Anti-CTLA antibody compositions and related methods

Abstract

The present disclosure provides compositions comprising anti-CTLA-4 antibodies and related methods for treating cancer and other disorders that respond to anti-CTLA-4 antagonism.

Description

[Technical Field]

[0001] The present disclosure relates to compositions comprising anti-CTLA antibodies and related methods for treating cancer.

[0002] (Description of Sequence Listing) A computer-readable format of the Sequence Listing has been submitted electronically with this application and is incorporated by reference in its entirety. The Sequence Listing is contained in a file with the file name "22-1658-US-PRO.xml", created on October 20, 2022, and is 19kb in size. [Background technology]

[0003] Cancer continues to be a major global health burden. It is the second leading cause of death in the United States after heart disease, accounting for nearly one in four deaths. The five-year survival rate for all cancers diagnosed between 1999 and 2006 was 68%, which is 18% higher than the rate reported between 1975 and 1977, likely reflecting advances in earlier diagnosis of certain cancers and improvements in treatment.

[0004] It is estimated that approximately 12.7 million cancer cases and 7.6 million cancer deaths occurred worldwide in 2008. In most cases, these cancers are diagnosed at an advanced or metastatic stage, when life expectancy is very short. Despite recent advances in chemotherapy and understanding the molecular biology of cancer, progress in treatment options for advanced and metastatic disease has been limited. The poor prognosis reflects the limited effectiveness of available treatment options and highlights the need for the development of newer treatment options.

[0005] The role of the immune system, particularly T cell-mediated cytotoxicity, in tumor and infection control is well recognized. There is increasing evidence that T cells control tumor growth and survival in cancer patients, both early and late in the disease. However, it has been difficult to initiate and sustain tumor-specific T cell responses in cancer patients.

[0006] CTLA4 is expressed on activated T cells and functions as a co-inhibitor to suppress T cell responses after CD28-mediated T cell activation. CTLA4 is thought to regulate the magnitude of initial activation of naive and memory T cells after TCR engagement and to be part of a central inhibitory pathway that influences both antitumor immunity and autoimmunity. CTLA4 is expressed exclusively on T cells, while expression of its ligands, CD80 (B7.1) and CD86 (B7.2), is primarily restricted to antigen-presenting cells, T cells, and other immune mediators. Antagonist anti-CTLA4 antibodies that block the CTLA4 signaling pathway have been reported to enhance T cell activation in cancer and infectious conditions and disorders.

[0007] Tremelimumab is a humanized immunoglobulin (Ig) G2 monoclonal antibody directed against the human T-cell receptor protein cytotoxic T-lymphocyte-associated protein 4 (CTLA4) with potential immune checkpoint inhibitory and antitumor activity. Summary of the Invention

[0008] The present disclosure relates generally to compositions comprising anti-CTLA antibodies and related methods for treating cancer.

[0009] In one aspect, the disclosure described herein provides a composition comprising an anti-CTLA antibody, the anti-CTLA antibody comprising a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6; and one or more deamidated variants of an anti-CTLA-4 antibody, wherein the composition comprises no more than 45%, no more than 40%, no more than 35%, 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% deamidated variants.

[0010] In another aspect, the disclosure described herein provides a composition comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, and one or more oxidized variants of the anti-CTLA antibody, wherein the composition comprises 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 of the oxidized variants.

[0011] In another aspect, the disclosure described herein provides a composition comprising an anti-CTLA antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6; and one or more aggregation variants of the anti-CTLA-4 antibody, wherein the composition comprises 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 of aggregation variants.

[0012] In another aspect, the disclosure described herein provides a composition comprising an anti-CTLA antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, and a fragmentation variant of the anti-CTLA-4 antibody, wherein the composition comprises less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the fragmentation variant.

[0013] In another aspect, the disclosure described herein provides a composition comprising an anti-CTLA antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6; and up to 100% heavy chain N-terminal pyroglutamic acid variants and / or up to 100% heavy chain C-terminal lysine truncation variants of the anti-CTLA-4 antibody.

[0014] In another aspect, the disclosure described herein provides a composition comprising an anti-CTLA antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition further comprises 34 ng / mg or less of host cell protein (HCP).

[0015] In another aspect, the disclosure described herein provides a composition comprising an anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 9, wherein the composition comprises (i) no more than 35%, 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 of the anti-CTLA-4 antibody; and (ii) no more than 26%, no more than 25%, or no more than 3% oxidized variants of the anti-CTLA-4 antibody. (iii) less than 45%, less than 40%, less than 35%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the anti-CTLA-4 antibody aggregate variants; (iv) less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the anti-CTLA-4 antibody fragment variants.

[0016] In another aspect, the disclosure described herein provides a composition comprising an anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 9, and the composition further comprises (i) 30% or less oxidized variants at heavy chain Trp-52 of the anti-CTLA-4 antibody, (ii) 35% or less deamidated variants at heavy chain Met-256 and / or heavy chain Met-432 of the anti-CTLA-4 antibody, (iii) 34 ng / mg or less host cell protein, (iv) 4% or less aggregation variants of the anti-CTLA-4 antibody, and / or (iv) 10% or less fragmentation variants of the anti-CTLA-4 antibody.

[0017] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken in conjunction with the appended claims, which should be noted that the claims are defined by the description herein, and not by the specific discussion of the features and advantages set forth herein. [Brief explanation of the drawings]

[0018] [Figure 1] The IEC profiles of fractions B, D, and E are shown. [Figure 2] cIEF analysis of IEC fractions is shown. [Figure 3] 1 shows the effect of LC Asn-30 deamidation on potency. [Figure 4] The fragmentation sites of tremelimumab are shown. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present disclosure relates to compositions comprising anti-CTLA antibodies and related methods for treating cancer.

[0020] 1.General definition The term "antibody," as used herein in the broadest sense, 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.

[0021] The terms "full antibody," "whole antibody," or "intact antibody," used interchangeably herein, refer to a heterotetrameric glycoprotein with a molecular weight of approximately 150,000 daltons. An intact antibody consists 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 "Fab" fragments and an "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, a CH1 (heavy chain) and a CL (light chain). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. The Fc fragment 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 distinct heavy chain amino acid sequences, termed m, a, g, e, and d, respectively, and each heavy chain can pair with either a kappa or l light chain. The majority of antibodies in serum belong to the IgG class, and there are four isotypes of human IgG: IgG1, IgG2, IgG3, and IgG4, whose sequences differ primarily in their hinge regions.

[0022] Fully human antibodies can be obtained using a variety of methods, for example, using yeast-based libraries or transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies. Yeast cells displaying human antibodies on their surface that bind to the antibody of interest can be selected using FACS (fluorescence-activated cell sorting)-based methods or by capture on beads using labeled antigen. Transgenic animals engineered to express human immunoglobulin genes can be immunized with the antigen of interest and antigen-specific human antibodies 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.

[0023] Monoclonal antibodies can be produced by eukaryotic or prokaryotic cell clones expressing the antibody. Monoclonal antibodies can also be produced by eukaryotic cell lines capable of recombinantly expressing the 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.

[0024] Antibodies can be derived from, for example, rat, mouse, primate (e.g., cynomolgus monkey, Old World monkey, or great ape), human, or from other sources such as nucleic acids encoding antibody molecules generated using molecular biology techniques known to those of skill in the art.

[0025] The antibody can be either a fully human antibody, a humanized antibody, or a chimeric antibody. In one aspect, the antibody is a humanized antibody. In one embodiment, the antibody is a monoclonal antibody.

[0026] 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.

[0027] 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 (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain may comprise about 100-110 or more variable regions primarily responsible for antigen recognition. The variable regions may be initially expressed linked to a cleavable signal peptide.

[0028] A variable region without a signal peptide may be 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.

[0029] The terms "VH" and "VL" are used herein to refer to the heavy and light chain variable regions, respectively, of an antibody.

[0030] 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 can be found on a standard antibody. Thus, an intact antibody can have, for example, two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites can be the same. All chains exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or "CDRs."

[0031] Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 or more amino acids. The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or CDRs.

[0032] The CDRs from the two chains of each pair can be aligned by the framework regions, enabling binding to a specific epitope. Thus, in one example, from N- to C-terminus, both the light and heavy chains comprise the following domains: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0033] Acceptable heavy 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 regions (including hinge regions) and light chain constant regions are also readily recognized by those skilled in the art. Acceptable antibody isotypes are likewise readily recognized by those skilled in the art.

[0034] "CDRs" are defined as the complementarity determining region amino acid sequences of an antibody. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0035] 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 indicate any antibody variant positions or post-translationally modified variant positions.

[0036] The term "antigen-binding fragment" refers to a portion of an intact antibody and / or to the antigen-determining variable domain of an intact antibody. It is known that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, linear antibodies, single-chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0037] 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, through post-translational modification, chemical change, or sequence alteration by at least one deletion, substitution, or addition. Some post-translational modifications result in chemical changes that do not alter the sequence (e.g., Met and oxidized Met, or Asp and isomerized / isoAsp, or aggregation), while other post-translational modifications result in sequence changes, such as conversion of one amino acid residue to another (e.g., Asn conversion to Asp via deamidation, or lysine deletion). Additional post-translationally modified variants are described below. Variant antibody sequences containing sequence changes can be the result of engineered sequence changes or post-translational modifications.

[0038] Amino acid replacements or substitutions 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."

[0039] In one embodiment, the substitution is a conservative substitution. 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 the group that maintain all or substantially all of the binding affinity of the antibody are considered conservative substitutions.

[0040] For example, groups of amino acids 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 chain orientation-influencing residues 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 alternative embodiments, antibody variants contain at least one substitution while retaining the antibody identity.

[0041] "Semi-conservative variations" include amino acid substitutions of amino acids within a larger group (i.e., aromatic or aliphatic), but do not include amino acid substitutions within the same side chain subgroup. For example, substitutions of asparagine with aspartic acid or lysine with asparagine involve amino acids within the same group (i.e., aliphatic), but from different subgroups. "Non-conservative variations" include amino acid substitutions between different groups (e.g., substitutions of tryptophan with lysine, or serine with phenylalanine, etc.).

[0042] In one embodiment, the antibody variant is an antibody that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical (i.e., has sequence identity) to an 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%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO: 10 and / or a light chain amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO: 9.

[0043] The "percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the "identity" value, expressed as a percentage, calculated over the entire length of the query sequence after performing a pairwise global sequence alignment using an appropriate algorithm or software, such as BLASTN, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign.

[0044] Importantly, the query sequence may be described by a nucleic acid sequence identified in one or more claims herein.

[0045] The "percent identity" between a query amino acid sequence and a subject amino acid sequence is the "identity" value, expressed as a percentage, calculated over the entire length of the query sequence after performing a pairwise global sequence alignment using an appropriate algorithm / software, such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign.

[0046] Importantly, the query sequence may be described by an amino acid sequence identified in one or more claims herein.

[0047] The query sequence may be 100% identical to the subject sequence, or may contain up to an 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 changes include at least one amino acid deletion, substitution (including conservative and non-conservative substitutions), or insertion, which may occur at the amino- or carboxy-terminal position of the query sequence, or anywhere between these terminal positions, and may be dispersed either individually among the amino acids or nucleotides in the query sequence, or within one or more contiguous groups within the query sequence.

[0048] The % identity can be determined over the entire length of the query sequence, including the CDRs. Alternatively, the % identity can 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, so that the CDR sequences are fixed and intact.

[0049] Amino acid sequences useful 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 the present disclosure (e.g., for an antibody heavy chain or antibody light chain). In the present disclosure, the percent identity between the recited amino acid sequences may include any discrete subrange of the percent identity ranges recited above (e.g., any range of integer values ​​within a specified range, or discrete subvalues ​​within a specified range).

[0050] The terms "specifically binds" or "binds specifically," as used herein with respect to antibodies, mean that the antibody binds to the target antigen as well as to a distinct domain or distinct amino acid sequence within the target antigen, and does not bind, or binds only weakly, 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., molecules with a high degree of sequence identity or molecules from another genus or species). The antibodies described herein may bind to human CTLA-4 with at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold greater affinity than they bind to closely related molecules.

[0051] Affinity, also called "binding affinity," is the strength of binding at a single interaction site, i.e., the strength of binding of one molecule, e.g., an antibody, to another molecule, e.g., its target antigen, at a single binding site. 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).

[0052] The binding affinity (KD) of the antibody-target antigen interaction can be, for example, about 1 picomolar (pM) to about 100 micromolar (μM) (e.g., about 1 picomolar (pM) to about 1 nanomolar (nM), about 1 nM to about 1 micromolar (μM), or about 1 pM to about 100 pM). In some forms, the anti-CTLA-4 antibody can bind to a CTLA-4 protein with a KD of 1 nanomolar or less (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values). In some embodiments, the anti-CTLA-4 antibody can bind to CTLA-4 with a KD of 200 pM or less (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values).

[0053] Alternatively, KD can be 1 pM to 1000 pM, for example, 10 pM to 800 pM, for example, about 700 pM. The binding affinity of an antibody is determined by the association constant (Ka) and the dissociation constant (Kd) (KD = Kd / Ka). Binding affinity can be measured by BIACORE (surface plasmon resonance), for example, by capturing a test antibody on a protein A-coated sensor surface and flowing a target antigen over this surface. Alternatively, binding affinity can be measured by FORTEBIO, for example, by using a test antibody receptor captured on a protein A-coated needle and flowing a target antigen over this surface.

[0054] The K can be 1 x 10 Ms 1 or less. The K can be 1 x 10 Ms 1 to 1 x 10 Ms 1, or 1 x 10 Ms 1 to 1 x 10 Ms 1. A slow K can result in slow dissociation of the antibody-target antigen complex and improved neutralization of the target antigen.

[0055] The term "specific antigen binding activity" as used herein refers to antigen binding activity measured by surface plasmon resonance (SPR). CTLA-4 specific binding activity can be determined, for example, by SPR using a BIACORE instrument performed in binding mode. This is the binding activity divided by the total protein (e.g., tremelimumab) content in the sample. The term "FcRn binding activity" as used herein refers to fetal Fc (FcRn) receptor binding activity 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.

[0056] The SPR method for specific antigen binding and FcRn binding uses a tremelimumab reference standard. The tremelimumab reference standard can be used in the assay to provide system suitability and sample comparability data to ensure that the method is performing properly. The reference standard can allow for the establishment of a calibration curve, and sample concentrations can be interpolated from the curve.

[0057] Potency is defined herein as the inhibitory activity of an anti-CTLA-4 antibody or composition described herein that inhibits ligand (CTLA-4) binding to CTLA-4. This can be measured by specific binding to the antigen CTLA-4, by a potency assay (e.g., an IL-2 reporter assay), or by a potency reporter gene bioassay. The potency assay can be a cell-based competitive binding assay that measures the dose-dependent ability of the antibody or composition to inhibit CTLA-4 ligand binding to B7 ligand (CD80, CD86). Results can be reported as percent potency relative to a reference material (e.g., a control sample).

[0058] 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.

[0059] As used herein, when referring to a measurable value such as an amount, duration, etc., reference to "about" is meant to encompass variations of ±5%, ±1%, ±0.1%, ±20% or ±10% from the specified value, where such variations are appropriate for practicing the disclosed methods.

[0060] 2. Anti-CTLA-4 antibody In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof is tremelimumab. Tremelimumab and antigen-binding fragments thereof for use in the methods, compositions, and combinations provided herein comprise a heavy chain and a light chain, or a heavy chain variable region and a light chain variable region. In some embodiments, tremelimumab or antigen-binding fragments thereof for use in the methods, compositions, and combinations provided herein comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, tremelimumab or antigen-binding fragments thereof for use in the methods, compositions, and combinations provided herein comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4-6, and the light chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1-3. Those of skill in the art can readily identify the Chothia definition, the Abm definition, or other CDR definitions known to those of skill in the art. In some embodiments, tremelimumab or antigen-binding fragments thereof for use in the methods, compositions, and combinations provided herein comprise the variable heavy and variable light chain CDR sequences of the 11.2.1 antibody disclosed in U.S. Patent No. 6,682,736, which is incorporated herein by reference in its entirety.

[0061] Tremelimumab light chain (LC) variable region: PSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKV (SEQ ID NO: 7)

[0062] Tremelimumab light chain (LC) variable region (deamidated LCDR1): PSSLSASVGDRVTITCRASQSIDSYLDWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKV (SEQ ID NO: 13)

[0063] Tremelimumab heavy chain (HC) variable region: GVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VH (SEQ ID NO: 8)

[0064] Tremelimumab heavy chain (HC) variable region (oxidized HC-CDR-2): GVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIXYDGSNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VH (SEQ ID NO: 14) (X is an oxidized methionine)

[0065] Tremelimumab heavy chain CDR HC-CDR1: GFTFSSYGMH (SEQ ID NO: 4) HC-CDR2: VIWYDGSNKYYADSV (SEQ ID NO: 5) HC-CDR3:DPRGATLYYYYYGMD V (SEQ ID NO: 6) HC-CDR2 (oxidized): VIXYDGSNKYYADSV (SEQ ID NO: 12) (X is oxidized tryptophan)

[0066] Tremelimumab light chain CDR1 LC-CDR1: RASQSINSYLD (SEQ ID NO: 1) LC-CDR2: AASSLQS (SEQ ID NO: 2) LC-CDR3: QQYYSTPFT (SEQ ID NO: 3) LC-CDR1 (deamidated): RASQSIDSYLD (SEQ ID NO: 11)

[0067] Tremelimumab complete light chain DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASS LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9)

[0068] Tremelimumab complete heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV IWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYY YYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

[0069] Tremelimumab complete heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV IXiYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLY YYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEX2TKNQVSLTCLVKGFYPSDIAVEWESX3GQPEX3NYKTTPPX2LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX2HEALHNHYTQKSLSLSPG K (SEQ ID NO: 15) (wherein X3 is an asparagine that can be deamidated, X2 is methionine or oxidized methionine, and X1 is tryptophan or oxidized tryptophan).

[0070] Compositions of the disclosure may include anti-CTLA-4 antibodies comprising one or more CDRs described herein, or one or both of the heavy chain variable regions or light chain variable regions described herein, or one or both of the heavy chains or light chains described herein.

[0071] 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: 4, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain sequence comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 3.

[0072] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR2 ("CDRH2") comprising an amino acid sequence having one or two amino acid mutations ("CDR variants") relative to the amino acid sequence set forth in SEQ ID NO:5.

[0073] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR2 ("CDRH2") comprising an amino acid sequence ("CDR variant") that has no more than five amino acid mutations, such as no more than four, no more than three, no more than two, or no more than one, relative to the amino acid sequence set forth in SEQ ID NO: 5. 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: 4.

[0074] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR2 (“CDRH2”) comprising the amino acid sequence set forth in SEQ ID NO: 12, wherein X can be oxidized tryptophan.

[0075] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR3 ("CDRH3") comprising an amino acid sequence having one or two amino acid mutations ("CDR variants") relative to the amino acid sequence set forth in SEQ ID NO:6.

[0076] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR1 ("CDRL1") comprising an amino acid sequence having no more than three amino acid mutations, such as one or two, relative to the amino acid sequence set forth in SEQ ID NO:1 (a "CDR variant").

[0077] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR1 (“CDRL1”) comprising the amino acid sequence set forth in SEQ ID NO:11.

[0078] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR2 ("CDRL2") comprising an amino acid sequence having one or two amino acid mutations ("CDR variants") relative to the amino acid sequence set forth in SEQ ID NO:2.

[0079] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR3 ("CDRL3") comprising an amino acid sequence having no more than three amino acid mutations, such as one or two, relative to the amino acid sequence set forth in SEQ ID NO:3 (a "CDR variant").

[0080] In one embodiment, the anti-CTLA-4 antibody comprises a CDRH1 comprising an amino acid sequence having up to one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO:3, a CDRH2 comprising an amino acid sequence having up to five amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:4, a CDRH3 comprising an amino acid sequence having up to one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO:5, a CDRL1 comprising an amino acid sequence having up to three amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:1, a CDRL2 comprising an amino acid sequence having up to one amino acid mutation relative to the amino acid sequence set forth in SEQ ID NO:2, and / or a CDRL3 comprising an amino acid sequence having up to three amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO:3.

[0081] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region ("VH") comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. 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, such as 1 to 5, such as 1 to 3, particularly up to 2 amino acid mutations, relative to the amino acid sequence set forth in SEQ ID NO: 8.

[0082] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region ("VL") comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 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: 8, such as 1 to 3, 1 to 5, particularly up to 2 amino acid mutations, relative to the amino acid sequence set forth in SEQ ID NO: 7.

[0083] In some embodiments, the anti-CTLA-4 antibody comprises a VH having the amino acid sequence set forth in SEQ ID NO:8 and a VL having the amino acid sequence set forth in SEQ ID NO:7.

[0084] In one embodiment, the anti-CTLA-4 antibody comprises a VH comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8, and a VL comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.

[0085] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain sequence ("HC") comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. 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: 10, such as 1 to 10, such as 1 to 7, particularly up to 6 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 10. 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: 10.

[0086] In one embodiment, the anti-CTLA-4 antibody comprises a light chain region ("LC") comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. 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: 9, such as 1 to 10, such as 1 to 5, particularly up to 3 amino acid mutations relative to the amino acid sequence set forth in SEQ ID NO: 9. 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: 9.

[0087] In one embodiment, the anti-CTLA-4 antibody comprises an HC comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and an LC comprising an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. Thus, the antibody is an antibody having a heavy chain that is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 10 and / or a light chain that is at least about 90% identical to the light chain amino acid sequence of SEQ ID NO:9.

[0088] In one embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9. In one embodiment, the antibody is tremelimumab, which comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9.

[0089] 3. Post-translational modification products Those skilled in the art will understand that post-translational modifications can occur during antibody production to produce post-translationally modified products. A "post-translationally modified variant" of an antibody described herein is an antibody composition in which all or a portion of the composition comprises a "post-translational modification." A post-translational modification is a chemical change to an antibody that can result from the production of the antibody in a host cell, upstream and / or downstream manufacturing processes, and / or storage time and conditions (e.g., effects of exposure to light, temperature, pH, water, or reaction with excipients and / or excipient container closure systems). Thus, a composition of the present disclosure can be formed from the production or storage of an antibody of the present disclosure. Exemplary post-translational modifications include antibody sequence alteration ("antibody variants" above), cleavage of specific leader sequences, addition of various sugar moieties in various glycosylation patterns, such as non-enzymatic glycosylation or glycation; deamidation; oxidation; disulfide bond scrambling and other cysteine ​​variants, such as free sulfhydryls, racemic disulfides, thioethers, and trisulfide bonds; isomerization; C-terminal lysine truncation or clipping; and / or N-terminal glutamine cyclization.

[0090] In one example, post-translationally modified products include "product-related impurities" that include chemical changes that result in a decrease in function and / or activity. In another example, post-translationally modified products include "product-related substances" that include chemical changes that do not result in a decrease in function and / or activity. Product-related impurities of the anti-CTLA-4 antibodies described herein include oxidized and aggregated variants. Product-related substances of the anti-CTLA-4 antibodies described herein include deamidated variants, isomerized variants, C-terminal truncated variants, and N-terminal pyroglutamate variants.

[0091] In one embodiment, the anti-CTLA-4 antibody is tremelimumab, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 10 and a light chain having the amino acid sequence set forth in SEQ ID NO: 9, including all functional post-translational modifications thereof.

[0092] The percent variants provided herein are expressed as a percentage of the total amount of antibody (e.g., "population" of antibodies) in the composition. For example, 50% or less oxidized variants refers to a situation where the total antibody in the composition is 100%, of which 50% or less is oxidized, and does not include any other non-antibody material present in the composition, which may or may not be oxidized.

[0093] Antibody variants are commonly observed when the composition of an 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).

[0094] Post-translational modifications can increase or decrease the net charge of an antibody, resulting in an increase or decrease in pI value, thereby resulting in acidic and basic variants (collectively "charge variants") of the major isoform. A "major isoform" is an antibody population that elutes as a major peak on a chromatogram or electropherogram. When antibodies are analyzed using IEF-based methods, acidic species are variants with a lower apparent pI, and basic species are variants with a higher 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 main peak from CEX or later than the main peak from AEX, and basic species are variants that elute later than the main peak from CEX or earlier than the main peak from AEX. These methods separate the major isoforms of antibodies from acidic isoforms (acidic variants) and basic isoforms (basic variants).

[0095] Charge variants can be detected by various methods, such as ion exchange chromatography, e.g., IEF (isoelectric focusing). The percentage of charge variants can be determined using capillary isoelectric focusing (cIEF). Capillary isoelectric focusing (cIEF) was used to measure the pI of tremelimumab and separate the charge variants. This method can be used to quantify acidic and basic species as a percentage of the total area peak. The terms "species," "isoform," "form," and "peak" are used interchangeably to refer to the major isoforms and charge variants (acidic and basic variants).

[0096] In one aspect, a composition comprises an acidic variant of an anti-CTLA-4 antibody, the acidic variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 100% of the acidic variant.

[0097] In one embodiment, the composition comprises an acidic variant of anti-CTLA-4, wherein the acidic variant comprises a heavy chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7, and the composition comprises less than 100% of the acidic variant.

[0098] In another embodiment, the composition comprises an acidic variant of an anti-CTLA-4 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: 10 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, and the composition comprises less than 100% of the acidic variant. In yet a further embodiment, the composition comprises an acidic variant of an anti-CTLA-4 antibody, wherein the acidic variant comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, and the composition comprises less than 100% of the acidic variant. In one embodiment, the composition comprises an acidic variant of tremelimumab, wherein the acidic variant comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, and the composition comprises less than 100% of the acidic variant.

[0099] In one aspect, the composition comprises less than 100% acidic variant. In one embodiment, the composition comprises less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 35%, less than 30%, or less than 25% 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% 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% 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.

[0100] 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.

[0101] In one aspect, the composition comprises a basic variant of an anti-CTLA-4 antibody, the basic variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 100% basic variant.

[0102] In another embodiment, the composition comprises a basic variant of an anti-CTLA-4 antibody, wherein the basic variant comprises a heavy chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 8 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7, and the composition comprises less than 100% of the basic variant.

[0103] In one aspect, the composition contains less than 100% basic variant. In one embodiment, the composition contains less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 35%, less than 30%, or less than 25% basic variant. Alternatively, the composition contains 5-100%, 5-90%, 5-80%, 5-70%, 5-60%, 5-50%, 5-40%, 5-35%, 5-30%, or 5-25% basic variant. Alternatively, the composition contains 10-100%, 10-97%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-35%, 10-30%, or 10-25% basic 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 basic variant.

[0104] 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 basic variant.

[0105] In one embodiment, the composition has a bioassay potency of at least 70% compared to a reference standard bioassay potency.

[0106] In another embodiment, the composition comprises a basic variant of an anti-CTLA-4 antibody, wherein the basic variant comprises a heavy chain that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10 and / or a light chain that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, and the composition comprises less than 100% basic variant. In yet a further embodiment, the composition comprises a basic variant of an anti-CTLA-4 antibody, wherein the basic variant comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, and the composition comprises less than 100% basic variant. In one embodiment, the composition comprises a basic variant of tremelimumab, wherein the basic variant comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, and the composition comprises less than 100% basic variant.

[0107] In one aspect, the composition comprises a major isoform of an anti-CTLA-4 antibody, the major isoform comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 3, CDRH2 of SEQ ID NO: 4, and CDRH3 of SEQ ID NO: 5, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the composition comprises more than 1% of the major isoform.

[0108] In another embodiment, the composition comprises a major isoform of an anti-CTLA-4 antibody, wherein the major isoform 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, and the composition comprises more than 1% of the major isoform.

[0109] In another embodiment, the composition comprises a major isoform of an anti-CTLA-4 antibody, the major isoform comprising a heavy chain that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, and the composition comprises more than 1% of the major isoform. In yet a further embodiment, the composition comprises a major isoform of an anti-CTLA-4 antibody, the major isoform comprising the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, and the composition comprises more than 1% of the major isoform.

[0110] In one embodiment, the composition comprises a major isoform of tremelimumab, wherein the major isoform comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, and wherein the composition comprises more than 1% of the major isoform.

[0111] In one aspect, the composition comprises >1% of a major isoform. In one embodiment, the composition comprises greater than 2.6%, greater than 3%, greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90% of a 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 a major isoform. In one embodiment, the composition comprises 5-80%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, or 60-80% of a 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.

[0112] The percent acidic variants, percent basic variants, and percent major isoforms can be determined using capillary isoelectric focusing (cIEF). It is understood that these isoform / charge variant embodiments can be combined with any one of the antibody variants described herein, or combinations thereof.

[0113] In one aspect, a composition comprises charge variants of an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 3, CDRH2 of SEQ ID NO: 4, and CDRH3 of SEQ ID NO: 5, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 100% acidic variants, and / or less than 100% basic variants, and / or more than 1% of the major isoform.

[0114] In another aspect, a composition comprises charge variants of an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 3, CDRH2 of SEQ ID NO: 4, and CDRH3 of SEQ ID NO: 5, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, wherein the composition comprises 4-97% acidic variants, and / or 10-97% basic variants, and / or 2-80% of the major isoform.

[0115] In another aspect, a composition comprises charge variants of an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 3, CDRH2 of SEQ ID NO: 4, and CDRH3 of SEQ ID NO: 5, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 35% acidic variants, and / or less than 35% basic variants, and / or more than 55% of the major isoform.

[0116] In another aspect, a composition comprises charge variants of an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 3, CDRH2 of SEQ ID NO: 4, and CDRH3 of SEQ ID NO: 5, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, wherein the composition comprises 4-30% acidic variants, and / or 10-30% basic variants, and / or 60-80% of the major isoform.

[0117] In one aspect, a composition comprises charge variants of an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising a VH of SEQ ID NO: 8 and a light chain amino acid sequence comprising a VL of SEQ ID NO: 7, wherein the composition comprises less than 100% acidic variants and / or less than 100% basic variants and / or more than 1% of the major isoform. In one embodiment, the composition comprises 10-97% acidic variants and / or 10-97% basic variants and / or 2-80% of the major isoform. In an alternative embodiment, the composition comprises 10-30% acidic variants and / or 10-30% basic variants and / or 60-80% of the major isoform. In a further embodiment, the composition comprises less than 35% acidic variants and / or less than 35% basic variants and / or more than 55% of the major isoform. In one aspect, a composition comprises charge variants of an anti-CTLA-4 antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 10 and the light chain amino acid sequence of SEQ ID NO: 9, wherein the composition comprises less than 100% acidic variants and / or less than 100% basic variants and / or more than 1% of the major isoform. In one embodiment, the composition comprises 10-97% acidic variants and / or 10-97% basic variants and / or 2-80% of the major isoform. In an alternative embodiment, the composition comprises 10-30% acidic variants and / or 10-30% basic variants and / or 60-80% of the major isoform. In a further embodiment, the composition comprises less than 35% acidic variants and / or less than 35% basic variants and / or more than 55% of the major isoform.

[0118] In one embodiment, the composition has a bioassay potency of at least 70% compared to a reference standard bioassay potency.

[0119] Oxidation can occur during production and / or storage (i.e., in the presence of oxidizing conditions) and results in covalent modification of proteins induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation occurs primarily at methionine residues, but can also occur at tryptophan residues and free cysteine ​​residues. Oxidation can occur in the CDR regions, Fab (non-CDR) regions, or Fc regions.

[0120] In one aspect, the composition comprises an antibody comprising an oxidative post-translational modification ("oxidation" or "oxidized"), also referred to herein as an "oxidative variant." The variant may comprise oxidized amino acid residues in the heavy and / or light chain sequences, such as the CDRs of the heavy chain sequence and / or the CDRs of the light chain sequence. The oxidative variant may be present in one or both of the heavy or light chains.

[0121] In one aspect, the composition comprises an oxidized variant of an anti-CTLA-4 antibody, the oxidized variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 3, a CDRH2 of SEQ ID NO: 4, and a CDRH3 of SEQ ID NO: 5, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 40% oxidized variant.

[0122] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies comprising antibodies having heavy chain amino acid sequences comprising SEQ ID NO:3 (CDRH1), SEQ ID NO:4 (CDRH2), and SEQ ID NO:5 (CDRH3), and light chain amino acid sequences comprising SEQ ID NO:1 (CDRL1), SEQ ID NO:2 (CDRL2), and SEQ ID NO:3 (CDRL3), as well as oxidized variants thereof, wherein less than 40% of the antibody population consists of oxidized variants.

[0123] In one embodiment, the oxidation variant comprises oxidation at methionine and / or tryptophan residues in the CDRs of the heavy chain sequence and / or the light chain sequence. In one embodiment, the oxidation variant comprises oxidation at methionine and / or tryptophan residues in any one of SEQ ID NOS: 1-6. In a further aspect, the antibody comprises oxidation at methionine residues in the CDRs of the heavy chain sequence, such as CDRH1 and / or CDRH3. In a further embodiment, the antibody comprises oxidation at tryptophan residues in the CDRs of the light chain sequence, such as CDRL2. In some embodiments, the oxidation variant comprises one or a combination of oxidation at W52 of CDRH2, M256, M362, M401, and / or M432 of the complete heavy chain.

[0124] It will be understood that reference to a position in a CDR (e.g., W52) provides a position number relative to the entire antibody sequence (consecutive numbering), and thus W52 of CDRH2 will be understood to refer to the third residue of SEQ ID NO:5.

[0125] 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, hi some embodiments, the oxidation variant comprises one or a combination of oxidations at M256, M362, M401, and / or M432 in the Fc region of the heavy chain sequence.

[0126] In one aspect, a composition comprises an antibody that is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 10 and / or at least about 90% identical to the light chain sequence of SEQ ID NO: 9, and comprises an oxidation in the heavy chain sequence, e.g., an oxidation at amino acid W52 of CDRH2, M256 of the Fc, M362 in the Fc region, M401 in the Fc region, and / or M432 in the Fc region. In one embodiment, a composition comprises an antibody that is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 10 and / or at least about 90% identical to the light chain sequence of SEQ ID NO:9.

[0127] 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: 8 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7. In a further embodiment, the antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 10 and / or at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 9. In yet a further embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9.

[0128] In one aspect, a composition comprises an anti-CTLA-4 antibody having a heavy chain sequence comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain sequence comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 3, wherein the composition comprises less than 35% oxidized variants.

[0129] In one aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 8 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 7, wherein the composition comprises less than 35% oxidation variants. In a further aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain variable region of SEQ ID NO: 8 and / or a light chain variable region of SEQ ID NO: 7, wherein the composition comprises less than 35% oxidation variants.

[0130] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies comprising an antibody having a heavy chain variable region set forth in SEQ ID NO: 8 and a light chain variable region set forth in SEQ ID NO: 7, as well as oxidized variants thereof, wherein less than 35% of the antibody population consists of oxidized variants.

[0131] In one aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 10 and / or a light chain at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, wherein the composition comprises less than 35% oxidized variants.

[0132] In one embodiment, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 35% oxidized variants.

[0133] In one embodiment, the composition comprises an oxidized variant of tremelimumab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, and the composition comprises less than 45% oxidized variant.

[0134] In one embodiment, the composition comprises an oxidized variant of tremelimumab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, and the composition comprises the oxidized variant in an amount ranging from 0.1% to 35%.

[0135] In one aspect, a composition comprises (a) an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, and (b) an antibody having a heavy chain sequence that is at least 90% identical to SEQ ID NO: 10 and a light chain sequence that is at least 90% identical to SEQ ID NO: 9, wherein the composition comprises less than 35% oxidized variants.

[0136] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies comprising an antibody having a heavy chain amino acid sequence set forth in SEQ ID NO: 10 and a light chain amino acid sequence set forth in SEQ ID NO: 9, as well as oxidized variants thereof, wherein less than 35% of the antibody population consists of oxidized variants.

[0137] In one aspect, the composition comprises (a) an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, (b) an antibody having a heavy chain sequence that is at least 90% identical to SEQ ID NO: 10 and a light chain sequence that is at least 90% identical to SEQ ID NO: 9, 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: less than 30% oxidation at W52 of the heavy chain; less than 35% oxidation at M256 of the heavy chain; less than 12.5% ​​oxidation at M362 of the heavy chain; less than 16% oxidation at M401 of the heavy chain; and / or less than 35% oxidation at M432 of the heavy chain.

[0138] In one aspect, the composition comprises less than 35% oxidation variants. In one embodiment, the composition comprises less than 35%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, or less than 3% oxidation variants. In one embodiment, the composition comprises 0.01-35%, 0.01-30%, 0.01-20%, 0.01-15%, 0.01-10%, 0.01-5%, 0.01-4%, or 0.01-3% oxidation variants.

[0139] Alternatively, the composition contains 0.05-35%, 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 contains 0.5-35%, 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 contains 1-35%, 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, or 2-3% of the oxidation 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 one of the antibody variants described herein.

[0140] In one embodiment, the composition comprises any one or combination of less than 35% oxidation at M256 of the heavy chain, less than 12.5% ​​oxidation at M362 of the heavy chain, less than 16% oxidation at M401 of the heavy chain, less than 35% oxidation at M432 of the heavy chain, and / or less than 30% oxidation at W52 of CDRH2.

[0141] In one aspect, the composition comprises (a) an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, and (b) an oxidized variant of the antibody selected from any one or combination of: less than 25% oxidation at W52 of the heavy chain, less than 30% oxidation at M256 of the heavy chain, less than 10% oxidation at M362 of the heavy chain, less than 12% oxidation at M401 of the heavy chain, and / or less than 30% oxidation at M432 of the heavy chain.

[0142] In one embodiment, the composition comprises less than 25% oxidation at W52 of the heavy chain sequence. In one embodiment, the composition comprises less than 25%, less than 20%, less than 15%, less than 10%, less than 7.5%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% oxidation at W52 of the heavy chain sequence. Alternatively, the composition comprises 0-25%, 0-20%, 0-15%, 0-10%, 0-7.5%, 0-5%, 0-4%, 0-3%, 0-2%, or 0-1% oxidation at W52 of the heavy chain sequence. In one embodiment, the composition comprises 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 at W52 of the heavy chain sequence. In one embodiment, the composition comprises 0.05 to 25%, 0.05 to 20%, 0.05 to 15%, 0.05 to 10%, 0.05 to 7.5%, 0.05 to 5%, 0.05 to 4%, 0.05 to 3%, 0.05 to 2%, or 0.05 to 1% oxidation at W52 of the heavy chain sequence. Alternatively, the composition comprises 0.5-25%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-7.5%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, or 0.5-1% oxidation at W52 of the heavy chain sequence. Alternatively, the composition comprises 0.1% or more and 25% or less oxidation at W52 of the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation at W52 of the heavy chain sequence. As demonstrated by the data presented herein, heat stress-forced degradation resulting in up to 4.5% oxidation at W52 results in 98% efficacy.

[0143] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 25% oxidation at W52 of the heavy chain sequence. In one embodiment, a composition comprises an oxidation variant of tremelimumab, wherein the oxidation variant comprises a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 25% oxidation at W52 of the heavy chain sequence.

[0144] In one embodiment, the composition comprises less than 35% oxidation at M256 of the heavy chain sequence. In one embodiment, the composition comprises less than 35%, less than 30%, less than 25%, less than 20%, less than 16%, less than 15%, less than 12.5%, less than 10%, less than 7.5%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% oxidation at M256 of the heavy chain sequence. Alternatively, the composition comprises 0 to 35%, 0 to 30%, 0 to 25%, 0 to 20%, 0 to 16%, 0 to 15%, 0 to 12.5%, 0 to 10%, 0 to 7.5%, 0 to 5%, 0 to 4%, 0 to 3%, 0 to 2%, or 0 to 1% oxidation at M256 of the heavy chain sequence. In one embodiment, the composition comprises 0.01 to 35%, 0.01 to 30%, 0.01 to 25%, 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 at M256 of the heavy chain sequence. Alternatively, the composition may comprise 0.5-35%, 0.5-30%, 0.5-25%, 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%, or 0.5-1% oxidation at M256 of the heavy chain sequence. Alternatively, the composition may comprise 0.1% or more and 35% or less oxidation at M256 of the heavy chain sequence. Alternatively, the composition may comprise about 30%, about 25%, about 20%, 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation at M256 of the heavy chain sequence. As demonstrated by the data presented herein, forced HO degradation resulting in a maximum of 68.5% oxidation at M34 results in a potency of 83 in the bioassay. Extrapolating from the forced HO degradation data for oxidation at HC Met256, a maximum of 13.4% oxidation can result in a potency of at least 88%. In one embodiment, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 13.4% oxidation at M256 of the heavy chain sequence.In one embodiment, the composition comprises an oxidized variant of tremelimumab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, and the composition comprises less than 13.4% oxidation at M256 of the heavy chain sequence.

[0145] In one embodiment, the composition comprises less than 16% oxidation at M401 of the heavy chain sequence. In one embodiment, the composition comprises less than 16%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% oxidation at M401 of the heavy chain sequence. In one embodiment, the composition comprises 0-16%, 0-15%, 0-10%, 0-5%, 0-4%, 0-3%, 0-2%, or 0-1% oxidation at M401 of the heavy chain sequence. In one embodiment, the composition comprises 0.01-16%, 0.01-15%, 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, or 0.01-1% oxidation at M401 of the heavy chain sequence. Alternatively, the composition comprises 0.5-16%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, or 0.5-1% oxidation at M401 of the heavy chain sequence. Alternatively, the composition comprises 0.1% or more, or 16% or less oxidation at M401 of the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation at M401 of the heavy chain sequence. As demonstrated by the data presented herein, forced H2O2 degradation resulting in up to 55.2% oxidation at M401 results in 83% efficacy in bioassays.

[0146] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 16% oxidation at M401 of the heavy chain sequence. In one embodiment, a composition comprises an oxidation variant of tremelimumab, wherein the oxidation variant comprises a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 16% oxidation at M401 of the heavy chain sequence.

[0147] In one embodiment, the composition comprises less than 12.5% ​​oxidation at M362 of the heavy chain sequence. In one embodiment, the composition comprises less than 12.5%, less than 10%, less than 5%, less than 4%, or less than 3% oxidation at M362 of the heavy chain sequence. In one embodiment, the composition comprises 0.01-12.5%, 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, or 0.01-1% oxidation at M3628 of the heavy chain sequence. Alternatively, the composition comprises 0.5-12.5%, 0.5-10%, 0.5-5%, 0.5-4%, or 0.5-3% oxidation at M362 of the heavy chain sequence. Alternatively, the composition comprises 1-12.5%, 1-10%, 1-5%, 1-4%, 1-3%, 2-4%, or 2-3% oxidation at M362 of the heavy chain sequence. Alternatively, the composition comprises 1% or more and 12.5% ​​or less oxidation at M362 of the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation at M362 of the heavy chain sequence. As demonstrated by the data presented herein, forced HO degradation resulting in up to 26.5% oxidation at M362 results in 83% potency in bioassays. Therefore, it is expected that oxidation at M362 can be higher than 26.5% without affecting relative potency.

[0148] In one embodiment, the composition comprises less than 35% oxidation at M432 of the heavy chain sequence. In one embodiment, the composition comprises less than 35%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% oxidation at M432 of the heavy chain sequence. In one embodiment, the composition comprises 0.01 to 35%, 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 at M432 of the heavy chain sequence. Alternatively, the composition comprises 0.5-35%, 0.5-30%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-4%, or 0.5-3% oxidation at M432 of the heavy chain sequence. Alternatively, the composition comprises 0.1% or more and 35% or less oxidation at M432 of the heavy chain sequence. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation at M432 of the heavy chain sequence. As demonstrated by the data presented herein, HO-forced degradation resulting in up to 42.1% oxidation at M432 results in 83% efficacy in the bioassay (i.e., within assay variability—full function), and photolytically forced degradation resulting in up to 20.7% oxidation at M432 results in 88% efficacy in the bioassay. Therefore, it is expected that oxidation at M432 can be higher than 42.1% without affecting relative potency.

[0149] In one embodiment, the composition comprises an oxidized variant of tremelimumab, the oxidized variant comprising a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 35% oxidation at M256 sequence and / or M362 and / or M401 and / or M432 of the heavy chain sequence.

[0150] In one aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 100% acidic variants, and / or less than 100% basic variants, and / or more than 1% of the major isoform, and / or less than 35% oxidized variants.

[0151] In another aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises 5-45% acidic variants, and / or 5-97% basic variants, and / or 2-80% of the major isoform, and / or less than 35% oxidized variants.

[0152] In one example, oxidation can be determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS).

[0153] 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 can include dimers or higher-order structures formed from an antibody monomer and its subunits. Thus, high molecular weight (HMW) species can consist of a dimerized antibody and a monomer with an additional subunit (such as a monomer with two light chain subunits, or an LC-LC dimer non-covalently bound to a monomer). Aggregation variants can be, for example, covalent or non-covalent, reducible or non-reducible, and visible or invisible aggregates of the antibodies disclosed herein. Aggregation variants can be characterized based on their size and distinguished from antibodies. For example, the size distribution of an antibody composition can be detected using size exclusion chromatography (SEC). In one aspect, a composition comprises an anti-CTLA-4 antibody having a heavy chain sequence comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain sequence comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 3, wherein the composition comprises less than 26% aggregation variants. It will be understood that these aggregation variant embodiments may be combined with any one of the antibody variants described herein.

[0154] In one aspect, a composition comprises aggregation variants of an anti-CTLA-4 antibody, the aggregation variants comprising a heavy chain sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 26% aggregation variants.

[0155] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies comprising antibodies having heavy chain amino acid sequences comprising SEQ ID NO:4 (CDRH1), SEQ ID NO:5 (CDRH2), and SEQ ID NO:6 (CDRH3), and light chain amino acid sequences comprising SEQ ID NO:1 (CDRL1), SEQ ID NO:2 (CDRL2), and SEQ ID NO:3 (CDRL3), and aggregation variants thereof, wherein less than 26% of the antibody population consists of aggregation variants.

[0156] 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: 8 and / or a light chain variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9. In a further embodiment, the antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 10 and / or at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 9. In yet a further embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9.

[0157] In one aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 8 and / or a light chain variable region at least about 90% identical to the amino acid sequence of SEQ ID NO: 7, wherein the composition comprises less than 26% aggregation variants. In a further aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain variable region of SEQ ID NO: 8 and / or a light chain variable region of SEQ ID NO: 7, wherein the composition comprises less than 26% aggregation variants.

[0158] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies comprising an antibody having a heavy chain variable region set forth in SEQ ID NO: 8 and a light chain variable region set forth in SEQ ID NO: 7, as well as aggregation variants thereof, wherein less than 26% of the antibody population consists of aggregation variants.

[0159] In one aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10 and / or a light chain that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, and the composition comprises less than 26% aggregation variants.

[0160] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, and an antibody having a heavy chain sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10 and / or a light chain sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, wherein the composition comprises less than 26% aggregation variants.

[0161] In one aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 26% aggregation variants. In one embodiment, a composition comprises aggregation variants of tremelimumab, wherein the aggregation variants comprise a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 26% aggregation variants. In one embodiment, a composition comprises aggregation variants of tremelimumab, wherein the aggregation variants comprise a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises between 0.01% and 26% aggregation variants.

[0162] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies comprising an antibody having a heavy chain amino acid sequence set forth in SEQ ID NO: 10 and a light chain amino acid sequence set forth in SEQ ID NO: 9, as well as aggregation variants thereof, wherein less than 26% of the antibody population consists of aggregation variants.

[0163] The antibody composition may contain less than 26% aggregation variants, such as less than 26%, less than 25%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% aggregation variants. In other embodiments, the composition may contain 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 contain more than 1% and less than 26% aggregation variants. Alternatively, the composition may contain about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% aggregation variants. As shown by the data presented herein, the combination of acid treatment and heat-forced degradation, which produces up to 25.9% of the composition containing aggregated antibodies, results in 91% efficacy in the bioassay (i.e., within assay variability—full function).

[0164] In one aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises less than 100% acidic variants, and / or less than 100% basic variants, and / or more than 1% of the major isoform, and / or less than 35% oxidized variants, and / or less than 26% aggregated variants.

[0165] In another aspect, a composition comprises an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition comprises 5-97% acidic variants, and / or 5-97% basic variants, and / or 2-80% of the major isoform, and / or less than 35% oxidized variants, and / or less than 26% aggregated variants.

[0166] Fragmentation variants ("fragment variants") are variants that comprise a portion of a full-length antibody. For example, such fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and immunoglobulin single variable domains. An antibody composition may contain less than 10% antibody fragments, such as less than 5%, less than 4.6%, less than 4.5%, less than 4.4%, less than 4.3%, less than 4.2%, less than 4.1%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.05% antibody fragments. 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% of the antibody fragment. In other embodiments, the composition may comprise 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% of the antibody fragment. Alternatively, the composition may comprise about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% of the antibody fragment. It will be understood that these embodiments of the antibody fragment variants may be combined with any one of the antibody variants described herein.

[0167] For example, deamidation, which may occur during production and / or storage, may be an enzymatic or chemical reaction. Deamidation may occur via a simple chemical reaction in which the amide nitrogen of the next amino acid in the chain nucleophilically attacks the amide (N+1 attacks N), forming a succinimide intermediate. Deamidation may primarily convert asparagine (N) to isoaspartic acid (isoaspartate) and aspartic acid (aspartate) (D) in a ratio of approximately 3:1. Thus, this deamidation reaction may involve the isomerization of aspartate (D) to isoaspartate. Both the deamidation of asparagine and the isomerization of aspartate may involve a succinimide intermediate. To a lesser extent, deamidation may also occur with glutamine residues. Deamidation may occur in the CDR, Fab (non-CDR region), or Fc region.

[0168] Isomerization is the conversion of aspartate (D) to isoaspartate, which contains an intermediate succinimide (succinimide-aspartic acid residue).

[0169] In one aspect, the composition comprises an antibody that comprises a deamidation post-translational modification ("deamidation" or "deamidated"), also referred to herein as a "deamidation variant."

[0170] 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 either or both the heavy and light chains. It will be understood that these deamidation variant embodiments can be combined with any one of the antibody variants described herein. In some embodiments, the deamidation variants comprise one or a combination of deamidation at N30 of CDRL1 and / or N388 and / or N393 in the Fc region of the heavy chain sequence.

[0171] 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.

[0172] In one aspect, the composition comprises a sequence at least about 90% identical to the light chain amino acid sequence of SEQ ID NO:9, and optionally a sequence at least about 90% identical to the heavy chain sequence of SEQ ID NO:10, and comprises deamidation in the light chain sequence, e.g., deamidation at amino acid residue N30 and / or N388 and / or N393 in the Fc heavy chain region. In some embodiments, the deamidated variant comprises up to 45% deamidation at N30 of SEQ ID NO:1 or SEQ ID NO:7, and / or N388 of SEQ ID NO:10, and / or N393 of SEQ ID NO:10.

[0173] 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 variant comprises the light chain sequence of SEQ ID NO: 11 (i.e., the CDRL1 sequence having N30D).

[0174] The composition may comprise up to 45% deamidated variants. In one embodiment, the composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises up to 45% deamidated variants.

[0175] In one embodiment, the composition comprises up to 45% deamidation at N30 of CRDL1 and / or N388 and / or N393 of the heavy chain sequence. In one embodiment, the composition comprises 0-45%, 0-40%, 0-30%, 0-20%, or 0-10% deamidation at N30. Alternatively, the composition comprises 0.1-45%, 0.1-40%, 0.1-30%, 0.1-20%, or 0.1-10% deamidation at N30. Alternatively, the composition comprises 1-45%, 1-40%, 1-30%, 1-20%, or 1-10% deamidation at N30. Alternatively, the composition comprises 2-45%, 3-45%, 4-45%, 5-45%, 6-45%, 7-45%, 8-45%, 9-45%, 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 N30. 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 in N30. As shown in the data presented herein, treated samples resulting in a maximum of 34.8% deamidation at N30 resulted in 76% potency; therefore, deamidation at N30 at levels higher than 34.8% would have a significant effect on relative potency (less than 76%).

[0176] In one embodiment, the composition comprises 0-45%, 0-40%, 0-30%, 0-20%, or 0-10% deamidation in N388. Alternatively, the composition comprises 0.1-45%, 0.1-40%, 0.1-30%, 0.1-20%, or 0.1-10% deamidation in N388.

[0177] Alternatively, the composition comprises 1-45%, 1-40%, 1-30%, 1-20%, or 1-10% deamidation at N388. Alternatively, the composition comprises 0.5% or more, 1% or more, or 2% or more deamidation at N388. As shown by the data presented herein, base-treated samples resulting in up to 28.2% deamidation at N388 do not affect potency in the bioassay (i.e., within assay variability—full function), and therefore it is expected that deamidation at N388 can be higher than the reported level of 28.2% without any effect on relative potency or FcRn binding.

[0178] In one embodiment, the composition comprises 0-45%, 0-40%, 0-30%, 0-20%, or 0-10% deamidation in N393. Alternatively, the composition comprises 0.1-45%, 0.1-40%, 0.1-30%, 0.1-20%, or 0.1-10% deamidation in N393.

[0179] Alternatively, the composition comprises 1-45%, 1-40%, 1-30%, 1-20%, or 1-10% deamidation in N393. Alternatively, the composition comprises 0.5% or more, 1% or more, or 2% or more deamidation in N393. As shown by the data presented herein, base-treated samples resulting in up to 28.2% deamidation in N393 do not affect potency in the bioassay (i.e., within assay variability—full function), and therefore it is expected that deamidation in N393 can be higher than the reported level of 28.2% without any effect on relative potency or FcRn binding.

[0180] 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 45% deamidation at N388 and / or N393 of the heavy chain.

[0181] In one example, deamidation can be determined using Lys-C and / or tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS).

[0182] The antibody composition may comprise (i) an antibody (e.g., an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 10 and the light chain amino acid sequence of SEQ ID NO: 11, 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 variant or a C-terminal lysine clip variant), an oxidation variant, an aggregation variant, and / or a fragmentation variant.

[0183] Thus, in one aspect, a composition is provided comprising an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises (i) less than 35% oxidation variants and (ii) less than 26% aggregation variants.

[0184] 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: 10 and / or SEQ ID NO: 12, and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 35% oxidized variants.

[0185] In one aspect, a composition comprises an antibody comprising a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO: 10 and / or SEQ ID NO: 12, and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises less than 26% aggregation variants.

[0186] 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-CTLA-4 antibody, the variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 4, a CDRH2 of SEQ ID NO: 5, and a CDRH3 of SEQ ID NO: 6, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 1, a CDRL2 of SEQ ID NO: 2, and a CDRL3 of SEQ ID NO: 3, wherein the composition has at least 60% of the potency of a composition comprising the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, 10-97% acidic variants, 10-97% basic variants, 2-80% major isoforms, 30% or less HC W52 oxidation variants, 35% or less HC M256 oxidation variants, 12.5% ​​or less HC M362 oxidation variants, 16% or less HC M401 oxidation variants, 35% or less HC M432 oxidation variants, 26% or less aggregation variants, 45% or less LC N30 deamidation variants, 45% or less HC N388 deamidation variants, 45% or less HC N393 deamidation variants, and / or 10% or less fragmentation variants. Potency can be determined using the bioassays described herein.

[0187] Glycation is a post-translational modification involving a non-enzymatic chemical reaction between reducing sugars, such as glucose, and free amine groups in proteins, typically observed at the epsilon amine of lysine side chains or the N-terminus of proteins. Glycation can occur during production and / or storage in the presence of reducing sugars.

[0188] Disulfide bond scrambling can occur during production and / or storage conditions. Under certain circumstances, disulfide bonds can be broken or formed incorrectly, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryls (-SH) can facilitate shuffling.

[0189] Thioether formation and disulfide bond racemization can occur under basic conditions during production or storage by beta-elimination of the disulfide bridge back to a cysteine ​​residue via a dehydroalanine and persulfide intermediate. Subsequent cross-linking of dehydroalanine and cysteine ​​can result in the formation of a thioether bond, or the free cysteine ​​residue can reform the disulfide bond with a mixture of D- and L-cysteine.

[0190] Trisulfides can result from the insertion of a sulfur atom into a disulfide bond (Cys-SS-S-Cys) and can be formed due to the presence of hydrogen sulfide in the production cell culture.

[0191] N-terminal glutamine (Q, Gln) and glutamate (glutamic acid) (E, Glu) in the heavy and / or light chains can form pyroglutamic acid (pGlu) through cyclization. While pGlu formation can occur in the production bioreactor, it can also be formed non-enzymatically, for example, depending on the pH and temperature of processing and storage conditions. Cyclization of the N-terminal Q or E is commonly observed in natural human antibodies.

[0192] 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. A variation of this process involves the removal of lysines from one or both heavy chains by cellular enzymes from recombinant host cells. Administration to a human subject / patient will likely result in the removal of any remaining C-terminal lysines.

[0193] Binding of fetal Fc receptors (FcRn) to anti-CTLA-4 antibodies can be measured using surface plasmon resonance (SPR).

[0194] Antibody compositions containing the above-described antibodies and antibody variants retain specific antigen binding and / or FcRn binding and / or potency. For example, antibody compositions containing the above-described antibodies, antibody variants, and post-translationally modified variants have CTLA-4-specific antigen binding of greater than 0.70 and / or FcRn binding of greater than 70% and / or potency of greater than 70%. Thus, these levels (%) of variants 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 CTLA-4 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 more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, or more than 50%.

[0195] The composition may comprise a mixture of antibody variants and post-translationally modified variants, for example, the antibody composition may comprise two or more of acidic variants, basic variants, oxidized variants, deamidated variants, aggregation variants, and fragmentation variants.

[0196] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises any one or combination of: (i) up to 100% acidic variants; (ii) up to 100% basic variants; (iii) less than 35% oxidation at W52 of the heavy chain; (iv) less than 35% oxidation at M256 of the heavy chain; (v) less than 12.5% ​​oxidation at M362 of the heavy chain; (vi) less than 16% oxidation at M401; (vii) less than 35% oxidation at M432; and / or (viii) less than 26% aggregation variants.

[0197] In one aspect, a composition comprises an antibody having a heavy chain sequence of SEQ ID NO: 10 and a light chain sequence of SEQ ID NO: 9, wherein the composition comprises any one or combination of: (i) up to 100% acidic variants, (ii) up to 100% basic variants, (iii) less than 35% oxidation at W52 of the heavy chain, (iv) less than 35% oxidation at M256 of the heavy chain, (v) less than 12.5% ​​oxidation at M362 of the heavy chain, (vi) less than 16% oxidation at M401 of the heavy chain, (vii) less than 35% oxidation at M432 of the heavy chain, (viii) less than 26% aggregation variants, (ix) less than 45% deamidation at N30 of the light chain, (x) less than 45% deamidation at N388 of the heavy chain, (xi) less than 45% deamidation at N393 of the heavy chain, and / or (xii) less than 10% fragmentation variants.

[0198] The present disclosure encompasses antibodies that may be subject to or may undergo 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 (one or more, or two or more) described herein. Thus, a composition may include a population of antibodies with and without the post-translational modification.

[0199] The described compositions may be subject to or undergo one or more post-translational modifications. Modifications may occur in the CDRs, variable framework regions, or constant regions. Modifications may result in a change in the charge of the molecule.

[0200] In one embodiment, the post-translational modifications described herein, except as identified and described as product-associated impurities, do not result in significant changes in antigen binding affinity, biological activity, pharmacokinetics (PK), aggregation, immunogenicity, and / or binding to Fc receptors.

[0201] 4. Composition impurities Composition impurities can include host cell proteins (HCPs). HCPs are process-related impurities produced by the host organism. HCP impurities can be reported as ng of HCP per mg of drug (ppm).

[0202] In one embodiment, a composition is provided comprising an anti-CTLA antibody, the anti-CTLA antibody comprising a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition comprises 13.54 ppm or less of the endoplasmic reticulum chaperone BiP.

[0203] In another embodiment, a composition is provided comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition comprises 5.21 ppm or less of elongation factor 1-gamma.

[0204] In another embodiment, a composition is provided comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition comprises 3.41 ppm or less of T complex protein 1 subunit eta.

[0205] In another embodiment, a composition is provided comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, and the composition comprises 3.24 ppm or less of importin subunit beta-1.

[0206] In another embodiment, a composition is provided comprising an anti-CTLA antibody, the anti-CTLA antibody comprising a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition comprises 2.75 ppm or less of glyceraldehyde-3-phosphate dehydrogenase.

[0207] In another embodiment, a composition is provided comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, and the composition comprises 2.52 ppm or less of an RNA splicing ligase RtcB homolog.

[0208] In another embodiment, a composition is provided comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, and the composition comprises 1.53 ppm or less of RuvB-like 2 OS=Mus musculus.

[0209] In another embodiment, a composition is provided comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition comprises 1.46 ppm or less of T-complex protein 1 subunit zeta.

[0210] In a further embodiment, a composition is provided comprising an anti-CTLA antibody, the anti-CTLA antibody comprising a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition also comprises less than 13.5 ppm of endoplasmic reticulum chaperone BiP, less than 5.21 ppm of elongation factor 1-gamma, less than 3.41 ppm of T complex protein 1 subunit eta, less than 3.24 ppm of importin subunit beta-1, less than 2.75 ppm of glyceraldehyde-3-phosphate dehydrogenase, less than 2.52 ppm of RNA splicing ligase RtcB homolog, less than 1.53 ppm of RuvB-like 2 OS=Mus musculus, and less than 1.46 ppm of T complex protein 1 subunit zeta.

[0211] 5. Manufacturing method The antibodies and compositions described herein can be obtained by any means, including via in vitro sources (e.g., hybridomas or cell lines that recombinantly produce the antibodies) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described, for example, in U.S. Patent No. 6,682,736.

[0212] For example, the composition can be expressed in and purified from a recombinant expression system. 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.

[0213] Many different expression systems and purification regimens can be used to produce the compositions. Generally, host cells are transformed with a recombinant expression vector encoding the antibody. A wide range of host cells can be used, including eukaryotic cell lines of mammalian origin (e.g., CHO, Perc6, HEK293, HeLa, NS0).

[0214] 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.

[0215] Suitable cloning and expression vectors and cloning methods for use with eukaryotic or mammalian cell hosts are known in the art.

[0216] The host cells are cultured to allow for expression of the recombinant expression vector encoding the antibody.

[0217] The composition can be recovered and purified by conventional protein purification procedures. For example, the composition can be harvested directly from the culture medium. Harvesting of the cell culture medium can be via clarification, for example, by centrifugation and / or depth filtration. Following recovery of the composition, it is purified to ensure sufficient purity. Thus, in one aspect, a cell culture medium is provided comprising the composition described herein. In one embodiment, the cell culture medium comprises CHO cells.

[0218] The composition can then be purified from the cell culture medium. This can 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 that binds the antibody molecules), and eluting the antibody molecules from the purification medium to produce an eluate. Thus, in one aspect, an eluate is provided that comprises a composition described herein.

[0219] Purification can involve one or more chromatography steps (e.g., one or more chromatography resin runs and / or one or more filtration steps). For example, affinity chromatography using resins such as Protein A, Protein G, or Protein 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.

[0220] Alternatively, the purification step comprises an affinity chromatography resin step followed by a cation exchange resin step.

[0221] 6. Pharmaceutical Compositions and Formulations The compositions described herein may be in the form of a pharmaceutical composition.

[0222] In one aspect, a pharmaceutical composition is provided that includes the composition and at least one pharmaceutically acceptable excipient.

[0223] A "pharmaceutical composition" can include 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, be capable of being formulated into a pharmaceutical formulation, and not be harmful to the recipient thereof and / or interfere with the efficacy of the active ingredient. Thus, the pharmaceutical compositions of the present disclosure are suitable for administration to a patient.

[0224] As used herein, a "pharmaceutically acceptable excipient" can include one or more buffers, such as water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. Often, it will preferably include an isotonicity agent, e.g., a polyol, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride; a preservative; a cosolvent; an antioxidant, including ascorbic acid and methionine; a chelating agent, such as EDTA; a metal complex, e.g., a Zn2+-protein complex; a biodegradable polymer; and / or a salt-forming counterion, such as sodium or potassium.

[0225] The precise nature of the excipient or other material may 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 intranasal. It will be understood that preferred excipients may vary with, for example, the condition of the recipient and the disease being treated.

[0226] The mixture of excipients and each concentrate together form a "pharmaceutical formulation" (or "formulation"). Such compositions are suitably 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, aliquots of the frozen formulation comprising the composition may be lyophilized. The lyophilisate may be reconstituted by the addition of water or other aqueous solution to produce a reconstituted formulation comprising the composition.

[0227] In one embodiment, the composition is a liquid formulation. In some embodiments, the formulation comprises from about 10 mg / mL to about 125 mg / mL of antibody. In further embodiments, the formulation comprises from about 15 mg / mL to about 100 mg / mL, particularly from about 15 mg / mL to about 125 mg / mL of antibody, such as from about 15 mg / mL to about 50 mg / mL. In a further embodiment, the formulation comprises from about 20 mg / mL of antibody. In another embodiment, the formulation comprises from about 50 mg / mL of antibody.

[0228] In one aspect, a formulation is provided comprising a pharmaceutical composition described herein, comprising about 10 mg / mL to about 125 mg / mL (e.g., about 20 mg / mL to about 50 mg / mL, e.g., 20 mg / mL) of an antibody and a buffer at a pH of about 5.0 to about 6.5, e.g., pH 5.5. In one embodiment, the composition is a liquid formulation.

[0229] 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 histidine buffer). In some embodiments, the composition comprises a CTLA-4 antibody and two or more of citrate buffer, histidine buffer, arginine, trehalose, sodium chloride, and polysorbate 80. In certain embodiments, the buffer is a citrate buffer. Citrate buffers can be obtained, 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 5.5 to about 6.0, particularly about 5.5 or about 6.0, from about 5.0 to about 6.5.

[0230] In another embodiment, the buffer is a histidine buffer. In one embodiment, the histidine buffer has a pH of about 6.0 to about 6.5, particularly about 5.5 to about 7.0, such as about 6.0 or about 6.5, about 5.5 to about 6.5.

[0231] In some embodiments, the formulation includes a surfactant. A "surfactant" is a surface-active agent that can exert its effect at solid-solid, solid-liquid, liquid-liquid, and liquid-air interfaces due to its chemical composition containing both hydrophilic and hydrophobic groups. Surfactants can reduce the concentration of proteins in dilute solutions at air-water and / or water-solid interfaces, where proteins may adsorb and potentially aggregate. Surfactants can bind to hydrophobic interfaces in protein formulations. Some parenterally acceptable nonionic surfactants contain either polysorbate or polyether groups. Polysorbate 20 and polysorbate 80, particularly polysorbate 80 (PS80), are suitable surfactant stabilizers in the formulations of the present disclosure. In one embodiment, the formulation further includes PS80. In some embodiments, the formulation includes about 0.01% to about 0.1% w / v, such as about 0.01% to about 0.05%, or about 0.01% to about 0.03%, 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.

[0232] The formulation may include a chelating agent. As used herein, the term "chelating agent" generally refers to an excipient that can form at least one bond (e.g., covalent, ionic, or other) with a metal ion. Chelating agents are typically multidentate ligands that can be used in liquid compositions as selected stabilizers to form complexes with species that may promote instability.

[0233] Suitable chelating agents for use in the present invention include, but are not limited to, aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, 2-(2-amino-2-oxoethyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, niacinamide, and descholate. Examples of suitable aminopolycarboxylic acids include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycol ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid, and aspartic acid. Examples of suitable hydroxyaminocarboxylic acids include N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethyl-glycine (bicine), and N-(trishydroxymethylmethyl)glycine (tricine). An example of a suitable N-substituted glycine is glycylglycine. An example of a suitable deoxycholate is sodium deoxycholate. Mixtures of two or more chelating agents are also encompassed by the present invention. In some embodiments, the chelating agent is EDTA. In another embodiment, the chelating agent is edetate disodium dihydrate.

[0234] In some embodiments, the formulation comprises water. 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 composition comprises about 5% w / v trehalose.

[0235] In some embodiments, the formulation comprises arginine and / or trehalose, such as about 80 mM to about 120 mM (particularly 100 mM) arginine or about 2% to about 10% w / v (particularly 5% w / v) trehalose. In one embodiment, the formulation comprises 222 mM trehalose dihydrate.

[0236] In one embodiment, a formulation is provided that comprises a pharmaceutical composition comprising about 5 mg / mL to about 125 mg / mL of an antibody, about 10 mM to about 40 mM of histidine buffer, about 200 mM to about 250 mM of trehalose dihydrate, about 0.2 mM to about 0.3 mM of edetate disodium dihydrate, and about 0.01% to about 0.03% w / v of polysorbate 80, and having a pH of about 5.0 to about 5.8.

[0237] In one aspect, a formulation is provided comprising about 5 to 125 mg / mL of an antibody, about 20 mM histidine buffer, about 222 mM trehalose dihydrate, about 0.27 mM edetate disodium dihydrate, and about 0.02% (w / v) polysorbate 80, and having a pH of about 5.5.

[0238] In one aspect, a formulation is provided comprising 20 mg / mL antibody, 20 mM histidine buffer, about 222 mM trehalose dihydrate, 0.27 mM edetate disodium dihydrate, and 0.02% (w / v) polysorbate 80, having a pH of about 5.5.

[0239] 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 by differential scanning calorimetry; the higher the transition temperature, the more stable the product formulation. A "stable" formulation can be one in which the phase transition does not occur at temperatures above about 50°C, above about 55°C, or above about 60°C. Stability can be measured at a selected temperature over a selected period of time. For example, for a product stored at a recommended temperature of 2°C to 8°C, the formulation can 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 can be one in which no more than about 10%, no more than about 5%, e.g., no more than about 4% aggregate variants of the antibody are present in the formulation. A "stable" formulation can be one in which no more than about 60%, no more than about 50%, e.g., no more than about 45% acidic variants of the antibody are present in the formulation. A "stable" formulation can be one in which no more than about 35%, no more than about 10%, e.g., no more than about 20% basic variants of the antibody are present in the formulation.

[0240] In certain aspects of the disclosure, 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 can be one in which no more than about 4% aggregate variants, no more than about 45% acidic variants, and no more than about 20% basic variants of the antibody are present in the formulation.

[0241] In yet another aspect, the present disclosure relates to articles of manufacture, embodiments, and kits comprising a container holding a composition in a formulation described herein. In one aspect, an injection device containing the formulation is provided. The injection device may include a pen injector device or an autoinjector device. In one embodiment, the formulation is contained in a pre-filled syringe.

[0242] 7. Methods of Treatment and Compositions for Use The present disclosure further provides methods of treating any disease or disorder in which inappropriate expression (e.g., overexpression) or increased activity of CTLA-4 protein causes or contributes to the pathological effects of the disease, or in which a decrease in CTLA-4 protein levels or activity has a therapeutic benefit in a mammal, preferably a human.

[0243] In one aspect, there is provided a pharmaceutical formulation as described herein for use in therapy. Such treatment may be for any disease or disorder in which inappropriate expression (e.g., overexpression) or increased activity of CTLA-4 protein causes or contributes to the pathological effects of the disease, or in which a decrease in CTLA-4 protein levels or activity has a therapeutic benefit in a mammal, preferably a human.

[0244] The composition may be used in a method of increasing T cell activation or T cell effector function in a subject, the method comprising administering a therapeutically effective dose of an agent capable of inhibiting CTLA-4 signaling. The composition may be used in a method of inducing an immune response in a subject, the method comprising administering a therapeutically effective dose of an agent capable of inhibiting CTLA-4 signaling. The composition may 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 dose of an agent capable of inhibiting CTLA-4 signaling.

[0245] In one aspect, a composition for use in treating cancer is provided.

[0246] 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.

[0247] As used herein, the term "treatment" and its grammatical variations refer to therapeutic therapy. With respect to a particular condition, treatment means (1) improving the condition of one or more biological manifestations of the condition, (2) a) inhibiting one or more points in the biological cascade that leads to or is responsible for the condition, or b) inhibiting one or more biological manifestations of the condition, (3) alleviating one or more symptoms, effects, or side effects associated with the condition or its treatment, (4) slowing the progression of the condition or one or more biological manifestations of the condition, or (5) preventing the onset of one or more biological manifestations of the condition.

[0248] Treatment can be therapeutic, prophylactic, or preventative. A subject is one in need thereof. Subjects in need of treatment can include individuals already suffering from a particular medical disorder as well as individuals who may develop the disorder in the future.

[0249] Therefore, preventive treatment is also contemplated. Those skilled in the art will understand that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to substantially reduce the likelihood or severity of a condition or its biological manifestations, or to delay the onset of such a condition or its biological manifestations. Preventive treatment 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 has been exposed to a carcinogen.

[0250] Thus, 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 a disease. The subject can be asymptomatic. The subject can have a genetic predisposition to a disease. Such individuals are administered a prophylactically effective amount of the composition. A prophylactically effective amount is an amount that prevents or delays the onset of one or more aspects or symptoms of a disease described herein.

[0251] 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 is recognized in the art, an agent used as a therapeutic agent in a therapy may reduce the severity of a given disease state, but need not eliminate every sign of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically need not be completely effective in preventing the onset of a disease to constitute a viable prophylactic agent. It is sufficient to merely reduce the effects of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by providing another beneficial effect), or to reduce the likelihood that the disease will occur (e.g., by delaying the onset of the disease) or worsen in a subject.

[0252] 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 be a non-human animal. The antibodies, compositions, and methods of the disclosure also have veterinary applications. The subject to be treated may be a livestock animal, e.g., a cow or bull, a sheep, a pig, a bull, a goat, or a horse, or may be a domestic animal, such as a dog or cat. The animal may be of any age or a mature adult animal.

[0253] The present disclosure also provides a method of treating cancer in a mammal.

[0254] 8. Cancer treatment The present disclosure provides a method for reducing tumors or inhibiting tumor cell growth in a subject, the method comprising administering a therapeutically effective dose of an agent capable of inhibiting CTLA-4 signaling. The method may comprise administering the aforementioned composition to a mammal suffering from cancer, followed by treating the cancer in the mammal. As discussed herein, CTLA-4 is aberrantly expressed in various cancers, and CTLA-4 expression in some cancer (e.g., renal cell carcinoma) patients correlates with tumor aggressiveness. The method can be used to detect and treat tumors, including, for example, adenocarcinoma, lung adenocarcinoma, acute myeloid leukemia ("AML"), acute lymphoblastic leukemia ("ALL"), adrenocortical tumors, 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, choriocarcinoma, chronic myeloid leukemia, CNS tumors, colon adenocarcinoma, colon cancer, colorectal cancer, diffuse intrinsic pontine glioma (DIP) G), diffuse large B-cell lymphoma ("DLBCL"), embryonal rhabdomyosarcoma (ERMS), endometrial cancer, epithelial carcinoma, 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, clear cell renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma , monocytic 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, recurrent 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 area (e.g., The present invention can be used to treat any type of cancer known in the art, such as squamous cell carcinoma of the esophagus, squamous cell carcinoma of the head and neck (SCHNC), squamous cell carcinoma of the lung, 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. In one embodiment, the cancer is hepatocellular carcinoma. In another embodiment, the cancer is NSCLC.

[0255] In some embodiments, cancers treated with the compositions described herein are characterized by microsatellite instability or the lack thereof. Microsatellite instability ("MSI") is or contains a change in the DNA of a particular cell (such as a tumor cell) in which the number of repeats of a microsatellite (a short, repetitive sequence of DNA) differs from the number of repeats contained in the inherited DNA. Microsatellite instability results from the inability 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.

[0256] 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 microsatellite stability (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, microsatellite instability is detected by NGS. In some embodiments, microsatellite instability is detected by IHC. In some embodiments, microsatellite instability is detected by PCR.

[0257] In some 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.

[0258] In some embodiments, the cancer is mismatch repair deficient (dMMR) cancer. Microsatellite instability can result from the inability of defective DNA mismatch repair (MMR) system to repair replication-related errors. This failure allows mismatch mutations to persist throughout the genome, especially in regions of repetitive DNA known as microsatellites, resulting in an increased mutation burden, which can improve response to certain anti-CTLA-4 agents.

[0259] In some embodiments, the cancer is a hypermutated cancer. In some embodiments, the cancer harbors a mutation in polymerase epsilon (POLE). In some embodiments, the cancer harbors a mutation in polymerase delta (POLD).

[0260] 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 containing a mutation in POLE or POLD (e.g., an MSI-H non-endometrial cancer containing a mutation in POLE or POLD).

[0261] In one aspect, a method of treating cancer is provided, the method comprising administering to a subject in need of cancer treatment a therapeutically effective amount of a composition (e.g., a pharmaceutical composition or formulation) described herein.

[0262] As used herein, the terms "cancer" and "tumor" are used interchangeably and, in either 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 easily distinguished from non-cancerous 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 ancestors. This includes metastasized cancer cells, as well as 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 the tumor mass, for example, by procedures such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound, or palpation during a physical examination, and / or due to the expression of one or more cancer-specific antigens in samples obtainable from a patient.

[0263] In some embodiments, the cancer is head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), renal cancer, bladder cancer, melanoma, Merkel cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, breast cancer, prostate cancer, salivary gland tumor, thymoma, adrenocortical tumor, esophageal cancer, gastric cancer, colorectal cancer, appendix cancer, urothelial cell carcinoma, or squamous cell carcinoma (e.g., of the lung; or of the anogenital area, such as the anus, penis, cervix, vagina, or vulva; or of the esophagus).

[0264] In some embodiments, the cancer is a hematological cancer, hi some embodiments, the hematological cancer is 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 some embodiments, the cancer is a blood-borne 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 myelogenous leukemia ("CML"), chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, and multiple myeloma; acute and chronic leukemia such as lymphoblastic leukemia, myeloid leukemia, lymphocytic leukemia, and myelocytic leukemia.

[0265] 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.

[0266] 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., of the anus, penis, cervix, vagina, or vulva).

[0267] 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, uterine 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.

[0268] In some embodiments, the cancer is a CNS or brain cancer such as neuroblastoma (NB), glioma, diffuse intrinsic 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 some embodiments, the cancer is a CNS tumor.

[0269] In some embodiments, the tumor is a solid tumor. In some embodiments, the cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma, colon cancer, colorectal cancer, renal cancer, pancreatic cancer, osteosarcoma, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, nipple cancer, and the like.

[0270] In some embodiments, the tumor is a solid tumor, such as carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, 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 stage solid tumor. In some embodiments, the tumor is a metastatic solid tumor.

[0271] In some embodiments, the cancer is a gynecological cancer (i.e., 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.

[0272] In some embodiments, the cancer is ovarian cancer (e.g., serous ovarian cancer or clear cell ovarian cancer). In some embodiments, the cancer is fallopian tube cancer (e.g., serous fallopian tube cancer or clear cell fallopian tube cancer). In some embodiments, the cancer is primary peritoneal cancer (e.g., primary serous peritoneal cancer or clear cell primary peritoneal cancer).

[0273] In some embodiments, the ovarian cancer is an epithelial cancer. Epithelial cancer accounts for 85% to 90% of ovarian cancers. While historically thought to begin on the surface of the ovaries, new evidence suggests that at least some ovarian cancers begin 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 normal female reproductive system, there are two fallopian tubes, one on each side of the uterus. Cancer cells that begin in the fallopian tubes may progress early to the surface of the ovaries. The term "ovarian cancer" is used to describe epithelial cancers that begin 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 begins in the cells that produce eggs in the ovaries. In some aspects, the cancer is or comprises a stromal tumor. A stromal tumor begins in the connective tissue cells that hold the ovaries together (sometimes the tissue that produces the female hormone called estrogen). In some embodiments, the cancer is or comprises a granulosa cell tumor. Granulosa cell tumors secrete estrogen and may result in 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 at some point shown a partial or complete response to platinum-based therapy (e.g., a partial or complete response to the last or penultimate platinum-based therapy). In some embodiments, the gynecological cancer is currently resistant to platinum-based therapy.

[0274] In some embodiments, the cancer is breast cancer. Breast cancer usually begins in either the cells or ducts of the milk-producing glands known as lobules. Less commonly, breast cancer can begin in the stromal tissues. These include the fatty and fibrous connective tissues of the breast. Over time, breast cancer cells can invade nearby tissues, such as the axillary lymph nodes or lungs, in a process known as metastasis. The stage of the breast cancer, the size of the tumor, and its growth rate are all factors that determine the type of treatment provided.

[0275] Treatment options include surgery to remove the tumor, drug treatments including chemotherapy and hormone 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 involved. 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 death, accounting for approximately 521,000 deaths. Of these cases, approximately 15% are triple-negative, meaning they do not express estrogen receptors, progesterone receptors (PRs), 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.

[0276] 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 some embodiments, the cancer is TNBC.

[0277] In some embodiments, the cancer is metastatic breast cancer. In some embodiments, the 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.

[0278] In some embodiments, the cancer is endometrial cancer ("EC"). In some embodiments, the cancer is metastatic endometrial.

[0279] 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 occurring 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 currently, no targeted therapies are approved for use in EC. There is a need for agents and regimens that improve survival rates for advanced and recurrent EC in the first-line (1L) and second-line (2L) settings. The most common histological form 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%).

[0280] From a pathogenic perspective, EC is classified into two distinct types, so-called type I and type II. Type I tumors are low-grade estrogen-associated endometrioid carcinomas (EEC), whereas type II tumors are non-endometrioid carcinomas (NEEC) (mainly serous and clear cell). 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 occurs in pre- and postmenopausal patients and is an estrogen-associated cancer preceded by precursor lesions (endometrial hyperplasia / endometrioid intraepithelial neoplasia). Microscopically, low-grade EEC (EEC 1-2) contains tubular glands somewhat resembling proliferative endometrium, with architectural complexity involving merging of glandular and cribriform patterns. High-grade EEC exhibits a solid pattern of growth. In contrast, SC occurs in postmenopausal patients in the absence of hyperestrogenism. Microscopically, SC shows thick fibrous or edematous papillae with prominent stratification of tumor cells, cell budding, and undifferentiated cells with large eosinophilic cytoplasm. The majority of EECs are low-grade tumors (grades 1 and 2), which, when confined to the uterus, are associated with a favorable prognosis. Grade 3 EECs (EEC3) are invasive tumors with an increased frequency of lymph node metastasis. SCs are highly aggressive, independent of estrogen stimulation, and occur primarily in older women. EEC3s and SCs are considered high-grade tumors. SCs and EEC3s were compared using Surveillance, Epidemiology, and End Results (SEER) program data from 1988 to 2001. They accounted for 10% and 15% of ECs, respectively, but 39% and 27% of cancer deaths, respectively.

[0281] 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 non-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).

[0282] In some embodiments, the cancer is a metastatic cancer.

[0283] In some embodiments, the cancer is a recurrent cancer (eg, a recurrent gynecological cancer such as recurrent epithelial ovarian cancer, recurrent fallopian tube cancer, recurrent primary peritoneal cancer, or recurrent endometrial cancer).

[0284] Subjects in need of cancer treatment can include patients from various stages, 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 are deemed transplant ineligible.

[0285] 9. Route of Administration The subject may have previously received at least one cancer therapy before treatment with the compositions of the present disclosure. In one embodiment, the subject has previously 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 cancer therapies before treatment with the compositions of the present disclosure. In another embodiment, the subject has been newly diagnosed with cancer and has previously received zero therapies before treatment with the compositions of the present disclosure.

[0286] The compositions of the present disclosure may be administered by any suitable route. For some compositions, suitable routes include oral, rectal, nasal, topical (such as buccal and sublingual), vaginal, parenteral (such as subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), and intratumoral.

[0287] It will be appreciated that the preferred route may vary with, for example, the condition of the recipient and the cancer to be treated.

[0288] In some embodiments, the composition is administered intravenously (e.g., by intravenous (IV) infusion). In further embodiments, the composition is administered by 30 minute IV infusion.

[0289] 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 present disclosure. The injection device may include a pen injector device or an autoinjector device.

[0290] In one embodiment, the composition is contained in a pre-filled syringe.

[0291] The desired dosage can be delivered by a single bolus of the composition, by multiple boluses of the composition, or by continuous infusion of the composition.

[0292] In certain embodiments, the compositions of the present disclosure are administered as pharmaceutical compositions.

[0293] As used herein, the term "administering" is meant to refer to the delivery of a composition described herein to achieve a therapeutic objective. The composition may be administered at intervals sufficient in duration to achieve a clinical benefit.

[0294] The composition may be administered to the subject in such a way as to target the treatment to a specific site.

[0295] In certain embodiments, the composition may be co-administered to a subject in conjunction with one or more additional therapeutic agents. In other embodiments, the composition may be co-administered to a subject in conjunction with one or more additional cancer therapeutic agents. The additional cancer therapeutic agents may include, but are not limited to, other immunomodulatory agents, therapeutic antibodies, CAR-T therapeutics, BiTEs, HDAC inhibitors, proteasome inhibitors, anti-inflammatory compounds, and immunomodulatory imid drugs (IMiDs).

[0296] "Co-administered" refers to the administration of two or more different pharmaceutical compositions or treatments (e.g., radiation therapy) that are administered to a subject in combination in the same or separate pharmaceutical compositions. Thus, co-administration includes the simultaneous administration of a single pharmaceutical composition containing two or more pharmaceutical agents, or the administration of two or more different compositions to the same subject at the same or different times.

[0297] In some embodiments, the compositions may be administered in combination with other agents that inhibit immune checkpoint pathways, for example, the compositions may be administered in combination with agents that inhibit or antagonize the PD-1 pathway.

[0298] In some embodiments, the method comprises administering the composition in combination with durvalumab. In some embodiments, the combination is for administration to a patient afflicted with liver cancer (hepatocellular carcinoma), ovarian cancer, head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), renal 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 tumor, esophageal cancer, gastric cancer, colorectal cancer, appendix cancer, urothelial cell carcinoma, or squamous cell carcinoma (e.g., of the lung, of the anogenital region such as the anus, penis, cervix, vagina, or vulva, or of the esophagus). In further embodiments, the cancer is selected from liver cancer, ovarian cancer, or lung cancer (e.g., NSCLC).

[0299] 10. Dosage The compositions described herein may be administered in therapeutically effective amounts.

[0300] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" of a composition refers to an amount of an agent (such as 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.

[0301] 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 are within the purview of the attending physician.

[0302] The ranges provided herein are of any kind and include all values ​​within the particular ranges recited, as well as values ​​relating to the endpoints of the particular ranges.

[0303] In some embodiments, the therapeutically effective dose is about 10-500 mg fixed dose (e.g., about 10 mg fixed dose, about 25 mg fixed dose, about 50 mg fixed dose, about 75 mg fixed dose, about 100 mg fixed dose, about 200 mg fixed dose, about 300 mg fixed dose, about 400 mg fixed dose, or about 500 mg fixed dose). 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 about 15 mg / kg. In some embodiments, the therapeutically effective dose is about 50 mg fixed dose. In some embodiments, the therapeutically effective dose is about 75 mg. In some embodiments, the therapeutically effective dose is about 100 mg.

[0304] In one embodiment, the composition is administered once every 2 to 6 weeks (eg, every 2, 3, or 4 weeks, particularly every 3 weeks).

[0305] In one embodiment, the composition is administered once every three weeks for two to six administration cycles (e.g., the first three, four, or five administration cycles, particularly the first four administration cycles).

[0306] In some embodiments, the first dose and the second dose are different, ie, the first dose is about 75 mg and the second dose is about 100 mg.

[0307] In some embodiments, the composition is administered at a dosing interval (or treatment cycle) of 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 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, or more weeks. 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.

[0308] In some embodiments, the composition is administered at a dose of 75 mg every three weeks. In another embodiment, the composition is administered at a dose of 75 mg every three weeks for four cycles. In another embodiment, the composition is administered at a dose of 75 mg every three weeks for four cycles, followed by an additional 75 mg dose at week 16.

[0309] In some embodiments, the composition is administered as a single 300 mg dose. In another embodiment, the composition is administered as a single 300 mg dose on day 1 of a treatment cycle in combination with 1500 mg of durvalumab, with durvalumab monotherapy continued every 4 weeks.

[0310] In some embodiments, the composition is administered according to Table 1.

[0311] [Table 1] aPatients weighing 40 kg or less should receive weight-based dosing equivalent to tremelimumab 1 mg / kg and durvalumab 20 mg / kg until their weight improves to greater than 30 kg. b Tremelimumab was administered first, followed by durvalumab and, if applicable, chemotherapy on the day of administration. c When tremelimumab is administered in combination with durvalumab and chemotherapy, see the durvalumab prescribing information for dosing information. d When tremelimumab is administered in combination with durvalumab and platinum-based chemotherapy, see the prescribing information for nab-paclitaxel, gemcitabine, pemetrexed, and carboplatin or cisplatin for dosing information.

[0312] In some embodiments, the compositions described herein are administered according to a dosing regimen demonstrated to provide clinical benefit to the patient. 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 embodiments, the clinical benefit is partial response ("PR").

[0313] In 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.

[0314] All patent and literature references disclosed herein are expressly and entirely incorporated by reference. [Example]

[0315] Example 1: Tremelimumab impurities Tremelimumab was evaluated to determine product-related and process-related impurities. The test methods used to evaluate the impurities are described herein.

[0316] method Amino acid analysis Amino acid analysis was used to determine the amino acid composition of the protein. Samples were acid hydrolyzed, labeled, and analyzed by liquid chromatography. Quantitation of each amino acid was determined against an external standard curve.

[0317] Analytical Ultracentrifugation Analytical ultracentrifugation was used to measure purity, fragmentation, and aggregation. Different size species were separated based on their sedimentation behavior under a strong centrifugal field. Species that separated with a lower sedimentation coefficient than the monomer peak were recorded as fragments. Species that separated with a higher sedimentation coefficient than the monomer peak were recorded as aggregates.

[0318] competitive binding ELISA A competitive enzyme-linked immunosorbent assay (ELISA) was used as the binding assay. This assay measures the ability of tremelimumab to block CTLA-4 binding to the B7.2 fusion protein. Binding activity was assessed as a dose-response normalized to a reference standard, yielding potency values ​​relative to the tremelimumab reference standard.

[0319] Capillary isoelectric focusing (cIEF) The charge heterogeneity of tremelimumab was determined by imaging capillary electrophoresis (iCE). A tremelimumab sample was mixed with carrier ampholytes and injected into a capillary. When a potential was applied, the carrier ampholytes created a pH gradient within the capillary, causing the charge isoforms to separate. Tremelimumab charge isoforms concentrated at points of neutral net charge (pI) within the capillary.

[0320] Reduced capillary gel electrophoresis Proteins were subjected to a denaturant, sodium dodecyl sulfate (SDS), under reducing conditions. When SDS-coated proteins were placed in an electric field within a capillary, the proteins migrated toward the anode through a replaceable SDS polymer sieving matrix, separating them according to their apparent molecular weight.

[0321] Non-reducing capillary gel electrophoresis In non-reducing capillary gel electrophoresis, proteins are subjected to the denaturing agent, SDS, under non-reducing conditions. When SDS-coated proteins are placed in an electric field within a capillary, the proteins migrate toward the anode through a replaceable SDS polymer sieving matrix, separating them according to their apparent molecular weight.

[0322] FcRn binding assay by SPR FcRn binding was measured using a surface plasmon resonance binding assay. This method is based on the ability of test samples to bind to recombinant FcRn immobilized on an SPR sensor surface. A reference flow cell was prepared using the same standard amine coupling procedure without the addition of FcRn.

[0323] High-Performance Size Exclusion Chromatography The purity of tremelimumab (monomer) and product-related impurities (aggregates) were determined by high performance size exclusion chromatography (HPSEC).

[0324] The HPSEC method separates size variants according to their relative molecular size and shape using differential exclusion from stationary phase pores in a column. As the mobile phase carries the sample through the column, the solute diffuses into the pores at a rate inversely proportional to the hydrodynamic volume, which is based on the solute size and the pore exclusion limit. The separated variants were detected and quantified by a UV detector.

[0325] Ion Exchange Chromatography (IEC) Charge heterogeneity was assessed using ion exchange chromatography. Samples were injected onto an ion exchange column and eluted with a salt gradient. Eluted proteins were detected using UV absorbance at 220 (or 280) nm. Results were reported as the percentage of the 0-Lys peak, the 1-Lys peak, and the 2-Lys peak. To assess charge heterogeneity in the absence of C-terminal lysine, samples were digested with carboxypeptidase B (CBP) prior to analysis. Results were reported as the area percentage of the pre-peak, main peak, and post-peak.

[0326] Ligand binding assay using SPR The assay was performed using surface plasmon resonance (SPR) to measure the association (ka) and dissociation (kd) of the interaction between tremelimumab and CTLA-4. The ratio kd / ka is the equilibrium dissociation constant of the interaction between tremelimumab and CTLA-4. K Provides information on the K of reference standards D The binding affinity of the test sample was calculated by comparing the results with the corresponding values ​​of the ATP-dependent ATPase activity.

[0327] Methionine oxidation by Lys-C mapping Methionine oxidation of tremelimumab samples was quantitatively determined by peptide mapping. The resulting peptide fragments were separated by reverse-phase HPLC and detected using an ultraviolet detector. One methionine-containing peptide fragment and its respective oxidized form were monitored. The percent oxidation of this methionine-256-containing fragment was reported.

[0328] N-terminal sequencing N-terminal sequencing by Edman degradation was used to determine the amino acid sequences at the N-termini of the heavy and light chains of tremelimumab. The results were compared with the theoretical sequences of the light and heavy chains.

[0329] Peptide mapping using mass spectrometry Peptide mapping was used to verify primary sequence and assess post-translational modifications. Samples were denatured, reduced, alkylated, and digested with trypsin. Peptides were separated by reverse-phase liquid chromatography and measured using a UV detector and mass spectrometer. Results were reported in terms of the consistency of peptide fragmentation patterns of test samples compared to reference standards.

[0330] Q-TOF mass spectrometry Q-TOF mass spectrometry (MS) was used to confirm the primary structure of the molecules based on differences in mass-to-charge ratio (m / z). The measured mass of the molecules was obtained by deconvolution of the m / z data and compared with the theoretical mass of the molecules based on their amino acid composition. Results are reported in units of molecular weight (Da).

[0331] Reporter gene bioassay CTLA4 is a cell surface receptor on activated T cells. The reporter gene bioassay measures the ability of tremelimumab to attenuate CTLA4-mediated inhibitory signals during T cell activation by blocking the binding of CTLA4 to its natural ligands, B7.1 (CD80) and B7.2 (CD86), on antigen-presenting cells (APCs). Tremelimumab was incubated with cell lines at various concentrations in the presence of the superantigen SEE. The amount of luminescence produced is proportional to T cell activity and is quantified in a luminometer after reaction with a luciferase substrate. The potency of test samples was determined by comparison with a reference standard.

[0332] Reversed-phase HPLC with mass spectrometry The RP-HPLC / MS method was used only for peak identification. Samples were deglycosylated using PNGase F. Both non-reduced and reduced samples were separated by RP-HPLC and analyzed by Q-TOF mass spectrometer. Mass spectrometry data were collected and analyzed. Each peak in the RP-HPLC profile was identified and reported.

[0333] Product-related impurities Product-related impurities, such as aggregates and fragments, are typically found in monoclonal antibody products as a result of degradation or modification. Key quality attributes identified for tremelimumab include those listed below (Table 2).

[0334] [Table 2]

[0335] agglomeration Aggregation is one of the major degradation pathways for therapeutic antibodies. Several aggregation mechanisms have been observed in protein solutions, including association of native monomers, aggregation of conformationally modified monomers, aggregation of chemically modified monomers, nucleation-controlled aggregation, and surface-induced aggregation. Aggregates can be broadly divided into two classes: covalent and noncovalent. Noncovalent aggregates can be further classified as reversible and irreversible.

[0336] Tremelimumab aggregates, including both dimers and high molecular weight (BMW) aggregates, were measured by high performance size exclusion chromatography (HPSEC).

[0337] Characterization of aggregates To assess the impact on biological activity, tremelimumab aggregation was induced by low pH (2.5-3.0) and high temperature (45°C) treatment in combination with shear force (shaking). The stressed material was fractionated by preparative SEC to generate concentrated aggregates. The aggregate and monomer fractions were subsequently characterized by physicochemical and biological analyses (Table 4). The results surprisingly indicate that aggregation has only a moderate impact on biological activity, as a decrease in relative potency was observed only in the high aggregate fraction.

[0338] [Table 3]

[0339] Abstract The results showed that tremelimumab remained active at relatively high levels of aggregation.

[0340] Aggregation increased after the material was subjected to stress conditions, i.e., low pH, high temperature, and shear force. The impact of aggregates on bioactivity was moderate. When materials had more than 70% aggregates, a decrease in relative potency was observed.

[0341] CDR deamidation (light chain Asn-30) Asparagine deamidation is another degradation pathway for therapeutic antibodies. When deamidation occurs in the CDR (complementarity-determining region), the impact of deamidation on biological activity can vary. The impact of deamidation on activity can vary from product to product. CDR deamidation at either Asn-33, Asn-55, or Asn-102 in IgG has been reported to reduce antigen binding and biological activity. Furthermore, succinimide formation (a deamidated intermediate) at Asn-55 in the CDR2 region of IgG1 has been reported to reduce biological activity by 70% and ligand binding affinity by 50%. Subsequent hydrolysis of this succinimide resulted in a further decrease in potency. The impact of deamidation on protein activity cannot be predicted with the degree of certainty required for pharmaceutical compositions.

[0342] Tremelimumab (IgG2) has one CDR deamidation site at Asn-30 on the light chain. LC Asn-30 deamidation, measured by peptide mapping, ranged from 6.5 to 6.9% for the samples tested (Table 5).

[0343] [Table 4]

[0344] Characterization of CDR deamidation (LC Asn-30) When tremelimumab samples stressed at 40°C during stability testing were analyzed, a significant increase in deamidation at LC Asn-30 was observed by peptide mapping.

[0345] These data are consistent with the increase in the acidic peak by cIEF (Table 6).

[0346] [Table 5]

[0347] To assess the impact of deamidation on biological activity, tremelimumab was incubated at pH 9 for 7 days and then fractionated by ion exchange chromatography (IEC, Figure 1) to enrich for the LC Asn-30 deamidated species. The main peak (M) fraction (Fraction B), along with two acidic fractions containing LC Asn-30 deamidation (Fraction D and Fraction E), were subsequently characterized by physicochemical and biological analyses. cIEF analysis of the IEC fractions is shown in Figure 2.

[0348] Table 7 summarizes the LC Asn-30 deamidation levels in IEC fractions by peptide mapping and cIEF, and their effect on biological activity (reporter gene bioassay), CTLA-4 binding, and FcRn-binding activity. IEC fraction D contained 41.2% Asn-30 deamidation and eluted primarily as acidic peak 1 (A1) in cIEF. IEC fraction D showed reduced biological activity (78%) and reduced CTLA-4 binding (62%). CTLA-4 binding was lower (54%) for IEC fraction E, which had higher LC Asn-30 deamidation and eluted primarily as acidic peak 2 (A2) in cIEF.

[0349] [Table 6] A1 = acidic peak 1; A2 = acidic peak 2; A3 = acidic peak 3; A4 = acidic peak 4; M = main peak; NT = not tested

[0350] Further characterization was performed after incubation of tremelimumab in PBS and human serum at 37°C to assess changes in deamidation levels under physiological conditions. Deamidation at LC Asn-30 was the major degradation product detected in these studies (Table 8).

[0351] [Table 7]

[0352] The correlation between biological activity and LC Asn-30 deamidation level (measured by cIeF acidic peak and peptide mapping) is shown in Figure 3. The effect of LC Asn-30 deamidation on biological activity was moderate: greater than 75% relative potency was maintained when LC Asn-30 deamidation was greater than 80% as measured by cIeF acidic peak or greater than 40% as measured by peptide mapping.

[0353] Abstract Deamidation increased under heat stress conditions at 40°C (37.7% by peptide mapping after 3 months) and during serum or PBS incubation at 37°C. The impact of CDR deamidation at LC Asn-30 on bioactivity was moderate: material with >80% cIeF acidic peak retained >75% relative potency.

[0354] fragmentation Fragmentation is a further degradation pathway for monoclonal antibodies. Fragmentation can occur at sites adjacent to Asp and His residues and has been shown to be affected by the presence of metal ions, oxidative free radical impurities, or residual host cell proteases. The hinge region of IgG2 is less susceptible to cleavage than IgG1. The total fragmentation of IgG2 is generally less than that of IgG1 under similar conditions.

[0355] Fragment characterization Tremelimumab is resistant to fragmentation even under prolonged stress conditions. As shown in Table 10, fragments in tremelimumab stressed at 40°C for up to 6 months were 6.3% or less by non-reducing gel electrophoresis and 5.0% or less by reducing gel electrophoresis. The low levels of fragments are consistent with literature reports that IgG2 is less susceptible to fragmentation due to the lack of peptide bond hydrolysis in its hinge region.

[0356] [Table 8]

[0357] To identify the fragments observed by gel electrophoresis, control samples and samples subjected to heat stress (40°C for 6 months) were analyzed by RP-LC / MS. Fragments were detected in the heat-stressed samples compared to undetectable fragment levels in the control samples. The major fragments were determined to be heavy chain fragments (HC1-99, HC330-450), light chain fragments (LC1-212, LC1-213), and light chain fragments with succinimides. Succinimides are deamidation or isomerization intermediates. Succinimide intermediates are identified in heat-stressed IgG, which exhibits increased levels of deamidation or isomerization. Trace amounts of heavy chain fragments HC239-450 and HC241-450 were also detected. A schematic diagram of tremelimumab labeled with fragmentation sites is shown in Figure 4.

[0358] The tremelimumab fragmentation pattern was fully characterized by orthogonal physicochemical methods, as summarized in Table 11. The expected fragments and other product-related species detected by RP-LC / MS in heat-stressed tremelimumab are listed. The identification of gel electrophoresis peaks was based on the results of RP-LC / MS, deglycosylation studies, and partial reduction studies. Peptide mapping was further performed to verify the fragments and aglycosylated species.

[0359] [Table 9] HC: heavy chain; LC: light chain; Nd: non-detectable; NR: non-reduced; R: reduced; Su-LC: LC with succinimide

[0360] To assess the impact of fragmentation on biological activity, the relative potency (reporter gene assay) was determined for tremelimumab samples stressed for 0, 2, and 3 months at 40°C (Table 12). The results demonstrate that greater than 95% relative potency was maintained after 3 months of incubation at 40°C in the presence of low levels of fragments.

[0361] [Table 10]

[0362] Abstract The fragmentation pattern was characterized by RP-LC / MS. The predominant fragment species were the heavy chain N-terminal fragment (HC1-99), the heavy chain C-terminal fragment (HC330-450), and the light chain C-terminal fragments (LC1-212 and LC1-213).

[0363] No hinge region fragments were detected. Tremelimumab fragmentation occurred very slowly under stress conditions (40°C). The presence of low levels of fragments (2.7% after 3 months at 40°C) had little effect on biological activity. Fragmentation is not the major degradation pathway for tremelimumab.

[0364] oxidation Amino acid oxidation is known to affect protein structure, activity, and degradation rate. Oxidation of Met and Trp is a chemical modification that occurs during the purification, formulation, and storage of monoclonal antibodies. Oxidation of other residues (Cys and Tyr) has also been observed. Fe oxidation (Met-252 and Met-428; EU numbering) has been reported to decrease FcRn binding and cause a shorter serum half-life. Loss of biological activity has been shown to correlate with oxidation in the CDR regions.

[0365] Tremelimumab oxidation was measured by tryptic peptide mapping as a characterization test at Trp-52 in the CDR and Met-256, Met-362, Met-401, and Met-432 in the Fc region. As the most susceptible site for oxidation, Met-256 oxidation was monitored by Lys-C peptide mapping.

[0366] Characterization of oxidative thermal stress After heat stress at 40°C for 3 months, a slight increase in oxidation of tryptophan and methionine residues was observed (Table 14).

[0367] [Table 11]

[0368] chemical oxidation Prior to peptide mapping and biological analysis, tremelimumab samples were subjected to chemical oxidation by treatment with hydrogen peroxide (100 ppm) at room temperature (Table 15). Met-256 and Met-401 were more readily oxidized than Met-432 and Met-362. Met-256 oxidation up to 79.8% did not affect potency. However, at oxidation levels above 35.5%, there was a decrease in FcRn binding.

[0369] [Table 12]

[0370] Photooxidation In addition to chemical oxidation, light stress studies were performed at IX and 3X exposure levels as outlined by the ICH Guidance QIB. That is, light radiation exposure exceeds 200 watt-hours per square meter for UV-A and 12,000,000 lux-hours for cool white light (CWL). Another study was performed on tremelimumab samples exposed to a CWL intensity of approximately 2,000 lux for 14 days (672,000 lux-hours total).

[0371] Table 16 summarizes the level of oxidation in light-stressed samples by peptide mapping and its effect on potency and FcRn binding. No significant effect on potency or FcRn binding was observed under exposure at IX ICH guidelines or 14-day CWL exposure. Decreases in potency and FcRn binding occurred after exposure at 3X ICH guidelines. Oxidation of Trp-52, Met-256, and Met-432 was observed under IX ICH guidelines and 14-day CWL exposure, and significantly increased after 3X CH guidelines exposure. Trp-52 is located in the heavy chain CDR region, and oxidation at this site may cause loss of potency under excessive light exposure (3X ICH). This study demonstrated that light-stress oxidation of tryptophan in the CDR at 36.7% affected biological activity.

[0372] [Table 13]

[0373] Abstract Chemical oxidation and light stress studies demonstrated that up to 35.5% Met-256 oxidation did not affect potency or FcRn binding. Trp-52 oxidation at 36.7% reduced potency and FcRn binding.

[0374] Host Cell Proteins (HCPs) LC-MS characterization of HCPs was performed on the tremelimumab sample and the pre-purification sample. The results are summarized in Tables 17-21. Fewer than 20 HCPs were identified in the tremelimumab sample. Meanwhile, 569-660 HCPs were detected in three different pre-purification samples (3-5), respectively. This method achieves high sensitivity for HCP identification by depleting the majority of the mAb molecules using native digestion. The concentration of each identified HCP was estimated using a label-free quantification approach. The results confirmed HCP clearance throughout the purification process, although a few low-abundance HCPs remained at low levels.

[0375] [Table 14] * semi-quantitative

[0376] [Table 15]

[0377] [Table 16] ppm: parts per million of active substance, ng / mg protein

[0378] A useful upper limit using a tolerance of approximately 5 times the standard deviation of the data set yields an upper limit of approximately 34 ng / mg, which is well below the range of currently approved monoclonal antibodies (<100 ng / mg protein) (Champion K. et al., ioprocess International; 2005; 3(8):52-7).

[0379] A useful upper limit for each HCP can also be established based on the same methodology, e.g., the upper limit for the endoplasmic reticulum chaperone BiP is approximately 14 ng / mg.

[0380] [Table 17]

Claims

1. 1. A composition comprising an anti-CTLA antibody, wherein the anti-CTLA antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6; and one or more deamidated variants of the anti-CTLA-4 antibody, wherein the composition comprises 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 of the deamidated variants.

2. 2. The composition of claim 1, wherein the amount of the deamidated variant of the anti-CTLA antibody is between the lower limit of detection of a method used to identify the deamidated variant and 45%, 40%, 35%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

3. The amount of the deamidated variant of the anti-CTLA antibody is (i) 0.01-45%, 0.01-40%, 0.01-35%, 0.01-30%, 0.01-25%, 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%, 0.0 1-1%, 0.05-45%, 0.05-40%, 0.05-35%, 0.05-30%, 0.05-25%, 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%, 0.05-2%, 0 . 05-1%, 0.1-45%, 0.1-40%, 0.1-35%, 0.1-30%, 0.1-25%, 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%, 0.5-45% , 0.5-40%, 0.5-35%, 0.5-30%, 0.5-25%, 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) The composition of any one of claims 1 or 2, wherein the concentration is about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

4. The composition of claim 3, wherein the amount of the deamidated variant of the anti-CTLA antibody is 0.01 to 45%.

5. 5. The composition of any one of claims 1 to 4, wherein the deamidated variant of the anti-CTLA-4 antibody comprises a deamidated residue that is an aspartic acid residue, a succinimido-aspartic acid residue, or an isoaspartic acid residue.

6. The composition of any one of claims 1 to 5, wherein one or more of the deamidated residues are within one or more CDRs of the anti-CTLA-4 antibody.

7. The composition of any one of claims 1 to 6, wherein the one or more deamidated residues of the anti-CTLA antibody are within any one of the amino acid sequences of SEQ ID NOs: 1 to 6.

8. The composition of any one of claims 1 to 7, wherein the deamidated variant of the anti-CTLA-4 antibody comprises deamidation at N30 of the CDRL1 of the anti-CTLA-4 antibody.

9. The composition of any one of claims 1 to 8, wherein the deamidated variant of the anti-CTLA-4 antibody comprises deamidation at N30 of the amino acid sequence of SEQ ID NO:

1.

10. The composition of any one of claims 1 to 9, wherein the deamidated mutant of the anti-CTLA-4 antibody comprises the amino acid sequence of SEQ ID NO:

11.

11. 11. The composition of any one of claims 1 to 10, wherein the deamidated variant of the anti-CTLA-4 antibody comprises 0.1-45%, 0.1-35%, 0.1-30%, 0.1-25%, 0.1-20%, 0.1-10%, 0.1-5%, 0.1-1%, 1-45%, 1-35%, 1-30%, 1-25%, 1-20%, 1-10%, 1-5%, 5-45%, 5-35%, 5-30%, 5-25%, 5-20%, or 5-10% deamidation in N30 of the anti-CTLA-4 antibody.

12. 12. The composition of any one of claims 1 to 11, wherein the deamidated variant of the anti-CTLA-4 antibody 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 in N30 of the anti-CTLA-4 antibody.

13. 13. The composition of any one of claims 1 to 12, wherein the deamidated variant of the anti-CTLA-4 antibody comprises 0.1 to 45%, 0.1 to 35%, 0.1 to 30%, 0.1 to 25%, 0.1 to 20%, 0.1 to 10%, 0.1 to 5%, 0.1 to 1%, 1 to 45%, 1 to 35%, 1 to 30%, 1 to 25%, 1 to 20%, 1 to 10%, 1 to 5%, 5 to 45%, 5 to 35%, 5 to 30%, 5 to 25%, 5 to 20%, or 5 to 10% deamidation at N388 of the anti-CTLA-4 antibody.

14. 14. The composition of any one of claims 1 to 13, wherein the deamidated variant of the anti-CTLA-4 antibody 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 at N388 of the anti-CTLA-4 antibody.

15. 15. The composition of any one of claims 1 to 14, wherein the deamidated variant of the anti-CTLA-4 antibody comprises 0.1 to 45%, 0.1 to 35%, 0.1 to 30%, 0.1 to 25%, 0.1 to 20%, 0.1 to 10%, 0.1 to 5%, 0.1 to 1%, 1 to 45%, 1 to 35%, 1 to 30%, 1 to 25%, 1 to 20%, 1 to 10%, 1 to 5%, 5 to 45%, 5 to 35%, 5 to 30%, 5 to 25%, 5 to 20%, or 5 to 10% deamidation at N393 of the anti-CTLA-4 antibody.

16. 16. The composition of any one of claims 1 to 15, wherein the deamidated variant of the anti-CTLA-4 antibody 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 at N393 of the anti-CTLA-4 antibody.

17. The composition comprises: (i) deamidation at N30 of the anti-CTLA-4 antibody; (ii) deamidation at N388 of the anti-CTLA-4 antibody, and / or (iii) the composition of any one of claims 1 to 16, comprising deamidation at N393 of the anti-CTLA-4 antibody.

18. The composition comprises: (i) deamidation of 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at N30 of the anti-CTLA-4 antibody; (ii) deamidation of 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less in N388 of the anti-CTLA-4 antibody; and / or (iii) the composition of any one of claims 1 to 17, comprising 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less deamidation in N393 of the anti-CTLA-4 antibody.

19. The composition of any one of claims 1 to 18, wherein the anti-CTLA-4 antibody and the acidic variant of the anti-CTLA-4 antibody comprise a light chain variable domain having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:7 and a heavy chain variable domain having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

8.

20. 20. The composition of claim 19, wherein the anti-CTLA-4 antibody comprises a light chain variable domain having the amino acid sequence of SEQ ID NO:7 and a heavy chain variable domain having the amino acid sequence of SEQ ID NO:

8.

21. 21. The composition of any one of claims 1 to 20, wherein the anti-CTLA-4 antibody and the acidic variant of the anti-CTLA-4 antibody comprise a light chain having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and a heavy chain having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

10.

22. 22. The composition of claim 21, wherein the anti-CTLA-4 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:9 and a heavy chain having the amino acid sequence of SEQ ID NO:

10.

23. 22. The composition of claim 21, wherein the acidic variant of the anti-CTLA-4 antibody comprises deamidation at one or more of N388 and / or N393 of the amino acid sequence of SEQ ID NO:

10.

24. The composition of any one of claims 1 to 23, wherein the deamidated variant of the anti-CTLA-4 antibody further comprises deamidation at N388 and / or N393 of the heavy chain of the anti-CTLA-4 antibody.

25. 25. The composition of any one of claims 1 to 24, wherein the method used to identify the deamidated variants or deamidation in residues of the anti-CTLA-4 antibody is capillary isoelectric focusing (cIeF).

26. The composition of any one of claims 1 to 25, wherein the composition further comprises an oxidized variant of the anti-CTLA-4 antibody.

27. A composition comprising an anti-CTLA antibody, the anti-CTLA antibody comprising a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, and one or more oxidized variants of the anti-CTLA antibody; The composition comprises no more than 35%, 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 said oxidation variants.

28. 28. The composition of claim 27, wherein the amount of the oxidized variant of the anti-CTLA antibody is between the lower limit of detection of the method used to identify the oxidized variant and 35%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

29. The amount of the oxidized variant of the anti-CTLA antibody is (i) 0.01-35%, 0.01-30%, 0.01-25%, 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%, 0.05~35%, 0.05~30%, 0.05~25%, 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%, 0.05~2%, 0.0 or selected from any one of the ranges: 5-1%, 0.1-35%, 0.1-30%, 0.1-25%, 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%, 0.5-35%, 0.5-30%, 0.5-25%, 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) The composition of any one of claims 27 or 28, wherein the concentration is about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

30. 30. The composition of claim 29, wherein the amount of the oxidized amidated variant of the anti-CTLA antibody is 0.01 to 35%.

31. The composition of any one of claims 24 to 30, wherein one or more of the oxidized residues are within one or more CDRs of the anti-CTLA antibody.

32. The composition of any one of claims 24 to 31, wherein the oxidized variant of the anti-CTLA antibody comprises an oxidation at a tryptophan or methionine residue.

33. The composition of any one of claims 24 to 32, wherein the one or more oxidized residues of the anti-CTLA antibody are within any one of the amino acid sequences of SEQ ID NOs: 1 to 6.

34. The composition of any one of claims 24 to 33, wherein the oxidized variant of the anti-CTLA antibody comprises an oxidation at W52 of the CDRH2 of the anti-CTLA-4 antibody.

35. 35. The composition of claim 34, wherein the composition comprises less than 30%, less than 25%, less than 20%, less than 16%, less than 15%, less than 12.5%, less than 10%, less than 5%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% oxidation at W52 of CDRH2 of the anti-CTLA antibody.

36. The composition of claim 35, wherein the composition comprises oxidation at W52 of the CDRH2 of the anti-CTLA antibody in an amount between the lower limit of detection of a method used to identify oxidation at W52 of the CDRH2 and 30%, 25%, 20%, 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

37. The composition comprises: (i) 0.01-30%, 0.01-25%, 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%, 0.05-30%, 0.05-25%, 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%, 0.05-2%, 0.05 or selected from any one of the ranges of up to 1%, 0.1-30%, 0.1-25%, 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%, 0.5-30%, 0.5-25%, 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%; (ii) the composition of any one of claims 24 to 36, comprising an oxidation at W52 of the CDRH2 of the anti-CTLA antibody in an amount of about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

38. The composition of any one of claims 24 to 37, wherein the oxidized variant of the anti-CTLA antibody comprises the amino acid sequence of SEQ ID NO:

12.

39. 39. The composition of any one of claims 24 to 38, wherein the oxidized mutant of the anti-CTLA antibody comprises oxidation at one or more of M256, M362, M401, and / or M432 of the heavy chain of the anti-CTLA antibody.

40. The composition comprises: (i) an oxidation of 35% or less, 30% or less, 25% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at M256 of the heavy chain of the anti-CTLA antibody; (ii) an oxidation of 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at M362 of the heavy chain of the anti-CTLA antibody; (iii) an oxidation of 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at M401 of the heavy chain of the anti-CTLA antibody; and / or (iv) the composition of any one of claims 24-38, comprising 35% or less, 30% or less, 25% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less oxidation at M432 of the heavy chain of the anti-CTLA antibody.

41. The composition of any one of claims 24 to 39, wherein the composition comprises oxidation at M256 of the heavy chain of the anti-CTLA antibody in an amount between the lower limit of detection of a method used to identify oxidation at M256 of the heavy chain of the anti-CTLA antibody and 35%, 30%, 25%, 20%, 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

42. The composition comprises: (i) 0.01-35%, 0.01-30%, 0.01-25%, 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%, 0.05~35%, 0.05~30%, 0.05~25%, 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%, 0.05~2%, 0.0 or selected from any one of the ranges: 5-1%, 0.1-35%, 0.1-30%, 0.1-25%, 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%, 0.5-35%, 0.5-30%, 0.5-25%, 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) the composition of any one of claims 24 to 41, comprising an oxidation at M256 of the heavy chain of the anti-CTLA antibody in an amount that is about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

43. The composition of any one of claims 24 to 42, wherein the composition comprises oxidation at M401 of the heavy chain of the anti-CTLA antibody in an amount between the lower limit of detection of a method used to identify oxidation at M401 of the heavy chain of the anti-CTLA antibody and 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

44. The composition comprises: (i) 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 %, 0.05-16%, 0.05-15%, 0.05-12.5%, 0.05-10%, 0.05-7.5%, 0.05-5%, 0.05-4%, 0.05-3%, 0.05-2%, 0.05-1% , 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%, 0.5-16%, 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) the composition of any one of claims 24 to 43, comprising an oxidation at M401 of the heavy chain of the anti-CTLA antibody in an amount of about 16%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

45. The composition of any one of claims 24 to 44, wherein the composition comprises oxidation at M362 of the heavy chain of the anti-CTLA antibody in an amount between the lower limit of detection of a method used to identify oxidation at M362 of the heavy chain of the anti-CTLA antibody and 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

46. The composition comprises: (i) 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% , 0.05-12.5%, 0.05-10%, 0.05-7.5%, 0.05-5%, 0.05-4%, 0.05-3%, 0.05-2%, 0.05-1%, 0 or selected from any one of the ranges of 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%, 0.5-12.5%, 0.5-10%, 0.5-7.5%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, and 0.5-1%; (ii) the composition of any one of claims 24 to 45, comprising an oxidation at M362 of the heavy chain of the anti-CTLA antibody in an amount of about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

47. The composition of any one of claims 24 to 46, wherein the composition comprises oxidation at M432 of the heavy chain of the anti-CTLA antibody in an amount between the lower limit of detection of a method used to identify oxidation at M432 of the heavy chain of the anti-CTLA antibody and 35%, 30%, 25%, 20%, 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

48. The composition comprises: (i) 0.01-35%, 0.01-30%, 0.01-25%, 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%, 0.05~35%, 0.05~30%, 0.05~25%, 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%, 0.05~2%, 0.0 or selected from any one of the ranges: 5-1%, 0.1-35%, 0.1-30%, 0.1-25%, 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%, 0.5-35%, 0.5-30%, 0.5-25%, 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) the composition of any one of claims 24 to 47, comprising an oxidation at M432 of the heavy chain of the anti-CTLA antibody in an amount that is about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

49. The composition of any one of claims 26 to 48, wherein the amount of oxidation at M256 of the oxidized variant of the anti-CTLA-4 antibody is between the lower limit of detection of a method used to identify oxidation at M256 of the oxidized variant of the anti-CTLA-4 antibody and 35%, 30%, 25%, 20%, 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

50. The composition comprises: (i) 0.01-35%, 0.01-30%, 0.01-25%, 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%, 0.05~35%, 0.05~30%, 0.05~25%, 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%, 0.05~2%, 0.0 or selected from any one of the ranges: 5-1%, 0.1-35%, 0.1-30%, 0.1-25%, 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%, 0.5-35%, 0.5-30%, 0.5-25%, 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) the composition of any one of claims 26 to 49, comprising oxidation at M256 of the oxidation variant of the anti-CTLA-4 antibody in an amount that is about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

51. 51. The composition of any one of claims 26 to 50, wherein the amount of oxidation at M401 of the anti-CTLA-4 antibody is between the lower limit of detection of a method used to identify oxidation at M401 of the anti-CTLA-4 antibody and 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

52. The composition comprises: (i) 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 %, 0.05-16%, 0.05-15%, 0.05-12.5%, 0.05-10%, 0.05-7.5%, 0.05-5%, 0.05-4%, 0.05-3%, 0.05-2%, 0.05-1% , 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%, 0.5-16%, 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) the composition of any one of claims 26 to 51, comprising an oxidation at M401 of the anti-CTLA-4 antibody in an amount of about 16%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

53. 53. The composition of any one of claims 26 to 52, wherein the amount of oxidation at M362 of the anti-CTLA-4 antibody is between the lower limit of detection of a method used to identify oxidation at M362 of the anti-CTLA-4 antibody and 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

54. The composition comprises: (i) 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% , 0.05-12.5%, 0.05-10%, 0.05-7.5%, 0.05-5%, 0.05-4%, 0.05-3%, 0.05-2%, 0.05-1%, 0 or selected from any one of the ranges of 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%, 0.5-12.5%, 0.5-10%, 0.5-7.5%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, and 0.5-1%; (ii) the composition of any one of claims 26 to 53, comprising an amount of about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% oxidation of the anti-CTLA-4 antibody at M362.

55. 55. The composition of any one of claims 26-54, wherein the amount of oxidation at M432 of the anti-CTLA-4 antibody is between the lower limit of detection of a method used to identify oxidation at M432 and 35%, 30%, 25%, 20%, 16%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, or 1%.

56. The composition comprises: (i) 0.01-35%, 0.01-30%, 0.01-25%, 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%, 0.05~35%, 0.05~30%, 0.05~25%, 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%, 0.05~2%, 0.0 or selected from any one of the ranges: 5-1%, 0.1-35%, 0.1-30%, 0.1-25%, 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%, 0.5-35%, 0.5-30%, 0.5-25%, 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) the composition of any one of claims 26 to 55, comprising oxidation at M432 of the anti-CTLA-4 antibody in an amount that is about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

57. 57. The composition of any one of claims 26 to 56, wherein the method used to identify the oxidation variants or oxidation in residues of the anti-CTLA-4 antibody is capillary isoelectric focusing (cIeF).

58. 1. A composition comprising an anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6; and one or more aggregation mutants of the anti-CTLA-4 antibody, wherein the composition comprises 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% of said aggregation mutants.

59. 59. The composition of claim 58, wherein the amount of aggregation variants of the anti-CTLA-4 antibody is between the lower limit of detection of the method used to identify the aggregation variants of the anti-CTLA-4 antibody and 26%, 25%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%.

60. The composition comprises: (i) 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 %, 0.05-26%, 0.05-25%, 0.05-20%, 0.05-10%, 0.05-5%, 0.05-4%, 0.05-3%, 0.05-2%, 0.05-1%, or selected from any one of the ranges 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%, 0.5-26%, 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%; (ii) the composition of any one of claims 58 and 59, comprising an aggregated variant of the anti-CTLA-4 antibody in an amount of about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

61. The composition of any one of claims 58 to 60, wherein the method used to identify the aggregation variants of the anti-CTLA-4 antibody is size exclusion chromatography.

62. 62. The composition of claim 61, wherein the size exclusion chromatography is size exclusion high performance liquid chromatography (SE-HPLC).

63. A composition comprising an anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6; and a fragmentation variant of the anti-CTLA-4 antibody, wherein the composition comprises 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% of fragmentation variants.

64. 64. The composition of claim 63, wherein the amount of fragmentation variants of the anti-CTLA-4 antibody is between the lower limit of detection of the method used to identify the aggregation variants of the anti-CTLA-4 antibody and 10%, 5%, 4%, 3%, 2%, or 1%.

65. The composition comprises: (i) selected from any one of the ranges of 0.01-10%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.05-10%, 0.05-5%, 0.05-4%, 0.05-3%, 0.05-2%, 0.05-1%, 0.1-10%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, and 0.5-1%; or (ii) the composition of any one of claims 63 and 64, comprising the fragmented variant of the anti-CTLA-4 antibody in an amount of about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%.

66. The composition of any one of claims 63 to 65, wherein the fragmentation variant comprises a heavy chain N-terminal fragment (HC1-99), a heavy chain C-terminal fragment (HC330-450), and a light chain C-terminal fragment (LC1-212 and LC1-213).

67. The composition of any one of claims 63 to 66, wherein the anti-CTLA-4 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO:

9.

68. 68. The composition of any one of claims 63 to 67, wherein the method used to identify the aggregation variants of the anti-CTLA-4 antibody is reducing or non-reducing capillary gel electrophoresis (CGE).

69. A composition comprising an anti-CTLA antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3; a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6; and up to 100% heavy chain N-terminal pyroglutamic acid mutants and / or up to 100% heavy chain C-terminal lysine truncation mutants of the CTLA-4 antibody.

70. 1. A composition comprising an anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain comprising CDRL1 having the amino acid sequence of SEQ ID NO: 1, CDRL2 having the amino acid sequence of SEQ ID NO: 2, and CDRL3 having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable domain comprising CDRH1 having the amino acid sequence of SEQ ID NO: 4, CDRH2 having the amino acid sequence of SEQ ID NO: 5, and CDRH3 having the amino acid sequence of SEQ ID NO: 6, wherein the composition further comprises 34 ng / mg or less of host cell protein (HCP).

71. 71. The composition of claim 70, further comprising 34 ng / mg or less of HCP.

72. The HCPs include elongation factor 1-alpha1, glyceraldehyde-3-phosphate dehydrogenase, RuvB-like 2 (OS = Mus musculus), peroxiredoxin 1, T-complex protein 1 subunit theta, elongation factor 1-gamma, T-complex protein 1 subunit zeta, importin subunit beta-1, 60S acidic ribosomal protein P0, 40S ribosomal protein SA, T-complex protein 1 subunit eta, RNA splicing ligase RtcB homolog, perilipin-3, mitotic checkpoint protein BUB3, methylosome protein 50, aminoacyl-tRNA synthase complex-interacting multifunctional protein 2, fatty acid synthase, hypoxia-upregulated protein 1, endoplasmic reticulum chaperone BiP, staphylococcal nuclease domain-containing protein 1, cytoplasmic dynein 1 light intermediate chain 1, and HEP AC.

72. The composition of claim 70 or 71, wherein the enzyme is selected from the group consisting of AM family member 2 and pyrroline-5-carboxylate reductase 2.

73. 73. The composition of claim 72, wherein the composition further comprises less than or equal to 13.5 ppm of endoplasmic reticulum chaperone BiP, less than or equal to 5.21 ppm of elongation factor 1-gamma, less than or equal to 3.41 ppm of T-complex protein 1 subunit eta, less than or equal to 3.24 ppm of importin subunit beta-1, less than or equal to 2.75 ppm of glyceraldehyde-3-phosphate dehydrogenase, less than or equal to 2.52 ppm of RNA splicing ligase RtcB homolog, less than or equal to 1.53 ppm of RuvB-like 2 OS=Mus musculus, and less than or equal to 1.46 ppm of T-complex protein 1 subunit zeta.

74. The composition of any one of claims 1 to 73, wherein the anti-CTLA-4 antibody comprises a light chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable domain having the amino acid sequence of SEQ ID NO:

8.

75. The composition of any one of claims 1 to 74, wherein the anti-CTLA-4 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 9 and a heavy chain having the amino acid sequence of SEQ ID NO:

10.

76. 76. The composition of any one of claims 1 to 75, wherein the acidic variant of the anti-CTLA-4 antibody and / or the oxidative variant of the anti-CTLA-4 antibody comprises a light chain having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and a heavy chain having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:

15.

77. 77. The composition of claim 76, wherein the acidic variant of the anti-CTLA-4 antibody and / or the oxidative variant of the anti-CTLA-4 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:9 and a heavy chain having the amino acid sequence of SEQ ID NO:

15.

78. The composition of claim 75, wherein the anti-CTLA-4 antibody comprises an oxidation at one or more of M256, M362, and / or M432 of the amino acid sequence of SEQ ID NO:

10.

79. The composition comprises: (i) an oxidation of 35% or less, 30% or less, 25% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at M256 of the amino acid sequence of SEQ ID NO: 10; (ii) an oxidation of 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at M362 of the amino acid sequence of SEQ ID NO: 10; (iii) oxidation of 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at M401 of the amino acid sequence of SEQ ID NO: 10; and / or (iv) the composition of claim 76 or claim 77, comprising an oxidation of 35% or less, 30% or less, 25% or less, 20% or less, 16% or less, 15% or less, 12.5% ​​or less, 10% or less, 5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.5% or less at M432 of the amino acid sequence of SEQ ID NO:

10.

80. 1. A composition comprising an anti-CTLA-4 antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 9, wherein the composition comprises: (i) no more than 35%, 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% oxidation variants of the anti-CTLA-4 antibody; (ii) 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 of the aggregation variants of the anti-CTLA-4 antibody; (iii) a deamidated variant of the anti-CTLA-4 antibody that is 45% or less, 40% or less, 35% or less, 30%, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, or 3% or less; and (iv) less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% fragmentation variants of the anti-CTLA-4 antibody.

81. 1. A composition comprising an anti-CTLA-4 antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 9, wherein the composition comprises: (i) 30% or less of the variants oxidized at Trp-52 of the heavy chain of the anti-CTLA-4 antibody; (ii) 35% or less of the mutants of the anti-CTLA-4 antibody are deamidated at heavy chain Met-256 and / or heavy chain Met-432; (iii) 34 ng / mg or less of host cell protein; (ii) 4% or less of the aggregation variants of the anti-CTLA-4 antibody; and / or (iv) The composition further comprising 10% or less fragmented variants of the anti-CTLA-4 antibody.

82. The composition of any one of claims 1 to 81, wherein the anti-CTLA-4 antibody is a full-length antibody.

83. The composition of any one of claims 1 to 82, wherein the anti-CTLA-4 antibody is human.

84. The composition of any one of claims 1 to 83, wherein the composition is formed during manufacture or storage of the antibody.

85. A pharmaceutical composition comprising the composition of any one of claims 1 to 84 and at least one pharmaceutically acceptable excipient.

86. 85. A formulation comprising the pharmaceutical composition of claim 84, the formulation comprising about 5 mg / mL to about 125 mg / mL of an anti-CTLA-4 antibody and a buffer at a pH of about 5.0 to about 6.

5.

87. 87. The formulation of claim 86, wherein the buffering agent is a histidine buffer.

88. 88. The formulation of claim 87, wherein the buffering agent is a histidine buffer at a pH of about 5.

5.

89. 89. The formulation of any one of claims 86 to 88, further comprising trehalose dihydrate.

90. 90. The formulation of any one of claims 86 to 89, further comprising polysorbate 80.

91. 91. The formulation of any one of claims 86 to 90, further comprising a chelating agent.

92. 86. A formulation comprising the pharmaceutical composition of claim 85, the formulation comprising: (a) about 20 mg / mL of an anti-CTLA-4 antibody; (b) about 20 mM histidine buffer; (c) about 222 mM trehalose dihydrate; (d) about 0.27 mM edetate disodium dihydrate; (e) about 0.02% w / v polysorbate 80 at a pH of about 5.

5.

93. An injection device comprising a composition according to any one of claims 1 to 84, a pharmaceutical composition according to claim 85, or a formulation according to any one of claims 86 to 92.

94. 85. A cell culture medium comprising the composition of any one of claims 1 to 84.

95. An eluate comprising the composition of any one of claims 1 to 84.

96. 93. A method of treating cancer, said method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1 to 84, the pharmaceutical composition of claim 85, or the formulation of any one of claims 86 to 92.

97. 97. The method of claim 96, wherein the composition is administered in a dose of 50 mg to 400 mg, for example, 75 mg or 300 mg.

98. A composition according to any one of claims 1 to 84, a pharmaceutical composition according to claim 85, or a formulation according to any one of claims 86 to 92 for use in therapy.

99. A composition according to any one of claims 1 to 84, a pharmaceutical composition according to claim 85, or a formulation according to any one of claims 86 to 92 for use in the treatment of cancer.

100. 94. Use of a composition according to any one of claims 1 to 84, a pharmaceutical composition according to claim 85, or a formulation according to any one of claims 86 to 92 in the manufacture of a medicament for use in the treatment of cancer.

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