In one embodiment, the anti-PD-1 antibody comprises a light chain region ("LC") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In one embodiment, the LC comprises an amino acid sequence having at least one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO: 10, e.g., 1 to 10, e.g., 1 to 5, particularly up to 3 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 10. In a further embodiment, the LC comprises 1, 2, or 3 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 10.

[0083] In one embodiment, the anti-PD-1 antibody comprises an HC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; and an LC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. Thus, the antibody is an antibody having a heavy chain at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or a light chain at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 10.

[0084] In one embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10. In one embodiment, the antibody is dostarlimab, comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10.

[0085] Post-translational modification products Those skilled in the art will recognize that post-translational modifications can occur during antibody production, producing post-translationally modified products. A "post-translationally modified variant" of an antibody described herein is an antibody composition in which all or part of the composition comprises a "post-translational modification." A post-translational modification is a chemical change in the antibody that can result from the production of the antibody in a host cell, upstream and / or downstream manufacturing processes, and / or the length and conditions of storage (e.g., the effects of temperature, pH, water, exposure to light, or reaction with excipients and / or excipient container closure systems). Thus, a composition of the invention can be formed from the "production or storage" of an antibody of the invention. Exemplary post-translational modifications include alteration of the antibody sequence ("antibody variants" as described above), cleavage of specific leader sequences, addition of various sugar moieties in various glycosylation patterns including non-enzymatic glycosylation or glycation; deamidation; oxidation; disulfide bond scrambling and other cysteine ​​variants, e.g., free sulfhydryls, racemic disulfides, thioethers, and trisulfide bonds; isomerization; truncation or clipping of C-terminal lysines; and / or N-terminal glutamine cyclization.

[0086] In one example, post-translationally modified products comprise "product-related impurities" that comprise chemical changes that result in a decrease in function and / or activity. In another example, post-translationally modified products comprise "product-related substances" that comprise chemical changes that do not result in a decrease in function and / or activity. Product-related impurities of the anti-PD-1 antibodies described herein include oxidation variants and aggregation variants. Product-related substances of the anti-PD-1 antibodies described herein include deamidation variants, isomerization variants, C-terminal truncation variants, and N-terminal pyroglutamate variants.

[0087] In one embodiment, the anti-PD-1 antibody is dostarlimab, which comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:9 and a light chain having the amino acid sequence set forth in SEQ ID NO:10, including all functional post-translational modifications thereof.

[0088] The percent variants provided herein are expressed as a percentage of the total amount of antibody in a composition (e.g., a "population" of antibodies). For example, a 65% or less oxidized variant is in the context of 100% total antibody in a composition, of which 65% or less is oxidized, this does not include any other non-antibody material present in the composition, which may or may not be oxidized.

[0089] Antibody variants are commonly found when the composition of the antibody is analyzed by charge-based separation techniques such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX).

[0090] Post-translational modifications can increase or decrease the net charge of an antibody, resulting in a decrease or increase in pI value, thereby resulting in acidic and basic variants (collectively referred to as "charge variants") of the major isoform. A "major isoform" is an antibody population that elutes as a major peak on a chromatogram. When antibodies are analyzed using IEF-based methods, acidic species are variants with a low apparent pI, and basic species are variants with a high apparent pI. When analyzed by chromatography-based methods, acidic and basic species are defined based on their retention time relative to the major peak. Acidic species are variants that elute earlier than the major CEX peak or later than the major AEX peak, and basic species are variants that elute later than the major CEX peak or earlier than the major AEX peak. These methods separate the major isoform of an antibody from the acidic and basic isoforms (acidic variants) and basic isoforms (basic variants).

[0091] Charge variants can be detected by various methods, such as ion exchange chromatography (e.g., WCX-10 HPLC (weak cation exchange chromatography)) or IEF (isoelectric focusing). The percent charge variant can be determined using capillary isoelectric focusing (cIEF). Capillary isoelectric focusing (cIEF) was used to measure the pI of dostarlimab and separate the charge variants (see Figure 1). Using this method, acidic and basic species can be quantified as a percentage of the total area peak. The terms "species," "isoform," "form," and "peak" are used interchangeably to refer to the major isoform and charge variants (acidic and basic variants).

[0092] In one aspect, a composition comprises an acidic variant of an anti-PD-1 antibody, wherein the acidic variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 100% of the acidic variant.

[0093] In one embodiment, the composition comprises an acidic variant of an anti-PD-1 antibody, wherein the acidic variant comprises a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:7 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:8; the composition comprises no more than 100% of the acidic variant.

[0094] In another embodiment, the composition comprises an acidic variant of an anti-PD-1 antibody, wherein the acidic variant comprises a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:10; and the composition comprises no more than 100% of the acidic variant. In yet a further embodiment, the composition comprises an oxidized variant of an anti-PD-1 antibody, wherein the acidic variant comprises a heavy chain sequence of SEQ ID NO:9 and a light chain sequence of SEQ ID NO:10; and the composition comprises no more than 100% of the acidic variant. In one embodiment, the composition comprises an acidic variant of dostarlimab, wherein the acidic variant comprises a heavy chain sequence of SEQ ID NO:9 and a light chain sequence of SEQ ID NO:10; and the composition comprises no more than 100% of the acidic variant.

[0095] In one aspect, the composition comprises 100% or less of the acidic variant. In one embodiment, the composition comprises 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 35% or less, 30% or less, or 25% or less of the acidic variant. Alternatively, the composition comprises 5-100%, 5-90%, 5-80%, 5-70%, 5-60%, 5-50%, 5-40%, 5-35%, 5-30%, or 5-25% of the acidic variant. Alternatively, the composition comprises 10-100%, 10-97%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-35%, 10-30%, or 10-25% of the acidic variant. Alternatively, the composition comprises 20-100%, 20-97%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-35%, 20-30%, or 20-25% of the acidic variant. Alternatively, the composition comprises about 60%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, or about 10% of the acidic variant.

[0096] In one aspect, the composition comprises basic variants of an anti-PD-1 antibody, wherein the basic variants comprise a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 35% basic variants.

[0097] In one embodiment, the composition comprises a basic variant of an anti-PD-1 antibody, wherein the basic variant comprises a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:7 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:8; the composition comprises 35% or less of the basic variant. In one embodiment, the composition has a bioassay potency that is at least 60% of the reference standard bioassay potency.

[0098] In another embodiment, the composition comprises a basic variant of an anti-PD-1 antibody, wherein the basic variant comprises a heavy chain at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and / or a light chain at least about 90% identical to the amino acid sequence of SEQ ID NO:10; and the composition comprises 35% or less of the basic variant. In yet a further embodiment, the composition comprises a basic variant of an anti-PD-1 antibody, wherein the basic variant comprises the heavy chain sequence of SEQ ID NO:9 and the light chain sequence of SEQ ID NO:10; and the composition comprises 35% or less of the basic variant. In one embodiment, the composition comprises a basic variant of dostarlimab, wherein the basic variant comprises the heavy chain sequence of SEQ ID NO:9 and the light chain sequence of SEQ ID NO:10; and the composition comprises 35% or less of the basic variant.

[0099] In one aspect, the composition comprises 35% or less of the basic variant. In one embodiment, the composition comprises 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 8% or less, 7.5% or less, 7% or less, 6% or less, or 5% or less of the basic variant. In one embodiment, the composition comprises 0.1-35%, 0.1-30%, 0.1-25%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-8%, 0.1-7.5%, 0.1-7%, 0.1-6%, or 0.1-5% of the basic variant. In one embodiment, the composition comprises 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 1-8%, 1-7.5%, 1-7%, 1-6%, or 1-5% of the basic variant. Alternatively, the composition comprises about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 7.5%, or about 5% of the basic variant.

[0100] In one aspect, a composition comprises a major isoform of an anti-PD-1 antibody, the major isoform comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; the composition comprises 1% or more of the major isoform.

[0101] In one embodiment, the composition comprises a major isoform of an anti-PD-1 antibody, wherein the major isoform comprises a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:7 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO:8; the composition comprises 1% or more of the major isoform.

[0102] In another embodiment, the composition comprises a major isoform of an anti-PD-1 antibody, wherein the major isoform comprises a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 9 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 10; and the composition comprises 1% or more of the major isoform. In yet a further embodiment, the composition comprises a major isoform of an anti-PD-1 antibody, wherein the major isoform comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10; and the composition comprises 1% or more of the major isoform. In one embodiment, the composition comprises a major isoform of dostarlimab, wherein the major isoform comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10; and the composition comprises 1% or more of the major isoform.

[0103] In one aspect, the composition comprises 1% or more of the major isoform. In one embodiment, the composition comprises 2.6% or more, 3% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 90% or more of the major isoform. In one embodiment, the composition comprises 2-90%, 2-80%, 2-75%, 5-90%, 10-90%, 20-90%, 30-90%, 40-90%, 50-90%, or 60-90% of the major isoform. In one embodiment, the composition comprises 5-80%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, or 60-80% of the major isoform. Alternatively, the composition comprises about 80%, about 75%, about 70%, about 65%, about 60%, about 50% or about 55% of the major isoform.

[0104] The percent acidic variant, percent basic variant, and percent major isoform can be determined using capillary isoelectric focusing (cIEF). It is understood that these isoform / charge variant embodiments may be combined with any one or combination of the antibody variants described herein.

[0105] In one aspect, a composition comprises charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 100% acidic variants; and / or no more than 35% basic variants; and / or 1% or more of the major isoform.

[0106] In another aspect, a composition comprises charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; the composition comprises 10-97% acidic variants; and / or 0.1-35% basic variants; and / or 2-80% of the major isoform.

[0107] In another aspect, a composition comprises charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 35% acidic variants; and / or no more than 5% basic variants; and / or no less than 55% major isoforms.

[0108] In another aspect, a composition comprises charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; the composition comprises 10-30% acidic variants; and / or 0.1-10% basic variants; and / or 60-80% of the major isoform.

[0109] In one aspect, a composition comprises charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a VH of SEQ ID NO:7 and a light chain amino acid sequence comprising a VL of SEQ ID NO:8; the composition comprises 100% or less acidic variants, and / or 35% or less basic variants, and / or 1% or more of the major isoform. In one embodiment, the composition comprises 10-97% acidic variants, and / or 0.1-35% basic variants, and / or 2-80% of the major isoform. In another embodiment, the composition comprises 10-30% acidic variants, and / or 0.1-10% basic variants, and / or 60-80% of the major isoform. In a further embodiment, the composition comprises 35% or less acidic variants, and / or 5% or less basic variants, and / or 55% or more of the major isoform. In one aspect, a composition comprises a charge variant of an anti-PD-1 antibody comprising the heavy chain amino acid sequence of SEQ ID NO:9 and the light chain amino acid sequence of SEQ ID NO:10; the composition comprises 100% or less acidic variants; and / or 35% or less basic variants; and / or 1% or more of the major isoform. In one embodiment, the composition comprises 10-97% acidic variants; and / or 0.1-35% basic variants; and / or 2-80% of the major isoform. In another embodiment, the composition comprises 10-30% acidic variants; and / or 0.1-10% basic variants; and / or 60-80% of the major isoform. In a further embodiment, the composition comprises 35% or less acidic variants; and / or 5% or less basic variants; and / or 55% or more of the major isoform. In one embodiment, the composition has a bioassay potency that is at least 60% of the reference standard bioassay potency.

[0110] Oxidation can occur during manufacturing and / or storage (i.e., in the presence of oxidizing conditions) and results in covalent modification of proteins induced directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation can occur primarily through methionine residues, but can also occur at tryptophan and free cysteine ​​residues. Oxidation can occur in the CDRs, Fab (non-CDR) regions, or Fc regions.

[0111] In one aspect, the composition comprises an antibody comprising an oxidative post-translational modification ("oxidized" or "oxidized"), also referred to herein as an "oxidized variant." The variant may comprise oxidized amino acid residues in the heavy and / or light chain sequences, e.g., in the CDRs of the heavy chain sequence and / or the CDRs of the light chain sequence. The oxidized variant may be present in one or both of the heavy and light chains.

[0112] In one aspect, the composition comprises an oxidized variant of an anti-PD-1 antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 65% oxidized variant.

[0113] In one aspect, the composition comprises: an antibody having a heavy chain amino acid sequence comprising SEQ ID NO:1 (CDRH1), SEQ ID NO:2 (CDRH2), and SEQ ID NO:3 (CDRH3) and a light chain amino acid sequence comprising SEQ ID NO:4 (CDRL1), SEQ ID NO:5 (CDRL2), and SEQ ID NO:6 (CDRL3); and their oxidized variants, wherein no more than 65% of the antibody population is composed of oxidation variants.

[0114] In one embodiment, the oxidation variant comprises oxidation of methionine and / or tryptophan residues within the CDRs of the heavy chain sequence and / or the light chain sequence. In one embodiment, the oxidation variant comprises oxidation of methionine and / or tryptophan residues in any one of SEQ ID NOS: 1-6. In a further embodiment, the antibody comprises oxidation of methionine residues within the CDRs of the heavy chain sequence, such as CDRH1 and / or CDRH3. In a further embodiment, the antibody comprises oxidation of tryptophan residues within the CDRs of the light chain sequence, such as CDRL2. In some embodiments, the oxidation variant comprises one or a combination of oxidations at M34 of CDRH1, M103 of CDRH3, and / or W50 of CDRL2.

[0115] References to positions within a CDR (e.g., M34, M103, or W50) are understood to give the position number relative to the entire antibody sequence (consecutive numbering). Thus, M34 of CDRH1 corresponds to the fourth residue of SEQ ID NO: 1, i.e., the underlined SYD M S (SEQ ID NO: 1). Similarly, M103 of CDRH3 refers to the fourth residue of SEQ ID NO: 3, i.e., the underlined PYYA M DY (SEQ ID NO: 3), and W50 of CDRL2 refers to the first residue of SEQ ID NO: 5, i.e., the underlined W Refers to ASTLHT (SEQ ID NO: 5).

[0116] In one embodiment, the antibody comprises oxidation of methionine and / or tryptophan residues in the Fc region of the heavy chain sequence and / or the Fc region of the light chain sequence. In some embodiments, the oxidation variant comprises one or a combination of oxidations at M248, M354, and / or M424 in the Fc region of the heavy chain sequence.

[0117] In one aspect, a composition comprises an antibody that is at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or at least about 90% identical to the light chain sequence of SEQ ID NO: 10, and comprises an oxidation in the heavy chain sequence, e.g., an oxidation at amino acid M34 of CDRH1, M103 of CDRH3, M248 in the Fc region, M354 in the Fc region, and / or M424 in the Fc region. In one embodiment, a composition comprises an antibody that is at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or at least about 90% identical to the light chain sequence of SEQ ID NO: 10, and comprises an oxidation in the light chain sequence, e.g., an oxidation at amino acid W50 of CDRL2.

[0118] In one embodiment, the antibody comprises a heavy chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8. In a further embodiment, the antibody is at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 10. In yet a further embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10.

[0119] In one aspect, a composition comprises an anti-PD-1 antibody having a heavy chain sequence comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain sequence comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:4, a CDRL2 comprising the amino acid sequence of SEQ ID NO:5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; the composition comprises no more than 65% oxidation variants.

[0120] In one aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 8, wherein the composition comprises no more than 65% oxidation variants. In a further embodiment, a composition comprises an anti-PD-1 antibody comprising a heavy chain variable region of SEQ ID NO: 7 and / or a light chain variable region of SEQ ID NO: 8, wherein the composition comprises no more than 65% oxidation variants.

[0121] In one aspect, the composition comprises: an antibody having a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 8; and Their oxidized variants wherein no more than 65% of the antibody population is composed of oxidation variants.

[0122] In one aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and / or a light chain sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:10, wherein the composition comprises no more than 65% oxidation variants.

[0123] In one aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises no more than 65% oxidation mutants.

[0124] In one embodiment, the composition comprises an oxidized variant of dostarlimab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; the composition comprises no more than 65% oxidized variant.

[0125] In one embodiment, the composition comprises an oxidized variant of dostarlimab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; the composition comprises the oxidized variant in an amount ranging from 0.1% to 65%.

[0126] In one aspect, the composition comprises (a) an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and (b) an antibody having a heavy chain sequence at least 90% identical to SEQ ID NO: 9 and a light chain sequence at least 90% identical to SEQ ID NO: 10, wherein the composition comprises no more than 65% oxidized variants.

[0127] In one aspect, the composition comprises: an antibody having a heavy chain amino acid sequence as set forth in SEQ ID NO: 9 and a light chain amino acid sequence as set forth in SEQ ID NO: 10; and Their oxidized variants wherein no more than 65% of the antibody population is composed of oxidation variants.

[0128] In one aspect, the composition comprises: (a) an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; and (b) an antibody having a heavy chain sequence at least 90% identical to SEQ ID NO: 9 and a light chain sequence at least 90% identical to SEQ ID NO: 10; and (c) an oxidation variant of the antibody of (a) and / or (b), wherein the oxidation variant is selected from any one or combination of 34% or less oxidation at W50 of the light chain, 21% or less oxidation at M34 of the heavy chain, and / or 64% or less oxidation at M103 of the heavy chain.

[0129] In one aspect, the composition comprises 65% or less of the oxidation variant. In one embodiment, the composition comprises 65% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, or 3% or less of the oxidation variant. In one embodiment, the composition comprises 0.01-65%, 0.01-60%, 0.01-50%, 0.01-40%, 0.01-30%, 0.01-20%, 0.01-15%, 0.01-10%, 0.01-5%, 0.01-4%, or 0.01-3% of the oxidation variant. Alternatively, the composition comprises 0.05-65%, 0.05-60%, 0.05-50%, 0.05-40%, 0.05-30%, 0.05-20%, 0.05-15%, 0.05-10%, 0.05-5%, 0.05-4%, or 0.05-3% of the oxidation variant. Alternatively, the composition comprises 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, or 0.5-3% of the oxidation variant. Alternatively, the composition comprises 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, or 2-3% of the oxidized variant. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% of the oxidized variant. It will be understood that these oxidized variant embodiments may be combined with any of the antibody variants described herein.

[0130] In one embodiment, the composition comprises one or a combination of no more than 21% oxidation in M34 of CDRH1, no more than 64% oxidation in M103 of CDRH3, and / or no more than 34% oxidation in W50 of CDRL2. In another embodiment, the composition comprises one or a combination of no more than 16% oxidation in M34 of CDRH1, no more than 47% oxidation in M103 of CDRH3, and / or no more than 25% oxidation in W50 of CDRL2.

[0131] In one aspect, the composition comprises (a) an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and (b) an oxidized variant of the antibody selected from any one or combination of 34% or less oxidation at W50 of the light chain, 21% or less oxidation at M34 of the heavy chain, and / or 64% or less oxidation at M103 of the heavy chain.

[0132] In one embodiment, the composition comprises 34% or less oxidation of the W50 of the light chain sequence. In one embodiment, the composition comprises 34% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less oxidation of the W50 of the light chain sequence. Alternatively, the composition comprises 0-34%, 0-30%, 0-25%, 0-20%, 0-15%, 0-10%, 0-7.5%, 0-5%, 0-4%, 0-3%, 0-2%, or 0-1% oxidation of the W50 of the light chain sequence. In one embodiment, the composition comprises 0.01 to 34%, 0.01 to 30%, 0.01 to 25%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 7.5%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.01 to 2%, or 0.01 to 1% oxidation to W50 of the light chain sequence. In one embodiment, the composition comprises 0.05-34%, 0.05-30%, 0.05-25%, 0.05-20%, 0.05-15%, 0.05-10%, 0.05-7.5%, 0.05-5%, 0.05-4%, 0.05-3%, 0.05-2%, or 0.05-1% oxidation of the W50 of the light chain sequence. Alternatively, the composition comprises 0.5-34%, 0.5-30%, 0.5-25%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-7.5%, 0.5-5%, 0.5-4%, or 0.5-3%, 0.5-2%, or 0.5-1% oxidation of the W50 of the light chain sequence. Alternatively, the composition comprises 0.1% or more and 34% or less oxidation of the W50 of the light chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation of the W50 of the light chain sequence. As demonstrated by the data provided herein, photolytic forced degradation resulting in a maximum of 4.8% oxidation of the W50 provides 90% efficacy in the bioassay (i.e., within the range of assay variation to full functionality), and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) forced degradation resulting in a maximum of 38.9% oxidation of the W50 results in 44% efficacy (bioassay).Extrapolating from the AAPH-forced degradation data for oxidation at LC Trp 50, a maximum of 34% oxidation could yield at least 60% efficacy, and a maximum of 25% oxidation could yield at least 70% efficacy. This was calculated using the linear slope of AAPH-oxidized samples of <1% (control), <1% (TO), 38.9% (day 1), 74.3% (day 3), and 86.8% (day 5), with potencies of 98%, 102%, 44%, 14%, and 5%, respectively (bioassay).

[0133] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises no more than 34% oxidation in the W50 of the light chain sequence. In one embodiment, a composition comprises an oxidized variant of dostarlimab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; and the composition comprises no more than 34% oxidation in the W50 of the light chain sequence.

[0134] In one embodiment, the composition comprises 21% or less oxidation of M34 of the heavy chain sequence. In one embodiment, the composition comprises 21% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less oxidation of M34 of the heavy chain sequence. Alternatively, the composition comprises 0-21%, 0-20%, 0-16%, 0-15%, 0-12.5%, 0-10%, 0-7.5%, 0-5%, 0-4%, 0-3%, 0-2%, or 0-1% oxidation of M34 of the heavy chain sequence. In one embodiment, the composition comprises 0.01 to 21%, 0.01 to 20%, 0.01 to 16%, 0.01 to 15%, 0.01 to 12.5%, 0.01 to 10%, 0.01 to 7.5%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.01 to 2%, or 0.01 to 1% oxidation of M34 of the heavy chain sequence. Alternatively, the composition comprises 0.5 to 21%, 0.5 to 20%, 0.5 to 16%, 0.5 to 15%, 0.5 to 12.5%, 0.5 to 10%, 0.5 to 7.5%, 0.5 to 5%, 0.5 to 4%, 0.5 to 3%, 0.5 to 2%, or 0.5 to 1% oxidation of M34 of the heavy chain sequence. Alternatively, the composition comprises 0.1% or more and 21% or less oxidation of M34 of the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation of M34 of the heavy chain sequence. As shown by the data provided herein, forced HO degradation resulting in a maximum of 28.8% oxidation of M34 provides 47% efficacy in bioassays. Extrapolating from the HO degradation data for oxidation of HC Met 34, a maximum of 21% oxidation can provide at least 60% efficacy, and a maximum of 16% oxidation can provide at least 70% efficacy. This is calculated using the linear slope of HO oxidation samples of <1% (control), <1% (TO), and 28.8% (2-week HO), resulting in 98%, 94%, and 47% efficacy, respectively (bioassay). In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises no more than 21% oxidation to M34 of the heavy chain sequence.In one embodiment, the composition comprises an oxidized variant of dostarlimab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; and the composition comprises no more than 21% oxidation at M34 of the heavy chain sequence.

[0135] In one embodiment, the composition comprises 64% or less oxidation of M103 of the heavy chain sequence. In one embodiment, the composition comprises 64% or less, 60% or less, 50% or less, 47% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less oxidation of M103 of the heavy chain sequence. In one embodiment, the composition comprises 0 to 64%, 0 to 60%, 0 to 50%, 0 to 47%, 0 to 40%, 0 to 30%, 0 to 20%, 0 to 15%, 0 to 10%, 0 to 5%, 0 to 4%, 0 to 3%, 0 to 2%, or 0 to 1% oxidation of M103 of the heavy chain sequence. In one embodiment, the composition comprises 0.01 to 64%, 0.01 to 60%, 0.01 to 50%, 0.01 to 47%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.01 to 2%, or 0.01 to 1% oxidation of M103 of the heavy chain sequence. Alternatively, the composition may comprise 0.5-64%, 0.5-60%, 0.5-50%, 0.5-47%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, or 0.5-1% oxidation of M103 of the heavy chain sequence. Alternatively, the composition may comprise 0.1% or more but not more than 64% oxidation of M103 of the heavy chain sequence. Alternatively, the composition may comprise about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation of M103 of the heavy chain sequence. As demonstrated by the data provided herein, forced HO degradation resulting in up to 86.1% oxidation of M103 yields 47% efficacy in bioassays. Extrapolating from the H2O2 forced degradation data for oxidation in HC Met 103, a maximum of 64% oxidation could result in at least 60% efficacy, and a maximum of 47% oxidation could result in at least 70% efficacy. This was calculated using the linear slope of H2O2 oxidized samples of <1% (control), 1.2% (TO), and 86.1% (week 2 H2O2), with efficacy of 98%, 94%, and 47%, respectively (bioassay).

[0136] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises no more than 64% oxidation at M103 of the heavy chain sequence. In one embodiment, a composition comprises an oxidized variant of dostarlimab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; and the composition comprises no more than 64% oxidation at M103 of the heavy chain sequence.

[0137] In one embodiment, the composition comprises 65% or less oxidation of M248 in the heavy chain sequence. In one embodiment, the composition comprises 65% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, or 3% or less oxidation of M248 in the heavy chain sequence. In one embodiment, the composition comprises 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.01 to 2%, or 0.01 to 1% oxidation of M248 in the heavy chain sequence. Alternatively, the composition comprises 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, or 0.5-3% oxidation of M248 in the heavy chain sequence. Alternatively, the composition comprises 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, or 2-3% oxidation of M248 in the heavy chain sequence. Alternatively, the composition comprises 1% or more and 65% or less oxidation of M248 in the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation of M248 of the heavy chain sequence. As demonstrated by the data provided herein, forced HO degradation resulting in a maximum of 47.1% oxidation of M248 confers 94% potency in the bioassay (i.e., within the range of assay variation to full functionality), and forced photolytic degradation resulting in a maximum of 33.4% oxidation of M248 confers 90% potency in the bioassay (i.e., within the range of assay variation to full functionality). Therefore, it is expected that oxidation at M248 can be greater than 47.1% without affecting relative potency or FcRn binding.

[0138] In one embodiment, the composition comprises 65% or less oxidation of M354 in the heavy chain sequence. In one embodiment, the composition comprises 65% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less oxidation of M354 in the heavy chain sequence. In one embodiment, the composition comprises 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.01 to 2%, or 0.01 to 1% oxidation of M354 in the heavy chain sequence. Alternatively, the composition comprises 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, or 0.5-3% oxidation of M354 in the heavy chain sequence. Alternatively, the composition comprises 0.1% or more and 65% or less oxidation of M354 in the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation of M354 in the heavy chain sequence. As shown by the data provided herein, H2O2-forced degradation resulting in a maximum of 16.7% oxidation of M354 confers 94% potency in the bioassay (i.e., within the range of assay variation to full functionality), and photolytically-forced degradation of a maximum of 10.9% oxidation of M354 confers 90% potency in the bioassay (i.e., within the range of assay variation to full functionality). Therefore, it is expected that oxidation of M354 can be higher than 16.7% without affecting relative potency or FcRn binding.

[0139] In one embodiment, the composition comprises 65% or less oxidation of M424 in the heavy chain sequence. In one embodiment, the composition comprises 65% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less oxidation of M424 in the heavy chain sequence. In one embodiment, the composition comprises 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.01 to 2%, or 0.01 to 1% oxidation of M424 in the heavy chain sequence. Alternatively, the composition comprises 0-65%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%, 0-15%, 0-10%, 0-5%, 0-4%, or 0-3% oxidation of M424 in the heavy chain sequence. Alternatively, the composition comprises 0.1% or more and 65% or less oxidation of M424 in the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation of M424 in the heavy chain sequence. As demonstrated by the data provided herein, forced HO degradation resulting in a maximum 29.0% oxidation of M424 confers 94% potency in the bioassay (i.e., within the range of assay variation to full functionality), and forced photolysis degradation resulting in a maximum 27.0% oxidation of M424 confers 90% potency in the bioassay (i.e., within the range of assay variation to full functionality). Thus, it is expected that oxidation of M424 can be greater than 29.0% without affecting relative potency or FcRn binding.

[0140] In one embodiment, the composition comprises an oxidized variant of dostarlimab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; and the composition comprises no more than 65% oxidation at M248 and / or M354 and / or M424 of the heavy chain sequence.

[0141] In one aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 100% acidic variants; and / or no more than 35% basic variants; and / or 1% or more of the major isoform; and / or no more than 65% oxidized variants.

[0142] In another aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises 5-60% acidic variants; and / or 0.1-35% basic variants; and / or 20-90% major isoforms; and / or up to 65% oxidized variants.

[0143] In one example, oxidation can be determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). In one example, a sample comprising a composition described herein can be denatured with guanidine hydrochloride, reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and digested with endoproteinase Lys-C (Lys-C) or trypsin. Enzymatic digestion with either Lys-C or trypsin can be performed at 37°C for 4 hours. Sample digestion can be quenched with trifluoroacetic acid prior to liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis. The LC-MS / MS analysis system can employ reversed-phase ultra-high performance liquid chromatography (UHPLC) with a C18 column, UV detection at 214 nm, and electrospray ionization mass spectrometry (ESI-MS). Peptides can then be detected using a UV detector and a mass spectrometer (e.g., a Thermo Scientific LTQ Orbitrap XL). Extracted ion chromatograms of the unmodified and modified peptides are used to calculate the oxidation level by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0144] In one aspect, the composition comprises an antibody that is an aggregated antibody (high molecular weight (HMW) species), also referred to herein as an "aggregation variant." Aggregated antibodies may include dimers or higher-order structures formed from an antibody monomer and its subunits. Thus, high molecular weight (HMW) species may consist of dimerized antibodies and monomers with additional subunits (e.g., a monomer with two light chain subunits, or an LC-LC dimer noncovalently bound to a monomer). Aggregation variants can be, for example, covalent or noncovalent, reducible or nonreducible, and visible or invisible aggregates of the antibodies disclosed herein. Aggregation variants or fragmentation variants can be characterized based on their size and distinguishable from antibodies. For example, the size distribution of an antibody composition can be detected using size exclusion chromatography (SEC), such as SE-HPLC. In one aspect, a composition comprises an anti-PD-1 antibody having a heavy chain sequence comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain sequence comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 6; the composition comprises no more than 36% aggregation variants. It will be understood that embodiments of these aggregation variants may be combined with any of the antibody variants described herein.

[0145] In one aspect, a composition comprises aggregation variants of an anti-PD-1 antibody, wherein the aggregation variants comprise a heavy chain sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 36% aggregation variants.

[0146] In one aspect, the composition comprises: an antibody having a heavy chain amino acid sequence comprising SEQ ID NO:1 (CDRH1), SEQ ID NO:2 (CDRH2), and SEQ ID NO:3 (CDRH3) and a light chain amino acid sequence comprising SEQ ID NO:4 (CDRL1), SEQ ID NO:5 (CDRL2), and SEQ ID NO:6 (CDRL3); and their aggregation mutants wherein 36% or less of the antibody population is composed of aggregation variants.

[0147] In one embodiment, the antibody comprises a heavy chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8. In a further embodiment, the antibody is at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 10. In yet a further embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10.

[0148] In one aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 8, wherein the composition comprises 36% or less aggregation variants. In a further embodiment, a composition comprises an anti-PD-1 antibody comprising a heavy chain variable region of SEQ ID NO: 7 and / or a light chain variable region of SEQ ID NO: 8, wherein the composition comprises 36% or less aggregation variants.

[0149] In one aspect, the composition comprises: an antibody having a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 8; and their aggregation mutants and a population of anti-PD-1 antibodies comprising 36% or less of the antibody population consisting of aggregation variants.

[0150] In one aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and / or a light chain sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:10, wherein the composition comprises no more than 36% aggregation variants.

[0151] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and an antibody having a heavy chain sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 9 and / or a light chain sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 36% or less aggregation variants.

[0152] In one aspect, a composition comprises an anti-PD-1 antibody comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, wherein the composition comprises 36% or less aggregation variants. In one embodiment, a composition comprises aggregation variants of Dostarlimab, wherein the aggregation variants comprise the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10; the composition comprises 36% or less aggregation variants. In one embodiment, a composition comprises aggregation variants of Dostarlimab, wherein the aggregation variants comprise the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10; the composition comprises aggregation variants in the range of 0.01% to 36% aggregation variants.

[0153] In one aspect, the composition comprises: an antibody having a heavy chain amino acid sequence as set forth in SEQ ID NO: 9 and a light chain amino acid sequence as set forth in SEQ ID NO: 10; and their aggregation mutants wherein 36% or less of the antibody population is composed of aggregation variants.

[0154] The antibody composition may contain 36% or less aggregation variants, for example, 35% or less, 30% or less, 26% or less, 25% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less aggregation variants. In another embodiment, the composition may contain 0.01-36%, 0.01-35%, 0.01-30%, 0.01-26%, 0.01-25%, 0.01-20%, 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, or 0.01-1% aggregation variants. Alternatively, the composition may comprise more than 1% but less than 36% aggregation variants. Alternatively, the composition may contain about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% aggregation variants. As demonstrated by the data provided herein, temperature-forced degradation resulting in compositions of up to 11.2% comprising aggregated antibodies provides 86% potency in the bioassay (i.e., within the assay variation to full functionality). Acid-treated samples resulting in compositions of up to 15.2% comprising aggregated antibodies provide 125% potency in the bioassay or 88% potency with an MSD (i.e., within the assay variation to full functionality). Extrapolating from the temperature-forced degradation data for the aggregation mutants, up to 36% can provide at least 60% potency, and up to 26% can provide at least 70% potency. This is calculated using the linear slope of 0.9% (control), 1.4% (40°C at 3 weeks), and 11.2% (50°C at 3 weeks) aggregation mutant temperature-treated samples, which have potencies of 98%, 94%, and 86%, respectively (bioassay).

[0155] In one aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 100% acidic variants; and / or no more than 35% basic variants; and / or 1% or more major isoforms; and / or no more than 65% oxidation variants; and / or no more than 36% aggregation variants.

[0156] In another aspect, a composition comprises an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises 5-60% acidic variants; and / or 0.1-35% basic variants; and / or 20-90% major isoforms; and / or 65% or less oxidized variants; and / or 36% or less aggregated variants.

[0157] Fragmentation variants ("fragment variants") are variants comprising a portion of a full-length antibody. For example, such fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and immunoglobulin single variable domains. An antibody composition may contain 10% or less fragmented antibodies, e.g., 5% or less, 4.6% or less, 4.5% or less, 4.4% or less, 4.3% or less, 4.2% or less, 4.1% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, or 0.05% or less fragmented antibodies. In another embodiment, the composition may contain 0.01 to 10%, 0.01 to 5%, 0.01 to 4.6%, 0.01 to 4.5%, 0.01 to 4%, 0.01 to 3.5%, 0.01 to 3%, 0.01 to 2.5%, 0.01 to 2%, 0.01 to 1.5%, 0.01 to 1%, 0.01 to 0.5%, 0.01 to 0.1%, or 0.01 to 0.05% antibody fragment. In other embodiments, the composition may contain 0.5-10%, 0.5-5%, 0.5-4.6%, 0.5-4.5%, 0.5-4%, 0.5-3.5%, 0.5-3%, 0.5-2.5%, 0.5-2%, 0.5-1.5%, 0.5-1%, 0.6-1.5%, or 0.6-1.0% antibody fragments. Alternatively, the composition may contain about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% antibody fragments. It will be understood that these fragment variant embodiments may be combined with any of the antibody variants described herein.

[0158] Deamidation, which may occur during manufacturing and / or storage, can be an enzymatic or chemical reaction. Deamidation can occur through a simple chemical reaction involving intramolecular cyclization, in which the amide nitrogen of the next amino acid in the chain nucleophilically attacks the amide (N+1 attacks N) to form a succinimide intermediate. Deamidation can primarily convert asparagine (N) to isoaspartic acid (isoaspartic acid group) and aspartic acid (aspartic acid group) (D) in an approximately 3:1 ratio. Thus, this deamidation reaction may involve the isomerization of aspartic acid group (D) to isoaspartic acid. Both the deamidation of asparagine and the isomerization of aspartic acid groups may involve the intermediate succinimide. Deamidation of glutamine residues can also occur in a similar manner, albeit to a much lesser extent. Deamidation can occur in the CDRs, Fab (non-CDR regions), or Fc regions. The isomerization is the conversion of an aspartic acid group (D) to an isoaspartic acid group, involving an intermediate succinimide (succinimide-aspartic acid residue).

[0159] In one aspect, the composition comprises an antibody comprising a deamidation post-translational modification ("deamidation" or "deamidated"), also referred to herein as a "deamidation variant."

[0160] In one embodiment, the antibody comprises deamidation of asparagine residues in the CDRs of the heavy chain sequence and / or the CDRs of the light chain sequence. In a further embodiment, the antibody comprises deamidation of asparagine residues in the CDRs of the heavy chain sequence. In one embodiment, the antibody comprises deamidation of asparagine residues in the Fc region of the heavy chain sequence and / or the Fc region of the light chain sequence. The deamidation variants can be present in one or both of the heavy and light chains. It will be understood that these deamidation variant embodiments can be combined with any of the antibody variants described herein. In some embodiments, the deamidation variants comprise one or a combination of deamidation at N380 and / or N385 in the Fc region of the heavy chain sequence.

[0161] In one embodiment, the deamidated variant comprises a deamidated residue selected from an aspartic acid residue, a succinimido-aspartic acid residue, or an isoaspartic acid residue.

[0162] In one aspect, a composition comprises an antibody that comprises a sequence that is at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 (and optionally comprises a sequence that is at least 90% identical to the light chain sequence of SEQ ID NO:10), and that comprises deamidation in the heavy chain sequence, e.g., deamidation at amino acid residues N380 and / or N385 of the Fc region. In some embodiments, the deamidation variant comprises up to 100% deamidation at N380 and / or N385 of SEQ ID NO:9.

[0163] Deamidation can result in a sequence change in which an asparagine residue (N) is converted to an aspartic acid residue (D). Thus, in one embodiment, the deamidated mutant comprises the heavy chain sequence of SEQ ID NO: 11 (i.e., the heavy chain sequence having N380D). In another embodiment, the deamidated mutant comprises the heavy chain sequence of SEQ ID NO: 12 (i.e., the heavy chain sequence having N385D). In yet another embodiment, the deamidated mutant comprises the heavy chain sequence of SEQ ID NO: 13 (i.e., the heavy chain sequence having N380D and N385D).

[0164] The composition may comprise up to 100% deamidated variants. In one aspect, the composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and the composition comprises up to 100% deamidated variants.

[0165] In one embodiment, the composition comprises up to 100% deamidation at N380 and / or N385 of the heavy chain sequence. In one embodiment, the composition comprises 0-100%, 0-90%, 0-80%, 0-70%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%, or 0-10% deamidation at N380. Alternatively, the composition comprises 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, or 0.1-10% deamidation at N380. Alternatively, the composition comprises 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, or 1-10% deamidation in N380. Alternatively, the composition comprises 2-100%, 3-100%, 4-100%, 5-100%, 6-100%, 7-100%, 8-100%, 9-100%, 2-30%, 3-30%, 4-30%, 5-30%, 2-40%, 3-40%, 4-40%, 5-40%, 2-10%, 3-10%, 4-10%, or 5-9% deamidation in N380. Alternatively, the composition comprises 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more deamidation of N380. As shown in the data provided herein, base-treated samples resulting in up to 27.8% deamidation of N380 yielded 96% potency in the bioassay (i.e., within the range of assay variation to full functionality), and therefore it is expected that deamidation in N380 can be higher than the reported level of 27.8% without affecting relative potency or FcRn binding.

[0166] In one embodiment, the composition comprises 0-100%, 0-90%, 0-80%, 0-70%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%, or 0-10% deamidation of N385. Alternatively, the composition comprises 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, or 0.1-10% deamidation of N385. Alternatively, the composition comprises 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, or 1-10% deamidation of N385. Alternatively, the composition comprises 0.5% or more, 1% or more, or 2% or more deamidation of N385. As shown in the data provided herein, base-treated samples resulting in up to 27.2% deamidation of N385 conferred 96% potency in the bioassay (i.e., within the range of assay variation to full functionality), and therefore, it is expected that deamidation of N385 can be higher than the reported level of 27.2% without affecting relative potency or FcRn binding.

[0167] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises up to 100% deamidation at N380 and / or N385 of the heavy chain.

[0168] In some embodiments, the composition comprises about 0.5-2%, about 0.5%, about 1%, about 1.5%, or about 2% deamidation at N84 of SEQ ID NO: 9. In some embodiments, the composition comprises about 0.5-2%, about 0.5%, about 1%, about 1.5%, or about 2% deamidation at N137 of SEQ ID NO: 9. In some embodiments, the composition comprises about 5-8%, about 5%, about 6%, about 7%, or about 8% deamidation at N311 of SEQ ID NO: 9. In some embodiments, the composition comprises about 0.5-3%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, or about 3% deamidation at N430 of SEQ ID NO: 9.

[0169] In one example, deamidation can be determined using Lys-C and / or tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described herein above.

[0170] In one aspect, the composition comprises an antibody comprising an isomerization post-translational modification ("isomerization" or "isomerized"), also referred to herein as an "isomerization variant." The variant may comprise an isomerized amino acid residue in the heavy chain sequence and / or the light chain sequence, e.g., in the CDR of the heavy chain sequence and / or the CDR of the light chain sequence. The isomerization variant may be present in one or both of the heavy and / or light chains. The isomerization post-translational modification may result in isoaspartic acid and / or succinimido-aspartic acid residues. In one example, aspartic acid (Asp) isomerization can be determined using Lys-C and / or tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described herein above. It will be understood that these isomerization variant embodiments may be combined with any of the antibody variants described herein.

[0171] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises up to 100% isomerization variants. In one embodiment, a composition comprises an isomerization variant of dostarlimab, wherein the isomerization variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10; and the composition comprises no more than 100% isomerization variants.

[0172] In some embodiments, the composition comprises an isomerization variant. In some embodiments, the composition comprises up to 100% isomerization variant. The composition may comprise 0-100%, 0-90%, 0-80%, 0-70%, 0-60%, 0-50%, 0-40%, 0-30%, 0-15%, 0-20%, or 0-10% isomerization variant. Alternatively, the composition may comprise 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, or 0.1-10% isomerization variant. Alternatively, the composition may contain 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15% or 1-10% of the isomerized variant.

[0173] In one embodiment, the composition comprises up to 100% isomerization at D147 of SEQ ID NO: 9. The composition may comprise 0-100%, 0-90%, 0-80%, 0-70%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%, 0-15%, or 0-10% isomerization at D147 of the heavy chain sequence. Alternatively, the composition may comprise 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0-15%, or 0.1-10% isomerization at D147 of the heavy chain sequence. In some embodiments, the composition comprises 1% or more isomerization at D147 of the heavy chain sequence. As shown in the data provided herein, a base-treated sample resulting in 20.8% isomerization at D147 gave 96% potency in the bioassay (i.e., within the range of assay variation to full functionality), and therefore it is expected that isomerization at D147 can be higher than the reported level of 20.8% without affecting relative potency or FcRn binding.

[0174] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises up to 100% isomerization at D147 of the heavy chain.

[0175] In some embodiments, the composition comprises 0-15%, 0.1-15%, 1-15%, 1% or more, 1.5% or more, or 2% or more isomerization at D151, D167, D261, D266, D276, D395, D397, and / or 409 of SEQ ID NO:9. For example, the composition comprises about 2.3% isomerization at D62 of SEQ ID NO:9. For example, the composition comprises about 13.1% isomerization at D261 / 266 / 276 of SEQ ID NO:9. For example, the composition comprises about 3.1% isomerization at D151 / 167 of SEQ ID NO:9. For example, the composition comprises about 2.7% isomerization at D395 / 397 / 409 of SEQ ID NO:9.

[0176] The antibody composition may comprise (i) an antibody (e.g., an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, as described herein); and (ii) an antibody variant comprising one or more or a combination of an amino acid sequence variant (e.g., a deamidation or C-terminal lysine clipping variant), an oxidation variant, an isomerization variant, an aggregation variant, and / or a fragmentation variant.

[0177] Thus, in one aspect, a composition is provided comprising an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises (i) no more than 65% oxidation variants; and (ii) no more than 36% aggregation variants.

[0178] In one aspect, the composition comprises an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and / or SEQ ID NO:13, and a light chain sequence of SEQ ID NO:10, wherein the composition comprises no more than 65% oxidized variants.

[0179] In one aspect, the composition comprises an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and / or SEQ ID NO:13, and a light chain sequence of SEQ ID NO:10, wherein the composition comprises 36% or less aggregation variants.

[0180] In another embodiment, the composition comprising the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the potency of a reference standard having 100% potency. In one aspect, a composition comprises a variant of an anti-PD-1 antibody, the variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition has at least 60% of the potency of a composition comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, 10-97% acidic variants, 0.1-35% basic variants, 2-80% major isoforms, 4.8% or less LC W50 oxidation variants, 1% or less HC M34 oxidation variants, 1.2% or less HC M103 oxidation variants, 15.2% or less aggregation variants, 16.7% or less HC M354 oxidation variants, 29.0% or less HC M424 oxidation variants, 47.1% or less HC M248 oxidation variants, 20.8% or less HC D147 isomerization variants, 13.1% or less HC The antibody has D151 or D167 isomerization mutants, 3.1% or less of HC D261, D266, or D276 isomerization mutants, 4.6% or less of fragmentation mutants, 27.8% or less of HC N380 deamidation mutants, 27.2% or less of HC N385 deamidation mutants, about 7.4% or less of HC N311 deamidation mutants, about 2.0% or less of N430 deamidation mutants, 90% or more of heavy chain (HC) C-terminal lysine deletion mutants (ΔK443), and 1% or less of HC N-terminal pyroglutamic acid mutants. Potency can be determined using the bioassays described herein.

[0181] Glycation is a post-translational modification that involves the non-enzymatic chemical reaction of reducing sugars, such as glucose, with free amine groups in proteins, commonly found at the epsilon amine of lysine side chains or the N-terminus of proteins. Glycation can occur during manufacture and / or storage in the presence of reducing sugars.

[0182] Disulfide bond scrambling can occur during manufacturing and / or storage conditions. Under certain circumstances, disulfide bonds can break or form improperly, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryls (-SH) can facilitate shuffling.

[0183] Thioether formation and disulfide bond racemization can occur under basic conditions during manufacturing or storage via beta-elimination of the disulfide bridge back to the cysteine ​​residue via dehydroalanine and a persulfide intermediate. Subsequent cross-linking of dehydroalanine with cysteine ​​can result in thioether bond formation, or the free cysteine ​​residue can reform the disulfide bond with a mixture of D- and L-cysteine.

[0184] Trisulfides can result from the insertion of sulfur atoms into disulfide bonds (Cys-SS-S-Cys) and can also be formed due to the presence of hydrogen sulfide in the production cell culture.

[0185] The N-terminal glutamine (Q, Gln) and glutamic acid (E, Glu) groups of the heavy and / or light chains can undergo cyclization to form pyroglutamic acid (pGlu) groups. While pGlu formation can occur in the production bioreactor, it can also occur non-enzymatically, depending on, for example, the pH and temperature of processing and storage conditions. Cyclization of the N-terminal Q or E is commonly observed in natural human antibodies.

[0186] C-terminal lysine clipping (also called C-terminal lysine cleavage) is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in recombinant and native human antibodies. Variations of this process include the removal of lysines from one or both heavy chains by cellular enzymes from recombinant host cells. Administration to a human subject / patient can result in the removal of the remaining C-terminal lysines.

[0187] In one embodiment, the post-translational modification is an antibody variant (e.g., a sequence variant). Exemplary post-translationally modified antibody variants include an asparagine (N, Asn) switched to aspartic acid (D, Asp) ("deamidation"), an N-terminal pyroglutamic acid group, and / or a C-terminal lysine truncation. In one example, antibody variants, such as N380D or N385D in the heavy chain sequence, can be determined using Lys-C and / or tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described hereinabove. Extracted ion chromatograms of the unmodified and modified peptides are used to calculate the level of the antibody variant, e.g., N380D or N385D, by dividing by the total area under the curve for both the modified and unmodified peptides.

[0188] In one aspect, a composition comprises an antibody comprising an N-terminal pyroglutamic acid ("pyroglutamic acid") post-translational modification in its heavy chain amino acid sequence ("N-terminal pyroglutamic acid variant"). In one embodiment, the composition comprises an antibody comprising a sequence at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 (and optionally comprising a sequence at least 90% identical to the light chain sequence of SEQ ID NO:10), and comprising pyroglutamic acid at the N-terminus of the heavy chain.

[0189] In one embodiment, the composition comprises up to 100% heavy chain N-terminal pyroglutamic acid variants. The composition may comprise 0-100%, 0-90%, 0-80%, 0-70%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%, or 0-10% heavy chain N-terminal pyroglutamic acid variants. Alternatively, the composition may comprise 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, or 0.1-10% heavy chain N-terminal pyroglutamic acid variants. Alternatively, the composition may contain 1 to 100%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10% of heavy chain N-terminal pyroglutamic acid variants. Alternatively, the composition may contain 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 2% or less, or 1% or less of heavy chain N-terminal pyroglutamic acid variants.

[0190] In one aspect, the composition comprises an antibody comprising a deletion of the C-terminal lysine (K443) in the heavy chain amino acid sequence. In one embodiment, the composition comprises an antibody comprising a sequence at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 (and optionally a sequence at least 90% identical to the light chain sequence of SEQ ID NO:10), and comprising a deletion of the C-terminal lysine residue (K443) in the heavy chain.

[0191] In one embodiment, the composition comprises up to 100% heavy chain C-terminal lysine-truncated mutants. In another embodiment, the composition comprises 10% or more heavy chain C-terminal lysine-truncated mutants. In another embodiment, the composition comprises 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more heavy chain C-terminal lysine-truncated mutants. The composition may comprise 1 to 100%, 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, or 90 to 100% heavy chain C-terminal lysine-truncated mutants. Alternatively, the composition may contain about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 95-99%, about 96-99%, or about 97-99% heavy chain C-terminal lysine truncation mutants.

[0192] In one embodiment, the composition comprises up to 100% heavy chain N-terminal pyroglutamic acid variants and up to 100% heavy chain C-terminal lysine truncation variants.

[0193] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises up to 100% heavy chain N-terminal pyroglutamic acid variants and / or up to 100% heavy chain C-terminal lysine truncation variants.

[0194] In one example, N-terminal pyroglutamic acid and C-terminal lysine truncations can be determined using Lys-C and / or tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described herein above.

[0195] Neonatal Fc receptor (FcRn) binding to anti-PD-1 antibodies can be measured using surface plasmon resonance (SPR). The antibody can be captured by FcRn immobilized on a nitrilotriacetic acid (NTA) sensor chip. The FcRn-bound concentration of a sample can be determined by interpolation of the binding response on a standard curve. Specific binding activity (%) is calculated by dividing the FcRn-bound concentration by the total protein concentration.

[0196] Antibody compositions comprising the above-described antibodies and antibody variants retain specific antigen binding and / or FcRn binding and / or potency. For example, antibody compositions comprising the above-described antibodies, antibody variants, and post-translationally modified variants have PD-1-specific antigen binding of greater than 0.70; and / or FcRn binding of greater than 70% and / or potency of greater than 70%. Thus, variants at these levels (%) can be tolerated in antibody compositions without significantly affecting function (i.e., without reducing activity). In one embodiment, "reduced function" or "reduced activity" refers to a percentage reduction in PD-1 binding, FcRn binding, or potency compared to a reference standard that is significant relative to assay variability. For example, a reduction in function, activity, or potency can be described as a reduction of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more.

[0197] For example, the reference standard (or reference substance or control or unstressed control) is a composition comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10. In one aspect, the reference standard comprises 10-97% acidic variants, and / or 0.1-35% basic variants, and / or 2-80% of the major isoform. In one embodiment, the reference standard comprises 4.8% or less of LC W50 oxidized variants. In another embodiment, the reference standard comprises 1% or less of HC M34 oxidized variants. In another embodiment, the reference standard comprises 1.2% or less of HC M103 oxidized variants. In another embodiment, the reference standard comprises 10-97% acidic variants, and / or 0.1-35% basic variants, and / or 2-80% of the major isoforms, and / or 4.8% or less of LC W50 oxidized variants, and / or 1% or less of HC M34 oxidized variants, and / or 1.2% or less of HC M103 oxidized variants. In another embodiment, the reference standard comprises 15.2% or less aggregation variants. In another embodiment, the reference standard comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, 10-97% acidic variants, and / or 0.1-35% basic variants, and / or 2-80% of the major isoform, and / or 4.8% or less LC W50 oxidation variants, and / or 1% or less HC M34 oxidation variants, and / or 1.2% or less HC M103 oxidation variants, and / or 15.2% or less aggregation variants.

[0198] In another embodiment, the reference standard further comprises 16.7% or less of HC M354 oxidation variants. In another embodiment, the reference standard further comprises 29.0% or less of M424 oxidation variants. In another embodiment, the reference standard further comprises 47.1% or less of HC M248 oxidation variants. In another embodiment, the reference standard further comprises 20.8% or less of HC D147 isomerization variants. In another embodiment, the reference standard further comprises 13.1% or less of HC D151 or D167 isomerization variants. In another embodiment, the reference standard further comprises 3.1% or less of HC D261, D266, or D276 isomerization variants. In another embodiment, the reference standard further comprises 4.6% or less of fragmentation variants. In another embodiment, the reference standard further comprises 27.8% or less of HC N380 deamidation variants and / or 27.2% or less of HC N385 deamidation variants. In a further embodiment, the reference standard further comprises about 7.4% or less of HC N311 deamidation mutants and / or about 2.0% or less of N430 deamidation mutants. In another embodiment, the reference standard further comprises 90% or more of heavy chain (HC) C-terminal lysine deletion mutants (ΔK443) and 1% or less of HC N-terminal pyroglutamic acid mutants.In another embodiment, the reference standard comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, 10-97% acidic variants, and / or 0.1-35% basic variants, and / or 2-80% major isoforms, and / or 4.81% or less LC W50 oxidation variants, and / or 1% or less HC M34 oxidation variants, and / or 1.2% or less HC M103 oxidation variants, and / or 15.2% or less aggregation variants, 16.7% or less HC M354 oxidation variants, and / or 29.0% or less HC M424 oxidation variants, and / or 47.1% or less HC M248 oxidation variants, and / or 20.8% or less HC D147 isomerization variants, and / or 13.1% or less HC D151 or D167 isomerization variants, and / or 3.1% or less HC D261, D266 or D276 isomerization mutants, and / or 4.6% or less fragmentation mutants, and / or 27.8% or less HC N380 deamidation mutants, and / or 27.2% or less HC N385 deamidation mutants, and / or about 7.4% or less HC N311 deamidation mutants, and / or about 2.0% or less N430 deamidation mutants, and / or 90% or more heavy chain (HC) C-terminal lysine deletion mutants (ΔK443), and / or 1% or less HC N-terminal pyroglutamic acid mutants.

[0199] In one embodiment, the reference standard comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, 10-30% acidic variants; and / or 0.1-10% basic variants; and / or 60-80% of the major isoform, and / or about 1% or less LC W50 oxidation variants, and / or about 1% or less HC M34 oxidation variants, and / or about 1% or less HC M103 oxidation variants, and / or about 1% aggregation variants, about 1% or less HC M354 oxidation variants, and / or about 1% or less HC M424 oxidation variants, and / or about 2-3% HC M248 oxidation variants, and / or about 1% or less HC D147 isomerization variants, and / or about 1% HC D151 or D167 isomerization variants, and / or about 0.6-1% fragmentation variants, and / or 5-9% HC N380 deamidation variants, and / or about 1% or less HC N385 deamidated mutants, and / or about 5.8% HC N311 deamidated mutants, and / or about 1.2% N430 deamidated mutants, and / or about 97-99% heavy chain (HC) C-terminal lysine deletion mutants (ΔK443), and / or about 1% or less HC N-terminal pyroglutamic acid mutants.

[0200] A reference standard as proposed herein is a composition comprising an anti-PD-1 antibody.

[0201] The composition may comprise a mixture of antibody variants and post-translationally modified variants, for example, an antibody composition may comprise two or more of acidic variants, basic variants, oxidized variants, deamidated variants, isomerized variants, aggregation variants, fragmentation variants, N-terminal pyroglutamic acid variants, and C-terminal lysine truncation variants.

[0202] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises any one or combination of: (i) up to 100% acidic variants; (ii) up to 35% basic variants; (iii) no more than 34% oxidation at W50 of the light chain; (iv) no more than 21% oxidation at M34 of the heavy chain; (v) no more than 64% oxidation at M103 of the heavy chain; (vi) no more than 65% oxidation at M248; (vii) no more than 65% oxidation at M354; (viiii) no more than 65% oxidation at M424; and / or (ix) no more than 36% aggregation variants.

[0203] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, wherein the composition comprises: (i) up to 100% acidic variants; (ii) up to 35% basic variants; (iii) no more than 64% oxidation at M103 of the heavy chain; (iv) no more than 34% oxidation at W50 of the light chain; (v) no more than 21% oxidation at M34 of the heavy chain; (vi) no more than 65% oxidation at M248; (vii) no more than 65% oxidation at M354. (viii) up to 65% oxidation at M424; (ix) up to 36% aggregation mutants, (x) up to 100% isomerization at D147 of the heavy chain; (xi) up to 100% deamidation at N380, (xii) up to 100% deamidation at N385, (xiii) up to 100% heavy chain N-terminal pyroglutamic acid mutants, and / or (xiv) up to 100% heavy chain C-terminal lysine truncation mutants.

[0204] In one aspect, a composition comprises an antibody having a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, and / or SEQ ID NO:13, and a light chain sequence of SEQ ID NO:10, wherein the composition comprises any one or combination of: (i) up to 100% acidic variants; (ii) up to 35% basic variants; (iii) no more than 34% oxidation at W50 of the light chain; (iv) no more than 21% oxidation at M34 of the heavy chain; (v) no more than 64% oxidation at M103 of the heavy chain; (vi) no more than 65% oxidation at M248; (vii) no more than 65% oxidation at M354; (viii) no more than 65% oxidation at M424; and / or (ix) no more than 36% aggregation variants.

[0205] The present invention encompasses antibodies that may have or have had one or more of the post-translational modifications described herein. Exemplary compositions may include: 1) a mixture or blend of antibodies with and without the post-translational modifications(s) described herein. Thus, a composition may include a population of antibodies with a post-translational modification and a population of antibodies without the post-translational modification.

[0206] The described compositions may have or have had one or more post-translational modifications. The modifications may occur in the CDR, variable framework region, or constant region. The modifications may result in a change in the charge of the molecule.

[0207] In one embodiment, the post-translational modifications described herein, except as designated and described as product-related impurities, do not result in significant changes in antigen binding affinity, biological activity, pharmacokinetics (PK) / pharmacodynamics (PD), aggregation, immunogenicity, and / or binding to Fc receptors.

[0208] Manufacturing method The antibodies and compositions described herein can be obtained by any means, including through in vitro sources (e.g., hybridomas or cell lines that recombinantly produce antibodies) and in vivo sources (e.g., rodents). Methods for producing antibodies are known in the art and are described, for example, in WO2018 / 085468.

[0209] For example, the composition can be expressed in a recombinant expression system and purified therefrom. In one embodiment, the composition is produced by a method of culturing host cells under conditions suitable for expression of an antibody comprising SEQ ID NO: 9 and SEQ ID NO: 10, which is expressed, optionally purified, and optionally formulated into a pharmaceutical composition.

[0210] Several different expression systems and purification methods can be used to produce these compositions. Generally, host cells are transformed with a recombinant expression vector encoding the antibody. A wide variety of host cells can be used, including eukaryotic cell lines of mammalian origin (e.g., CHO, Perc6, HEK293, HeLa, NS0). Suitable host cells include mammalian cells such as Chinese hamster ovary CHO cells (e.g., CHOK1 and CHO-DG44). In one embodiment, the antibody is produced by Chinese hamster ovary cells.

[0211] The host cell can be an isolated host cell. The host cell is not normally part of a multicellular organism (e.g., a plant or animal). The host cell can be a non-human host cell.

[0212] Suitable cloning and expression vectors and cloning methods for use with eukaryotic or mammalian cell hosts are known in the art.

[0213] The host cells are cultured to allow for expression of the recombinant expression vector encoding the antibody.

[0214] The composition can be recovered and purified by conventional protein purification procedures. For example, the composition can be directly collected from the culture medium. The cell culture medium can be collected by, for example, clarification by centrifugation and / or depth filtration. After the composition is collected, purification is performed to ensure sufficient purity. Thus, in one aspect, a cell culture medium comprising the composition described herein is provided. In one embodiment, the cell culture medium comprises CHO cells.

[0215] The composition can then be purified from the cell culture medium. This may include harvesting the cell culture supernatant, contacting the cell culture supernatant with a purification medium (e.g., a Protein A resin or a Protein G resin to bind the antibody molecules), and eluting the antibody molecules from the purification medium to obtain an eluate. Thus, in one aspect, an eluate comprising the composition described herein is provided.

[0216] Purification can involve one or more chromatography steps, such as one or more chromatography resins; and / or one or more filtration steps. For example, affinity chromatography using resins such as Protein A, G, or L can be used to purify the composition. Alternatively, or in addition, ion exchange resins, such as cation exchange, can be used to purify the composition.

[0217] Alternatively, the purification step comprises an affinity chromatography resin step followed by a cation exchange resin step.

[0218] Pharmaceutical Compositions and Formulations The compositions described herein may be in the form of a pharmaceutical composition.

[0219] In one aspect, a pharmaceutical composition is provided comprising the composition and at least one pharmaceutically acceptable excipient.

[0220] A "pharmaceutical composition" may comprise a composition described herein (i.e., an active ingredient) and one or more pharmaceutically acceptable excipients. The excipients must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of being formulated, not harmful to the recipient thereof, and / or not interfere with the efficacy of the active ingredient. Thus, pharmaceutical compositions of the present invention are suitable for administration to a patient.

[0221] As used herein, a "pharmaceutically acceptable excipient" can include one or more of a buffer, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. Often, isotonicity agents, such as polyols, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, preservatives, cosolvents, antioxidants, including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (e.g., Zn 2+ -protein complex); a biodegradable polymer; and / or a salt-forming counterion such as sodium or potassium.

[0222] The precise nature of the excipient or other material will depend on the route of administration, which may be, for example, oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), and intratumoral. It will be appreciated that preferred excipients may vary with, for example, the condition of the recipient and the disease being treated.

[0223] The mixture of excipients and their respective concentrations together form a "pharmaceutical formulation" (or "formulation"). Such compositions are preferably free of visible particulate matter. The formulation may be in liquid or lyophilized form. The composition in a liquid formulation may be filled into a container and frozen. In certain embodiments, an aliquot of the frozen formulation comprising the composition may be lyophilized. The lyophilizate may be reconstituted by adding water or other aqueous solution to produce a reconstituted formulation comprising the composition.

[0224] In one embodiment, the composition is a liquid formulation. In some embodiments, the formulation comprises about 10 mg / mL to about 125 mg / mL of antibody. In a further embodiment, the formulation comprises about 20 mg / mL to about 125 mg / mL, e.g., about 20 mg / mL to about 100 mg / mL, particularly about 20 mg / mL to about 50 mg / mL of antibody. In a further embodiment, the formulation comprises about 20 mg / mL of antibody. In another embodiment, the formulation comprises about 50 mg / mL of antibody.

[0225] In one aspect, a formulation is provided comprising a pharmaceutical composition described herein, comprising about 20 mg / mL to about 125 mg / mL (e.g., about 20 mg / mL to about 50 mg / mL) of an antibody and a buffer having a pH of about 5.5 to about 6.5. In one embodiment, the composition is a liquid formulation.

[0226] In some embodiments, the composition is formulated as a sterile liquid. In some embodiments, the composition is free of visible particles. In some embodiments, the composition is formulated in a buffer (e.g., a citrate buffer). In some embodiments, the composition comprises a PD-1 antibody and two or more of the following: a citrate buffer, a histidine buffer, arginine, trehalose, sodium chloride, and polysorbate 80.

[0227] In certain embodiments, the buffer is a citrate buffer. Citrate buffering can be achieved, for example, by using a conjugate acid / conjugate base system (sodium citrate / citric acid) or by titrating a sodium citrate solution with HCl. In one embodiment, the citrate buffer has a pH of about 5.0 to about 6.5, for example, about 5.5 to about 6.0, particularly about 5.5 or about 6.0.

[0228] In another embodiment, the buffer is a histidine buffer, which has a pH of about 5.5 to about 7.0, about 5.5 to about 6.5, for example, about 6.0 to about 6.5, particularly about 6.0 or about 6.5.

[0229] In some embodiments, the formulation comprises a surfactant. A "surfactant" is a surface active agent that, due to its chemical composition containing both hydrophilic and hydrophobic groups, can exert its effects at solid-solid, solid-liquid, liquid-liquid, and liquid-air interfaces. In dilute solutions, surfactants can reduce protein concentration at air-water and / or water-solid interfaces, where proteins may adsorb and aggregate. Surfactants can bind to hydrophobic interfaces in protein formulations. Some parenterally acceptable nonionic surfactants comprise either polysorbate or polyether groups. Polysorbate 20 and 80, particularly polysorbate 80 (PS80), are suitable surfactant stabilizers in the formulations of the present invention. In one embodiment, the formulation further comprises PS80. In some embodiments, the formulation comprises about 0.01% to about 0.1%, e.g., about 0.01% to about 0.05%, or about 0.01% to about 0.03% w / v of PS80 or PS20. In some embodiments, the formulation comprises about 0.02% w / v of PS80 or PS20. In a preferred embodiment, the formulation comprises about 0.02% w / v of PS80.

[0230] In some embodiments, the formulation further comprises sodium chloride at a concentration to adjust the osmolality of the formulation to about 290-325 mOsm / kg, e.g., about 290 mOsm / kg. In one embodiment, the formulation comprises about 20 mM to about 40 mM, e.g., about 25 mM to about 35 mM sodium chloride. In a further embodiment, the formulation comprises about 31 mM sodium chloride.

[0231] The formulation may also include a solubilizing agent, such as arginine, e.g., L-arginine-HCl. In some embodiments, the formulation comprises arginine in the range of about 80 mM to about 120 mM, e.g., about 90 mM to about 110 mM, particularly about 95 mM to about 105 mM. In some embodiments, the formulation comprises about 100 mM arginine.

[0232] In some embodiments, the formulation comprises a polyol. In some embodiments, the polyol is a sugar, preferably a non-reducing sugar. In some embodiments, the non-reducing sugar is trehalose. In some embodiments, the formulation comprises trehalose in the range of about 2% to about 10% w / v. In some embodiments, the formulation comprises about 5% w / v trehalose.

[0233] In some embodiments, the formulation comprises arginine and / or trehalose, for example, about 80 mM to about 120 mM (particularly 100 mM) arginine or about 2% to about 10% w / v (particularly 5% w / v) trehalose.

[0234] In one aspect, a formulation is provided comprising a pharmaceutical composition comprising about 20 mg / mL to about 125 mg / mL of an antibody, about 10 mM to about 40 mM citrate buffer or histidine buffer, about 80 mM to about 120 mM arginine or about 2% to about 10% w / v trehalose, about 20 mM to about 40 mM sodium chloride, and about 0.01% to about 0.03% w / v polysorbate 80, at a pH of about 5.5 to about 6.5.

[0235] In one aspect, a formulation is provided comprising about 20-125 mg / mL antibody, about 25 mM citrate buffer, about 100 mM arginine (e.g., L-arginine-HCl), about 31 mM sodium chloride, and about 0.02% (w / v) polysorbate 80 at about pH 6.

[0236] In one aspect, a formulation is provided comprising about 20 mg / mL antibody, about 25 mM citrate buffer, about 100 mM arginine, about 31 mM sodium chloride, and about 0.02% (w / v) polysorbate 80 at about pH 6.

[0237] In one aspect, a formulation comprising about 50 mg / mL antibody, about 25 mM citrate buffer, about 100 mM arginine, about 31 mM sodium chloride, and about 0.02% w / v polysorbate 80 at about pH 6 is provided.

[0238] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability during manufacture, transportation, storage, and administration. Stability can be measured at a selected temperature for a selected period of time. For example, for a product stored at a recommended temperature of 2°C to 8°C, the formulation may be stable at room temperature, about 30°C, or 40°C for at least one month, and / or at about 2°C to 8°C for at least one year, preferably at least two years. For example, the degree of aggregation, acidic variants, and / or basic variants during storage can be used as an indicator of protein stability. Thus, a "stable" formulation may be one in which the antibody aggregate variants present in the formulation are about 10% or less, about 5% or less, e.g., about 4% or less. A "stable" formulation may be one in which the antibody acidic variants present in the formulation are about 60% or less, about 50% or less, e.g., about 35% or less. A "stable" formulation may be one in which the antibody basic variants present in the formulation are about 35% or less, about 10% or less, e.g., about 15% or less.

[0239] In certain aspects of the invention, the formulation allows the composition to remain stable to storage, freezing, thawing, and / or mixing for at least 18 months at about 2-8° C. In one embodiment, a “stable” formulation may be one in which no more than about 4% aggregate variants, no more than about 35% acidic variants, and no more than about 15% basic variants of the antibody are present in the formulation.

[0240] In yet another aspect, the invention is directed to an article of manufacture, e.g., a kit, comprising a container holding a composition in a formulation described herein. In one aspect, an injection device is provided comprising the formulation. The injection device may comprise a pen injector device or an autoinjector device. In one embodiment, the formulation is contained in a pre-filled syringe.

[0241] Methods of Treatment and Compositions for Use The present invention further provides methods of treating any disease or disorder in which inappropriate expression (e.g., overexpression) or increased activity of PD-1 protein causes or contributes to the pathological effects of the disease, or in which decreased levels or activity of PD-1 protein has a therapeutic benefit in a mammal, preferably a human.

[0242] In one aspect, provided are compositions described herein for use in therapy. Such therapy can relate to any disease or disorder in which inappropriate expression (e.g., overexpression) or increased activity of PD-1 protein causes or contributes to the pathological effects of the disease, or in which reduced levels or activity of PD-1 protein has a therapeutic benefit in a mammal, preferably a human.

[0243] The composition can be used in a method of enhancing T cell activation or T cell effector function in a subject, the method comprising administering a therapeutically effective amount of an agent capable of inhibiting PD-1 signaling. The composition can be used in a method of inducing an immune response in a subject, the method comprising administering a therapeutically effective amount of an agent capable of inhibiting PD-1 signaling. The composition can be used in a method of enhancing an immune response or increasing immune cell activity in a subject, the method comprising administering a therapeutically effective amount of an agent capable of inhibiting PD-1 signaling.

[0244] In one aspect, compositions are provided for use in the treatment of cancer. Alternatively, compositions are provided for use in the treatment of infectious diseases.

[0245] In one aspect, there is provided a use of a composition described herein in the manufacture of a medicament for use in the treatment of cancer. Alternatively, there is provided a composition described herein in the manufacture of a medicament for use in the treatment of infectious disease.

[0246] As used herein, the term "treat" and grammatical variations thereof refer to therapeutic therapy. With respect to a particular condition, treatment means (1) improving the condition of one or more biological manifestations of a pathology, (2) a) interfering with one or more points in the biological cascade that leads to or contributes to the pathology, or b) interfering with one or more biological manifestations of the pathology, (3) alleviating one or more symptoms, effects, or side effects associated with the pathology or its treatment, (4) slowing the progression of the pathology or one or more biological manifestations of the pathology, or (5) preventing the onset of one or more biological manifestations of the pathology.

[0247] Treatment can be therapeutic, prophylactic, or preventative. The subject is one in need thereof. Those in need of treatment can include those already suffering from a particular medical disorder as well as those who may develop the disorder in the future.

[0248] Therefore, preventive treatment is also contemplated.Those skilled in the art will recognize that "prevention" is not an absolute term.In medicine, "prevention" is understood to refer to the prophylactic administration of drugs to substantially reduce the likelihood or severity of pathology or its biological manifestations, or to delay the onset of such pathology or its biological manifestations.Preventive therapy is appropriate when a subject is considered to be at high risk of developing cancer, for example, when the subject has a strong family history of cancer, or when the subject is exposed to a carcinogen.

[0249] Therefore, the methods, antibodies, and compositions described herein can be used for prophylactic or preventative treatment, where specified. In this case, the described methods, antibodies, and compositions can be used to prevent or delay the onset of one or more aspects or symptoms of the disease. The subject may be asymptomatic. The subject may have a genetic predisposition to the disease. A prophylactically effective amount of the composition is administered to such an individual. A prophylactically effective amount is an amount that prevents or delays the onset of one or more aspects or symptoms of the disease described herein.

[0250] The methods, antibodies, and compositions need not effect a complete cure or eradicate all symptoms or signs of a disease to constitute a viable therapeutic treatment. As recognized in the art, drugs used as therapeutic agents in therapy may reduce the severity of a given condition, but need not eliminate all signs of the disease to be considered a useful therapeutic agent. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a disease to constitute a viable prophylactic agent. It is sufficient to simply reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by enhancing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of the disease occurring in a subject (e.g., by delaying the onset of the disease) or of its worsening.

[0251] The terms "individual," "subject," and "patient" are used interchangeably herein and may be broadly defined to include any person in need of treatment, for example, a person in need of cancer treatment. The subject is typically a human. The subject may also be a mammal, such as a mouse, rat, or a primate (e.g., a marmoset or monkey). The subject may also be a non-human animal. The antibodies, compositions, and methods of the present disclosure also have veterinary applications. The subject to be treated may be an agricultural animal, e.g., a cow or bull, a sheep, a pig, a cow, a goat, or a horse, or a domestic animal, such as a dog or cat. The animal may be of any age, or a mature adult animal.

[0252] The present invention also provides a method for treating cancer, an infectious disease, or an autoimmune disease in a mammal.

[0253] cancer treatment The present disclosure provides a method for reducing tumors or inhibiting tumor cell growth in a subject, comprising administering a therapeutically effective amount of an agent capable of inhibiting PD-1 signaling. The method may comprise administering the aforementioned composition to a mammal having cancer or an infectious disease, after which the cancer or infectious disease is treated in the mammal. As discussed herein, PD-1 is aberrantly expressed in various cancers, and PD-L1 expression in some cancer (e.g., renal cell carcinoma) patients correlates with tumor aggressiveness. The method can be used to treat tumors, including, for example, adenocarcinoma, lung adenocarcinoma, acute myeloid leukemia ("AML"), acute lymphoblastic leukemia ("ALL"), adrenocortical carcinoma, anal cancer, appendix cancer, B-cell derived leukemia, B-cell derived lymphoma, bladder cancer, brain cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), fallopian tube cancer, testicular cancer, brain cancer, cervical cancer, choroidal cancer, chronic myeloid leukemia, CNS tumors, colon adenocarcinoma, colon cancer, colorectal cancer, diffuse pontine glioma ( DIPG), Diffuse Large B-cell Lymphoma ("DLBCL"), Embryonic Rhabdomyosarcoma (ERMS), Endometrial Cancer, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma, Follicular Lymphoma ("FL"), Gallbladder Cancer, Gastric Cancer, Gastrointestinal Cancer, Glioma, Head and Neck Cancer, Blood Cancer, Hepatocellular Carcinoma, Hodgkin's Lymphoma / Primary Mediastinal B-cell Lymphoma, Renal Cancer, Renal Clear Cell Carcinoma, Laryngeal Cancer, Leukemia, Liver Cancer, Lung Cancer, Lymphoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Monoclonal Antibody Myeloid leukemia, multiple myeloma, myeloma, neuroblastoma-derived CNS tumors, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), oral cancer, osteosarcoma, ovarian cancer, ovarian carcinoma, pancreatic cancer, peritoneal cancer, primary peritoneal cancer, prostate cancer, relapsed or refractory classical Hodgkin's lymphoma (cHL), renal cell carcinoma, rectal cancer, salivary gland cancer (e.g., salivary gland tumors), sarcoma, skin cancer, small cell lung cancer, small intestine cancer, squamous cell carcinoma of the anogenital region (e.g., It can be used to treat any type of cancer known in the art, such as squamous cell carcinoma of the anus, penis, cervix, vagina, or vulva), esophageal squamous cell carcinoma, squamous cell carcinoma of the head and neck (SCHNC), lung squamous cell carcinoma, gastric cancer, T-cell derived leukemia, T-cell derived lymphoma, thymic carcinoma, thymoma, thyroid cancer, uveal melanoma, urothelial cell carcinoma, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0254] In some embodiments, the cancers treated with the compositions described herein are characterized by microsatellite instability or lack thereof. Microsatellite instability ("MSI") is an alteration in or contains a DNA mutation in a particular cell (e.g., a tumor cell) in which the number of microsatellite repeats (short, repeated sequences of DNA) differs from that contained in the inherited DNA. Microsatellite instability results from a failure of a defective DNA mismatch repair (MMR) system to repair replication-related errors. This failure allows mismatch mutations to persist throughout the genome, particularly in regions of repetitive DNA known as microsatellites, resulting in an increased mutation burden. At least some tumors characterized by MSI-H have demonstrated improved response to certain anti-PD-1 agents (Le et al., (2015) N. Engl. J. Med. 372(26):2509-2520; Westdorp et al., (2016) Cancer Immunol. Immunother. 65(10): 1249-1259).

[0255] In some embodiments, the cancer has a microsatellite instability status of high microsatellite instability (e.g., MSI-H status). In some embodiments, the cancer has a microsatellite instability status of low microsatellite instability (e.g., MSI-L status). In some embodiments, the cancer has a microsatellite instability status of stable microsatellite (e.g., MSS status). In some embodiments, the microsatellite instability status is assessed by next-generation sequencing (NGS)-based assays, immunohistochemistry (IHC)-based assays, and / or PCR-based assays. In some embodiments, the microsatellite instability is detected by NGS. In some embodiments, the microsatellite instability is detected by IHC. In some embodiments, the microsatellite instability is detected by PCR.

[0256] In embodiments, the cancer is associated with high tumor mutation burden (TMB). In some embodiments, the cancer is associated with high TMB and MSI-H. In some embodiments, the cancer is associated with high TMB and MSI-L or MSS. In some embodiments, the cancer is endometrial cancer associated with high TMB. In some related embodiments, the endometrial cancer is associated with high TMB and MSI-H. In some related embodiments, the endometrial cancer is associated with high TMB and MSI-L or MSS.

[0257] In some embodiments, the cancer is a mismatch repair-deficient (dMMR) cancer. Microsatellite instability can result from a defective DNA mismatch repair (MMR) system's failure to repair replication-related errors. This failure persists mismatch mutations throughout the genome, particularly in regions of repetitive DNA known as microsatellites, resulting in an increased mutation burden that can improve response to certain anti-PD-1 agents.

[0258] In some embodiments, the cancer is a hypermutated cancer. In some embodiments, the cancer has a mutation in polymerase ε (POLE). In some embodiments, the cancer has a mutation in polymerase δ (POLD).

[0259] In some embodiments, the cancer is an endometrial cancer (e.g., an MSI-H or MSS / MSI-L endometrial cancer). In some embodiments, the cancer is an MSI-H cancer comprising a mutation in POLE or POLD (e.g., an MSI-H non-endometrial cancer comprising a mutation in POLE or POLD).

[0260] In one aspect, methods of treating cancer are provided comprising administering to a subject in need thereof a therapeutically effective amount of a composition (including a pharmaceutical composition or formulation) described herein.

[0261] As used herein, the terms "cancer" and "tumor" are used interchangeably and, in the singular or plural, refer to cells that have undergone transformation, such as malignant transformation, that renders them pathological to the host organism. Primary cancer cells can be readily distinguished from noncancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells as used herein includes not only primary cancer cells but also any cells derived from cancer cell ancestors. This includes metastasized cancer cells and in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically manifest as solid tumors, a "clinically detectable" tumor is one that can be detected based on tumor mass by procedures such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound, or palpation during physical examination, and / or by the expression of one or more cancer-specific antigens in samples obtainable from a patient.

[0262] In embodiments, the cancer is head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, bladder cancer, melanoma, Merkel cell carcinoma (e.g., Bhatia et al., Curr. Oncol. Rep., 13(6): 488-497 (2011)), cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, breast cancer, prostate cancer, salivary gland tumor, thymoma, adrenocortical carcinoma, esophageal cancer, gastric cancer, colorectal cancer, appendix cancer, urothelial cell carcinoma, or squamous cell carcinoma (e.g., of the lung; the anogenital region, including the anus, penis, cervix, vagina, or vulva; or the esophagus).

[0263] In some embodiments, the cancer is a hematological cancer, hi some embodiments, the hematological cancer is selected from diffuse large B-cell lymphoma ("DLBCL"), Hodgkin's lymphoma ("HL"), non-Hodgkin's lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML"), acute lymphoblastic leukemia ("ALL"), or multiple myeloma ("MM"). In embodiments, the cancer is a hematogenous cancer such as acute lymphoblastic leukemia ("ALL"), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia ("AML"), acute promyelocytic leukemia ("APL"), acute monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute anaplastic leukemia, chronic myelocytic leukemia ("CML"), chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, and multiple myeloma; acute and chronic leukemias such as lymphoblastic, myeloid, lymphocytic, and myelocytic leukemia.

[0264] In some embodiments, the cancer is a lymphoma, such as Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and polycythemia vera.

[0265] In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma of the lung. In some embodiments, the cancer is squamous cell carcinoma of the esophagus. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is squamous cell carcinoma of the anogenital region (e.g., anus, penis, cervix, vagina, or vulva).

[0266] In some embodiments, the cancer is bladder cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), fallopian tube cancer, bile duct cancer, colon adenocarcinoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gastric cancer, renal clear cell carcinoma, lung cancer (e.g., lung adenocarcinoma or lung squamous cell carcinoma), mesothelioma, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer, endometrial cancer, or uveal melanoma. In some embodiments, the cancer is ovarian cancer, fallopian tube cancer, or peritoneal cancer. In some embodiments, the cancer is breast cancer (e.g., TNBC). In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer). In some embodiments, the cancer is prostate cancer.

[0267] In some embodiments, the cancer is a CNS or brain cancer, such as neuroblastoma (NB), glioma, diffuse pontine glioma (DIPG), pilocytic astrocytoma, astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, vestibular schwannoma, adenoma, metastatic brain tumor, meningioma, spinal cord tumor, or medulloblastoma. In several embodiments, the cancer is a CNS tumor.

[0268] In some embodiments, the cancer is a solid tumor. In multiple embodiments, the cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, and the like. The tumor is a solid tumor such as basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, skin cancer, melanoma, neuroblastoma (NB), or retinoblastoma. In some embodiments, the tumor is an advanced solid tumor. In some embodiments, the tumor is a metastatic solid tumor.

[0269] In some embodiments, the cancer is a gynecological cancer (i.e., a cancer of the female reproductive system, such as ovarian, fallopian tube, cervical, vaginal, vulvar, uterine, or primary peritoneal, or breast cancer). In some embodiments, cancers of the female reproductive system include, but are not limited to, ovarian, fallopian tube, peritoneal, and breast cancer.

[0270] In some embodiments, the cancer is ovarian cancer (e.g., serologic or clear cell ovarian cancer). In some embodiments, the cancer is fallopian tube cancer (e.g., serologic or clear cell fallopian tube cancer). In some embodiments, the cancer is primary peritoneal cancer (e.g., serologic or clear cell primary peritoneal cancer).

[0271] In some embodiments, the ovarian cancer is an epithelial cancer. Epithelial cancer accounts for 85% to 90% of ovarian cancers. While historically thought to arise from the surface of the ovaries, new evidence suggests that at least some ovarian cancers arise in specialized cells in the fallopian tubes. The fallopian tubes are small tubes that connect a woman's ovaries to the uterus, part of the female reproductive system. In a typical female reproductive system, there are two fallopian tubes, one on each side of the uterus. Cancer cells that begin in the fallopian tubes may travel to the surface of the ovaries early on. The term "ovarian cancer" is often used to refer to epithelial cancers that arise in the ovaries, fallopian tubes, and the lining of the abdominal cavity, called the peritoneum. In some embodiments, the cancer is or comprises a germ cell tumor. A germ cell tumor is a type of ovarian cancer that develops in the egg-producing cells of the ovaries. In some embodiments, the cancer is or comprises a stromal tumor. Stromal tumors arise in the connective tissue cells that hold the ovaries together, which may produce the female hormone called estrogen. In some embodiments, the cancer is or comprises a granulosa cell tumor. Granulosa cell tumors secrete estrogen and may cause abnormal vaginal bleeding at the time of diagnosis. In some embodiments, the gynecological cancer is associated with homologous recombination repair deficiency / homologous repair deficiency ("HRD") and / or a BRCA1 / 2 mutation. In some embodiments, the gynecological cancer is platinum-sensitive. In some embodiments, the gynecological cancer has responded to platinum-based therapy. In some embodiments, the gynecological cancer has developed resistance to platinum-based therapy. In some embodiments, the gynecological cancer has previously shown a partial or complete response to platinum-based therapy (e.g., a partial or complete response to the last platinum-based therapy or the penultimate platinum-based therapy). In some embodiments, the gynecological cancer is currently resistant to platinum-based therapy.

[0272] In some embodiments, the cancer is breast cancer. Breast cancer typically arises in either the cells of the milk-producing glands, known as lobules, or in the ducts. Less commonly, breast cancer can also arise in stromal tissue. These include the fatty and fibrous connective tissue of the breast. Over time, breast cancer cells can invade nearby tissues, such as the axillary lymph nodes or lungs, a process known as metastasis. The stage of breast cancer, tumor size, and its rate of growth all determine the type of treatment offered. Treatment options include surgery to remove the tumor, drug treatments, including chemotherapy and hormonal therapy, radiation therapy, and immunotherapy. Prognosis and survival rates vary widely, with 5-year relative survival rates ranging from 98% to 23%, depending on the type of breast cancer present. Breast cancer is the second most common cancer worldwide, with approximately 1.7 million new cases in 2012 and the fifth leading cause of cancer deaths, accounting for approximately 521,000 deaths. Of these cases, approximately 15% are triple-negative, meaning they do not express estrogen receptors, progesterone receptors (PR), or HER2. In some embodiments, triple-negative breast cancer (TNBC) is characterized as breast cancer cells that are estrogen receptor-negative (fewer than 1% of cells), progesterone receptor-negative (fewer than 1% of cells), and HER2-negative.

[0273] In some embodiments, the cancer is ER-positive breast cancer, ER-negative breast cancer, PR-positive breast cancer, PR-negative breast cancer, HER2-positive breast cancer, HER2-negative breast cancer, BRCA1 / 2-positive breast cancer, BRCA1 / 2-negative cancer, or TNBC. In multiple embodiments, the cancer is TNBC.

[0274] In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is advanced breast cancer. In some embodiments, the cancer is stage II, stage III, or stage IV breast cancer. In some embodiments, the cancer is stage IV breast cancer.

[0275] In some embodiments, the cancer is endometrial cancer ("EC"). In some embodiments, the endometrial cancer is metastatic endometrial cancer.

[0276] Endometrial cancer is the most common cancer of the female reproductive tract. The annual number of new cases of endometrial cancer (EC) is estimated to be approximately 325,000 worldwide. EC is the most common cancer in postmenopausal women. Approximately 53% of endometrial cancer cases occur in developed countries. In 2015, approximately 55,000 cases of EC were diagnosed in the United States, and no targeted therapies are currently approved for use in EC. Drugs and regimens that improve survival rates for advanced and recurrent EC in the first-line (1L) and second-line (2L) treatment settings are needed. The most common histological type is endometrioid adenocarcinoma, accounting for approximately 75–80% of diagnosed cases. Other histological forms include uterine papillary serous (<10%), clear cell (4%), mucinous (1%), squamous (<1%), and mixed (approximately 10%).

[0277] From an etiological perspective, EC is classified into two distinct types: type I and type II. Type I tumors are low-grade estrogen-associated endometrioid carcinomas (EEC), while type II tumors are non-endometrioid (NEEC) (primarily serous and clear cell) carcinomas. The World Health Organization recently updated the pathological classification of EC, recognizing nine distinct subtypes of EC, with EEC and serous carcinoma (SC) accounting for the majority of cases. EEC is an estrogen-associated cancer that occurs in perimenopausal patients and is preceded by precursor lesions (endometrial hyperplasia / endometrial intraepithelial neoplasia). Microscopically, low-grade EEC (EEC 1-2) contains tubular glands and resembles proliferative endometrium to some extent, but has architectural complexity due to the fusion of glandular and cribriform structures. High-grade EEC exhibits a solid morphology. In contrast, SC occurs in postmenopausal patients without hyperestrogenism. Microscopically, SC shows thick, fibrous, or edematous papillae with prominent stratification of tumor cells, cellular budding, and undifferentiated cells with large eosinophilic cytoplasm. The majority of EECs are low-grade tumors (grades 1 and 2) and, when confined to the uterus, are associated with a favorable prognosis. Grade 3 EECs (EEC3) are aggressive tumors with frequent lymph node metastasis. SCs are highly invasive, independent of estrogen stimulation, and primarily occur in older women. EEC3 and SCs are considered high-grade tumors. SCs and EEC3s have been compared using Surveillance, Epidemiology, and End Results (SEER) program data from 1988 to 2001. They accounted for 10% and 15% of ECs, respectively, and 39% and 27% of cancer-related deaths, respectively.

[0278] In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is squamous cell carcinoma of the lung. In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as squamous cell NSCLC. In some embodiments, the lung cancer is ALK-translocated lung cancer (e.g., ALK-translocated NSCLC). In some embodiments, the lung cancer is EGFR-mutated lung cancer (e.g., EGFR-mutated NSCLC).

[0279] In some embodiments, the cancer is a metastatic cancer.

[0280] In some embodiments, the cancer is a recurrent cancer (eg, a recurrent gynecological cancer, eg, recurrent epithelial ovarian cancer, recurrent fallopian tube cancer, recurrent primary peritoneal cancer, or recurrent endometrial cancer).

[0281] Subjects in need of cancer treatment can include patients with various stages of disease, including newly diagnosed, relapsed, refractory, progressive disease, remission, etc. Subjects in need of cancer treatment can also include patients who have undergone stem cell transplantation or who have been deemed transplant ineligible.

[0282] Treating infections This method can be used to treat any type of infectious disease (i.e., a disease or disorder caused by bacteria, viruses, fungi, or parasites). Examples of infectious diseases that can be treated by this method include, but are not limited to, diseases caused by human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), influenza virus, dengue virus, hepatitis B virus (HBV, or hepatitis C virus (HCV)).

[0283] Administration of the composition can induce an immune response against cancer or infectious disease in a mammal. An "immune response" can involve, for example, antibody production and / or activation of immune effector cells (e.g., T cells).

[0284] Treatment of autoimmune diseases This method can be used to treat any type of autoimmune disease (i.e., a disease or disorder caused by the overactivity of the immune system, which attacks and damages the body's own tissues), such as those described in MacKay IR and Rose NR (eds.), The Autoimmune Diseases, Fifth Edition, Academic Press, Waltham, MA (2014). Examples of autoimmune diseases that can be treated with these compositions include, but are not limited to, multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, scleroderma, Crohn's disease, psoriasis, systemic lupus erythematosus (SLE), and ulcerative colitis. When this method is used to treat autoimmune diseases, anti-TIM-3 antibody drugs can be used in combination with anti-inflammatory drugs, including, for example, corticosteroids (e.g., prednisone and fluticasone) and nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, and naproxen).

[0285] Administration route The subject may have received at least one previous cancer therapy before being treated with the compositions of the present invention. In one embodiment, the subject has been treated with at least one, at least two, at least three, at least four, at least five, at least six, or at least seven previous cancer therapies before being treated with the compositions of the present invention. In another embodiment, the subject is newly diagnosed with cancer and has not received any previous therapy before being treated with the compositions of the present invention.

[0286] The compositions of the present invention may be administered by any suitable route. For some compositions, suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), and intratumoral. It will be appreciated that the preferred route may vary depending, for example, on the condition of the recipient and the cancer being treated.

[0287] In some embodiments, the composition is administered intravenously (e.g., by intravenous (IV) infusion). In further embodiments, the composition is administered by a 30-minute IV infusion injection.

[0288] In some embodiments, the composition is administered by injection. Thus, in one aspect, an injection device is provided comprising the composition, pharmaceutical composition, or formulation of the invention. The injection device may include a pen injector device or an auto-injector device.

[0289] In one embodiment, the composition is contained in a pre-filled syringe.

[0290] The desired dose can be delivered by a single bolus of the composition, by multiple boluses of the composition, or by continuous infusion of the composition.

[0291] In certain embodiments, the compositions of the invention are administered as pharmaceutical compositions.

[0292] The term "administering," as used herein, refers to the delivery of a composition described herein to achieve a therapeutic objective. The composition may be administered for a period of time and at intervals sufficient to achieve a clinical benefit.

[0293] The composition can be administered to the subject in such a way as to target the therapy to a particular site.

[0294] In certain embodiments, the composition can be co-administered to a subject with one or more additional therapeutic agents. In another embodiment, the composition can be co-administered to a subject with one or more additional cancer therapeutic agents. The additional cancer therapeutic agents can include, but are not limited to, other immunomodulatory agents, therapeutic antibodies, CAR-T therapeutic agents, BiTEs, HDAC inhibitors, proteasome inhibitors, anti-inflammatory compounds, and immunomodulatory imid drugs (IMiDs).

[0295] "Co-administering" refers to the administration of two or more different pharmaceutical compositions or treatments (e.g., radiation therapy) administered to a subject in the same pharmaceutical composition or in a combination of separate pharmaceutical compositions. Thus, co-administration includes the simultaneous administration of a single pharmaceutical composition comprising two or more pharmaceutical agents, or the administration of two or more different compositions to the same subject at the same or different times.

[0296] For example, anti-PD-1 antibodies can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein, such as agents that are cytotoxic to cancer cells, modulate the immunogenicity of cancer cells, or promote an immune response to cancer cells. In this regard, for example, the compositions can be used in combination with at least one other anti-cancer agent, including, for example, any chemotherapeutic agent known in the art, ionizing radiation, small molecule anti-cancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy, and adoptive T-cell transfer), and / or surgery. In some embodiments, a subject (e.g., a mammal, e.g., a human) for treatment with an anti-PD-1 antibody is being treated or will be treated with chemotherapy (e.g., platinum-based chemotherapy). In some embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, or vinorelbine. In some such embodiments, the chemotherapeutic agent is a platinum-based chemotherapeutic agent such as cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin, hi some such embodiments, the chemotherapeutic agent is a formate antimetabolite such as pemetrexed.In some embodiments, the subject (e.g., a mammal, e.g., a human) for treatment with an anti-PD-1 antibody is receiving an anti-angiogenic agent, such as bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, fumagillin, CM101, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, angiogenic antisteroids, heparin, cartilage-derived angiogenesis inhibitor or (e.g., the peptides troponin I and chondromodulin I), matrix metalloproteinase inhibitors, angiostatin, endostatin, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thrombospondin, thalidomide, prolactin, ανβ 3 inhibitors, lenalidomide, linomide, ramucirumab, tasquinimod, ranibizumab, sorafenib, sunitinib, pazopanib, everolimus, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), bFGF soluble receptor, transforming growth factor beta, interferon alpha, interferon beta, soluble KDR and FLT-1 receptors, placental proliferin-related protein, pazopanib, sunitinib, sorafenib, axitinib, ponatinib, cabozantinib, regorafenib, vandetanib, lenvatinib, semaxanib, SU6668, vatalanib, tivozanib, cediranib, protamine, heparin, steroids, ascorbic acid ether, polysaccharide sulfate DS 4152, AGM 12470, Neovastat, RO4929097, MRK-003, MK-0752, PF03084014, MEDI0639, curcumin, 3,3'-diindolylmethane (DIM), resveratrol, 3,5-bis(2,4-difluorobenzylidene)-4-piperidone (DiFiD), and epigallocatechin-3-gallate (EGCG), honokiol, Flt2-11, CBO-P11, Je-11, V1, and any combination thereof. In some embodiments, the compositions can be used in combination with anti-inflammatory drugs, including, for example, corticosteroids (e.g., prednisone and fluticasone) and nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, and naproxen).

[0297] In some embodiments, the composition is used to treat an infectious disease. When the method of the present invention treats an infectious disease, the anti-PD-1 antibody drug can be administered in combination with at least one antibacterial agent or at least one antiviral agent. In this regard, the antibacterial agent may be any suitable antibiotic known in the art. The antiviral agent may be any suitable type of vaccine that specifically targets a particular virus (e.g., live attenuated vaccines, subunit vaccines, recombinant vector vaccines, and small molecule antiviral therapies (e.g., viral replication inhibitors and nucleoside analogs)).

[0298] In some embodiments, the composition can be administered in combination with other agents that inhibit immune checkpoint pathways. For example, the composition can be administered in combination with agents that inhibit or antagonize the CTLA-4, TIM-3, or LAG-3 pathways. Combination therapies that simultaneously target two or more of these immune checkpoint pathways have shown improved, potentially synergistic, anti-tumor activity. In some embodiments, the composition is administered in combination with an antibody that binds to TIM-3 and / or an antibody that binds to LAG-3. In this regard, the methods of the present invention for treating cancer or infectious disease in a mammal may further comprise administering to the mammal a composition comprising (i) an antibody that binds to TIM-3 protein and / or (ii) an antibody that binds to LAG-3 protein, optionally in combination with a pharmaceutically acceptable carrier. Exemplary antibody drugs specific for LAG-3 and TIM-3 are described in WO2016 / 126858 and WO2016 / 161270, respectively, both of which are incorporated herein by reference. In some embodiments, anti-TIM-3 antibody agents can be used in combination with anti-inflammatory agents, including, for example, corticosteroids (e.g., prednisone and fluticasone) and nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, and naproxen).

[0299] In some embodiments, the subject is receiving or will be receiving one or more additional therapeutic agents in combination with the anti-PD-1 antibody drug. In some embodiments, the additional therapy is a PARP inhibitor. PARP inhibitors include, for example, ABT-767, AZD 2461, BGB-290, BGP 15, CEP 8983, CEP 9722, DR 2313, E7016, E7449, fluzoparib (SHR 3162), IMP 4297, INO1001, JPI 289, JPI 547, monoclonal antibody B3-LysPE40 conjugate, MP 124, niraparib (Zejula) (MK-4827), NU 1025, NU 1064, NU 1076, NU1085, olaparib (AZD2281), ONO2231, PD 128763, R 503, R 554, rucaparib (Rubraca) (AG-014699, PF-01367338), SBP 101, SC 101914, simmiparib, talazoparib (BMN-673), veliparib (ABT-888), WW 46, 2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-ol, and salts or derivatives thereof. In some embodiments, the PARP inhibitor is selected from the group consisting of niraparib, olaparib, rucaparib, talazoparib, and veliparib. In some embodiments, the PARP inhibitor is selected from niraparib (e.g., niraparib free base, niraparib tosylate, or niraparib tosylate monohydrate, or any combination thereof).

[0300] In some embodiments, the additional therapy includes treatment with a composition that delivers an agent that inhibits TIM-3 or LAG-3, and treatment with a PARP inhibitor, such that the subject receives all 3. In some embodiments, the additional therapy includes treatment with a composition that delivers an agent that inhibits TIM-3, treatment with a composition that delivers an agent that inhibits LAG-3, and treatment with a PARP inhibitor, such that the subject receives all 4.

[0301] In some embodiments, the method comprises administering the composition in combination with niraparib. Such methods may optionally include the administration of an anti-angiogenic agent such as bevacizumab. In some embodiments, the combination is intended for administration to patients with ovarian cancer, head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, bladder cancer, melanoma, Merkel cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, endometrial cancer, fallopian tube cancer, breast cancer, prostate cancer, salivary gland tumor, thymoma, adrenocortical carcinoma, esophageal cancer, gastric cancer, colorectal cancer, appendix cancer, urothelial cell carcinoma, or squamous cell carcinoma (e.g., lung; anogenital area including anus, penis, cervix, vagina, or vulva; or esophageal). In further embodiments, the cancer is selected from ovarian cancer or lung cancer (e.g., NSCLC).

[0302] In some embodiments, the method comprises administering a composition in combination with niraparib to a patient, particularly a patient with recurrent and / or platinum-sensitive cancer. In some embodiments, the recurrent and / or platinum-sensitive cancer is ovarian cancer, head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, bladder cancer, melanoma, Merkel cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, endometrial cancer, fallopian tube cancer, breast cancer, prostate cancer, salivary gland tumor, thymoma, adrenocortical carcinoma, esophageal cancer, gastric cancer, colorectal cancer, appendix cancer, urothelial cell carcinoma, or squamous cell carcinoma (e.g., lung; anogenital area including anus, penis, cervix, vagina, or vulva; or esophageal). In some specific embodiments, the recurrent and / or platinum-sensitive cancer is ovarian cancer, anal cancer, fallopian tube cancer, or lung cancer. In some specific embodiments, the recurrent and / or platinum-sensitive cancer is endometrial cancer, triple-negative breast cancer, ovarian cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the lung, or squamous cell carcinoma of the anogenital region (e.g., squamous cell carcinoma of the anus, penis, cervix, vagina, or vulva). In further embodiments, the recurrent and / or platinum-sensitive cancer is ovarian cancer. Such methods may optionally include administration of an anti-angiogenic agent, such as bevacizumab.

[0303] dose The compositions described herein may be administered in a therapeutically effective amount.

[0304] A "therapeutically effective amount" or "therapeutically effective dose" of a composition, as used herein, refers to the amount of an agent (e.g., an antibody or pharmaceutical composition) that provides a therapeutic benefit in the treatment or management of one or more symptoms of the condition being treated.

[0305] Therapeutically effective amounts and treatment regimens are generally determined empirically and may depend on factors such as the age, weight, and health of the patient, and the disease or disorder being treated, such factors being within the purview of the attending physician.

[0306] Ranges provided herein, of any type, include all values ​​within the particular range described, as well as the values ​​relative to the endpoints of the particular range.

[0307] In some embodiments, the therapeutically effective dose is about 100 to 2000 mg (e.g., about 100 mg; about 200 mg; about 300 mg; about 400 mg; about 500 mg; about 600 mg; about 700 mg; about 800 mg; about 900 mg; about 1000 mg; about 1100 mg; about 1200 mg; about 1300 mg; about 1400 mg; about 1500 mg; about 1600 mg; about 1700 mg; about 1800 mg; about 1900 mg; or about 2000 mg). In some embodiments, the therapeutically effective dose is about 1 mg / kg. In some embodiments, the therapeutically effective dose is about 3 mg / kg. In some embodiments, the therapeutically effective dose is about 10 mg / kg. In some embodiments, the therapeutically effective dose is a flat dose of about 500 mg. In some embodiments, the therapeutically effective dose is about 800 mg. In some embodiments, the therapeutically effective dose is about 1000 mg.

[0308] In one embodiment, the composition is administered once every 2 to 6 weeks (eg, 2, 3, or 4 weeks, particularly 3 weeks).

[0309] In one embodiment, the composition is administered once every three weeks for two to six administration cycles (eg, the first three, four, or five administration cycles, particularly the first four cycles).

[0310] In one embodiment, the composition is administered at 500 mg every 3 weeks for 4 doses, then 1000 mg every 6 weeks until disease progression.

[0311] If desired, the effective daily dose of the (therapeutic) composition may be administered as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.

[0312] The present disclosure provides a method of treating cancer comprising administering to a patient in need of treatment a first dose of a composition at a first interval for a first period of time, and administering to the patient a second dose of the composition at a second interval for a second period of time.

[0313] In some embodiments, the first dose and the second dose are different, ie, the first dose is about 500 mg and the second dose is about 1000 mg.

[0314] In some embodiments, the first interval and the second interval are different. In some embodiments, the first interval is once every three weeks, and the second interval is once every six weeks. In some embodiments, the composition is administered at a first dose of 500 mg once every three weeks for a first period of 2 to 6 administration cycles (e.g., the first three, four, or five administration cycles, particularly the first four administration cycles), and then at a second dose of 1000 mg once every six weeks until therapy is discontinued (e.g., due to disease progression, adverse events, or as determined by a physician). In some embodiments, the composition is administered at a first dose of 500 mg once every three weeks for the first three administration cycles, and then at a second dose of 1000 mg once every six weeks or more until therapy is discontinued (e.g., due to disease progression, adverse events, or as determined by a physician). In some embodiments, the composition is administered at a first dose of 500 mg once every three weeks for the first four administration cycles, and at a second dose of 1000 mg once every six weeks or more until therapy is discontinued (e.g., due to disease progression, adverse events, or as determined by a physician). In some embodiments, the composition is administered at a first dose of 500 mg once every three weeks for the first five administration cycles, and at a second dose of 1000 mg once every six weeks or more until therapy is discontinued (e.g., due to disease progression, adverse events, or as determined by a physician). In some embodiments, the second dose is administered once every six weeks.

[0315] In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of once per week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), or once every six weeks (Q6W). In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of once per week (Q1W). In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of once every two weeks (Q2W). In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of once every three weeks (Q3W). In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of once every four weeks (Q4W). In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of once every five weeks (Q5W). In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of once every six weeks (Q6W). In some embodiments, the composition is administered for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more. In some embodiments, the composition is administered on the first day of a treatment cycle or within 1, 2, or 3 days of the first day of a treatment cycle.

[0316] In some embodiments, the compositions described herein are administered according to a dosing regimen indicated to achieve clinical benefit in patients. In some embodiments, the clinical benefit is stable disease ("SD"), partial response ("PR"), and / or complete response ("CR"). In some embodiments, the clinical benefit is stable disease ("SD"). In multiple embodiments, the clinical benefit is a partial response ("PR"). In multiple embodiments, the clinical benefit is a complete response ("CR"). In some embodiments, PR or CR is determined according to Response Evaluation Criteria in Solid Tumors (RECIST). In some embodiments, the composition is administered for a longer period to maintain the clinical benefit.

[0317] All patent and literature references disclosed herein are expressly incorporated herein by reference in their entirety. [Example]

[0318] Dostarlimab is a humanized monoclonal antibody (mAb) of the IgG4κ isotype, composed of two heavy chains and two light chains, with a single N-linked glycosylation site on each heavy chain.

[0319] Two batches of dostarlimab drug substance (DS) manufactured at a commercial process and scale were used to fully characterize the dostarlimab antibody population. Primary, secondary, and higher-order structures were evaluated, along with physicochemical properties, heterogeneity, biological activity, immunochemical properties, and degradation pathways. The results are consistent with previous characterization results of Clinical Reference Standard material (CRS). A summary of the characterization data is provided below. Characterization methods are briefly described in the first mentioned section.

[0320] 1. Confirmation of amino acid sequence by peptide mapping The amino acid sequence was confirmed using a specific peptide mapping method for protein characterization. Dostarlimab samples were denatured with guanidine hydrochloride, reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and digested with endoproteinase Lys-C (Lys-C) or trypsin. Enzymatic digestion with either Lys-C or trypsin was performed at 37°C for 4 hours. Sample digestion was quenched with trifluoroacetic acid, followed by liquid chromatography with tandem mass spectrometry (LC-MS / MS). The LC-MS / MS analysis system used a reversed-phase ultra-high-performance liquid chromatography (UHPLC) equipped with a C18 column, UV detection at 214 nm, and electrospray ionization mass spectrometry (ESI-MS).

[0321] The Lys-C peptide map results confirmed 98.3% of the predicted amino acid sequences of the light and heavy chains. Several small peptides (LL12: H189-K190, LH9: V210-K212, LH17: C317-K318, LH18: V319-K322) were not identified, likely due to early elution before MS signal data collection. To identify these small peptides, a tryptic digest of dostarlimab was analyzed by LC-MS / MS. Incorrectly cleaved tryptic peptides (TL16-17: H189-K207, TH13-15: T196-R213, TH24-25: C317-K322) were identified, completing sequence coverage for the missing peptides in the Lys-C peptide map.

[0322] The combined Lys-C and tryptic peptide map data confirmed 100% of the predicted amino acid sequence.

[0323] 1.1 Terminal amino acid sequence The N- and C-terminal amino acid sequences were confirmed using tryptic peptide maps. Dostarlimab samples were subjected to denaturation, reduction, alkylation, and trypsin digestion before LC-MS / MS analysis.

[0324] The N-terminal or C-terminal amino acids of the peptide were not detected by MS / MS. This does not affect the confirmation of the terminal amino acid sequence. The combination of the fragmentation pattern of the MS / MS data and the measured molecular weight (MW) is sufficient to exclude other substitutions of the terminal amino acids. Therefore, the N-terminal and C-terminal sequences of the dostarlimab light and heavy chains are consistent with the theoretical sequences.

[0325] 2. Post-translational modifications 2.1 Glycan analysis The single glycosylation of dostarlimab was determined by comparing peptide LH14-15:T285-K313 with its deglycosylated form in the Lys-C peptide map. Deglycosylation with PNGase F increased the signal of the LH14-15:T285-K313 peptide peak, and MS data confirmed the peak and glycosylation site as asparagine 293. The peak areas (extracted ions) of the LH14-15:T285-K313 peptide with and without N-glycans were used to calculate a glycosylation occupancy of 99%.

[0326] 2.2 Other post-translational modifications Ion peak areas extracted from Lys-C and tryptic peptide maps were used to quantify post-translational modifications (PTMs) on the dostarlimab protein.

[0327] 3. Heterogeneity analysis 3.1 Characterization of size heterogeneity Dostarlimab size variants were evaluated by preparative size-exclusion HPLC (SE-HPLC). SE-HPLC fractions were characterized using analytical SE-HPLC, reduced and non-reduced capillary electrophoresis (reduced CE-SDS and non-reduced CE-SDS), capillary isoelectric focusing (cIEF), MSD binding assay, light scattering, and analytical ultracentrifugal sedimentation velocity (AUC-SV). The results are summarized below.

[0328] 3.1.1 Size Exclusion Chromatography By SE-HPLC analysis, dostarlimab eluted primarily as a monomer, although low levels of high molecular weight species (HMW) eluting earlier than the monomer peak were also observed (Table 2). Low molecular weight species (LMW) eluting later than the monomer peak were below the method limit of quantitation.

[0329] [Table 2]

[0330] 3.1.2 SEC-MALS Dostarlimab was analyzed by SE-HPLC and multi-angle light scattering (SEC-MALS). The measured MW indicates that the HMW species consists of dimerized Dostarlimab protein and a monomer with a molecule >50 kDa bound to it (presumably a monomer with two light chain (LC) subunits or an LC-LC dimer noncovalently bound to the monomer).

[0331] 3.1.3 cIEF Capillary isoelectric focusing (cIEF) was used to measure the pI of dostarlimab and separate the charge variants. This method quantifies acidic and basic species as a percentage of the total area peak. A representative electropherogram and charge variant distribution are shown in Figure 1 and Table 3, respectively.

[0332] [Table 3]

[0333] 4. Functional activity (efficacy) Dostarlimab is an IgG4 mAb that blocks the interaction between programmed death protein 1 (PD-1) and its ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). PD-1 is a cell surface receptor expressed on T cells that limits T cell activation through binding to PD-L1 and, to a lesser extent, PD-L2. PD-1 also limits tyrosine kinase signaling from the T cell antigen receptor and costimulatory receptors. The PD-1 / PD-L1 checkpoint functions as a negative regulator of T cell activity to help control local inflammatory responses and maintain autologous tolerance. The biological activity and binding characteristics of dostarlimab were assessed using various analytical methods, including the MSD potency assay, cellular potency assay (bioassay), and Fc receptor (FcRn) binding, as described below.

[0334] 4.1 MSD combination The Meso Scale Discovery (MSD) potency assay uses engineered CHOK1 cells that constitutively express the PD-1 protein. Dostarlimab activity is assessed using competitive binding, which measures the dose-dependent ability of dostarlimab to inhibit ligand binding to PD-1 on CHOK1 cells. A ligand (PD-L1) is used as the ligand in the assay (PD-L1-mFc). The readout is measured using a specific detection antibody mixture that emits a quantifiable electrochemiluminescence (ECL) signal. Results are reported as a percentage of potency relative to a reference substance (e.g., a control sample).

[0335] 4.2 Cellular bioassays A cellular potency bioassay was developed using the Promega PD-1 / PD-L1 Blockade Bioassay (Cat. No. J1250 or J1255) to closely mimic the mechanism of action of dostarlimab. Specifically, PD-1 effector cells (Jurkat T cells (Promega #J1155)) expressing human PD-1 and a luciferase reporter gene driven by the nuclear factor of activated T-cell response element (NFAT-RE) are co-cultured with PD-L1 aAPCs (artificial antigen-presenting cells) (CHO-K1 cells (Promega #J1095)) expressing PD-L1. When these two cell types are co-cultured, PD-1 / PD-L1 interaction inhibits TCR signaling and the resulting NFAT-RE-mediated luminescence. Addition of Dostarlimab (an anti-PD-1 antibody that blocks the PD-1 / PD-L1 interaction) releases an inhibitory signal, leading to TCR activation and NFAT-RE-mediated luminescence. Dostarlimab's blocking of the inhibitory signal is dose-dependent, and the resulting luminescence can be quantified using a plate reader. From these signal responses, four-parameter curves are generated for both the reference substance and the Dostarlimab sample by plotting relative luciferase units (RLU) on the y-axis and log2-transformed concentration on the x-axis. The median effective concentration (EC50) is interpolated and used to calculate the potency of the Dostarlimab sample relative to that of the reference substance.

[0336] 4.2.1 FcRn binding Neonatal FcRn binding plays a role in the metabolic fate of IgG molecules in the body: weak binding of IgG to FcRn significantly reduces its persistence in serum.

[0337] FcRn binding was assessed by BIACORE analysis. Dostarlimab reference material and Herceptin (IgG1) were each immobilized on a sensor chip. Various concentrations of the analyte (FcRn) were injected for analysis of the association and dissociation characteristics. The FcRn binding intensities of the three analyzed samples were similar, as expected for IgG1 and IgG4s (see Table 4).

[0338] [Table 4]

[0339] 5. Decomposition products and pathways To evaluate the degradation pathway of dostarlimab, a forced degradation study was performed on commercial-scale (batch #1) DS. The degraded samples were evaluated for purity by SE-HPLC, cIEF, reduced CE-SDS, and non-reduced CE-SDS. Additionally, samples were evaluated for functionality by MSD binding assay, cell bioassay, and FcRn binding assay. PTMs were evaluated by reduced Lys-C or tryptic peptide mapping. The results are summarized in the following sections.

[0340] 5.1 Pyrolysis conditions Dostarlimab samples were incubated at 40°C or 50°C for up to 3 weeks. Samples were kept frozen until testing.

[0341] The SE-HPLC results showed that the HMW showed a slight increase in species (Table 5).

[0342] Both reduced and non-reduced CE-SDS showed no changes other than analytical variations at 40 °C. These included LMW, medium molecular weight (MMW), and HMW for reduced CE-SDS; and "pre-peak" and "post-peak" regions for non-reduced CE-SDS (data not shown).

[0343] The cIEF data showed an increase in acidic species (up to 59.1%) compared to the unstressed control in samples incubated at 50°C for up to 3 weeks (Table 6).

[0344] Peptide map data showed isomerization at Asp261 / 266 / 276 (specific sites of isomerization were not identified), deamidation at Asn380 and Asn385, and a slight increase in oxidation at Met248 and Met354 at 40°C (Table 7). While these small changes are still within the analytical variability, this trend was confirmed by the 50°C data. Pyrolysis at 50°C for 3 weeks showed the same trends as those shown at 40°C, but the changes were more pronounced. A faster rate of increase in aggregates and fragments was also observed (data not shown).

[0345] The HMW species observed under 50°C conditions were primarily higher order aggregates, which differed from the dimerization observed in the unstressed and 40°C samples. Reduced or nonreduced CE-SDS did not show any change in HMW levels, although a slight increase in fragments was observed (data not shown).

[0346] As noted above, at 50°C, peptide map data showed increased isomerization at HC Asp261 / 266 / 276, deamidation at HC Asn380 and HC Asn385, and oxidation at HC Met248, HC Met354, and HC Met424. The increase in these PTMs resulted in a corresponding increase in the percentage of acidic species, consistent with forced degradation resulting from base hydrolysis and oxidative conditions. All other post-translational modification levels were within the analytical variability.

[0347] Relative potency and FcRn binding were unaffected by the changes seen under pyrolysis conditions at 40°C and 50°C for up to 3 weeks (Table 8). The small differences observed were within the expected analytical variability.

[0348] [Table 5]

[0349] [Table 6]

[0350] [Table 7]

[0351] [Table 8]

[0352] In summary, degradation products observed under pyrolysis conditions are HMW species, fragments, isomerization at Asp261 / 266 / 276, deamidation at Asn380 and Asn385, and oxidation at Met248, Met354, and Met424. The combination of the following attributes does not adversely affect the relative potency of dostarlimab (MSD binding (76%) and bioassay (86%)) and FcRn binding. Up to 11.2% HMW (from SE-HPLC) Fragment level of 4.5-4.6% (LMW%+MMW% in reduced CE-SDS and pre-peak% in non-reduced CE-SDS) -Maximum 13.1% isomerization at HC Asp261 / 266 / 276 (from peptide map) Deamidation of up to 14.7% at HC Asn380 and up to 5.9% at HC Asn385 (from peptide maps) Oxidation of up to 7.6% at HC Met248, up to 4.2% at HC Met354, and up to 3.5% at HC Met424 (from peptide maps) Up to 59.1% acidic species (from cIEF)

[0353] Extrapolating from HMW and potency (bioassay) data, up to 7.1% HC N-terminal pyroglutamic acid and up to 36% HMW can result in at least 60% potency (bioassay). Up to 26% HMW can result in at least 70% potency (bioassay). This was calculated using the slope of the line for 0.9% (control), 1.4% (40°C, 3 weeks), and 11.2% (50°C, 3 weeks) HMW samples, which have potencies (bioassay) of 98%, 94%, and 86%, respectively.

[0354] 5.2 Acid and Base Hydrolysis 5.2.1 Acid hydrolysis Dostarlimab samples were titrated to pH 4.0 with HCl and incubated at 25°C for up to 3 weeks. Upon completion, samples were stored frozen until testing. The first time point sample, TO, was exposed to low pH but not incubated at elevated temperature. Once the sample established the target pH, it was immediately frozen to prevent degradation.

[0355] The SE-HPLC results showed an increase in HMW species upon initial low pH exposure (Table 9). Addition of HCl to the sample initially exposed it to an extremely low pH locally, which caused dostarlimab to form higher-order aggregates. HMW species increased by approximately 2% over the 3-week incubation, while LMW species remained in trace amounts over the 3-week incubation. The HMW species observed under acid hydrolysis were primarily higher-order aggregates, which differed from the dimerization observed in the unstressed sample.

[0356] cIEF data showed no change in the percentage of acidic peaks between unstressed samples and samples incubated at pH 4.0 for up to 3 weeks (Table 10). The slight differences observed were within the expected analytical variability. A decrease in the percentage of the major peak was observed upon initial exposure to low pH, and this trend continued with extended incubation times. As the percentage of the major peak decreased, the percentage of basic peaks increased proportionally. The increased HMW levels shown in the SE-HPLC results of these acid-treated samples supports this finding, as one of the major components of the base species is protein aggregates. Reduced and non-reduced CE-SDS data showed no change (not shown). Peptide map data did not indicate differences in PTM levels associated with acid hydrolysis, and the slight differences observed were within the expected analytical variability (Table 11).

[0357] Relative potency and FcRn binding were unaffected by the changes seen at pH 4.0 for up to 3 weeks (Table 12), and the observed differences were within the expected analytical variability.

[0358] [Table 9]

[0359] [Table 10]

[0360] [Table 11]

[0361] [Table 12]

[0362] In summary, the degradation products observed under acid hydrolysis conditions are HMW species (mainly higher order aggregates). In conclusion, up to 15.2% HMW (by SE-HPLC) and up to 34.1% basic species do not adversely affect relative potency and FcRn binding.

[0363] 5.2.2 Base hydrolysis Dostarlimab samples were titrated to pH 9.0 with NaOH and incubated at 40°C for up to three weeks. The 40°C condition was selected based on an initial condition screen to ensure significant levels of degradation were observed. Upon completion, samples were stored frozen to prevent degradation until testing. The initial sample, T0, was also exposed to high pH but without the elevated temperature incubation. T0 samples were frozen immediately once the target pH was established in the sample.

[0364] SE-HPLC results showed an increase in HMW species upon initial high pH exposure (Table 13). Addition of NaOH to the sample initially exposed it locally to an extremely high pH, ​​which caused dostarlimab to form higher-order aggregates. HMW species increased by approximately 4% over the 3-week incubation. The HMW species observed under base hydrolysis were primarily higher-order aggregates, which differed from the dimerization observed in the unstressed sample. LMW species increased slightly and remained below 1% over the 3-week incubation. Note that the peak displayed as the major peak in the SE-HPLC plot is the monomer.

[0365] cIEF data showed a decrease in the percentage of the major peak upon initial exposure to high pH, ​​a trend that continued with increasing incubation time (Table 14). While the percentage of the major peak decreased, the percentage of the acidic peak increased accordingly. The percentage of the basic peak appeared to decrease slightly, but the change was still within the analytical variability. Both reduced and non-reduced CE-SDS data indicated no degradation upon initial exposure to high pH (not shown). After 3 weeks of incubation, an increase in fragment and HMW levels was observed. After 3 weeks of exposure of dostarlimab to high pH conditions, peptide map analysis showed increased isomerization at HC Asp 147, deamidation at HC Asn 380 and HC Asn 385, isomerization at LC Asp 151 / 167, and oxidation at Met 248 (Table 15). All other PTM levels were within the analytical variability. The combination of aggregation, fragmentation, isomerization, and deamidation shifted the charge profile of dostarlimab samples treated at high pH toward acidic species as the predominant species.

[0366] Even though the charge profile of dostarlimab shifted, the relative potency and FcRn binding were unaffected by the changes seen at pH 9.0 for up to 1 week (Table 16).

[0367] [Table 13]

[0368] [Table 14]

[0369] [Table 15]

[0370] [Table 16]

[0371] In summary, degradation products observed under base hydrolysis conditions were HMW species, fragments, isomerization at Asp147 and Asp151 / 167, deamidation at Asn380 and Asn385, and oxidation at Met248. The combination of the following attributes does not adversely affect relative potency and FcRn binding. Up to 9.2% HMW species (by SE-HPLC) Fragment 2.0-4.2% (LMW%+MMW% in reduced CE-SDS and pre-peak% in non-reduced CE-SDS) -Maximum 20.8% isomerization at HC Asp147 (from peptide map) Deamidation of up to 27.8% at HC Asn380 and up to 27.2% at HC Asn385 (from peptide maps) -Maximum 3.1% isomerization at LC Asp151 / 167 (from peptide map) Up to 7.1% oxidation at HC Met248 (from peptide map) Up to 96.8% acidic species (from cIEF) Up to 5.2% HC N-terminal pyroglutamic acid variants

[0372] It is anticipated that isomerization at HC Asp147 may be higher than the reported level of 20.8% without affecting relative potency or FcRn binding.

[0373] It is anticipated that deamidation at HC Asn380 and HC Asn385 may be higher than the reported levels of 27.8% and 27.2% without affecting relative potency or FcRn binding.

[0374] 5.3 Oxidation conditions 5.3.1 H2O2 treatment Hydrogen peroxide (H2O2) was added to dostarlimab samples to a final concentration of 0.1% (v:v) H2O2. Samples with H2O2 added were incubated at 25°C for up to 3 weeks. Upon completion, the H2O2 treatment was quenched with methionine, and samples were stored frozen until testing. T0 samples were exposed to H2O2 but not incubated at elevated temperatures. Methionine was added to the T0 samples immediately after H2O2 (to quench the reaction and prevent degradation).

[0375] The SE-HPLC results for the H2O2-treated samples showed no difference in the levels of HMW and LMW species (Table 17). Starting with a 1-week incubation time, the cIEF data showed a decrease in the percentage of the main peak with a corresponding increase in the percentage of acidic species (Table 18). The main peak also showed a front shoulder in the H2O2-treated samples.

[0376] No differences were observed in the CE-SDS reduction data for all treated samples (not shown). Non-reduced CE-SDS data showed an increase in fragment levels starting from the 3-day incubation time (not shown). Note that in the SE-HPLC plots, the peak displayed as the major peak is the monomer.

[0377] cIEF and non-reduced CE-SDS data indicated that the fragments generated by H2O2 migrated as acidic species in cIEF. Peptide map data showed slightly elevated oxidation levels at Met248, Met354, and Met424 in the Fc region of HC upon H2O2 exposure. The oxidation levels at all three Met sites reached nearly 100% after 2 weeks of incubation (Table 19). Oxidation at Met34 and Met103 in the CDR of HC began to show an increase after 1 week of incubation. All other PTM levels were within the analytical variability.

[0378] cIEF of the H2O2-treated sample showed a change in the shape of the major peak, and the charge profile shifted to more acidic species, indicating that the oxidized species were located in the acidic peak region and that cIEF could monitor the excessive oxidation of dostarlimab.

[0379] The relative potency of dostarlimab by MSD binding assay was not affected by the changes observed in the H2O2-treated samples, and the observed differences were within the expected analytical variability (Table 20). However, cell bioassays showed that the relative potency decreased to less than 50% after 2 weeks. Because the dostarlimab binding domain (ligand, PD-1) is located in the CDRs, this decrease in potency is related to oxidation of the CDRs. Furthermore, dostarlimab's mechanism of action does not involve effector function, which has been demonstrated to be low, so oxidation in the Fc region is unlikely to affect potency. Therefore, changes in the CDRs would have had the greatest impact on binding and therefore potency.

[0380] FcRn binding analysis was unaffected by immediate HO exposure but decreased as oxidation levels increased with incubation time (Table 20). After 2 weeks of incubation, oxidation was observed in the CDR regions (approximately 29% of HC Met34 and approximately 86% of HC Met103) as well as the Fc region (close to 100%). Since FcRn binding occurs via the Fc region, it can be assumed that oxidation in the Fc region contributed to the observed decrease in FcRn binding.

[0381] [Table 17]

[0382] [Table 18]

[0383] [Table 19]

[0384] [Table 20]

[0385] In summary, the degradation products observed under H2O2 oxidation conditions are fragments, oxidation in the Fc region (Met248, Met354, and Met424) and CDRs (Met34 and Met103). The combination of the following attributes does not adversely affect relative potency or FcRn binding. 0.5-1.0% fragments (LMW%+MMW% in reduced CE-SDS and pre-peak% in non-reduced CE-SDS) Oxidation of the Fc region of HC: Met248 up to 47.1%, Met354 up to 16.7%, and Met424 up to 29.0% (from peptide maps) Oxidation of the CDR regions of HC: Met34 less than 1.0% and Met103 up to 1.2% (from peptide map)

[0386] Extrapolating from the oxidation and potency (bioassay) data for HC Met34, up to 21% oxidation can result in at least 60% potency (bioassay), and up to 16% oxidation for Met34 can result in at least 70% potency (bioassay). This was calculated using the linear slope of the <1% (control), <1% (T0), and 28.8% (HO2 2 weeks) oxidation samples, which have potencies of 98%, 94%, and 47%, respectively (bioassay).

[0387] Extrapolating from the oxidation and potency (bioassay) data for HC Met103, up to 64% oxidation can result in at least 60% potency (bioassay). Up to 47% oxidation for Met103 can result in at least 70% potency (bioassay). This was calculated using the linear slopes for samples oxidized to <1% (control), 1.2% (T0), and 86.1% (HO 2 weeks), which have potencies of 98%, 94%, and 47%, respectively (bioassay).

[0388] It is anticipated that oxidation of the Fc region of HCs may be higher than reported levels of up to 47.1% for Met248, 16.7% for Met354, and 29.0% for Met424 without affecting relative potency or FcRn binding.

[0389] 5.3.2 AAPH treatment Because H2O2-treated dostarlimab did not show oxidation of Trp residues in the CDR region, the effect of Trp oxidation was assessed using 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). AAPH was added to dostarlimab samples to a final concentration of 5 mM AAPH. The AAPH-treated samples were incubated at 40°C for up to 7 days. The 40°C condition was selected based on initial screening conditions to ensure significant levels of degradation were observed. Upon completion, the AAPH treatment was quenched with methionine, and the samples were frozen and stored until testing. The TO samples were exposed to AAPH without incubation at elevated temperatures. Immediately after AAPH addition, methionine was added to the samples to quench the reaction and prevent degradation.

[0390] The SE-HPLC results showed an increase in HMW species in the AAPH-treated samples starting at 1 day of incubation. LMW species increased slightly but remained below 1% throughout the 7-day incubation period (Table 21).

[0391] The cIEF data showed a decrease in the percentage of the major peak starting with 1 day of incubation, a trend that continued with increasing incubation time (Table 22). As the major peak decreased, both acidic and basic species increased. Furthermore, the major peak in cIEF also shifted slightly toward the acidic side. Both reduced and non-reduced CE-SDS showed an increase in HMW species and fragments (not shown). This change appeared on day 1 for the non-reduced CE-SDS data and on day 3 for the reduced CE-SDS data. Peptide map data showed increased oxidation of HC Met248, HC Met354, HC Met424, and LC Trp50 starting on day 1. Oxidation levels at Met103 also showed a slight increasing trend on day 1, which was confirmed by the increase seen in the day 3 data (Table 23). All other PTM levels were within the analytical variability.

[0392] cIEF of the AAPH-treated sample showed a change in the shape of the major peak and a shift in the charge profile toward more acidic species, indicating that the oxidized species were located in the acidic peak region and that cIEF could monitor the excessive oxidation of dostarlimab.

[0393] Starting with the 3-day incubation samples, relative potency (MSD binding assay) decreased due to the changes seen in the AAPH-treated samples (Table 24). Relative potency began to decrease by cell bioassay after 1 day of incubation and by MSD binding assay after 3 days of incubation. Similar to the TO H2O2 samples, immediate exposure to AAPH (TO) did not induce a decrease in potency of dostarlimab samples, and both TO samples had low levels of CDR oxidation. With the exception of the TO H2O2 sample, the level of Fc oxidation was much higher. This finding demonstrates that increased Fc oxidation does not affect potency.

[0394] The AAPH day 1 samples had comparable methionine oxidation in both the CDR and Fc regions as the TO H2O2 samples. However, the AAPH day 1 samples showed decreased potency. The primary difference between the TO H2O2 samples and the day 1 AAPH-treated samples was the oxidation level of CDR LC Trp50. Thus, increased oxidation of CDR Trp50 reduces relative potency. A comparison of the potency results from the peroxide-treated and AAPH-treated samples is shown in Table 25.

[0395] Starting with the AAPH-incubated samples on day 3, FcRn binding decreased. Therefore, FcRn binding decreased with increasing oxidation, which is consistent with the findings in the H2O2-treated samples. After 3 days, Met in the CDR region (HC Met103 was approximately 7%) and Met in the Fc region (Met248 was approximately 89%, Met354 was approximately 45%, and Met424 was approximately 78%) were overoxidized. The decrease in FcRn binding may be due to oxidation of the Fc region involved in FcRn binding.

[0396] [Table 21]

[0397] [Table 22]

[0398] [Table 23]

[0399] [Table 24]

[0400] [Table 25]

[0401] In summary, the degradation products observed under AAPH oxidation conditions are HMW species, fragments, oxidation of Fc region Met (HC Met248, HC Met354, and HC Met424), CDR region Met (HC Met103), and LC CDR Trp50. The combination of the following attributes does not adversely affect relative potency and FcRn binding. Maximum 0.9% HMW species (by SE-HPLC) Fragment 0.8-1.2% (LMW%+MMW% in reduced CE-SDS and pre-peak% in non-reduced CE-SDS) Fc region: up to 3.3% oxidation at HC Met248, less than 1.0% oxidation at HC Met354, and less than 1.0% oxidation at HC Met424 (from peptide maps) Less than 1.0% oxidation of HC Met103 and less than 1.0% oxidation of LC Trp50 in the CDR region (from peptide maps)

[0402] Extrapolating from the oxidation and potency data for LC Trp50, up to 34% oxidation can result in at least 60% potency. Up to 25% oxidation can result in at least 70% potency. This was calculated using the linear slope of the <1% (control), <1% (TO), 38.9% (day 1), 74.3% (day 3), and 86.8% (day 5) oxidation samples, which have potencies of 98%, 102%, 44%, 14%, and 5%, respectively.

[0403] Because oxidation of LC Trp50 is the major oxidation variant under AAPH oxidation conditions, H2O2 oxidation conditions better reflect the tolerable oxidation levels of CDR region HC Met34, HC Met103 and Fc region HC Met248, HC Met354, and HC Met424.

[0404] 5.4 Photolysis Dostarlimab samples were exposed to white light (visible light, 10,000 lux hours) for 1 week at 25° C., followed by UV light for 1 week at 25° C. Once complete, samples were stored frozen until testing.

[0405] The SE-HPLC results showed an increase in HMW species at the end of the photolysis conditions (Table 26). The cIEF data showed an increase in the percentage of acidic species and a decrease in the percentage of the main peak (Table 27). The reduced CE-SDS results showed no changes beyond analytical variability. The non-reduced CE-SDS data showed a decrease in the main peak and a slight increase in both fragments and HMW species.

[0406] Peptide map data showed elevated oxidation at the Fc region Met (HC Met248, HC Met354, and HC Met424) and LC CDR Trp50 at the end of the photolysis study (Table 28). All other PTM levels were within analytical variability.

[0407] Relative potency and FcRn binding were unaffected by the photolysis conditions (Table 29). Thus, the moderate oxidation levels observed in the Fc region Met (<33% HC Met248, <11% HC Met354, <27% HC Met424) and LC CDR Trp50 (<5%) did not affect relative potency and FcRn binding.

[0408] [Table 26]

[0409] [Table 27]

[0410] [Table 28]

[0411] [Table 29]

[0412] In summary, the degradation products seen under photolysis conditions are HMW species, fragments, oxidation at the Fc region Met (Met248, Met354, and Met424) and LC CDR Trp50. Combinations of the following attributes did not adversely affect relative potency and FcRn binding. Up to 3.3% HMW species (by SE-HPLC) Fragment 1.1-1.5% (LMW%+MMW% in reduced CE-SDS and pre-peak% in non-reduced CE-SDS) Fc region: up to 33.4% oxidation at HC Met 248, up to 10.9% oxidation at HC Met 354, and up to 27.0% oxidation at Met 424 (from peptide maps) Up to 4.8% oxidation in CDR LC Trp50 (from peptide map)

[0413] 5.5 Continuous shaking Dostarlimab samples were subjected to shaking at 300 revolutions per minute (rpm) for up to 3 weeks at 25° C. Upon completion, samples were stored frozen until testing.

[0414] No changes in product quality were observed in SE-HPLC, cIEF, reduced CE-SDS, and non-reduced CE-SDS.

[0415] Relative potency and FcRn binding were unaffected by sustained shaking. Peptide map data showed that all PTM levels were within the variability of the assay.

[0416] In summary, under conditions of continuous stirring, no significant degradation products were observed and there was no effect on relative potency and FcRn binding.

[0417] 5.6 Overview of decomposition products The incidence of degradation products observed under different forced degradation conditions that do not affect the efficacy of dostarlimab is summarized below (Table 30).

[0418] [Table 30]

[0419] 6. Formulation testing 6.1 Analysis method 6.1.1 Appearance The appearance of the samples, including transparency and color, was examined under a black and white background using a YB-2 light box.

[0420] 6.1.2 pH pH values ​​were measured by a Seven Multi pH meter.

[0421] 6.1.3 UV280 Protein concentrations were determined by absorbance at 280 nm using a NANODROP2000 spectrophotometer, with an extinction coefficient of 1.615. Samples were diluted gravimetrically.

[0422] 6.1.4 SEC-HPLC Size exclusion chromatography was performed using an Agilent 1260 Infinity system and a TSKGel G3000SWXL column (300 × 7.8 mm, 5 μm). The mobile phase was 50 mM phosphate buffer (PB), 300 mM NaCl, pH 7.0 ± 0.1, with a flow rate of 1.0 ml / min. Samples were diluted to 1 mg / ml for injection and detected at 280 nm.

[0423] 6.1.5 cIEF Twenty micrograms of reference standard or sample was mixed with 0.5 μl of the pI 5.85 marker, 0.5 μl of the pI 8.40 marker, 2 μl of Pharmalyte 3-10, 2 μl of Pharmalyte 5-8, 35 μl of 1% methylcellulose (MC), and purified water to a final volume of 100 μl. This mixture was then analyzed on an iCE3 capillary isoelectric focusing analyzer equipped with a fluorocarbon (FC)-coated all-column detection capillary. Electrophoresis was performed in two steps: (1) 1.5 kV for 1 minute and (2) 3 kV for 8 minutes. The autosampler tray was maintained at 5°C throughout the experiment.

[0424] 6.1.6 Dynamic Light Scattering (DLS) Using a micropipette, 40 μl aliquots of undiluted sample were transferred to 40 μl disposable cuvettes in a safety hood. Triplicate measurements were performed for each sample. Data were analyzed automatically using Zetasizer software.

[0425] 6.1.7 DSC Capillary cell differential scanning calorimetry (DSC) is used to measure the thermal stability of proteins by detecting the difference in the amount of heat required to raise the temperature of a sample and a reference as a function of temperature. Specifically, it is used to measure the thermal transition midpoint (Tm), an indicator of the relative stability of a protein in solution. Samples were diluted to approximately 1 mg / ml in reference buffer. 400 μl of reference buffer was added to the odd-numbered wells of a 96-well plate, and 400 μl of sample was added to the even-numbered wells of the 96-well plate. Samples were scanned from 20 to 110°C, and data analysis was performed using MicroCal VPCapillary DSC automated data analysis software.

[0426] 6.2 pH / Buffer Screening Test Four buffer systems were prepared, each with three different pH values: 25 mM acetate buffer pH 4.5, 25 mM acetate buffer pH 5.0, 25 mM acetate buffer pH 5.5, citrate buffer pH 5.0, 25 mM citrate buffer pH 5.5, 25 mM citrate buffer pH 6.0, 25 mM histidine buffer pH 5.5, 25 mM histidine buffer pH 6.0, 25 mM histidine buffer pH 6.5, 25 mM phosphate buffer pH 6.5, 25 mM phosphate buffer pH 7.0, and 25 mM phosphate buffer pH 7.5 (summarized in Table 31).

[0427] [Table 31]

[0428] The bulk drug substance underwent buffer exchange via centrifugal diafiltration into one of the 12 buffer systems in Table 21. Sample protein concentrations were then adjusted to 20 mg / ml with the corresponding buffer. Each sample was then sterilized by filtration and filled into glass vials, which were immediately stoppered and capped.

[0429] Samples were stored statically at 40° C. and shaken at 250 rpm at 30° C. for up to 4 weeks. Samples were examined for appearance, pH, UV280, SEC-HPLC, cIEF, DLS, and particulate matter.

[0430] All samples remained clear or slightly opalescent when stored at 40°C for 4 weeks and shaken at 250 rpm at 30°C for 2 weeks.

[0431] The pH values ​​of all 12 buffer systems appeared to be stable during shaking and storage at 40°C.

[0432] The protein concentration of all samples was stable at around 20 mg / ml.

[0433] The particulate counts of all 12 buffer systems were stable during shaking and storage at 40°C.

[0434] SEC-HPLC results: When stored at 40°C for 4 weeks, candidates 1, 4, 11, and 12 showed a faster rate of decrease in the main peak content, with candidates 5, 6, and 7 appearing to be the most stable. When shaken at 250 rpm at 30°C for 4 weeks, candidates 10, 11, and 12 showed a faster rate of decrease in the main peak content, with candidates 7 and 8 appearing to be the most stable. When stored at 40°C for 4 weeks, candidates 4, 8, 9, 10, 11, and 12 showed a faster rate of acidic peak formation, and when shaken at 250 rpm at 30°C for 4 weeks, candidates 9, 11, and 12 showed a faster rate of acidic peak formation.

[0435] DLS results: Candidates 1, 2, and 4 showed a shift in hydrodynamic radius (Z-average) when samples were stored at 40°C for 4 weeks. This indicated that the proteins were likely unstable in the buffer system, and changes in folding and / or surface potential may have caused changes in intermolecular interactions, leading to different association or aggregation patterns as seen in DLS readings over the period. Candidates 2, 3, and 12 showed the same unstable performance in Z-average when samples were shaken at 250 rpm and 30°C for 4 weeks. The polydispersity index (PDI) value is another indicator that reflects the sample stability performance by DLS. Candidates 6, 7, and 10 maintained reasonably small PDI values ​​throughout the test, while candidates 1, 2, 3, 4, and 12 showed unstable and relatively large PDI values.

[0436] Therefore, the results show that citrate buffer at pH 5.5 or 6.0 and histidine buffer at pH 6.0 or 6.5 are the most stable buffer systems.

[0437] 6.3 Excipient Screening The preferred buffer system was tested using a range of excipients (various candidate formulations summarized in Table 32).

[0438] [Table 32]

[0439] Samples were stored statically at 40° C. and shaken at 250 rpm at 30° C. for up to 4 weeks. Samples were examined for appearance, pH, UV280, SEC-HPLC, cIEF, DLS, and particulate matter.

[0440] All samples remained colorless with a slight opalescence when stored at 40°C for 4 weeks and shaken at 250 rpm at 30°C for 2 weeks.

[0441] The pH values ​​and osmolality of all 11 formulations (F1-F11) appeared stable during shaking and storage at 40°C.

[0442] The protein concentration of all samples was stable at around 20 mg / ml.

[0443] The particulate counts of all 11 formulations were stable during shaking and storage at 40°C.

[0444] SEC-HPLC results: Candidate F2 appeared to be the most stable formulation among the 11 candidates when stored at 40°C for 4 weeks and shaken at 250 rpm at 30°C for 4 weeks.

[0445] cIEF results: When stored at 40°C for 4 weeks and shaken at 250 rpm at 30°C for 4 weeks, candidates F2 and F3 appeared to be the most stable formulations among the 11 candidates.

[0446] When the samples were stored at 40°C for 4 weeks and shaken at 250 rpm at 30°C for 4 weeks, all 11 candidates maintained stable hydrodynamic radii (Z-average).

[0447] With the exception of candidates 4 and 11, all other candidates maintained reasonably small PDI values ​​throughout the study.

[0448] 6.4 High concentration test Dostarlimab is currently formulated at 20 or 50 mg / mL in 25 mM citrate, 100 mM L-arginine-HCl, 31 mM NaCl, 0.02% (w / v) PS80, pH 6.0. To evaluate protein stability in high-concentration formulations, a study was conducted in which the antibody concentration was concentrated to 125 mg / mL. Five protein concentrations ranging from 50 to 125 mg / mL were evaluated in increments of 25 mg / mL. The results demonstrated that dostarlimab can be concentrated to 125 mg / mL and is generally stable.

[0449] Antibodies are formulated at the drug substance stage. The drug product (DP) has the same strength and formulation as the drug substance (DS), so the forced degradation test described above is applicable to the drug product.

[0450] 6.5 Shelf Life Stability Dostarlimab 50 mg / mL drug product (DP) is stable in selected formulations under recommended long-term storage conditions. Conclusions can be based on the studies performed. Under long-term storage conditions at 5°C, no changes in product quality were observed for up to 18 months. After 6 months of accelerated storage at 25±5°C, all drug batches showed only minor changes in product quality. Dostarlimab drug product exhibits a photostability equivalent to 30 days under ambient light conditions.

[0451] Results from the stability program to date indicate that dostarlimab DP is stable when stored at the recommended long-term storage conditions (5 ± 3°C). No discernible changes were observed when evaluated in analytical studies up to 18 months of storage. Trend analysis of DP stored at accelerated conditions (25 ± 2°C / 60 ± 5% relative humidity (RH)) for up to 6 months revealed only minor changes in product quality attributes. Currently, real-time, actual temperature data substantiates the recommended long-term storage of the drug product at 5 ± 3°C for up to 18 months, although we will continue to collect and evaluate stability data to further extend shelf life if necessary.

[0452] [Table 33]

[0453] The present invention includes the following items. 1. A composition comprising an oxidized variant of an anti-PD-1 antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 65%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 4%, or no more than 3% of the oxidized variant.

[0454] 2. The composition according to item 1, wherein the amount of oxidation variant in the composition is between the lower limit of detection of the method used to identify the oxidation variant and 65%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4% or 3%.

[0455] 3. The oxidation mutant (i) Any one of the following ranges: 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.05 to 65%, 0.05 to 60%, 0.05 to 50%, 0.05 to 40%, 0.05 to 30%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.1 to 65%, 0.1 to 60%, 0.1 to 50% selected from 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, and 2-3%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% Item 3. The composition according to item 1 or 2, comprising:

[0456] 4. The composition according to any one of items 1 to 3, wherein the oxidation mutant comprises an oxidation at the methionine and / or tryptophan residue of any one of SEQ ID NOs: 1 to 6.

[0457] 5. The composition according to any one of items 1 to 4, wherein the oxidation variant comprises one or a combination of oxidations at M34 of CDRH1, M103 of CDRH3 and / or W50 of CDRL2.

[0458] 6. (i) 21% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less oxidation in CDRH1 M34; (ii) 64% or less, 60% or less, 50% or less, 47% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less oxidation in M103 of CDRH3; and / or (iii) Oxidation of CDRL2 at W50 of 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less 6. The composition according to any one of items 1 to 5, comprising:

[0459] 7. The composition according to any one of items 1 to 6, comprising oxidation at M34 of CDRH1 in an amount of from the lower limit of detection of a method used to identify oxidation at M34 of CDRH1 to 21%, 20%, 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

[0460] 8. Oxidation of M34 in CDRH1 (i) Any one of the following ranges: 0.01-21%, 0.01-20%, 0.01-16%, 0.01-15%, 0.01-12.5%, 0.01-10%, 0.01-7.5%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.05-21%, 0.05-20%, 0.05-16%, 0.05-15%, 0.05-12.5%, 0.05-10%, 0.05-7.5%, 0.05-5%, 0.05-4%, 0.05-3%, 0. selected from 0.05-2%, 0.05-1%, 0.1-21%, 0.1-20%, 0.1-16%, 0.1-15%, 0.1-12.5%, 0.1-10%, 0.1-7.5%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-21%, 0.5-20%, 0.5-16%, 0.5-15%, 0.5-12.5%, 0.5-10%, 0.5-7.5%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, and 0.5-1%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% The composition according to any one of items 1 to 7, comprising:

[0461] 9. The composition according to any one of items 1 to 8, comprising oxidation at M103 of CDRH3 in an amount of from the lower limit of detection of a method used to identify oxidation at M103 of CDRH3 to 64%, 60%, 50%, 47%, 40%, 30%, 20%, 15%, 10%, 5%, 2%, or 1%.

[0462] 10. Oxidation of M103 in CDRH3 (i) Any one of the following ranges: 0.01-64%, 0.01-60%, 0.01-50%, 0.01-47%, 0.01-40%, 0.01-30%, 0.01-20%, 0.01-15%, 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.05-64%, 0.05-60%, 0.05-50%, 0.05-47%, 0.05-40%, 0.05-30%, 0.05-20%, 0.05-15%, 0.05-10%, 0.05-5%, 0.05-4%, 0.05-3%; selected from 0.05-2%, 0.05-1%, 0.1-64%, 0.1-60%, 0.1-50%, 0.1-47%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-64%, 0.5-60%, 0.5-50%, 0.5-47%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2% and 0.5-1%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 10. The composition according to any one of items 1 to 9, comprising:

[0463] 11. The composition of any one of items 1 to 10, comprising oxidation in W50 of CDRL2 in an amount of from the lower limit of detection of a method used to identify oxidation in W50 of CDRL2 to 30%, 25%, 20%, 15%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

[0464] 12. Oxidation of W50 of CDRL2 (i) Any one of the following ranges: 0.01-34%, 0.01-30%, 0.01-25%, 0.01-20%, 0.01-15%, 0.01-10%, 0.01-7.5%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.05-34%, 0.05-30%, 0.05-25%, 0.05-20%, 0.05-15%, 0.05-10%, 0.05-7.5%, 0.05-5%, 0.05-4%, 0.05-3%, 0.0 selected from 5-2%, 0.05-1%, 0.1-34%, 0.1-30%, 0.1-25%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-7.5%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-34%, 0.5-30%, 0.5-25%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-7.5%, 0.5-5%, 0.5-4%, or 0.5-3%, 0.5-2% and 0.5-1%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 12. The composition according to any one of items 1 to 11, comprising:

[0465] 13. The composition of any one of items 1 to 12, wherein the antibody comprises a heavy chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8.

[0466] 14. The composition of any one of items 1 to 13, wherein the antibody is at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 10.

[0467] 15. The composition of any one of items 1 to 14, wherein the antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10.

[0468] 16. The composition according to item 14 or item 15, comprising one or a combination of an oxidation at M248 of SEQ ID NO:9, an oxidation at M354 of SEQ ID NO:9 and / or an oxidation at M424 of SEQ ID NO:9.

[0469] 17. (i) 65% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, or 3% or less oxidation of M248 of SEQ ID NO: 9; (ii) an oxidation of 65% or less, 60%, 50%, 45%, 40%, 35%, 30%, 20%, 15%, 10%, 5%, 4%, or 3% or less at M354 of SEQ ID NO: 9; and / or (iii) an oxidation of 65% or less, 60%, 50%, 45%, 40%, 35%, 30%, 20%, 15%, 10%, 5%, 4%, or 3% in M424 of SEQ ID NO: 9 17. The composition according to any one of items 14 to 16, comprising:

[0470] 18. The composition according to any one of items 14 to 17, wherein the amount of oxidation in M248 in the composition is between the lower limit of detection of the method used to identify oxidation in M248 and 65%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, or 3%.

[0471] 19. Oxidation in M248 (i) Any one of the following ranges: 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.05 to 65%, 0.05 to 60%, 0.05 to 50%, 0.05 to 40%, 0.05 to 30%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.1 to 65%, 0.1 to 60%, 0.1 to 50% selected from 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, and 2-3%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 19. The composition according to any one of items 14 to 18, comprising in an amount of

[0472] 20. The composition of any one of items 14 to 19, wherein the amount of oxidation in M354 in the composition is between the lower limit of detection of the method used to identify oxidation in M354 and 65%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, or 3%.

[0473] 21. Oxidation in M354 (i) Any one of the following ranges: 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.05 to 65%, 0.05 to 60%, 0.05 to 50%, 0.05 to 40%, 0.05 to 30%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.1 to 65%, 0.1 to 60%, 0.1 to 50% selected from 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, and 2-3%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 21. The composition according to any one of items 14 to 20, comprising:

[0474] 22. The composition according to any one of items 14 to 21, wherein the amount of oxidation at M242 in the composition is between the lower limit of detection of the method used to identify oxidation at M424 and 65%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4% or 3%.

[0475] 23. Oxidation in M424 (i) Any one of the following ranges: 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.05 to 65%, 0.05 to 60%, 0.05 to 50%, 0.05 to 40%, 0.05 to 30%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.1 to 65%, 0.1 to 60%, 0.1 to 50% selected from 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, and 2-3%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 23. The composition according to any one of items 14 to 22, comprising:

[0476] 24. The composition of any one of items 2, 7, 9, 11, 18, 20 or 22, wherein the method used to identify oxidation variants or oxidations at specific positions is optionally peptide mapping tandem mass spectrometry as shown in Example 1.

[0477] 25. The method, (i) denaturing, reducing, alkylating, and digesting the anti-PD-1 antibody; and (ii) performing liquid chromatography with tandem mass spectrometry; 25. The composition according to item 24, comprising:

[0478] 26. The method, (i) denaturing the anti-PD-1 antibody with guanidine hydrochloride, reducing it with dithiothreitol, alkylating it with iodoacetamide, digesting it with endoproteinase Lys-C or trypsin at 37°C for 4 hours, and quenching it with trifluoroacetic acid; and (ii) performing ultra-high performance liquid chromatography with tandem electrospray ionization mass spectrometry; 26. The composition according to item 24 or item 25, comprising:

[0479] 27. A composition comprising aggregation variants of an anti-PD-1 antibody, wherein the aggregation variants comprise a heavy chain sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition comprises no more than 36%, no more than 35%, no more than 30%, no more than 26%, no more than 25%, no more than 20%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% aggregation variants.

[0480] 28. The composition according to item 27, wherein the amount of aggregation variants in the composition is between the lower limit of detection of the method used to identify aggregation variants and 36%, 35%, 30%, 26%, 25%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0481] 29. The aggregation mutant is: (i) Any one of the following ranges: 0.01-36%, 0.01-35%, 0.01-30%, 0.01-26%, 0.01-25%, 0.01-20%, 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.05-36%, 0.05-35%, 0.05-30%, 0.05-26%, 0.05-25%, 0.05-20%, 0.05-10%, 0.05-5%, 0.05-4%, 0.05-3%, 0. 0.05-2%, 0.05-1%; selected from 0.1-36%, 0.1-35%, 0.1-30%, 0.1-26%, 0.1-25%, 0.1-20%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-36%, 0.5-35%, 0.5-30%, 0.5-26%, 0.5-25%, 0.5-20%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, and 0.5-1%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% Item 29. The composition according to item 27 or 28, comprising an amount of

[0482] 30. The composition of any one of items 27 to 29, wherein the antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10.

[0483] 31. The composition according to any one of items 27 to 30, wherein the method used to identify the aggregation mutant is size exclusion chromatography.

[0484] 32. The composition according to any one of items 27 to 31, wherein the method used to identify the aggregation mutants is optionally SE-HPLC as shown in Example 5.1.

[0485] 33. A composition comprising an antibody having a heavy chain sequence of SEQ ID NO:9 and a light chain sequence of SEQ ID NO:10, wherein the composition comprises: (i) no more than 65%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, or no more than 3% oxidation variants; and / or (ii) no more than 36%, no more than 35%, no more than 30%, no more than 26%, no more than 25%, no more than 20%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% aggregation variants.

[0486] 34. The composition according to item 33, wherein the amount of oxidation variant in the composition is between the lower limit of detection of the method used to identify the oxidation variant and 65%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4% or 3%.

[0487] 35. The oxidized mutant is (i) Any one of the following ranges: 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.05 to 65%, 0.05 to 60%, 0.05 to 50%, 0.05 to 40%, 0.05 to 30%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.1 to 65%, 0.1 to 60%, 0.1 to 50% selected from 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, and 2-3%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 35. The composition according to item 33 or 34, comprising an amount of

[0488] 36. The composition according to item 34 or 35, wherein the method used to identify the oxidation variant is optionally peptide mapping tandem mass spectrometry as shown in Example 1.

[0489] 37. The method, (i) denaturing, reducing, alkylating, and digesting the anti-PD-1 antibody; and (ii) performing liquid chromatography with tandem mass spectrometry; 37. The composition according to any one of items 34 to 36, comprising:

[0490] 38. The method, (i) denaturing the anti-PD-1 antibody with guanidine hydrochloride, reducing it with dithiothreitol, alkylating it with iodoacetamide, digesting it with endoproteinase Lys-C or trypsin at 37°C for 4 hours, and quenching it with trifluoroacetic acid; and (ii) performing ultra-high performance liquid chromatography with tandem electrospray ionization mass spectrometry; 38. The composition according to any one of items 34 to 37, comprising:

[0491] 39. The composition of any one of items 33 to 38, wherein the amount of aggregation variant in the composition is between the lower limit of detection of the method used to identify the aggregation variant and 36%, 35%, 30%, 26%, 25%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0492] 40. The aggregation mutant is (i) Any one of the following ranges: 0.01-36%, 0.01-35%, 0.01-30%, 0.01-26%, 0.01-25%, 0.01-20%, 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.05-36%, 0.05-35%, 0.05-30%, 0.05-26%, 0.05-25%, 0.05-20%, 0.05-10%, 0.05-5%, 0.05-4%, 0.05-3%, 0. 0.05-2%, 0.05-1%; selected from 0.1-36%, 0.1-35%, 0.1-30%, 0.1-26%, 0.1-25%, 0.1-20%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-36%, 0.5-35%, 0.5-30%, 0.5-26%, 0.5-25%, 0.5-20%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, and 0.5-1%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 40. The composition according to any one of items 33 to 39, comprising an amount of

[0493] 41. The composition according to item 39 or 40, wherein the method used to identify the aggregation mutant is size exclusion chromatography.

[0494] 42. The composition according to any one of items 39 to 41, wherein the method used to identify the aggregation variants is optionally SE-HPLC as shown in Example 5.1.

[0495] 43. A composition comprising a charge variant of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; (i) no more than 100%, no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 35%, no more than 30%, or no more than 25% acidic variants; and / or (ii) 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 8% or less, 7.5% or less, 7% or less, 6% or less, or 5% or less of basic variants; and / or (iii) 1% or more, 2.6% or more, 3% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 90% or more of the major isoforms A composition comprising:

[0496] 44. The composition according to item 43, wherein the amount of acidic variant in the composition is between the lower limit of detection of the method used to identify the acidic variant and 100%, 90%, 80%, 70%, 60%, 50%, 40%, 35%, 30% or 25%.

[0497] 45. Acidic variants, (i) selected from any one of the following ranges: 5-100%, 5-90%, 5-80%, 5-70%, 5-60%, 5-50%, 5-40%, 5-35%, 5-30%, 5-25%, 10-100%, 10-97%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-35%, 10-30%, 10-25%, 20-100%, 20-97%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-35%, 20-30%, and 20-25%; or (ii) about 60%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, or about 10% 45. The composition according to item 43 or 44, comprising in an amount of

[0498] 46. ​​The composition according to any one of items 43 to 45, wherein the amount of basic variant in the composition is between the lower limit of detection of the method used to identify the basic variant and 35%, 30%, 25%, 20%, 15%, 10%, 8%, 7.5%, 7%, 6% or 5%.

[0499] 47. Basic mutants are (i) selected from any one of the following ranges: 0.1-35%, 0.1-30%, 0.1-25%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-8%, 0.1-7.5%, 0.1-7%, 0.1-6%, 0.1-5%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 1-8%, 1-7.5%, 1-7%, 1-6%, and 1-5%; or (ii) about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 7.5%, or about 5% 47. The composition according to any one of items 43 to 46, comprising in an amount of

[0500] 48. The main form is (i) selected from any one of the following ranges: 2-90%, 2-80%, 2-75%, 5-90%, 10-90%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 5-80%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, and 60-80%; or (ii) about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, or about 55% 48. The composition according to any one of items 43 to 47, comprising an amount of

[0501] 49. A composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; the composition comprising: 10 to 97% acidic variants; and / or 0.1 to 35% basic variants; and / or 2 to 80% of the major isoform.

[0502] 50. A composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; wherein the composition comprises: 35% or less acidic variants; and / or 5% or less basic variants; and / or 55% or more of the major isoform.

[0503] 51. A composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; the composition comprising: 10-30% acidic variants; and / or 0.1-10% basic variants; and / or 60-80% of the major isoform.

[0504] 52. The composition of any one of items 43 to 51, wherein the percent acidic variants, percent basic variants, and percent major isoforms of the composition are determined using capillary isoelectric focusing.

[0505] 53. The composition according to any one of items 1 to 52, comprising a deamidated mutant.

[0506] 54. The composition of item 53, wherein the deamidated variant comprises a deamidated residue selected from an aspartic acid residue, a succinimido-aspartic acid residue, and an isoaspartic acid residue.

[0507] 55. The composition of item 54, wherein the deamidated mutant comprises up to 100% deamidation at N380 and / or N385 of SEQ ID NO: 9.

[0508] 56. The deamidated mutant is N380 at 0.1 to 100%, 0.1 to 90%, 0.1 to 80%, 0.1 to 70%, 0.1 to 60%, 0.1 to 50%, 0.1 to 40%, 0.1 to 30%, 0.1 to 20%, 0.1 to 10%, 1 to 100%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 55. The composition of item 54, comprising 10%, 2 to 100%, 3 to 100%, 4 to 100%, 5 to 100%, 6 to 100%, 7 to 100%, 8 to 100%, 9 to 100%, 2 to 30%, 3 to 30%, 4 to 30%, 5 to 30%, 2 to 40%, 3 to 40%, 4 to 40%, 5 to 40%, 2 to 10%, 3 to 10%, 4 to 10%, or 5 to 9% deamidation.

[0509] 57. The composition of item 54, wherein the deamidated variants comprise 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more deamidation in N380.

[0510] 58. The deamidated mutant is N385 at 0.1 to 100%, 0.1 to 90%, 0.1 to 80%, 0.1 to 70%, 0.1 to 60%, 0.1 to 50%, 0.1 to 40%, 0.1 to 30%, 0.1 to 20%, 0.1 to 10%, 1 to 100%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 10%, 2 58. The composition of any one of items 54 to 57, comprising from 100%, 3 to 100%, 4 to 100%, 5 to 100%, 6 to 100%, 7 to 100%, 8 to 100%, 9 to 100%, 2 to 30%, 3 to 30%, 4 to 30%, 5 to 30%, 2 to 40%, 3 to 40%, 4 to 40%, 5 to 40%, 2 to 10%, 3 to 10%, 4 to 10%, or 5 to 9% deamidation.

[0511] 59. The composition of any one of items 54 to 58, wherein the deamidated variant comprises 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more deamidation in N385.

[0512] 60. The composition according to any one of items 53 to 59, wherein the deamidated mutant comprises the sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

[0513] 61. A composition according to any one of items 1 to 60, comprising an isomerized variant.

[0514] 62. The composition according to item 61, comprising up to 100% isomerization at D147 of SEQ ID NO: 9.

[0515] 63. The composition according to item 61 or 62, comprising 0.1 to 90%, 0.1 to 80%, 0.1 to 70%, 0.1 to 60%, 0.1 to 50%, 0.1 to 40%, 0.1 to 30%, 0.1 to 20%, 0.1 to 15%, 0.1 to 10%, 1 to 100%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 15%, or 1 to 10% of the isomerized variant.

[0516] 64. The composition according to any one of items 1 to 63, comprising up to 100% heavy chain N-terminal pyroglutamic acid variants and / or up to 100% heavy chain C-terminal lysine truncation variants.

[0517] 65. 0.1~100%, 0.1~90%, 0.1~80%, 0.1~70%, 0.1~60%, 0.1~50%, 0.1~40%, 0.1~30%, 0.1~20%, 0.1~10%, 0.1~9%, 0.1~8%, 0.1~7%, 0.1~6%, 0.1~5%, 0.1~4%, 0.1~3%, 0.1~2%, 0.1~1%, 65. The composition according to any one of items 1 to 64, comprising 1 to 100%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 10%, 1 to 9%, 1 to 8%, 1 to 7%, 1 to 6%, 1 to 5%, 1 to 4%, 1 to 3%, or 1 to 2% heavy chain N-terminal pyroglutamic acid variant.

[0518] 66. A composition comprising 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of a heavy chain C-terminal lysine truncation mutant.

[0519] 67. The composition according to any one of items 1 to 66, comprising 1 to 100%, 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 99%, 96 to 99%, or 97 to 99% of a heavy chain C-terminal lysine truncation mutant.

[0520] 68. A composition comprising an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and / or SEQ ID NO:13, and a light chain sequence of SEQ ID NO:10, wherein the composition comprises no more than 64%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 4%, or no more than 3% oxidized variants.

[0521] 69. The composition according to item 68, wherein the amount of oxidation variant in the composition is between the lower limit of detection of the method used to identify the oxidation variant and 65%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4% or 3%.

[0522] 70. The oxidized variant (i) Any one of the following ranges: 0.01 to 65%, 0.01 to 60%, 0.01 to 50%, 0.01 to 40%, 0.01 to 30%, 0.01 to 20%, 0.01 to 15%, 0.01 to 10%, 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.05 to 65%, 0.05 to 60%, 0.05 to 50%, 0.05 to 40%, 0.05 to 30%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.1 to 65%, 0.1 to 60%, 0.1 to 50% selected from 0.1-40%, 0.1-30%, 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.5-65%, 0.5-60%, 0.5-50%, 0.5-40%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 1-65%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, and 2-3%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 70. The composition according to item 68 or item 69, comprising in an amount of

[0523] 71. The composition according to any one of items 68 to 70, wherein the method used to identify the oxidation variants is optionally peptide mapping tandem mass spectrometry as shown in Example 1.

[0524] 72. The method, (i) denaturing, reducing, alkylating, and digesting the anti-PD-1 antibody; and (ii) performing liquid chromatography with tandem mass spectrometry; 72. The composition according to any one of items 68 to 71, comprising:

[0525] 73. The method, (i) denaturing the anti-PD-1 antibody with guanidine hydrochloride, reducing it with dithiothreitol, alkylating it with iodoacetamide, digesting it with endoproteinase Lys-C or trypsin at 37°C for 4 hours, and quenching it with trifluoroacetic acid; and (ii) performing ultra-high performance liquid chromatography with tandem electrospray ionization mass spectrometry; 73. The composition according to any one of items 68 to 72, comprising:

[0526] 74. A composition comprising an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and / or SEQ ID NO:13, and a light chain sequence of SEQ ID NO:10, wherein the composition comprises no more than 36%, no more than 35%, no more than 30%, no more than 26%, no more than 25%, no more than 20%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% aggregation variants.

[0527] 75. The composition of item 74, wherein the amount of aggregation variants in the composition is between the lower limit of detection of the method used to identify aggregation variants and 36%, 35%, 30%, 26%, 25%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0528] 76. The aggregation mutant is i) Any one of the following ranges: 0.01-36%, 0.01-35%, 0.01-30%, 0.01-26%, 0.01-25%, 0.01-20%, 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.05-36%, 0.05-35%, 0.05-30%, 0.05-26%, 0.05-25%, 0.05-20%, 0.05-10%, 0.05-5%, 0.05-4%, 0.05-3%, 0.0 selected from 5-2%, 0.05-1%; 0.1-36%, 0.1-35%, 0.1-30%, 0.1-26%, 0.1-25%, 0.1-20%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-36%, 0.5-35%, 0.5-30%, 0.5-26%, 0.5-25%, 0.5-20%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, and 0.5-1%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% 76. The composition according to item 74 or item 75, comprising an amount

[0529] 77. The composition of any one of items 74 to 76, wherein the method used to identify aggregation mutants is size exclusion chromatography.

[0530] 78. The composition according to any one of items 27 to 31, wherein the method used to identify aggregation variants is optionally SE-HPLC as shown in Example 5.1.

[0531] 79. A composition comprising a variant of an anti-PD-1 antibody, wherein the variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; the composition has at least 60% of the potency of a composition comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, 10 to 97% acidic variants, 0.1 to 35% basic variants, 2 to 80% major isoforms, 4.8% or less light chain W50 oxidation variants, 1% or less heavy chain M34 oxidation variants, 1.2% or less heavy chain M103 oxidation variants, 15.2% or less aggregation variants, and 1% or less heavy ... M103 oxidation variants. 1. A composition having 6.7% or less heavy chain M354 oxidation mutants, 29.0% or less heavy chain M424 oxidation mutants, 47.1% or less heavy chain M248 oxidation mutants, 20.8% or less heavy chain D147 isomerization mutants, 13.1% or less heavy chain D151 or D167 isomerization mutants, 3.1% or less heavy chain D261, D266 or D276 isomerization mutants, 4.6% or less fragmentation mutants, 27.8% or less heavy chain N380 deamidation mutants, 27.2% or less heavy chain N385 deamidation mutants, about 7.4% or less heavy chain N311 deamidation mutants, about 2.0% or less heavy chain N430 deamidation mutants, 90% or more heavy chain C-terminal lysine deletion mutants (ΔK443), and 1% or less heavy chain N-terminal pyroglutamic acid mutants.

[0532] 80. A composition comprising a variant of an anti-PD-1 antibody, wherein the variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; at least 60% of the potency of a composition comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, 10 to 97% acidic variants, 0.1 to 35% basic variants, 2 to 80% major isoforms, 0.01 to 4.8% light chain W50 oxidation variants, 0.01 to 1% heavy chain M34 oxidation variants, 0.01 to 1.2% heavy chain M103 oxidation variants, 0.01 to 15.2% aggregation variants, 0.01 to 16.7% Heavy chain M354 oxidation mutant, 0.01 to 29.0% heavy chain M424 oxidation mutant, 0.01 to 47.1% heavy chain M248 oxidation mutant, 0.01 to 20.8% heavy chain D147 isomerization mutant, 0.01 to 13.1% heavy chain D151 or D167 isomerization mutant, 0.01 to 3.1% heavy chain D261, D266 or D276 isomerization mutant, 0.01 to 4.6% fragmentation mutant , 0.01 to 27.8% heavy chain N380 deamidation mutants, 0.01 to 27.2% heavy chain N385 deamidation mutants, approximately 0.01 to 7.4% heavy chain N311 deamidation mutants, approximately 0.01 to 2.0% heavy chain N430 deamidation mutants, more than 90% heavy chain C-terminal lysine deletion mutants (ΔK443), and 0.01 to 1% heavy chain N-terminal pyroglutamic acid mutants.

[0533] 81. The composition of any one of items 1 to 80, wherein the antibody is a full-length antibody.

[0534] 82. The composition of any one of items 1 to 81, wherein the antibody is humanized.

[0535] 83. The composition of any one of items 1 to 82, formed during production or storage of the antibody.

[0536] 84. A pharmaceutical composition comprising the composition according to any one of items 1 to 83 and at least one pharmaceutically acceptable excipient.

[0537] 85. A formulation comprising the pharmaceutical composition of item 82, comprising about 20 mg / mL to about 125 mg / mL of antibody and a buffer having a pH of about 5.5 to about 6.5.

[0538] 86. The formulation of item 85, wherein the buffer is selected from a citrate buffer or a histidine buffer.

[0539] 87. The formulation of item 85 or item 86, wherein the buffer is a citrate buffer at a pH of about 6.0.

[0540] 88. The formulation of any one of items 85 to 87, further comprising arginine and / or trehalose.

[0541] 89. The formulation of any one of items 85 to 88, further comprising polysorbate 80.

[0542] 90. The formulation of any one of items 85 to 89, further comprising sodium chloride at a concentration to adjust the osmolality to about 290 to 325 mOsm / kg.

[0543] 91. A formulation comprising the pharmaceutical composition of item 84, comprising: (a) about 20 mg / mL to about 125 mg / mL of antibody, (b) about 10 mM to about 40 mM citrate buffer or histidine buffer, (c) about 80 mM to about 120 mM arginine or about 2 to about 10% w / v trehalose, (d) about 20 mM to about 40 mM sodium chloride, and (e) about 0.01% to about 0.1% w / v polysorbate 80 at a pH of about 5.5 to about 6.5.

[0544] 92. The formulation of item 91, comprising about 20 mg / mL of antibody, about 25 mM citrate buffer, about 100 mM arginine, about 31 mM sodium chloride, and about 0.02% w / v polysorbate 80 at about pH 6.

[0545] 93. The formulation of item 91, comprising about 50 mg / mL of antibody, about 25 mM citrate buffer, about 100 mM arginine, about 31 mM sodium chloride, and about 0.02% (w / v) polysorbate 80 at about pH 6.

[0546] 94. An injection device comprising the composition according to any one of items 1 to 83, the pharmaceutical composition according to item 84 or the formulation according to any one of items 85 to 94.

[0547] 95. A cell culture medium comprising the composition according to any one of items 1 to 83.

[0548] 96. An elution solution comprising the composition according to any one of items 1 to 83.

[0549] 97. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the composition according to any one of items 1 to 83, the pharmaceutical composition according to item 84, or the formulation according to any one of items 85 to 93.

[0550] 98. The method of item 97, wherein the composition is administered in a dose of about 500 mg.

[0551] 99. The method of item 98, wherein the composition is administered once every three weeks.

[0552] 100. The method according to item 98 or item 99, wherein the composition is administered in four cycles.

[0553] 101. The method of item 97, wherein the composition is administered at a first dose of about 500 mg once every 3 weeks for 4 cycles, followed by a second dose of about 1000 mg once every 6 weeks or more.

[0554] 102. The method according to item 101, wherein a second dose of about 1000 mg once every 6 weeks or more is continued to maintain clinical benefit.

[0555] 103. A composition according to any one of items 1 to 83, a pharmaceutical composition according to item 84, or a formulation according to any one of items 84 to 93 for use in therapy.

[0556] 104. A composition according to any one of items 1 to 83, a pharmaceutical composition according to item 84, or a formulation according to any one of items 85 to 93 for use in the treatment of cancer.

[0557] 105. Use of a composition according to any one of items 1 to 83, a pharmaceutical composition according to item 84, or a formulation according to any one of items 85 to 93 in the manufacture of a medicament for use in the treatment of cancer.

[0558] Sequence Listing SEQ ID NO: 1 - CDRH1 SYDMS

[0559] SEQ ID NO:2 - CDRH2 TISGGGSYTYYQDSVKG

[0560] SEQ ID NO:3 - CDRH3 PYYAMDY

[0561] SEQ ID NO:4 - CDRL1 KASQDVGTAVA

[0562] SEQ ID NO:5 - CDRL2 WASTLHT

[0563] SEQ ID NO:6 - CDRL3 QHYSSYPWT

[0564] SEQ ID NO: 7 - Heavy chain variable region (CDRs underlined) [ka]

[0565] SEQ ID NO:8 - Light chain variable region (CDRs underlined) [ka]

[0566] SEQ ID NO: 9 - Full heavy chain sequence (CDRs are underlined) [ka]

[0567] SEQ ID NO: 10 - Full light chain sequence (CDRs are underlined) [ka]

[0568] SEQ ID NO: 11 - Full heavy chain sequence with N380D modification (CDRs are underlined) [ka]

[0569] SEQ ID NO: 12 - Full heavy chain sequence with N385D modification (CDRs underlined) [ka]

[0570] SEQ ID NO: 13 - Full heavy chain sequence with N380D and N385D modifications (CDRs are underlined) [ka]

[0571] SEQ ID NO:14 -- CDRL3 Alternative QHYNSYPWT

Claims

1. 1. A composition comprising an oxidized variant of an anti-PD-1 antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; wherein the composition comprises no more than 65% oxidized variant.

2. 2. The composition of claim 1, wherein said oxidized variant comprises an oxidation at the methionine and / or tryptophan residues of any one of SEQ ID NOs: 1-6.

3. 3. The composition of claim 1, wherein the oxidation variant comprises one or a combination of oxidations at M34 of CDRH1, M103 of CDRH3 and / or W50 of CDRL2.

4. 4. The composition of any one of claims 1 to 3, comprising one or a combination of: no more than 21% oxidation at M34 of CDRH1; no more than 64% oxidation at M103 of CDRH3; and / or no more than 34% oxidation at W50 of CDRL2.

5. The composition of any one of claims 1 to 4, wherein the antibody comprises a heavy chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

8.

6. 6. The composition of any one of claims 1 to 5, wherein the antibody is at least 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or at least about 90% identical to the light chain amino acid sequence of SEQ ID NO:

10.

7. The composition of any one of claims 1 to 6, wherein the antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO:

10.

8. 8. The composition of claim 6 or claim 7, comprising one or a combination of 65% or less oxidation at M248 of SEQ ID NO:9, 65% or less oxidation at M354 of SEQ ID NO:9 and / or 65% or less oxidation at M424 of SEQ ID NO:

9.

9. 1. A composition comprising aggregation variants of an anti-PD-1 antibody, said aggregation variants comprising a heavy chain sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; said composition comprising no more than 36% aggregation variants.

10. The composition of claim 9, wherein the antibody comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO:

10.

11. A composition comprising an antibody having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, comprising (i) no more than 65% oxidation variants; and / or (ii) no more than 36% aggregation variants.

12. 1. A composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; wherein the composition comprises: 100% or less acidic variants; and / or 35% or less basic variants; and / or 1% or more of a major isoform.

13. 1. A composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; 10 to 97% acidic variants; and / or 0.1 to 35% basic variants; and / or 2 to 80% of the major isoform.

14. 1. A composition comprising charge variants of an anti-PD-1 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; wherein the composition comprises no more than 35% acidic variants; and / or no more than 5% basic variants; and / or 55% or more of the major isoform.

15. The composition of any one of claims 12 to 14, wherein the percent acidic variants, percent basic variants and percent major isoforms of the composition are determined using capillary isoelectric focusing.

16. The composition of any one of claims 1 to 15, comprising a deamidated mutant.

17. 17. The composition of claim 16, wherein the deamidated variant comprises a deamidated residue selected from an aspartic acid residue, a succinimido-aspartic acid residue, or an isoaspartic acid residue.

18. 18. The composition of claim 17, wherein the deamidated variant comprises up to 100% deamidation at N380 and / or N385 of SEQ ID NO:

9.

19. 19. The composition of claim 17 or claim 18, wherein the deamidated mutant comprises the sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:

13.

20. A composition according to any one of claims 1 to 19, comprising an isomerised variant.

21. 21. The composition of claim 20, comprising up to 100% isomerization at D147 of SEQ ID NO:

9.

22. 22. The composition of any one of claims 1 to 21, comprising up to 100% heavy chain N-terminal pyroglutamic acid variants and / or up to 100% heavy chain C-terminal lysine truncation variants.

23. A composition comprising an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO: 13, and a light chain sequence of SEQ ID NO: 10, and comprising no more than 64% oxidized variants.

24. A composition comprising an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO: 13, and a light chain sequence of SEQ ID NO: 10, and comprising 36% or less aggregation variants.

25. 1. A composition comprising a variant of an anti-PD-1 antibody, said variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; said composition having at least 60% of the potency of a composition comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, 10-97% acidic variants, 0.1-35% basic variants, 2-80% major isoforms, 4.8% or less light chain W50 oxidation variants, 1% or less heavy chain M34 oxidation variants, 1.2% or less heavy chain M103 oxidation variants, and 15.2% or less aggregation variants. 16.7% or less heavy chain M354 oxidation mutants, 29.0% or less heavy chain M424 oxidation mutants, 47.1% or less heavy chain M248 oxidation mutants, 20.8% or less heavy chain D147 isomerization mutants, 13.1% or less heavy chain D151 or D167 isomerization mutants, 3.1% or less heavy chain D261, D266 or D276 isomerization mutants, 4.6% or less fragmentation mutants, 27.8% or less heavy chain N380 deamidation mutants, 27.2% or less heavy chain N385 deamidation mutants, about 7.4% or less heavy chain N311 deamidation mutants, about 2.0% or less heavy chain N430 deamidation mutants, 90% or more heavy chain C-terminal lysine deletion mutants (ΔK443), and 1% or less heavy chain N-terminal pyroglutamic acid mutants.

26. The composition of any one of claims 1 to 25, wherein the antibody is a full-length antibody.

27. The composition of any one of claims 1 to 26, wherein the antibody is humanized.

28. The composition of any one of claims 1 to 27, formed during the manufacture or storage of the antibody.

29. A pharmaceutical composition comprising the composition of any one of claims 1 to 28 and at least one pharmaceutically acceptable excipient.

30. 30. A formulation comprising the pharmaceutical composition of claim 29, comprising about 20 mg / mL to about 125 mg / mL of antibody and a buffer at a pH of about 5.5 to about 6.

5.

31. 31. The formulation of claim 30, wherein the buffer is selected from a citrate buffer or a histidine buffer.

32. 32. The formulation of claim 30 or claim 31, wherein the buffer is a citrate buffer at a pH of about 6.

0.

33. The formulation of any one of claims 30 to 32, further comprising arginine and / or trehalose.

34. The formulation of any one of claims 30 to 33, further comprising polysorbate 80.

35. 35. The formulation of any one of claims 30 to 34, further comprising sodium chloride in a concentration to adjust the osmolality of the formulation to about 290 to 325 mOsm / kg.

36. 30. A formulation comprising the pharmaceutical composition of claim 29, comprising: (a) about 20 mg / mL to about 125 mg / mL of antibody; (b) about 10 mM to about 40 mM citrate buffer or histidine buffer; (c) about 80 mM to about 120 mM arginine or about 2 to about 10% w / v trehalose; (d) about 20 mM to about 40 mM sodium chloride; and (e) about 0.01% to about 0.1% w / v polysorbate 80, at a pH of about 5.5 to about 6.

5.

37. 37. The formulation of claim 36, comprising about 20 mg / mL antibody, about 25 mM citrate buffer, about 100 mM arginine, about 31 mM sodium chloride, and about 0.02% w / v polysorbate 80 at about pH 6.

38. 37. The formulation of claim 36, comprising about 50 mg / mL antibody, about 25 mM citrate buffer, about 100 mM arginine, about 31 mM sodium chloride, and about 0.02% (w / v) polysorbate 80 at about pH 6.

39. An injection device comprising a composition according to any one of claims 1 to 28, a pharmaceutical composition according to claim 29, or a formulation according to any one of claims 30 to 38.

40. A cell culture medium comprising the composition of any one of claims 1 to 28.

41. An elution solution comprising the composition of any one of claims 1 to 28.

42. 38. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1 to 28, the pharmaceutical composition of claim 29, or the formulation of any one of claims 30 to 38.

43. 43. The method of claim 42, wherein the composition is administered in a dose of about 500 mg.

44. 44. The method of claim 43, wherein the composition is administered once every three weeks.

45. 45. The method of claim 43 or claim 44, wherein the composition is administered in four cycles.

46. 43. The method of claim 42, wherein the composition is administered at a first dose of about 500 mg once every three weeks for four cycles, followed by a second dose of about 1000 mg once every six or more weeks.

47. 47. The method of claim 46, wherein a second dose of about 1000 mg once every six weeks or more is continued to maintain clinical benefit.

48. A composition according to any one of claims 1 to 28, a pharmaceutical composition according to claim 29, or a formulation according to any one of claims 30 to 38 for use in therapy.

49. A composition according to any one of claims 1 to 28, a pharmaceutical composition according to claim 29, or a formulation according to any one of claims 30 to 38 for use in the treatment of cancer.

50. 38. Use of a composition according to any one of claims 1 to 28, a pharmaceutical composition according to claim 29, or a formulation according to any one of claims 30 to 38 in the manufacture of a medicament for use in the treatment of cancer.

51. 38. A method of treating an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1 to 28, the pharmaceutical composition of claim 29, or the formulation of any one of claims 30 to 38.

52. 38. A method of treating an infectious disease, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1 to 28, the pharmaceutical composition of claim 29, or the formulation of any one of claims 30 to 38.

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