Anti-CTLA antibody compositions and related methods

Abstract

The present disclosure provides compositions comprising an anti-CTLA-4 antibody and related methods for treating cancer and other conditions 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] Sequence Listing Statement

[0003] A sequence listing in computer-readable form has been submitted electronically with this application and is hereby incorporated by reference in its entirety. The sequence listing is contained in a file named "22-1658-US-PRO.xml" created on October 20, 2022, and is 19 kb in size. Background Art

[0004] Cancer remains a major global health burden. In the United States, it is the second most common cause of death after heart disease, accounting for 1 in 4 deaths. The 5-year survival rate for all cancers diagnosed between 1999 and 2006 was 68%, 18% higher than the 5-year survival rate reported between 1975 and 1977, which may reflect progress in the early diagnosis of certain cancers and improvements in treatment.

[0005] It is estimated that approximately 12.7 million cancer cases and 7.6 million cancer deaths occurred worldwide in 2008. These cancers are typically diagnosed at an advanced or metastatic stage and have a very short life expectancy. Despite recent progress in chemotherapy and understanding of cancer molecular biology, there has been limited progress in treatment options for advanced and metastatic diseases. The poor prognosis reflects the limited efficacy of existing treatment options and highlights the need to develop newer treatment options.

[0006] The role of the immune system, particularly T cell-mediated cytotoxicity, in tumor and infection control is well recognized. Increasing evidence indicates that T cells control tumor growth and survival in cancer patients in both the early and late stages of the disease. However, it is difficult to establish and maintain tumor-specific T cell responses in cancer patients.

[0007] CTLA4 is expressed on activated T cells and serves as a co-inhibitor to control T cell responses after CD28-mediated T cell activation. CTLA4 is thought to regulate the amplitude of early activation of naive and memory T cells after TCR engagement and is part of a central inhibitory pathway that affects anti-tumor immunity and autoimmunity. CTLA4 is expressed only on T cells, and the expression of its ligands CD80 (B7.1) and CD86 (B7.2) is mainly limited to antigen-presenting cells, T cells, and other immune-mediated cells. It has been reported that antagonistic anti-CTLA4 antibodies that block the CTLA4 signaling pathway enhance T cell activation in cancer and infectious disease conditions and disorders.

[0008] Tremelimumab is a humanized immunoglobulin (Ig) G2 monoclonal antibody against the human T cell receptor protein cytotoxic T lymphocyte-associated protein 4 (CTLA4), with potential immune checkpoint inhibitory and anti-tumor activities. SUMMARY OF THE INVENTION

[0009] The present disclosure generally relates to compositions comprising anti-CTLA antibodies and related methods for treating cancer.

[0010] In one aspect, the disclosure 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; 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 one or more deamidated variants of the anti-CTLA-4 antibody, wherein the composition comprises ≤45%, ≤40%, ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3% deamidated variants.

[0011] In another aspect, the disclosure 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; 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 one or more oxidized variants of the anti-CTLA antibody, wherein the composition comprises ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3% oxidized variants.

[0012] In another aspect, the disclosure 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; and one or more aggregation variants of the anti-CTLA-4 antibody, wherein the composition comprises ≤26%, ≤25%, ≤20%, ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of the aggregation variants.

[0013] In another aspect, the disclosure 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; and a fragmented variant of the CTLA-4 antibody, wherein the composition comprises ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of the fragmented variant.

[0014] In another aspect, the disclosure 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; and up to 100% of the heavy chain N-terminal pyroglutamate variant and / or up to 100% of the heavy chain C-terminal lysine cleavage variant of the CTLA-4 antibody.

[0015] In another aspect, the disclosure 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; and further comprising ≤34 ng / mg of host cell protein (HCP).

[0016] In another aspect, the disclosure herein provides 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) ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3% of an oxidized variant of the anti-CTLA-4 antibody; (ii) ≤26%, ≤25%, ≤20%, ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of an aggregated variant of the anti-CTLA-4 antibody; (iii) ≤45%, ≤40%, ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3% of a deamidated variant of the anti-CTLA-4 antibody; (iv) ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of a fragmented variant of the anti-CTLA-4 antibody.

[0017] In another aspect, the disclosure herein provides 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 further comprises: (i) ≤30% of a variant oxidized at Trp-52 of the heavy chain of the anti-CTLA-4 antibody; (ii) ≤35% of a variant deamidated at Met-256 and / or Met-432 of the heavy chain of the anti-CTLA-4 antibody; (iii) ≤34 ng / mg of host cell protein; (iv) ≤4% of an aggregated variant of the anti-CTLA-4 antibody; and / or (iv) ≤10% of a fragmented variant of the anti-CTLA-4 antibody.

[0018] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description in conjunction with the appended claims. It should be noted that the scope of the claims is defined by the recitations therein, rather than by the specific discussion of the features and advantages set forth in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Displays the IEC curves for fractions B, D, E.

[0020] Figure 2 Displays the cIEF analysis of the IEC fractions.

[0021] Figure 3 Displays the effect of LC Asn-30 deamidation on potency.

[0022] Figure 4 Displays the fragmentation sites of tremelimumab. DETAILED DESCRIPTION

[0023] The present disclosure relates to compositions comprising anti-CTLA antibodies and related methods for treating cancer.

[0024] 1. General Definitions

[0025] As used herein, the term "antibody" in the broadest sense refers to a molecule having immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of this type.

[0026] The terms whole antibody, full antibody, or complete antibody, which are used interchangeably herein, refer to a heterotetrameric glycoprotein having a molecular weight of approximately 150,000 daltons. A complete antibody is composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains, including two antigen-binding fragments, called "Fab" fragments and an "Fc" crystallizable fragment. The Fab fragment consists of the variable regions at the amino terminus, namely the variable heavy chain (VH) or variable light chain (VL), and the constant regions at the carboxyl terminus, namely CH1 (heavy chain) and CL (light chain). The Fc fragment consists of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc can initiate effector functions by binding to receptors on immune cells or by binding to the first component C1q of the classical complement pathway. The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by different heavy chain amino acid sequences called m, a, g, e, and d, respectively, and each heavy chain can pair with a K or L light chain. Most antibodies in serum belong to the IgG class, and there are four isotypes of human IgG, IgG1, IgG2, IgG3, and IgG4, which differ mainly in their hinge regions.

[0027] A variety of methods can be used to obtain fully human antibodies, such as using yeast-based libraries or transgenic animals (e.g., mice) capable of generating human antibody libraries. Yeast presenting human antibodies that bind to the antigen of interest on their surface can be selected using FACS (fluorescence-activated cell sorting)-based methods or by capture on beads using a labeled antigen. Transgenic animals modified to express human immunoglobulin genes can be immunized with the antigen of interest and antigen-specific human antibodies isolated using B cell sorting techniques. The desired properties of the human antibodies generated using these techniques can then be characterized, such as affinity, developability, and selectivity.

[0028] Monoclonal antibodies can be produced from eukaryotic cell clones or prokaryotic cell clones expressing the antibody. Monoclonal antibodies can also be produced from eukaryotic cell lines that can recombinantly express the heavy and light chains of the antibody by introducing nucleic acid sequences encoding the heavy and light chains of the antibody 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.

[0029] For example, the antibody can be derived from rats, mice, primates (e.g., cynomolgus monkeys, Old World monkeys, or great apes), humans, or other sources, such as nucleic acids encoding antibody molecules produced using molecular biology techniques known to those skilled in the art.

[0030] The antibody can be a fully human antibody, a humanized antibody, or a chimeric antibody. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a monoclonal antibody.

[0031] The antibody can comprise one or more modifications, including, for example, mutated constant domains such that the antibody has enhanced effector function / ADCC and / or complement activation.

[0032] The antibody can comprise two immunoglobulin (Ig) heavy chains (“HC”) and two Ig light chains (“LC”). The basic antibody structural unit can include, for example, a tetramer of subunits. Each tetramer can include two pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50 kDa - 70 kDa). The amino-terminal portion of each chain can include a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The variable region can initially be expressed in a manner linked to a cleavable signal peptide.

[0033] The variable region without the signal peptide can be referred to as the mature variable region. Thus, in one example, the light chain mature variable region can comprise the light chain variable region without the light chain signal peptide. The carboxyl-terminal portion of each chain can define a constant region. In one embodiment, the antibodies of the compositions described herein are full-length antibodies.

[0034] As used herein, the terms "VH" and "VL" refer to the variable heavy and variable light chains of an antibody, respectively.

[0035] The mature variable regions of each light / heavy chain pair can form an antibody binding site (also referred to as an antigen binding site). An "antigen binding site" refers to the site on an antibody that can specifically bind an antigen, which can be a single variable domain, or it can be the paired VH / VL domains found on a standard antibody. Thus, a full antibody can have, for example, two binding sites. Except in the case of bifunctional or bispecific antibodies, these two binding sites can be the same. Each of these chains can exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (also referred to as complementarity determining regions or "CDRs").

[0036] 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, where the heavy chain also includes 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 (FRs) connected by three hypervariable regions (also referred to as complementarity determining regions or CDRs).

[0037] The CDRs from the two chains of each pair can be aligned by the framework regions such that binding to a specific epitope is enabled. Thus, in one example, from the N-terminus to the C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0038] Acceptable heavy chain variable and light chain framework 1, framework 2, and framework 3 regions are readily recognizable to one of ordinary skill in the art. Acceptable heavy chain constant regions (including the hinge region) and light chain constant regions are also readily recognizable to one of ordinary skill in the art. Acceptable antibody isotypes are likewise readily recognizable to one of ordinary skill in the art.

[0039] "CDR" is defined as the amino acid sequence of the complementarity determining region of an antibody. These are the hypervariable regions of the 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, "CDR" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0040] Throughout the specification, the terms "CDR", "CDRL1", "CDRL2", "CDRL3",

[0041] "CDRH1", "CDRH2", and "CDRH3" follow the Kabat numbering convention. Amino acid residues in the variable region sequences and full-length antibody sequences are numbered sequentially to denote any antibody variant position or post-translational modification variant position.

[0042] The term "antigen-binding fragment" refers to a portion of a full antibody and / or to the antigen-determining variable domains of a full antibody. It is known that the antigen-binding function of an antibody can be performed by fragments of the full antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0043] The terms "variant", "antibody variant", "CDR variant", and "post-translational modification variant" refer to variant antibody sequences in which at least one amino acid sequence has been altered relative to the antibody sequence, for example, by post-translational modification, chemical change, or by sequence change 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 isomerization / iso-Asp; or aggregation), while others result in sequence changes, such as the conversion of one amino acid residue to another (e.g., Asn is converted to Asp by deamidation; or lysine is deleted). Further post-translational modification variants are described below. Variant antibody sequences containing sequence changes can be the result of designed sequence changes or post-translational modifications.

[0044] Amino acid substitutions or replacements can be conservative, semi-conservative, or non-conservative. Amino acids are roughly classified as "aromatic" or "aliphatic". Aromatic amino acids include aromatic rings (e.g., histidine, phenylalanine, tyrosine, and tryptophan). Non-aromatic amino acids are roughly classified as "aliphatic".

[0045] 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 grouped based on common side-chain characteristics, and substitutions that maintain all or substantially all of the binding affinity of the antibody within the group are considered conservative substitutions.

[0046] For example, the amino acid groups include: amino acid residues having hydrophobic side chains, such as methionine, alanine, valine, leucine, and isoleucine; amino acids having neutral hydrophilic side chains, such as cysteine, serine, and threonine; amino acids having acidic side chains, such as aspartic acid, glutamic acid; amino acids having basic side chains, such as asparagine, glutamine, histidine, lysine, and arginine; amino acids having residues that affect chain orientation, such as glycine and proline; and amino acids having aromatic side chains, such as tryptophan, tyrosine, and phenylalanine. The antibodies disclosed herein may contain such "conservative" amino acid substitutions. In an alternative embodiment, the antibody variant contains at least one substitution while retaining the norm of the antibody.

[0047] "Semi-conservative mutations" include amino acid substitutions of amino acids within a large group (i.e., aromatic or aliphatic) but not within the same side chain subgroup. For example, the substitution of aspartic acid for asparagine or asparagine for lysine involves amino acids within the same group (i.e., aliphatic) but different subgroups. "Non-conservative mutations" involve amino acid substitutions between different groups, such as lysine substituting for tryptophan, or phenylalanine substituting for serine, etc.

[0048] 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 to the antibody primary sequence (i.e., has sequence identity with the antibody primary sequence). In another embodiment, the antibody variant comprises an antibody that comprises 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.

[0049] 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 pairwise global sequence alignment using a suitable algorithm or software (such as BLASTN, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSSNeedle, T-Coffee, and DNASTAR Lasergene).

[0050] Importantly, the query sequence can be described by the nucleic acid sequences identified in one or more of the claims herein.

[0051] The "percent identity" between the query amino acid sequence and the subject amino acid sequence is the "identity" value, expressed as a percentage, calculated over the entire length of the query sequence after pairwise global sequence alignment using a suitable algorithm or software (such as BLASTP, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene), and using a suitable algorithm or software (such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign).

[0052] Importantly, the query sequence can be described by the amino acid sequences identified in one or more of the claims herein.

[0053] The query sequence can be 100% identical to the subject sequence, or, compared to the subject sequence, it can include up to a certain integer number of amino acid or nucleotide changes such that the % identity is less than 100%. For example, the query sequence is 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, and wherein the changes can occur at the amino-terminal or carboxyl-terminal positions of the query sequence or at any position between those terminal positions, singly scattered among the amino acids or nucleotides in the query sequence or scattered in one or more contiguous groups within the query sequence.

[0054] The % identity can be determined over the entire length of the query sequence including the CDRs. Alternatively, the % identity can exclude one or more CDRs or all CDRs, for example, all CDRs are 100% identical to the subject sequence, and the % identity varies in the remaining portion of the query sequence (e.g., the framework sequence) such that the CDR sequences are fixed and intact.

[0055] The amino acid sequences that can be used and are included in the compositions and related methods of the present disclosure can 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 with the amino acid sequences identified in the present disclosure (e.g., with an antibody heavy chain or an antibody light chain). In the present disclosure, the percentage identity between the amino acid sequences can include any discrete sub-ranges of the above identity percentage ranges (e.g., any range of integer values within a specific range or discrete sub-values within a specific range).

[0056] As used herein, the terms "specifically binds" or "specifically binds to" with respect to an antibody mean that the antibody binds to a target antigen and to a discrete domain or discrete amino acid sequence within the target antigen, and does not bind or binds insignificantly to other (e.g., unrelated) proteins. However, the term does not exclude the fact that the antibody may also cross-react with closely related molecules (e.g., those having a high degree of sequence identity or from another genus or species). The binding affinity of the antibodies described herein for human CTLA-4 is at least 2, 5, 10, 50, 100 or 1000 times that of their binding affinity for closely related molecules.

[0057] Affinity, also referred to as "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 for its target can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., surface plasmon resonance analysis using a BIACORE or similar instrument).

[0058] The binding affinity (KD) of the antibody - target antigen interaction can be, for example, from about 1 picomolar (pM) to about 100 micromolar (μM) (e.g., from about 1 pM to about 1 nanomolar (nM), from about 1 nM to about 1 μM, or from about 1 pM to about 100 pM). In some embodiments, the anti - CTLA - 4 antibody can bind to the CTLA - 4 protein with a KD less than or equal to 1 nanomolar (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 less than or equal to 200 pM (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).

[0059] Alternatively, the KD can be from 1 pM to 1000 pM, such as from 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). The binding affinity can be measured by BIACORE (surface plasmon resonance), for example, by capturing the test antibody on a protein A - coated sensor surface and flowing the target antigen over the surface. Alternatively, the binding affinity can be measured by FORTEBIO, for example, by capturing the test antibody receptor on a protein A - coated needle and flowing the target antigen over the surface.

[0060] K can be 1×103 M s−1 or less. Kd can be from 1×105 M s−1 to 1×103 M s−1; or from 1×104 M s−1 to 1×103 M s−1. A slow Kd can result in slow dissociation of the antibody - target antigen complex and improved neutralization of the target antigen.

[0061] As used herein, the term "specific antigen - binding activity" refers to antigen - binding activity measured by surface plasmon resonance (SPR). CTLA - 4 specific - binding activity can be determined by SPR using a BIACORE instrument, for example, in the binding mode. It is the binding activity divided by the total protein (e.g., tremelimumab) content in the sample. As used herein, the term "FcRn - binding activity" means neonatal Fc (FcRn) receptor - binding activity measured by surface plasmon resonance (SPR). FcRn binding can be determined using a BIACORE instrument. It is the binding activity to the FcRn receptor divided by the total protein concentration of the sample.

[0062] The SPR method for specific antigen binding and FcRn binding uses a reference standard of tremelimumab. The tremelimumab reference standard can be used for assays to obtain system suitability and sample comparability data to ensure that the method is properly performed. The reference standard allows for the establishment of a calibration curve and interpolation of the concentration of the sample from that curve.

[0063] Potency is defined herein as the inhibitory activity of an anti-CTLA-4 antibody or a composition as described herein to inhibit the ligand (CTLA-4) from binding to CTLA-4. This can be measured by specific binding to the antigen CTLA-4, by a potency assay (e.g., IL-2 reporter gene 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 the binding of the CTLA-4 ligand to the B7 ligands (CD80, CD86). The results can be reported as a percentage of potency relative to a reference material (e.g., a control sample).

[0064] The terms "peptide", "polypeptide", "protein", and "peptide chain" each refer to a molecule containing two or more amino acid residues. A peptide can be monomeric or polymeric.

[0065] As used herein, "about" when referring to a measurable value such as an amount, duration, etc., is intended to cover variations of ±20% or ±10% from the specified value, including ±5%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0066] 2. Anti-CTLA-4 Antibodies

[0067] In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof is tremelimumab. Tremelimumab and its antigen-binding fragments 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 its antigen-binding fragment for use in the methods, compositions, and combinations provided herein comprises a light chain variable region having the amino acid sequence of SEQ ID NO:7 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:8. In some embodiments, tremelimumab or its antigen-binding fragment for use in the methods, compositions, and combinations provided herein comprises 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 wherein the light chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1-3. One of ordinary skill in the art can readily identify Chothia-defined, Abm-defined, or other CDR definitions known to one of ordinary skill in the art. In some embodiments, tremelimumab or its antigen-binding fragment for use in the methods, compositions, and combinations provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 11.2.1 antibody, as disclosed in U.S. Patent No. 6,682,736, which is incorporated herein by reference in its entirety.

[0068] Trametinib light chain (LC) variable region :

[0069] PSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV(SEQID NO:7)

[0070] Trametinib light chain (LC) variable region (deamidated LCDR1) :

[0071] PSSLSASVGDRVTITCRASQSIDSYLDWYQQKPGKAPKLLIYAASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV(SEQID NO:13)

[0072] Trametinib heavy chain (HC) variable region :

[0073] GVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VH(SEQ ID NO:8)

[0074] Trametinib heavy chain (HC) variable region (oxidized HC-CDR-2) :

[0075] GVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIXYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VH(SEQ ID NO:14)(X is oxidized methionine)

[0076] Trametinib heavy chain CDR

[0077] HC-CDR1: GFTFSSYGMH(SEQ ID NO:4)

[0078] HC-CDR2: VIWYDGSNKYYADSV(SEQ ID NO:5)

[0079] HC-CDR3: DPRGATLYYYYYGMD V(SEQ ID NO:6)

[0080] HC-CDR2(oxidized): VIXYDGSNKYYADSV(SEQ ID NO:12)(X is oxidized tryptophan)

[0081] Trametinib light chain CDR1

[0082] LC-CDR1: RASQSINSYLD(SEQ ID NO:1)

[0083] LC-CDR2: AASSLQS(SEQ ID NO:2)

[0084] LC-CDR3: QQYYSTPFT(SEQ ID NO:3)

[0085] LC-CDR1 (deamidated): RASQSIDSYLD (SEQ ID NO:11)

[0086] Trametinib full light chain

[0087] DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:9)

[0088] Trametinib full-length heavy chain

[0089] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:10).

[0090] Trametinib full-length heavy chain

[0091] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIXiYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEX2TKNQVSLTCLVKGFYPSDIAVEWESX3GQPEX3NYKTTPPX2LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX2HEALHNHYTQKSLSLSPG K(SEQ ID NO:15) (wherein X3 is deamidated asparagine, X2 is methionine or oxidized methionine, and X1 is tryptophan or oxidized tryptophan).

[0092] The compositions of the present disclosure may comprise an anti-CTLA-4 antibody that comprises one or more of the CDRs described herein, or one or both of the heavy chain variable region or the light chain variable region described herein, or one or both of the heavy chain or the light chain described herein.

[0093] In one aspect, the composition comprises an antibody having a heavy chain sequence and a light chain sequence, the heavy chain sequence 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 light chain sequence 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.

[0094] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR2 (“CDRH2”) that comprises an amino acid sequence having one or two amino acid changes (“CDR variant”) compared to the amino acid sequence shown in SEQID NO:5.

[0095] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR2 ("CDRH2") that comprises an amino acid sequence having 5 or fewer, such as 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid change ("CDR variant") compared to the amino acid sequence set forth in SEQ ID NO:5. In another embodiment, CDRH1 comprises an amino acid sequence having one or two amino acid changes compared to the amino acid sequence set forth in SEQ ID NO:4.

[0096] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR2 ("CDRH2") that comprises the amino acid sequence set forth in SEQ ID NO:12, wherein X can be an oxidized tryptophan.

[0097] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR3 ("CDRH3") that comprises an amino acid sequence having one or two amino acid changes ("CDR variant") compared to the amino acid sequence set forth in SEQ ID NO:6.

[0098] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR1 ("CDRL1") that comprises an amino acid sequence having three or fewer, such as one or two amino acid changes ("CDR variant") compared to the amino acid sequence set forth in SEQ ID NO:1.

[0099] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR1 ("CDRL1") that comprises the amino acid sequence set forth in SEQ ID NO:11.

[0100] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR2 ("CDRL2") that comprises an amino acid sequence having one or two amino acid changes ("CDR variant") compared to the amino acid sequence set forth in SEQ ID NO:2.

[0101] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region CDR3 ("CDRL3") that comprises an amino acid sequence having three or fewer, such as one or two amino acid changes ("CDR variant") compared to the amino acid sequence set forth in SEQ ID NO:3.

[0102] In one embodiment, the anti-CTLA-4 antibody comprises: CDRH1, comprising an amino acid sequence having at most one amino acid change compared to the amino acid sequence shown in SEQ ID NO: 3; CDRH2, comprising an amino acid sequence having at most five amino acid changes compared to the amino acid sequence shown in SEQ ID NO: 4; CDRH3, comprising an amino acid sequence having at most one amino acid change compared to the amino acid sequence shown in SEQ ID NO: 5; CDRL1, comprising an amino acid sequence having at most three amino acid changes compared to the amino acid sequence shown in SEQ ID NO: 1; CDRL2, comprising an amino acid sequence having at most one amino acid change compared to the amino acid sequence shown in SEQ ID NO: 2; and / or CDRL3, comprising an amino acid sequence having at most three amino acid changes compared to the amino acid sequence shown in SEQ ID NO: 3.

[0103] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain variable region (“VH”) that comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, VH comprises an amino acid sequence having at least one amino acid change compared to the amino acid sequence shown in SEQ ID NO: 7, e.g., having 1 to 5 amino acid changes, such as 1 to 3 amino acid changes, particularly at most 2 amino acid changes, compared to the amino acid sequence shown in SEQ ID NO: 8.

[0104] In one embodiment, the anti-CTLA-4 antibody comprises a light chain variable region (“VL”) that comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 7. In one embodiment, VL comprises an amino acid sequence having at least one amino acid change compared to the amino acid sequence shown in SEQ ID NO: 8, e.g., having 1 to 5 amino acid changes, such as 1 to 3 amino acid changes, particularly at most 2 amino acid changes, compared to the amino acid sequence shown in SEQ ID NO: 7.

[0105] In one embodiment, the anti-CTLA-4 antibody comprises a VH having the amino acid sequence shown in SEQ ID NO: 8 and a VL having the amino acid sequence shown in SEQ ID NO: 7.

[0106] In one embodiment, the anti-CTLA-4 antibody comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:8; and the VL comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:7.

[0107] In one embodiment, the anti-CTLA-4 antibody comprises a heavy chain sequence ("HC") that comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:10. In one embodiment, the HC comprises an amino acid sequence having at least one amino acid change compared to the amino acid sequence shown in SEQ ID NO:10, such as 1 to 10 amino acid changes, such as 1 to 7 amino acid changes, particularly up to 6 amino acid changes, compared to the amino acid sequence shown in SEQ ID NO:10. In another embodiment, the HC comprises one, two, three, four, five, six or seven amino acid changes of the amino acid sequence shown in SEQ ID NO:10.

[0108] In one embodiment, the anti-CTLA-4 antibody comprises a light chain region ("LC") that comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:9. In one embodiment, the LC comprises an amino acid sequence having at least one amino acid change compared to the amino acid sequence shown in SEQ ID NO:9, such as 1 to 10 amino acid changes, such as 1 to 5 amino acid changes, particularly up to 3 amino acid changes, compared to the amino acid sequence shown in SEQ ID NO:9. In another embodiment, the LC comprises one, two or three amino acid changes of the amino acid sequence shown in SEQ ID NO:9.

[0109] In one embodiment, the anti-CTLA-4 antibody comprises an HC and an LC, wherein the HC comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:10; and the LC comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown 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.

[0110] 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 that comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9.

[0111] 3. Post-Translational Modification Products

[0112] Those skilled in the art will understand that post-translational modifications can occur after the antibody is produced, resulting in post-translational modification products. A "post-translational modification variant" of the antibodies described herein is an antibody composition, wherein all or a portion of the composition comprises a "post-translational modification". Post-translational modifications are chemical changes to the antibody that may be caused by the production of the antibody in a host cell, upstream and / or downstream manufacturing processes, and / or storage time and storage conditions (e.g., exposure to light, temperature, pH, effects of water, or reaction with excipients and / or the immediate container closure system). Thus, the compositions of the present disclosure can be formed by the manufacture or storage of the antibodies of the present disclosure. Exemplary post-translational modifications include antibody sequence alterations (the "antibody variants" as described above), cleavage of certain leader sequences, addition of various sugar moieties in various glycosylation patterns, including non-enzymatic glycosylation or glycation; deamidation; oxidation; disulfide scrambling and other cysteine variants, such as free sulfhydryls, meso-disulfides, thioethers, and trisulfides; isomerization; C-terminal lysine cleavage or clipping; and / or N-terminal glutamine cyclization.

[0113] In one example, the translationally modified product comprises "product-related impurities" which include chemical changes that result in a decrease in function and / or activity. In another example, the translationally modified product comprises "product-related substances" which include chemical changes that do not result in a decrease in function and / or activity. The product-related impurities of the anti-CTLA-4 antibody described herein include oxidized variants and aggregated variants. The product-related substances of the anti-CTLA-4 antibody described herein include deamidated variants, isomerized variants, C-terminal cleavage variants, and N-terminal pyroglutamate variants.

[0114] In one embodiment, the anti-CTLA-4 antibody is tremelimumab, which comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:10 and a light chain having the amino acid sequence shown in SEQ ID NO:9, including all of its functional post-translational modifications.

[0115] The percentages of variants provided herein are expressed as a percentage of the total amount of antibody in the composition (e.g., the "population" of the antibody). For example, 50% or less of the oxidized variant is in a case where the total antibody in the composition is 100%, where 50% or less is oxidized; it does not include any other non-antibody substances present in the composition that may or may not be oxidized.

[0116] Antibody variants are typically observed when analyzing the composition of an antibody 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).

[0117] Post-translational modifications can result in an increase or decrease in the net charge of the antibody and cause a decrease or increase in the pI value, resulting in acidic and basic variants (collectively referred to as "charged variants") relative to the major isotype. The "major isotype" is the population of antibodies that elutes as the main peak on chromatograms and electrophoretograms. When analyzing an antibody using an IEF-based method, acidic species are variants with a lower apparent pI and basic species are variants with a higher apparent pI. When analyzed by a chromatography-based method, acidic and basic species are defined based on their retention times relative to the main peak. Acidic species are variants that elute earlier than the main peak from CEX or later than the main peak from AEX, while 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 isotype of the antibody from acidic isotypes (acidic variants) and basic isotypes (basic variants).

[0118] Charged variants can be detected by various methods, such as ion exchange chromatography, e.g., IEF (isoelectric focusing). Capillary isoelectric focusing (cIEF) can be used to determine the percentage of charged variants. The pI of tremelimumab is measured and charge variants are separated using capillary isoelectric focusing (cIEF). This method can be used to quantify the percentage of acidic and basic species in the total peak area. The terms "species", "isotype", "form", and "peak" are used interchangeably and refer to the major isotype and charged variants (acidic variants and basic variants).

[0119] In one aspect, the composition comprises an acidic variant of an anti-CTLA-4 antibody, wherein the acidic variant comprises a heavy chain amino acid sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5, and CDRH3 of SEQ ID NO:6, and a light chain amino acid sequence having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3; wherein the composition comprises <100% of the acidic variant.

[0120] In one embodiment, the composition comprises an acidic variant of an anti-CTLA-4 antibody, 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; wherein the composition comprises <100% of the acidic variant.

[0121] In another embodiment, the composition comprises an acidic variant of an anti-CTLA-4 antibody, wherein the acidic 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 variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9; wherein the composition comprises <100% of the acidic variant. In yet another 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; wherein the composition comprises <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; wherein the composition comprises <100% of the acidic variant.

[0122] In one aspect, the composition comprises <100% of an acidic variant. In one embodiment, the composition comprises <95%, <90%, <80%, <70%, <60%, <50%, <40%, <35%, <30% or <25% of an acidic variant. Alternatively, the composition comprises 5%-100%, 5%-90%, 5%-80%, 5%-70%, 5%-60%, 5%-50%, 5%-40%, 5%-35%, 5%-30% or 5%-25% of an acidic variant. Alternatively, the composition comprises 10%-100%, 10%-97%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-35%, 10%-30% or 10%-25% of an 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 an acidic variant.

[0123] Alternatively, the composition comprises about 60%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20% or about 10% of an acidic variant.

[0124] In one aspect, the composition comprises a basic variant of an anti-CTLA-4 antibody, wherein the basic variant comprises a heavy chain amino acid sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5 and CDRH3 of SEQ ID NO:6, and a light chain amino acid sequence having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2 and CDRL3 of SEQ ID NO:3; wherein the composition comprises <100% of the basic variant.

[0125] In one 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; wherein the composition comprises <100% of the basic variant.

[0126] In one aspect, the composition comprises <100% of the basic variant. In one embodiment, the composition comprises <95%, <90%, <80%, <70%, <60%, <50%, <40%, <35%, <30% or <25% of the basic variant. Alternatively, the composition comprises 5%-100%, 5%-90%, 5%-80%, 5%-70%, 5%-60%, 5%-50%, 5%-40%, 5%-35%, 5%-30% or 5%-25% of the basic variant. Alternatively, the composition comprises 10%-100%, 10%-97%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-35%, 10%-30% or 10%-25% of the 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.

[0127] 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.

[0128] In one embodiment, the composition has at least 70% bioassay potency compared to a reference standard bioassay potency.

[0129] 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; wherein the composition comprises <100% of the basic variant. In yet another 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; wherein the composition comprises <100% of the 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; wherein the composition comprises <100% of the basic variant.

[0130] In one aspect, the composition comprises the major isotype of an anti-CTLA-4 antibody, wherein the major isotype comprises a heavy chain amino acid sequence having 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 having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3; wherein the composition comprises >1% of the major isotype.

[0131] In one embodiment, the composition comprises the major isotype of an anti-CTLA-4 antibody, wherein the major isotype 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; wherein the composition comprises >1% of the major isotype.

[0132] In another embodiment, the composition comprises the major isotype of an anti-CTLA-4 antibody, wherein the major isotype 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 variable region that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9; wherein the composition comprises >1% of the major isotype. In yet another embodiment, the composition comprises the major isotype of an anti-CTLA-4 antibody, wherein the major isotype comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9; wherein the composition comprises >1% of the major isotype.

[0133] In one embodiment, the composition comprises the major isotype of tremelimumab, wherein the major isotype comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9; wherein the composition comprises >1% of the major isotype.

[0134] In one aspect, the composition comprises >1% of the major isotype. In one embodiment, the composition comprises >2.6%, >3%, >5%, >10%, >20%, >30%, >40%, >50%, >55%, >60%, >65%, >70%, >75%, >80% or >90% of the major isotype. In one embodiment, the composition comprises 2%-90%, 2%-80%, 2%-75%, 5%-90%, 10%-90%, 20%-90%, 30%-90%, 40%-90%, 50%-90% or 60%-90% of the major isotype. In one embodiment, the composition comprises 5%-80%, 10%-80%, 20%-80%, 30%-80%, 40%-80%, 50%-80% or 60%-80% of the major isotype. Alternatively, the composition comprises approximately 80%, approximately 75%, approximately 70%, approximately 65%, approximately 60%, approximately 50% or approximately 55% of the major isotype.

[0135] The percentage of acidic variants, the percentage of basic variants, and the percentage of the major isotype can be determined using capillary isoelectric focusing (cIEF). It should be understood that these isotype / charged variant embodiments can be combined with any one or combination of the antibody variants described herein.

[0136] In one aspect, the composition comprises a charged variant of an anti-CTLA-4 antibody, the charged variant comprising a heavy chain amino acid sequence having 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 having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3; wherein the composition comprises <100% of acidic variants; and / or <100% of basic variants; and / or >1% of the major isotype.

[0137] In another aspect, the composition comprises a charged variant of an anti-CTLA-4 antibody, the charged variant comprising a heavy chain amino acid sequence having 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 having 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% of acidic variants; and / or 10%-97% of basic variants; and / or 2%-80% of the major isotype.

[0138] In another aspect, the composition comprises a charged variant of an anti-CTLA-4 antibody, the charged variant comprising a heavy chain amino acid sequence having 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 having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3; wherein the composition comprises: <35% acidic variant; and / or <35% basic variant; and / or >55% major isotype.

[0139] In another aspect, the composition comprises a charged variant of an anti-CTLA-4 antibody, the charged variant comprising a heavy chain amino acid sequence having 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 having 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 variant; and / or 10%-30% basic variant; and / or 60%-80% major isotype.

[0140] In one aspect, the composition comprises a charged variant of an anti-CTLA-4 antibody, the charged variant comprising a heavy chain amino acid sequence having VH of SEQ ID NO:8 and a light chain amino acid sequence having VL of SEQ ID NO:7; wherein the composition comprises: <100% acidic variant; and / or <100% basic variant; and / or >1% major isotype. In one embodiment, the composition comprises:

[0141] 10%-97% acidic variants; and / or 10%-97% basic variants; and / or 2%-80% of the major isotype. In an alternative embodiment, the composition comprises: 10%-30% acidic variants; and / or 10%-30% basic variants; and / or 60%-80% of the major isotype. In another embodiment, the composition comprises: <35% acidic variants; and / or <35% basic variants; and / or >55% of the major isotype. In one aspect, the composition comprises a charged variant of an anti-CTLA-4 antibody, the charged variant 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: <100% acidic variants; and / or <100% basic variants; and / or >1% of the major isotype. In one embodiment, the composition comprises: 10%-97% acidic variants; and / or 10%-97% basic variants; and / or 2%-80% of the major isotype. In an alternative embodiment, the composition comprises: 10%-30% acidic variants; and / or 10%-30% basic variants; and / or 60%-80% of the major isotype. In another embodiment, the composition comprises: <35% acidic variants; and / or <35% basic variants; and / or >55% of the major isotype.

[0142] In one embodiment, the composition has at least 70% bioassay potency compared to a reference standard bioassay potency.

[0143] Oxidation can occur during production and / or storage (i.e., in the presence of oxidative conditions) and results in covalent modification of the protein, which is induced directly by reactive oxygen species or indirectly by reaction with secondary by-products that react with oxidation compulsively. Oxidation can occur primarily on methionine residues, but can also occur on tryptophan and free cysteine residues. Oxidation can occur in the CDR, Fab (non-CDR) region or Fc region.

[0144] In one aspect, the composition comprises an antibody containing an oxidized post-translational modification (“oxidation” or “oxidized”), also referred to herein as an “oxidized variant”. The variant can contain oxidized amino acid residues in the heavy chain sequence and / or the light chain sequence, such as the CDR of the heavy chain sequence and / or the CDR of the light chain sequence. The oxidized variant can be present in one or both chains of the heavy or light chain.

[0145] In one aspect, the composition comprises an oxidized variant of an anti-CTLA-4 antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence having 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 having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3; wherein the composition comprises <40% of the oxidized variant.

[0146] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies, the anti-CTLA-4 antibody population comprising: a heavy chain amino acid sequence comprising SEQ ID NO:3 (CDRH1), SEQ ID NO:4 (CDRH2), and SEQ ID NO:5 (CDRH3) and a light chain amino acid sequence comprising SEQ ID NO:1 (CDRL1), SEQ ID NO:2 (CDRL2), and SEQ ID NO:3 (CDRL3), and oxidized variants thereof, wherein <40% of the antibody population consists of oxidized variants.

[0147] In one embodiment, the oxidized variant comprises oxidation at methionine and / or tryptophan residues in the CDRs of the heavy chain sequence and / or the CDRs of the light chain sequence. In one embodiment, the oxidized variant comprises oxidation at methionine and / or tryptophan residues in any of SEQ ID NOs: 1-6. In another embodiment, the antibody comprises oxidation at a methionine residue in the CDRs of the heavy chain sequence (e.g., CDRH1 and / or CDRH3). In another embodiment, the antibody comprises oxidation at a tryptophan residue in the CDRs of the light chain sequence (such as CDRL2). In some embodiments, the oxidized variant comprises one or a combination of oxidations at W52 of CDRH2, M256, M362, M401, and / or M432 of the full-length heavy chain.

[0148] It should be understood that reference to a position in a CDR (e.g., W52) provides a position number (consecutive numbering) relative to the entire antibody sequence. Thus, it should be understood that W52 of CDRH2 refers to the third residue of SEQ ID NO:5.

[0149] In one embodiment, the antibody comprises oxidation at methionine and / or tryptophan residues in the Fc region of the heavy chain sequence and / or the Fc region of the light chain sequence. In some embodiments, the oxidized variant comprises one or a combination of oxidations at M256, M362, M401, and / or M432 in the Fc region of the heavy chain sequence.

[0150] In one aspect, the 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 oxidation in the heavy chain sequence, e.g., oxidation at amino acid W52 in CDRH2, M256 in Fc, M362 in the Fc region, M401 in the Fc region, and / or M432 in the Fc region. In one embodiment, the 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.

[0151] 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 another 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 another embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9.

[0152] In one aspect, the composition comprises an anti-CTLA-4 antibody that has a heavy chain sequence and a light chain sequence, the heavy chain sequence 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 light chain sequence 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, wherein the composition comprises < 35% of the oxidized variant.

[0153] In one aspect, the composition comprises an anti-CTLA-4 antibody that 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, wherein the composition comprises < 35% of the oxidized variant. In another aspect, the composition comprises an anti-CTLA-4 antibody having the heavy chain variable region of SEQ ID NO:8 and / or the light chain variable region of SEQ ID NO:7, wherein the composition comprises < 35% of the oxidized variant.

[0154] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies, the anti-CTLA-4 antibody population comprising: an antibody having a heavy chain variable region shown in SEQ ID NO:8 and a light chain variable region shown in SEQ ID NO:7 and oxidized variants thereof, wherein <35% of the antibody population consists of oxidized variants.

[0155] In one aspect, the 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 sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9, wherein the composition comprises <35% oxidized variants.

[0156] In one aspect, the composition comprises an anti-CTLA-4 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 <35% oxidized variants.

[0157] 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; wherein the composition comprises <45% oxidized variants.

[0158] 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; wherein the composition comprises an amount of oxidized variants in the range of 0.1% to 35%.

[0159] 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 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 <35% oxidized variants.

[0160] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies, the anti-CTLA-4 antibody population comprising: an antibody having a heavy chain amino acid sequence shown in SEQ ID NO:10 and a light chain amino acid sequence shown in SEQ ID NO:9 and oxidized variants thereof, wherein <35% of the antibody population consists of oxidized variants.

[0161] 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 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 oxidized variant of the antibody of (a) and / or (b), wherein the oxidized variant is selected from <30% oxidation at W52 of the heavy chain,

[0162] <35% oxidation at M256 of the heavy chain, <12.5% oxidation at M362 of the heavy chain, <16% oxidation at M401 of the heavy chain, and / or <35% oxidation at M432 of the heavy chain, any one or combination thereof.

[0163] In one aspect, the composition comprises <35% oxidized variant. In one embodiment, the composition comprises <35%, <30%, <20%, <15%, <10%, <5%, <4% or <3% oxidized variant. 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% oxidized variant.

[0164] Alternatively, the composition comprises 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% oxidized variant. 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% oxidized variant. Alternatively, the composition comprises 1%-35%, 1%-30%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-4%, 1%-3%, 2%-4% or 2%-3% oxidized variant. Alternatively, the composition comprises about 10%, about 5%, about 4%, about 3%, about 2% or about 1% oxidized variant. It should be understood that these oxidized variant embodiments can be combined with any of the antibody variants described herein.

[0165] In one embodiment, the composition comprises one or a combination of the following: <35% oxidation at M256 of the heavy chain, <12.5% oxidation at M362 of the heavy chain, <16% oxidation at M401 of the heavy chain, <35% oxidation at M432 of the heavy chain, and / or <30% oxidation at W52 of CDRH2.

[0166] 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, the oxidized variant being selected from any one or combination of <25% oxidation at W52 of the heavy chain, <30% oxidation at M256 of the heavy chain, <10% oxidation at M362 of the heavy chain, <12% oxidation at M401 of the heavy chain, and / or <30% oxidation at M432 of the heavy chain).

[0167] In one embodiment, the composition comprises <25% oxidation at W52 of the heavy chain sequence. In one embodiment, the composition comprises <25%, <20%, <15%, <10%, <7.5%,

[0168] <5%, <4%, <3%, <2% or <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%-25%, 0.01%-20%, 0.01%-15%, 0.01%-10%, 0.01%-7.5%, 0.01%-5%, 0.01%-4%, 0.01%-3%, 0.01%-2% or 0.01%-1% oxidation at W52 of the heavy chain sequence. In one embodiment, the composition comprises 0.05%-25%, 0.05%-20%, 0.05%-15%, 0.05%-10%, 0.05%-7.5%, 0.05%-5%, 0.05%-4%, 0.05%-3%, 0.05%-2% or 0.05%-1% oxidation 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 shown by the data provided herein, heat forced degradation that produces up to 4.5% oxidation at W52 gives 98% potency.

[0169] In one aspect, 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 <25% oxidation at W52 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; wherein the composition comprises <25% oxidation at W52 of the heavy chain sequence.

[0170] In one embodiment, the composition comprises <35% oxidation at M256 of the heavy chain sequence. In one embodiment, the composition comprises <35%, <30%, <25%, <20%, <16%,

[0171] Oxidation at M256 of the heavy chain sequence of < 15%, < 12.5%, < 10%, < 7.5%, < 5%, < 4%, < 3%, < 2% or < 1%. Alternatively, the composition comprises 0%-35%, 0%-30%, 0%-25%, 0%-20%, 0%-16%, 0%-15%, 0%-12.5%, 0%-10%, 0%-7.5%, 0%-5%, 0%-4%, 0%-3%, 0%-2% or 0%-1% oxidation at M256 of the heavy chain sequence. In one embodiment, the composition comprises 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% or 0.01%-1% oxidation at M256 of the heavy chain sequence. Alternatively, the composition comprises 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 comprises 0.1% or more and 35% or less oxidation at M256 of the heavy chain sequence. Alternatively, the composition comprises approximately 30%, approximately 25%, approximately 20%, 10%, approximately 5%, approximately 4%, approximately 3%, approximately 2% or approximately 1% oxidation at M256 of the heavy chain sequence. As shown by the data provided herein, H2O2 forced degradation that produces up to 68.5% oxidation at M34 gives a potency of 83 in the bioassay. Extrapolating from the H2O2 forced degradation data of oxidation at HC Met256, up to 13.4% oxidation can produce at least 88% potency. In one aspect, 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 < 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; wherein the composition comprises < 13.4% oxidation at M256 of the heavy chain sequence.

[0172] In one embodiment, the composition comprises <16% oxidation at M401 of the heavy chain sequence. In one embodiment, the composition comprises <16%, <15%, <10%, <5%, <2% or <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 shown by the data provided herein, H2O2 forced degradation that produces up to 55.2% oxidation at M401 gives 83% potency in the bioassay.

[0173] In one aspect, 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 <16% oxidation at M401 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; wherein the composition comprises <16% oxidation at M401 of the heavy chain sequence.

[0174] In one embodiment, the composition comprises < 12.5% oxidation at M362 of the heavy chain sequence. In one embodiment, the composition comprises < 12.5%, < 10%, < 5%, < 4% or < 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 shown by the data provided herein, H2O2 forced degradation that produces up to 26.5% oxidation at M362 gives 83% potency in the bioassay. Thus, it is expected that the oxidation at M362 can be higher than 26.5% without any effect on the relative potency.

[0175] In one embodiment, the composition comprises <35% oxidation at M432 of the heavy chain sequence. In one embodiment, the composition comprises <35%, <30%, <20%, <15%, <10%, <5%, <2% or <1% oxidation at M432 of the heavy chain sequence. 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%, 0.01%-3%, 0.01%-2% or 0.01%-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 shown by the data provided herein, H2O2 forced degradation that produces up to 42.1% oxidation at M432 gives 83% potency in the bioassay (i.e., within-assay variability in the fully functional state), and photolysis forced degradation that produces up to 20.7% oxidation at M432 gives 88% potency in the bioassay. Thus, oxidation at M432 is expected to be higher than 42.1% with no effect on relative potency.

[0176] In one embodiment, the composition comprises an oxidized variant of tremelimumab, wherein the oxidized variant comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9; wherein the composition comprises <35% oxidation at M256 and / or M362 and / or M401 and / or M432 of the heavy chain sequence.

[0177] In one aspect, the composition comprises an anti-CTLA-4 antibody that comprises a heavy chain amino acid sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5, and CDRH3 of SEQ ID NO:6, and a light chain amino acid sequence having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3; wherein the composition comprises: <100% acidic variant; and / or <100% basic variant; and / or >1% major isotype; and / or <35% oxidized variant.

[0178] In another aspect, the composition comprises an anti-CTLA-4 antibody, the anti-CTLA-4 antibody comprising a heavy chain amino acid sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5 and CDRH3 of SEQ ID NO:6, and a light chain amino acid sequence having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2 and CDRL3 of SEQ ID NO:3; wherein the composition comprises: 5%-45% acidic variants; and / or 5%-97% basic variants; and / or 2%-80% major isotype; and / or <35% oxidized variants.

[0179] In one example, tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) can be used to determine oxidation.

[0180] 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". The aggregated antibody can comprise a dimer or higher-order structure formed from antibody monomers and their subunits. Thus, the high molecular weight (HMW) species can consist of dimeric antibodies and monomers with additional subunits (such as monomers with two light chain subunits, or LC-LC dimers non-covalently bound to the monomer). Aggregation variants can be, for example, covalent or non-covalent, reducible or non-reducible, and visible or subvisible aggregates of the antibodies disclosed herein. Aggregation variants can be characterized based on their size and distinguished from the antibodies. For example, size exclusion chromatography (SEC) can be used to detect the size distribution of the antibody composition. In one aspect, the composition comprises an anti-CTLA-4 antibody having a heavy chain sequence and a light chain sequence, the heavy chain sequence 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, the light chain sequence 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, wherein the composition comprises <26% aggregation variants. It should be understood that these aggregation variant embodiments can be combined with any of the antibody variants described herein.

[0181] In one aspect, the composition comprises an aggregated variant of an anti-CTLA-4 antibody, wherein the aggregated variant comprises a heavy chain sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5, and CDRH3 of SEQ ID NO:6, and a light chain sequence having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, and CDRL3 of SEQ ID NO:3; wherein the composition comprises <26% of the aggregated variant.

[0182] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies, the anti-CTLA-4 antibody population comprising: antibodies having a heavy chain amino acid sequence comprising SEQ ID NO:4 (CDRH1), SEQ ID NO:5 (CDRH2), and SEQ ID NO:6 (CDRH3) and a light chain amino acid sequence comprising SEQ ID NO:1 (CDRL1), SEQ ID NO:2 (CDRL2), and SEQ ID NO:3 (CDRL3), and aggregated variants thereof, wherein <26% of the antibody population consists of aggregated variants.

[0183] 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 another 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 another embodiment, the antibody comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9.

[0184] In one aspect, the composition comprises an anti-CTLA-4 antibody comprising 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, wherein the composition comprises <26% of the aggregated variant. In another aspect, the composition comprises an anti-CTLA-4 antibody having the heavy chain variable region of SEQ ID NO:8 and / or the light chain variable region of SEQ ID NO:7, wherein the composition comprises <26% of the aggregated variant.

[0185] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies, the anti-CTLA-4 antibody population comprising: antibodies having the heavy chain variable region shown in SEQ ID NO:8 and the light chain variable region shown in SEQ ID NO:7 and aggregated variants thereof, wherein <26% of the antibody population consists of aggregated variants.

[0186] In one aspect, the composition comprises an anti-CTLA-4 antibody, the 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 sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 9, wherein the composition comprises < 26% aggregated variants.

[0187] In one aspect, the composition comprises an antibody having the heavy chain sequence of SEQ ID NO: 10 and the 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 < 26% aggregated variants.

[0188] In one aspect, the composition comprises an anti-CTLA-4 antibody having the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9, wherein the composition comprises < 26% aggregated variants. In one embodiment, the composition comprises an aggregated variant of tremelimumab, wherein the aggregated variant comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9; wherein the composition comprises < 26% aggregated variants. In one embodiment, the composition comprises an aggregated variant of tremelimumab, wherein the aggregated variant comprises the heavy chain sequence of SEQ ID NO: 10 and the light chain sequence of SEQ ID NO: 9; wherein the composition comprises aggregated variants in the range of 0.01% to 26% aggregated variants.

[0189] In one aspect, the composition comprises a population of anti-CTLA-4 antibodies, the population of anti-CTLA-4 antibodies comprising: an antibody having the heavy chain amino acid sequence shown in SEQ ID NO: 10 and the light chain amino acid sequence shown in SEQ ID NO: 9 and its aggregated variants, wherein < 26% of the antibody population consists of aggregated variants.

[0190] The antibody composition may comprise <26% of aggregated variants, such as <26%, <25%, <20%, <10%, <5%, <4%, <3%, <2% or <1% of aggregated variants. In another embodiment, the composition may comprise 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% of aggregated variants. Alternatively, the composition comprises greater than 1% and less than 26% of aggregated variants. Alternatively, the composition may comprise about 10%, about 5%, about 4%, about 3%, about 2% or about 1% of aggregated variants. As shown by the data provided herein, the combined acid treatment and heat forced degradation of compositions containing up to 25.9% of aggregated antibodies gave 91% potency (i.e., within-assay variability in the fully functional state) in the bioassay.

[0191] In one aspect, the composition comprises an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5 and CDRH3 of SEQ ID NO:6, and a light chain amino acid sequence having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2 and CDRL3 of SEQ ID NO:3; wherein the composition comprises: <100% of acidic variants; and / or <100% of basic variants; and / or >1% of the major isotype; and / or <35% of oxidized variants; and / or <26% of aggregated variants.

[0192] In another aspect, the composition comprises an anti-CTLA-4 antibody comprising a heavy chain amino acid sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5 and CDRH3 of SEQ ID NO:6, and a light chain amino acid sequence having CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2 and CDRL3 of SEQ ID NO:3; wherein the composition comprises: 5%-97% of acidic variants; and / or 5%-97% of basic variants; and / or 2%-80% of the major isotype; and / or <35% of oxidized variants; and / or <26% of aggregated variants.

[0193] Fragmented variants (“fragment variants”) are variants that comprise a portion of a full-length antibody. For example, such fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and immunoglobulin single variable domains. An antibody composition can comprise <10% fragmented antibodies, such as <5%, <4.6%, <4.5%, <4.4%, <4.3%, <4.2%, <4.1%, <4%, <3.5%, <3%, <2.5%, <2%, <1.5%, <1%, <0.5%, or <0.05% fragmented antibodies. In another embodiment, the composition can comprise 0.01%-10%, 0.01%-5%, 0.01%-4.6%, 0.01%-4.5%, 0.01%-4%, 0.01%-3.5%, 0.01%-3%, 0.01%-2.5%, 0.01%-2%, 0.01%-1.5%, 0.01%-1%, 0.01%-0.5%, 0.01%-0.1%, or 0.01%-0.05% fragmented antibodies. In another embodiment, the composition can 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% fragmented antibodies. Alternatively, the composition can comprise about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% fragmented antibodies. It should be understood that these fragmented variant embodiments can be combined with any of the antibody variants described herein.

[0194] Deamidation can be an enzymatic or chemical reaction, which can occur, for example, during production and / or storage. Deamidation can occur by a simple chemical reaction of intramolecular cyclization, where the amide nitrogen of the next amino acid in the chain nucleophilically attacks the amide (N+1 attacks N); a succinimide intermediate is formed. Deamidation can predominantly convert asparagine (N) to isoaspartic acid (isoaspartate) and aspartic acid (aspartate) (D) in a ratio of about 3:1. Thus, this deamidation reaction may be related to the isomerization of aspartate (D) to isoaspartate. Both deamidation of asparagine and isomerization of aspartate can involve the intermediate succinimide. To a much lesser extent, glutamine residues can be deamidated in a similar manner. Deamidation can occur in the CDR, Fab (non-CDR regions), or Fc regions.

[0195] Isomerization is the conversion of aspartate (D) to isoaspartate, which involves the intermediate succinimide (succinimide-aspartic acid residue).

[0196] In one aspect, the composition comprises an antibody containing a deamidated post-translational modification (“deamidation” or “deamidated”), also referred to herein as a “deamidated variant”.

[0197] 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 another 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 deamidated variants can be present in one or both of the heavy and light chains. It should be understood that these deamidated variant embodiments can be combined with any of the antibody variants described herein. In some embodiments, the deamidated variant comprises one or a combination of deamidations at N30 of CDRL1 and / or N388 and / or N393 of the Fc region of the heavy chain sequence.

[0198] In one embodiment, the deamidated variant comprises a deamidated residue selected from: an aspartic acid residue, a succinimide-aspartic acid residue, or an isoaspartic acid residue.

[0199] In one aspect, the composition comprises an antibody that comprises a sequence that is at least about 90% identical to the light chain amino acid sequence of SEQ ID NO:9, and optionally comprises a sequence that is at least about 90% identical to the heavy chain sequence of SEQ ID NO:10, and comprises deamidation in the light chain sequence, such as deamidation at amino acid residues N30 and / or N388 and / or N393 of 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.

[0200] Deamidation can result in a sequence change where 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 with N30D).

[0201] The composition can comprise up to 45% deamidated variant. In one aspect, the composition comprises an antibody having the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9, wherein the composition comprises up to 45% deamidated variant.

[0202] 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 at 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 at N30. As the data provided herein show, processed samples that produce up to 34.8% deamidation at N30 give 76% potency and thus deamidation at N30 above the 34.8% level will have a significant impact on relative potency (below 76%).

[0203] In one embodiment, the composition comprises 0%-45%, 0%-40%, 0%-30%, 0%-20% or 0%-10% deamidation at N388. Alternatively, the composition comprises 0.1%-45%, 0.1%-40%, 0.1%-30%, 0.1%-20% or 0.1%-10% deamidation at N388.

[0204] 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 the data provided herein show, the base-treated samples that produce up to 28.2% deamidation at N388 have no effect on potency in the bioassay (i.e., within-assay variability in the fully functional state), and thus deamidation at N388 is expected to be above the reported level of 28.2% without any effect on relative potency or FcRn binding.

[0205] In one embodiment, the composition comprises 0%-45%, 0%-40%, 0%-30%, 0%-20% or 0%-10% deamidation at N393. Alternatively, the composition comprises 0.1%-45%, 0.1%-40%, 0.1%-30%, 0.1%-20% or 0.1%-10% deamidation at N393.

[0206] Alternatively, the composition comprises 1%-45%, 1%-40%, 1%-30%, 1%-20% or 1%-10% deamidation at N393. Alternatively, the composition comprises 0.5% or more, 1% or more, or 2% or more deamidation at N393. As shown by the data provided herein, the base-treated samples that produced up to 28.2% deamidation at N393 had no effect on potency in the bioassay (i.e., within-assay variability in the fully functional state), and thus deamidation at N393 is expected to be higher than the reported level of 28.2% without any effect on relative potency or FcRn binding.

[0207] In one aspect, the composition comprises an antibody having the heavy chain sequence of SEQ ID NO:9 and / or the 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.

[0208] In one example, Lys-C and / or tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) can be used to determine deamidation.

[0209] The antibody composition can comprise (i) an antibody (as described herein, 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); and (ii) antibody variants, including one or more or a combination of the following: amino acid sequence variants (e.g., deamidated or C-terminal lysine cleavage variants), oxidation variants, aggregation variants, and / or fragmentation variants.

[0210] Thus, in one aspect, there is provided a composition comprising an antibody having the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:9, wherein the composition comprises: (i) <35% oxidation variants; (ii) <26% aggregation variants.

[0211] In one aspect, the composition comprises an antibody that comprises a heavy chain sequence having one or a combination of sequences selected from SEQ ID NO:10 and / or SEQ ID NO:12 and the light chain sequence of SEQ ID NO:9, wherein the composition comprises <35% oxidation variants.

[0212] In one aspect, the composition comprises an antibody that comprises 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 <26% aggregated variants.

[0213] 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% potency compared to a reference standard having 100% potency. In one aspect, the composition comprises a variant of an anti-CTLA-4 antibody, wherein the variant comprises a heavy chain amino acid sequence having CDRH1 of SEQ ID NO:4, CDRH2 of SEQ ID NO:5 and CDRH3 of SEQ ID NO:6, 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 has at least 60% potency of a composition comprising a heavy chain sequence of SEQ ID NO:10 and a light chain sequence of SEQ ID NO:9, 10%-97% acidic variants, 10%-97% basic variants, 2%-80% major isotype, 30% or less HC W52 oxidized variants, 35% or less HCM256 oxidized variants, 12.5% or less HC M362 oxidized variants, 16% or less HC M401 oxidized variants, 35% or less HC M432 oxidized variants, 26% or less aggregated variants, 45% or less LC N30 deamidated variants, 45% or less HC N388 deamidated variants, 45% or less HC N393 deamidated variants and / or 10% or less fragmented variants. Potency can be determined using the bioassays described herein.

[0214] Glycation is a post-translational modification that involves a non-enzymatic chemical reaction between a reducing sugar (such as glucose) and a free amino group in a protein and is typically observed at the ε-amino of lysine side chains or at the N-terminus of the protein. Glycation can occur during production and / or storage in the presence of reducing sugars.

[0215] Disulfide scrambling can occur during production and / or storage conditions. In some cases, disulfide bonds may break or form incorrectly, generating unpaired cysteine residues (-SH). These free (unpaired) thiol (-SH) groups can promote shuffling.

[0216] During production or storage, the formation of thioethers and the racemization of disulfide bonds can occur under basic conditions by β-elimination of the disulfide bond back to cysteine residues via dehydroalanine and persulfide intermediates. Subsequent crosslinking of dehydroalanine and cysteine can lead to the formation of thioether bonds, or free cysteine residues can reform disulfide bonds with a mixture of D-cysteine and L-cysteine.

[0217] Trisulfides can be generated by the insertion of a sulfur atom into a disulfide bond (Cys-SS-S-Cys) and can form due to the presence of hydrogen sulfide in the production cell culture.

[0218] The N-terminal glutamine (Q, Gln) and glutamate (glutamic acid) (E, Glu) in the heavy and / or light chains can cyclize to form pyroglutamate (pGlu). pGlu formation can occur in the production bioreactor, but it can also form non-enzymatically, for example, depending on the pH and temperature of the processing and storage conditions. Cyclization of the N-terminal Q or E is typically observed in native human antibodies.

[0219] C-terminal lysine cleavage (also known as C-terminal lysine cutting) is an enzymatic reaction catalyzed by carboxypeptidase and is typically observed in recombinant and native human antibodies. Variants of this process include the removal of lysine from one or both heavy chains due to cellular enzymes from the recombinant host cell. Administration to a human subject / patient may result in the removal of any residual C-terminal lysine.

[0220] The binding of the neonatal Fc receptor (FcRn) to anti-CTLA-4 antibodies can be measured using surface plasmon resonance (SPR).

[0221] Antibody compositions comprising the above antibodies and antibody variants retain specific antigen binding and / or FcRn binding and / or potency. For example, antibody compositions comprising the above antibodies and antibody variants and post-translational modification variants have >0.70 CTLA-4 specific antigen binding; and / or >70% FcRn binding and / or >70% potency. Thus, these levels (%) of variants can be tolerated in the antibody composition without significantly affecting function (i.e., not resulting in a decrease in activity). In one embodiment, "decreased function" or "decreased activity" refers to a decrease in binding to CTLA-4 or binding to FcRn or potency in percentage form compared to a reference standard and is significant in terms of assay variability. For example, decreased function or activity or potency can be described as a decrease of >5%, >10%, >15%, >20%, >25%, >30%, >35%, >40%, >45% or >50%.

[0222] The composition may comprise a mixture of antibody variants and post - translational modification variants. For example, the antibody composition may comprise two or more of acidic variants, basic variants, oxidized variants, deamidated variants, aggregated variants, and fragmented variants.

[0223] In one aspect, 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 any one or combination of the following: (i) up to 100% acidic variants, (ii) up to 100% basic variants, (iii) < 35% oxidation at W52 of the heavy chain, (iv) < 35% oxidation at M256 of the heavy chain, (v) < 12.5% oxidation at M362 of the heavy chain, (vi) < 16% oxidation at M401, (vii) < 35% oxidation at M432, and / or (viii) < 26% aggregated variants.

[0224] In one aspect, 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 any one or combination of the following: (i) up to 100% acidic variants, (ii) up to 100% basic variants, (iii) < 35% oxidation at W52 of the heavy chain, (iv) < 35% oxidation at M256 of the heavy chain, (v) < 12.5% oxidation at M362 of the heavy chain, (vi) < 16% oxidation at M401 of the heavy chain, (vii) < 35% oxidation at M432 of the heavy chain, (viii) < 26% aggregated variants, (ix) < 45% deamidation at N30 of the light chain, (x) < 45% deamidation at N388 of the heavy chain; (xi) < 45% deamidation at N393 of the heavy chain, and / or (xii) < 10% fragmented variants.

[0225] The present disclosure includes antibodies that may have been subjected to or have undergone one or more of the post - translational modifications described herein. Exemplary compositions may comprise a mixture or blend of antibodies: 1) with and without the post - translational modification(s) described herein (one or more, or two or more). Thus, the composition may comprise a population of antibodies with post - translational modifications and a population of antibodies without post - translational modifications.

[0226] The composition may have been subjected to or have undergone one or more post - translational modifications. The modification may occur in the CDR, variable framework region, or constant region. The modification may result in a change in the molecular charge.

[0227] In one embodiment, the post-translational modifications described herein do not result in a significant alteration of antigen binding affinity, biological activity, pharmacokinetics (PK), aggregation, immunogenicity, and / or binding to Fc receptors, unless designated and described as product-related impurities.

[0228] 4. Constituent Impurities

[0229] Constituent impurities may include host cell proteins (HCPs). HCPs are process-related impurities produced by the host organism. HCP impurities may be reported as ng HCP / mg drug (ppm).

[0230] In one embodiment, a composition comprising an anti-CTLA antibody is provided, 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 ≤13.54 ppm of the endoplasmic reticulum chaperone BiP.

[0231] In another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 of elongation factor 1-γ.

[0232] In another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 of T-complex protein 1 subunit eta.

[0233] In another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 ≤ 3.24 ppm of importin subunit beta-1.

[0234] In another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 ≤ 2.75 ppm of glyceraldehyde-3-phosphate dehydrogenase.

[0235] In another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 ≤2.52 ppm of an RNA splicing ligase RtcB homolog.

[0236] In another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 ≤1.53 ppm of RuvB-like protein 2 of Mus musculus (RuvB-like 2 OS = Mus musculus).

[0237] In another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 of T-complex protein 1 subunit ζ.

[0238] In yet another embodiment, a composition comprising an anti-CTLA antibody is provided, 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 ≤13.5 ppm of endoplasmic reticulum chaperone BiP, ≤5.21 ppm of elongation factor 1-γ, ≤3.41 ppm of TCP-1 subunit eta, ≤3.24 ppm of importin subunit beta-1, ≤2.75 ppm of glyceraldehyde-3-phosphate dehydrogenase, ≤2.52 ppm of RNA splicing ligase RtcB homolog, ≤1.53 ppm of Mus musculus RuvB-like protein 2, and ≤1.46 ppm of TCP-1 subunit zeta.

[0239] 5. Preparation Methods

[0240] The antibodies and compositions described herein can be obtained by any means, including from in vitro sources (e.g., hybridomas or cell lines that recombinantly produce 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.

[0241] For example, the composition can be expressed in a recombinant expression system and purified from the recombinant expression system. In one embodiment, the composition is produced by a method of culturing a host cell under conditions suitable for expressing an antibody comprising SEQ ID NO:9 and SEQ ID NO:10, wherein the composition is expressed and optionally purified, and optionally formulated within a pharmaceutical composition.

[0242] Many different expression systems and purification protocols can be used to produce the composition. Generally, a host cell is transformed with a recombinant expression vector encoding the antibody. A variety of host cells can be used, including eukaryotic cell lines of mammalian origin (e.g., CHO, Perc6, HEK293, HeLa, NS0).

[0243] The host cell can be an isolated host cell. The host cell is generally not part of a multicellular organism (e.g., a plant or an animal). The host cell can be a non-human host cell.

[0244] Suitable cloning and expression vectors and cloning methods for eukaryotic or mammalian cell hosts are known in the art.

[0245] Culture host cells to express a recombinant expression vector encoding an antibody.

[0246] The composition can be recovered and purified by conventional protein purification procedures. For example, the composition can be directly harvested from the culture medium. The cell culture medium can be harvested via clarification, for example by centrifugation and / or depth filtration. After recovering the composition, purification is carried out to ensure sufficient purity. Thus, in one aspect, provided is a cell culture medium comprising the composition described herein. In one embodiment, the cell culture medium comprises CHO cells.

[0247] Subsequently, the composition can 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., protein A resin or protein G resin that binds antibody molecules), and eluting the antibody molecules from the purification medium to produce an eluate. Thus, in one aspect, provided is an eluate comprising the composition described herein.

[0248] One or more chromatography steps can be used in the purification, such as one or more chromatography resins; and / or one or more filtration steps. For example, affinity chromatography using resins such as protein A, G, or L can be used to purify the composition. Alternatively or in addition, ion exchange resins (such as cation exchange resins) can be used to purify the composition.

[0249] Alternatively, the purification step comprises: an affinity chromatography resin step followed by a cation exchange resin step.

[0250] 6. Pharmaceutical Compositions and Formulations

[0251] The composition described herein can be in the form of a pharmaceutical composition.

[0252] In one aspect, provided is a pharmaceutical composition comprising the composition and at least one pharmaceutically acceptable excipient.

[0253] A “pharmaceutical composition” can comprise the composition described herein (i.e., the active ingredient) and one or more pharmaceutically acceptable excipients. The excipients must be acceptable, i.e., compatible with the other ingredients in the formulation, capable of being used in pharmaceutical formulation, harmless to its recipient, and / or not interfering with the efficacy of the active ingredient. Thus, the pharmaceutical compositions of the present disclosure are suitable for administration to a patient.

[0254] As used herein, "pharmaceutically acceptable excipients" can include one or more of buffers, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations thereof. In many cases, it is preferred to include isotonic agents such as polyols, sugars, polyalcohols (such as mannitol, sorbitol) or sodium chloride, preservatives; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn2+-protein complexes); biodegradable polymers; and / or salt-forming counterions such as sodium or potassium.

[0255] The exact nature of the excipient or other materials can depend on the route of administration, which can be, for example, oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and intratumoral. It should be understood that the preferred excipients can vary, for example, with the condition of the recipient and the disease to be treated.

[0256] A mixture of excipients and the concentration of each excipient together form a "pharmaceutical formulation" (or "formulation"). Such compositions are suitably free of visible particulate matter. The formulation can be in liquid form or lyophilized form. A composition in liquid formulation can be filled into a container and frozen. In certain embodiments, aliquots of the frozen formulation containing the composition can be lyophilized. The lyophilizate can be reconstituted by adding water or other aqueous solutions to produce a reconstituted formulation containing the composition.

[0257] In one embodiment, the composition is a liquid formulation. In some embodiments, the formulation contains from about 10 mg / mL to about 125 mg / mL of an antibody. In another embodiment, the formulation contains from about 15 mg / mL to about 125 mg / mL, such as from about 15 mg / mL to about 100 mg / mL, particularly from about 15 mg / mL to about 50 mg / mL of an antibody. In yet another embodiment, the formulation contains about 20 mg / mL of an antibody. In an alternative embodiment, the formulation contains about 50 mg / mL of an antibody.

[0258] In one aspect, there is provided a formulation comprising the pharmaceutical composition described herein, the pharmaceutical composition containing from about 10 mg / mL to about 125 mg / mL (such as from about 20 mg / mL to about 50 mg / mL, for example, 20 mg / mL) of an antibody and a buffer having a pH of from about 5.0 to about 6.5 (for example, pH 5.5). In one embodiment, the composition is a liquid formulation.

[0259] 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 the following: a citrate buffer, a histidine buffer, arginine, trehalose, sodium chloride, and polysorbate 80. In certain embodiments, the buffer is a citrate buffer. The citrate buffer can be obtained, for example, by using a conjugate acid / conjugate base system (sodium citrate / citric acid) or by HCl titration of a sodium citrate solution. In one embodiment, the pH of the citrate buffer is from about 5.0 to about 6.5, such as from about 5.5 to about 6.0, particularly about 5.5 or about 6.0.

[0260] In alternative embodiments, the buffer is a histidine buffer. In one embodiment, the pH of the histidine buffer is from about 5.5 to about 7.0, from about 5.5 to about 6.5, such as from about 6.0 to about 6.5, particularly about 6.0 or about 6.5.

[0261] In some embodiments, the formulation comprises a surfactant. A "surfactant" is a surfactant that can act on the surface of solid-solid, solid-liquid, liquid-liquid, and liquid-air interfaces due to the simultaneous presence of a hydrophilic group and a hydrophobic group in its chemical composition. The surfactant can reduce the concentration of the protein in the dilute solution at the air-water and / or water-solid interfaces, where the protein can be adsorbed and potentially aggregated. The surfactant can bind to the hydrophobic interfaces in the protein formulation. Some parenterally acceptable nonionic surfactants contain a polysorbate group or a polyether group. Polysorbate 20 and 80, particularly polysorbate 80 (PS80), are suitable surfactant stabilizers in the formulations of the present disclosure. In one embodiment, the formulation further comprises PS80. In some embodiments, the formulation comprises from about 0.01% to about 0.1%, such as from about 0.01% to about 0.05% or from about 0.01% to about 0.03% w / v of PS80 or PS20. In some embodiments, the formulation comprises about 0.02% w / v of PS80 or PS20. In a preferred embodiment, the formulation comprises about 0.02% w / v of PS80.

[0262] The formulation can comprise a chelating agent. As used herein, the term "chelating agent" generally refers to an excipient that can form at least one bond (e.g., a covalent bond, an ionic bond, or other bond) with a metal ion. Chelating agents are generally polydentate ligands that can be used as stabilizers in a selected liquid composition to complex substances that may promote instability.

[0263] Chelating agents suitable for 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, nicotinamide, and deoxycholates. Examples of suitable aminopolycarboxylic acids include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-imino diacetic acid (ADA), bis(aminoethyl)ethylene glycol ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid, and aspartic acid. Examples of suitable hydroxyaminocarboxylic acids include N-hydroxy-ethyliminodiacetic acid (HIMDA), N,N-bis(hydroxyethyl)glycine (bicine), and N-(tris(hydroxymethyl)methyl)glycine (tricine). An example of a suitable N-substituted glycine is glycylglycine. An example of a suitable deoxycholate is sodium deoxycholate. The present invention also includes mixtures of two or more chelating agents. In some embodiments, the chelating agent is EDTA. In another embodiment, the chelating agent is disodium ethylenediaminetetraacetate dihydrate.

[0264] In some embodiments, the formulation contains a polyol. In some embodiments, the polyol is a sugar, preferably a non-reducing sugar. In some embodiments, the non-reducing sugar is trehalose. In some embodiments, the formulation contains trehalose in the range of about 2% to about 10% w / v. In some embodiments, the formulation contains about 5% w / v of trehalose.

[0265] In some embodiments, the formulation contains arginine and / or trehalose, such as about 80 mM to about 120 mM (especially 100 mM) of arginine or about 2% to about 10% w / v (especially 5% w / v) of trehalose. In one embodiment, the formulation contains 222 mM of trehalose dihydrate.

[0266] In one aspect, there is provided a formulation comprising a pharmaceutical composition, the pharmaceutical composition comprising about 5 mg / mL to about 125 mg / mL of an antibody, about 10 mM to about 40 mM of a histidine buffer, about 200 mM to about 250 mM of trehalose dihydrate, about 0.2 mM to about 0.3 mM of disodium ethylenediaminetetraacetate dihydrate, and about 0.01% to about 0.03% w / v of polysorbate 80, with a pH of about 5.0 to about 5.8.

[0267] In one aspect, there is provided a formulation comprising about 5 mg / mL - 125 mg / mL of an antibody, about 20 mM of a histidine buffer, about 222 mM of trehalose dihydrate, about 0.27 mM of disodium ethylenediaminetetraacetate dihydrate, and about 0.02% (w / v) of polysorbate 80, with a pH of about 5.5.

[0268] In one aspect, a formulation is provided that comprises an antibody at 20 mg / mL, 20 mM histidine buffer, 222 mM trehalose dihydrate, 0.27 mM disodium ethylenediaminetetraacetate dihydrate, and 0.02% (w / v) polysorbate 80, with a pH of about 5.5.

[0269] A "stable" formulation is one in which the protein substantially maintains its physical and / or chemical stability during manufacture, transportation, storage, and administration. Stability can be measured by differential scanning calorimetry, where a higher transition temperature is a more stable product formulation. A "stable" formulation can be one in which a phase change does not occur until about 50 °C or higher, about 55 °C or higher, about 60 °C or higher. Stability can be measured for a selected period of time at a selected temperature. For example, for a product stored at a recommended temperature of 2 °C to 8 °C, the formulation is stable at room temperature, about 30 °C or 40 °C for at least 1 month, and / or stable at about 2 °C to 8 °C for at least 1 year and preferably at least 2 years. For example, the degree of aggregation, acidic variants, and / or basic variants during storage can be used as indicators of protein stability. Thus, a "stable" formulation can be one in which about 10% or less, about 5% or less, such as about 4% or less of aggregated variants of the antibody are present in the formulation. A "stable" formulation can be one in which about 60% or less, about 50% or less, such as about 45% or less of acidic variants of the antibody are present in the formulation. A "stable" formulation can be one in which about 35% or less, about 10% or less, such as about 20% or less of basic variants of the antibody are present in the formulation..

[0270] In certain aspects of the present disclosure, the formulation allows the composition to remain stable when stored at about 2 °C to 8 °C for at least 18 months, frozen, thawed, and / or mixed. In one embodiment, a "stable" formulation can be one in which about 4% or less of aggregated variants, about 45% or less of acidic variants, and about 20% or less of basic variants of the antibody are present in the formulation.

[0271] In yet another aspect, the present disclosure relates to an article, exemplary form, kit that includes a container housing the composition in the formulation described herein. In one aspect, an injection device comprising the formulation is provided. The injection device can include a pen injector device or an autoinjector device. In one embodiment, the formulation is contained in a prefilled syringe.

[0272] 7. Treatment Methods and Compositions Used

[0273] The present disclosure also provides methods for treating any disease or disorder in which inappropriate expression (e.g., overexpression) or increased activity of the CTLA-4 protein causes or contributes to the pathological effects of the disease, or in which a decrease in CTLA-4 protein level or activity has a therapeutic benefit in a mammal, preferably a human.

[0274] In some embodiments, the compositions described herein are provided for use in treatment. Such treatment can relate to any disease or disorder in which inappropriate expression (e.g., overexpression) or increased activity of the CTLA-4 protein causes or contributes to the pathological effects of the disease, or in which a decrease in CTLA-4 protein level or activity has a therapeutic benefit in a mammal, preferably a human.

[0275] The composition can be used in a method for 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 can be used in a method for 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 can be used in a method for 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.

[0276] In one aspect, a composition for treating cancer is provided.

[0277] In one aspect, use of the composition described herein in the manufacture of a medicament for treating cancer is provided.

[0278] As used herein, the term "treatment" and its grammatical variants refer to therapeutic treatment. With respect to a particular disorder, treatment means: (1) ameliorating the disorder by one or more biological manifestations of the disorder, (2) interfering with a) one or more points in the biological cascade that causes or is responsible for the disorder or b) one or more biological manifestations of the disorder, (3) alleviating one or more of the symptoms, effects or side effects associated with the disorder or its treatment, (4) slowing the progression of one or more biological manifestations of the disorder or (5) preventing the onset of one or more biological manifestations of the disorder.

[0279] Treatment can be therapeutic, prophylactic or preventive. The subject will be a subject in need thereof. Persons in need of treatment can include those who already have a particular medical disease, as well as those who may develop such a disease in the future.

[0280] Therefore, prophylactic treatment is also contemplated. Those skilled in the art will understand that "prevention" is not an absolute term. In medicine, "prevention" should be understood to refer to the prophylactic administration of a drug to significantly reduce the likelihood or severity of a disorder or its biological manifestations, or to delay the onset of such a disorder or its biological manifestations. For example, prophylactic treatment is appropriate when a subject is considered to be at high risk of developing cancer, such as when the subject has a significant family history of cancer or when the subject has been exposed to a carcinogen.

[0281] Accordingly, the methods, antibodies, and compositions described herein can be used for prophylactic treatment or prophylactic measures (if specified). In such cases, the 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 the disease. A prophylactically effective amount of the composition is administered to such an individual. A prophylactically effective amount is an amount that prevents or delays the onset of one or more aspects or symptoms of the disease described herein.

[0282] The methods, antibodies, and compositions need not effect a complete cure of the disease, or eradicate every symptom or manifestation of the disease, to constitute a viable therapeutic treatment. As is well recognized in the art, a drug used as a therapeutic agent in a treatment method can reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a prophylactic treatment need not be completely effective in preventing the onset of a disease to constitute a viable prophylactic agent. It is sufficient to reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of disease occurrence (e.g., by delaying the onset of the disease) or progression in a subject.

[0283] The terms "individual," "subject," and "patient" are used interchangeably herein and can be broadly defined to include any person in need of treatment, such as a person in need of cancer treatment. The subject is typically a human. The subject can also be a mammal, such as a mouse, rat, or primate (e.g., marmoset or monkey). The subject can be a non-human animal. The antibodies, compositions, and methods of the present disclosure also have veterinary uses. The subject to be treated can be a farm animal, such as a cow or bull, sheep, pig, bovine, goat, or horse, or can be a domestic animal, such as a dog or cat. The animal can be of any age or a mature adult.

[0284] The present disclosure also provides methods of treating cancer in a mammal.

[0285] 8. Cancer Treatment

[0286] The present disclosure provides methods for reducing tumors or inhibiting the growth of tumor cells in a subject, the methods comprising administering a therapeutically effective dose of an agent capable of inhibiting CTLA-4 signaling. The methods can include administering the above composition to a mammal having cancer, thereby treating the cancer of the mammal. As discussed herein, CTLA-4 is aberrantly expressed in a variety of cancers, and the expression of CTLA-4 in patients with some cancers (e.g., renal cell carcinoma) is associated with tumor aggressiveness. The methods can be used to treat any type of cancer known in the art, such as adenocarcinoma, lung adenocarcinoma, acute myeloid leukemia (“AML”), acute lymphoblastic leukemia (“ALL”), adrenocortical carcinoma, anal cancer, appendiceal 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, cerebral cancer, cervical cancer, choriocarcinoma, chronic myeloid leukemia, central nervous system (CNS) tumors, colon adenocarcinoma, colon cancer, colorectal cancer, diffuse intrinsic pontine glioma (DIPG), diffuse large B cell lymphoma (“DLBCL”), embryonal rhabdomyosarcoma (ERMS), endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, follicular lymphoma (“FL”), gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, head and neck cancer, hematological cancer, hepatocellular carcinoma, Hodgkin lymphoma / primary mediastinal B cell lymphoma, kidney cancer, renal clear cell carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, monocytic leukemia, multiple myeloma, myeloma, neuroblast-derived CNS tumors, non-Hodgkin 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 lymphoma (cHL), renal cell carcinoma, rectal cancer, salivary gland adenocarcinoma (e.g., salivary gland tumors), sarcoma, skin cancer, small cell lung cancer, small intestine cancer, squamous cell carcinoma of the anogenital region (e.g., squamous cell carcinoma of the anus, penis, cervix, vagina, or vulva), esophageal squamous cell carcinoma, squamous cell carcinoma of the head and neck (SCHNC), lung squamous cell carcinoma, gastric cancer, T cell-derived leukemia, T cell-derived lymphoma, thymic carcinoma, thymoma, thyroid cancer, uveal melanoma, urothelial cell carcinoma, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Wilms tumor. In one embodiment, the cancer is hepatocellular carcinoma. In another embodiment, the cancer is NSCLC.

[0287] In some embodiments, the cancer treated with the compositions described herein is characterized by microsatellite instability or its absence. Microsatellite instability ("MSI") is or includes changes in the DNA of certain cells, such as tumor cells, where the number of repeats of a microsatellite (short repetitive sequences of DNA) differs from the number of repeats contained in the DNA from which it was inherited. Microsatellite instability results from a defect in the DNA mismatch repair (MMR) system that fails to repair replication-associated errors. This failure allows mismatched mutations to persist throughout the genome, but particularly in repetitive DNA regions called microsatellites, leading to an increased mutation burden.

[0288] 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 stable (e.g., MSS status). In some embodiments, the microsatellite instability status is evaluated by a next-generation sequencing (NGS)-based assay, an immunohistochemistry (IHC)-based assay, and / or a PCR-based assay. 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.

[0289] In embodiments, the cancer is associated with a 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, endometrial cancer is associated with high TMB and MSI-H. In some related embodiments, endometrial cancer is associated with high TMB and MSI-L or MSS.

[0290] In some embodiments, the cancer is a mismatch repair-deficient (dMMR) cancer. Microsatellite instability may result from a defect in the DNA mismatch repair (MMR) system that fails to repair replication-associated errors. This failure allows mismatched mutations to persist throughout the genome, but particularly in repetitive DNA regions called microsatellites, leading to an increased mutation burden, which may improve the response to certain anti-CTLA-4 agents.

[0291] In some embodiments, the cancer is a hypermutated cancer. In some embodiments, the cancer contains a polymerase ε (POLE) mutation. In some embodiments, the cancer contains a polymerase δ (POLD) mutation.

[0292] In some embodiments, the cancer is endometrial cancer (e.g., MSI-H or MSS / MSI-L endometrial cancer). In some embodiments, the cancer is an MSI-H cancer that contains a POLE or POLD mutation (e.g., an MSI-H non-endometrial cancer that contains a POLE or POLD mutation).

[0293] In one aspect, provided is a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition (including a pharmaceutical composition or formulation) described herein.

[0294] As used herein, the terms “cancer” and “tumor” are used interchangeably and, whether in the singular or plural, refer to cells that have undergone transformation (such as malignant transformation) such that they are pathologic to the host organism. Primary cancer cells can be readily distinguished from non-cancerous cells by well-recognized techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from cancer cell progenitors. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to cancer types that typically present as solid tumors, a “clinically detectable” tumor is a tumor detectable based on tumor mass; e.g., a tumor detectable by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or physical examination palpation, and / or a tumor detectable due to the expression of one or more cancer-specific antigens in a sample obtainable from the patient.

[0295] In embodiments, the cancer is head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, bladder cancer, melanoma, Merkel cell carcinoma, 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, appendiceal cancer, urothelial carcinoma, or squamous cell carcinoma (e.g., squamous cell carcinoma of the lung; squamous cell carcinoma of the anogenital region, including the anus, penis, cervix, vagina, or vulva; or squamous cell carcinoma of the esophagus).

[0296] In embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is selected from: diffuse large B-cell lymphoma (“DLBCL”), Hodgkin lymphoma (“HL”), non-Hodgkin lymphoma (“NHL”), follicular lymphoma (“FL”), acute myeloid leukemia (“AML”), acute lymphoblastic leukemia (“ALL”), or multiple myeloma (“MM”). In embodiments, the cancer is a hematogenous cancer, such as acute lymphoblastic leukemia (“ALL”), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute granulocytic leukemia (“AML”), acute promyelocytic leukemia (“APL”), acute monocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, acute myelomonocytic leukemia, acute non-lymphocytic leukemia, acute undifferentiated leukemia, chronic granulocytic leukemia (“CML”), chronic lymphocytic leukemia (“CLL”), hairy cell leukemia, and multiple myeloma; acute and chronic leukemias, such as lymphocytic leukemia, myeloid leukemia, lymphocytic leukemia, and granulocytic leukemia.

[0297] In some embodiments, the cancer is a lymphoma, such as Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and polycythemia vera.

[0298] In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma of the lung. In some embodiments, the cancer is squamous cell carcinoma of the esophagus. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is squamous cell carcinoma of the anogenital region (e.g., anus, penis, cervix, vagina, or vulva).

[0299] In some embodiments, the cancer is bladder cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), fallopian tube cancer, cholangiocarcinoma, colon adenocarcinoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gastric cancer, renal clear cell carcinoma, lung cancer (e.g., lung adenocarcinoma or lung squamous cell carcinoma), mesothelioma, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer, endometrial cancer, or uveal melanoma. In some embodiments, the cancer is ovarian cancer, fallopian tube cancer, or peritoneal cancer. In some embodiments, the cancer is breast cancer (e.g., TNBC). In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer). In some embodiments, the cancer is prostate cancer.

[0300] 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 an embodiment, the cancer is a CNS tumor.

[0301] In some embodiments, the cancer is a solid tumor. In an embodiment, the cancer is a solid tumor, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary

[0302] carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, 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 cancer, skin cancer, melanoma, neuroblastoma (NB), or retinoblastoma. In some embodiments, the tumor is an advanced solid tumor. In some embodiments, the tumor is a metastatic solid tumor.

[0303] In some embodiments, the cancer is a gynecological cancer (i.e., cancer of the female reproductive system, such as ovarian cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, or primary peritoneal cancer, or breast cancer). In some embodiments, cancers of the female reproductive system include, but are not limited to, ovarian cancer, fallopian tube cancer, peritoneal cancer, and breast cancer.

[0304] In some embodiments, the cancer is ovarian cancer (e.g., serous or clear cell ovarian cancer). In some embodiments, the cancer is fallopian tube cancer (e.g., serous or clear cell fallopian tube cancer). In some embodiments, the cancer is primary peritoneal cancer (e.g., serous or clear cell primary peritoneal cancer).

[0305] In some embodiments, ovarian cancer is epithelial cancer. Epithelial cancers account for 85% to 90% of ovarian cancers. Although ovarian cancer has traditionally been thought to begin on the surface of the ovary, new evidence suggests that at least some ovarian cancers begin in specialized cells in a part of the fallopian tube. The fallopian tubes are small tubes that connect a female's ovaries to her uterus, which is 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 tube may metastasize to the surface of the ovary very early. The term "ovarian cancer" is commonly used to describe epithelial cancers that begin in the ovary, fallopian tube, and the lining of the abdominal cavity (called the peritoneum). In some embodiments, the cancer is or includes a germ cell tumor. Germ cell tumors are a type of ovarian cancer that occur in the egg-producing cells of the ovary. In some embodiments, the cancer is or includes a stromal tumor. Stromal tumors occur in the connective tissue cells that hold the ovary together, which are sometimes the tissue that produces the female hormone called estrogen. In some embodiments, the cancer is or includes a granulosa cell tumor. Granulosa cell tumors may secrete estrogen, causing abnormal vaginal bleeding at the time of diagnosis. In some embodiments, the gynecologic cancer is associated with homologous recombination repair deficiency / homologous repair deficiency ("HRD") and / or BRCA1 / 2 mutations. In some embodiments, the gynecologic cancer is platinum-sensitive. In some embodiments, the gynecologic cancer responds to platinum-based therapies. In some embodiments, the gynecologic cancer has developed resistance to the resistance of platinum-based therapies. In some embodiments, the gynecologic cancer has at one time shown a partial or complete response to a platinum-based therapy (e.g., a partial or complete response to the last or the penultimate platinum-based therapy). In some embodiments, the gynecologic cancer is now resistant to platinum-based therapies.

[0306] In an embodiment, the cancer is breast cancer. Generally, breast cancer either begins in milk-producing cells called lobules or in the milk ducts. Less commonly, breast cancer can begin in the stromal tissue. This tissue includes the fatty and fibrous connective tissue of the breast. Over time, breast cancer cells invade nearby tissues, such as the lymph nodes in the armpit or the lungs, a process called metastasis. The stage of breast cancer, the size of the tumor, and its growth rate are all factors that determine the type of treatment provided.

[0307] Treatment options include surgical removal of the tumor, medical treatment (including chemotherapy and hormone therapy), radiation therapy, and immunotherapy. Prognosis and survival rates vary widely; five-year relative survival rates range from 98% to 23% depending on the type of breast cancer that occurs. Breast cancer is the second most common cancer in the world, with approximately 1.7 million new cases in 2012, and is the fifth most common cause of cancer death, resulting in approximately 521,000 deaths. Of these cases, approximately 15% are triple-negative, which do not express estrogen receptor, progesterone receptor (PR), or HER2. In some embodiments, triple-negative breast cancer (TNBC) is characterized by estrogen receptor-negative expression (<1% of cells), progesterone receptor-negative expression (<1% of cells), and HER2-negative breast cancer cells.

[0308] 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 an embodiment, the cancer is TNBC.

[0309] In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is advanced breast cancer. In some embodiments, the cancer is stage II, III, or IV breast cancer. In some embodiments, the cancer is stage IV breast cancer.

[0310] In some embodiments, the cancer is endometrial cancer (“EC”). In some embodiments, the endometrial cancer is metastatic endometrial cancer.

[0311] Endometrial cancer is the most common cancer in the female genital tract. The estimated number of new cases of endometrial cancer (EC) globally each year is approximately 325,000. In addition, EC is the most common cancer in postmenopausal women. Approximately 53% of endometrial cancer cases occur in developed countries. In 2015, approximately 55,000 cases of EC were diagnosed in the United States, and there are currently no approved targeted therapies for EC. Agents and regimens are needed to improve survival rates in advanced and recurrent EC in the first-line (1L) and second-line (2L) treatment settings. The most common histological form is endometrioid adenocarcinoma, accounting for approximately 75%-80% of diagnosed cases. Other histological forms include uterine papillary serous (less than 10%), clear cell 4%, mucinous 1%, squamous cell less than 1%, and mixed approximately 10%.

[0312] From a pathogenetic perspective, EC is divided into two different types, namely the so-called type I and type II. Type I tumors are low-grade and estrogen-related endometrioid carcinomas (EECs), while type II are non-endometrioid (NEEC) (mainly serous and clear cell) carcinomas. The World Health Organization recently updated the pathological classification of EC, recognizing nine different subtypes of EC, but EECs and serous carcinomas (SCs) account for the vast majority of cases. EECs are estrogen-related carcinomas that occur in perimenopausal patients and are preceded by precancerous lesions (endometrial hyperplasia / endometrioid intraepithelial neoplasia). Microscopically, low-grade EECs (EEC 1-2) contain tubular glands, some resembling proliferative-phase endometrium, with complex architecture, showing glandular fusion and cribriform patterns. High-grade EECs show a solid growth pattern. In contrast, SCs occur in postmenopausal patients without estrogen excess. Microscopically, SCs show thick, fibrotic, or edematous papillae, with marked tumor cell stratification, cell budding, and anaplastic cells with large eosinophilic cytoplasm. The vast majority of EECs are low-grade tumors (grades 1 and 2), and when confined to the uterus, they have a good prognosis. Grade 3 EECs (EEC3) are invasive tumors with an increased frequency of lymph node metastasis. SCs are highly invasive, are not related to estrogen stimulation, and mainly occur in elderly women. EEC3 and SC are considered high-grade tumors. SC and EEC3 were compared using data from the Surveillance, Epidemiology, and End Results (SEER) program from 1988 to 2001. They accounted for 10% and 15% of EC, respectively, but 39% and 27% of cancer deaths, respectively.

[0313] In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is squamous cell carcinoma of the lung. In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as squamous NSCLC. In some embodiments, the lung cancer is lung cancer with ALK translocation (e.g., ALK-translocated NSCLC). In some embodiments, the lung cancer is lung cancer with EGFR mutation (e.g., EGFR-mutated NSCLC).

[0314] In some embodiments, the cancer is metastatic cancer.

[0315] In some embodiments, the cancer is recurrent cancer (e.g., recurrent gynecological cancer, such as recurrent epithelial ovarian cancer, recurrent fallopian tube cancer, recurrent primary peritoneal cancer, or recurrent endometrial cancer).

[0316] Subjects in need of cancer treatment can include patients at multiple stages, including newly diagnosed, recurrent, refractory, progressive disease, remission, and other stages. Subjects in need of cancer treatment can also include patients who have received a stem cell transplant or patients who are considered ineligible for transplantation.

[0317] 9. Routes of Administration

[0318] Subjects may have received at least one prior cancer therapy before being treated with the compositions of the present disclosure. In one embodiment, before being treated with the compositions of the present disclosure, the subjects have received at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 prior cancer therapies. In another embodiment, the subjects are diagnosed with cancer and have not received any prior therapy before being treated with the compositions of the present disclosure.

[0319] The compositions of the present disclosure can be administered by any suitable route. For some compositions, suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), and intratumoral.

[0320] It should be understood that the preferred route may vary depending on, for example, the condition of the recipient and the cancer to be treated.

[0321] In some embodiments, the composition is administered intravenously (e.g., by intravenous (IV) infusion). In yet another embodiment, the composition is administered by a 30-minute IV infusion.

[0322] In some embodiments, the composition is administered by injection. Thus, in one aspect, there is provided an injection device comprising the composition, pharmaceutical composition, or formulation of the present disclosure. The injection device can include a pen-type syringe device or an autoinjector device.

[0323] In one embodiment, the composition is contained in a pre-filled syringe.

[0324] The desired dose can be delivered by administering the composition by a single bolus, by multiple boluses, or by continuous infusion.

[0325] In certain embodiments, the compositions of the present disclosure are administered as a pharmaceutical composition.

[0326] As used herein, the term "administer" means delivering the compositions described herein to achieve a therapeutic goal. The composition can be administered at intervals of administration for a period of time sufficient to achieve a clinical benefit.

[0327] The composition can be administered to a subject in a manner that targets the treatment to a specific site.

[0328] In certain embodiments, the composition can be co-administered to a subject with one or more additional therapeutic agents. In another embodiment, the composition can be co-administered to a subject with one or more additional cancer therapeutic agents. The additional cancer therapeutic agents can include, but are not limited to, other immunomodulatory drugs, therapeutic antibodies, CAR-T therapeutic agents, BiTE, HDAC inhibitors, proteasome inhibitors, anti-inflammatory compounds, and immunomodulatory imide drugs (IMiD).

[0329] "Co-administered" refers to the administration of two or more different pharmaceutical compositions or treatments (e.g., radiotherapy), where the two or more different pharmaceutical compositions or treatments are administered to a subject by combination in the same pharmaceutical composition or separate pharmaceutical compositions. Thus, co-administration involves the simultaneous administration of a single pharmaceutical composition containing two or more agents, or the administration of two or more different compositions to the same subject at the same or different times.

[0330] In some embodiments, the composition can be administered in combination with other agents that inhibit the immune checkpoint pathway. For example, the composition can be administered in combination with an agent that inhibits or antagonizes the PD-1 pathway.

[0331] In some embodiments, the method includes co-administering the composition with durvalumab. In some embodiments, the combination is used for administration to patients with the following cancers: liver cancer (hepatocellular carcinoma), ovarian cancer, head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, bladder cancer, melanoma, Merkel cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, endometrial cancer, fallopian tube cancer, breast cancer, prostate cancer, salivary gland tumors, thymoma, adrenocortical tumors, esophageal cancer, gastric cancer, colorectal cancer, appendiceal cancer, urothelial carcinoma, or squamous cell carcinoma (e.g., squamous cell carcinoma of the lung; squamous cell carcinoma of the anogenital region, including the anus, penis, cervix, vagina, or vulva; or squamous cell carcinoma of the esophagus). In additional embodiments, the cancer is selected from liver cancer, ovarian cancer, or lung cancer (e.g., NSCLC).

[0332] 10. Dosage

[0333] The compositions described herein can be administered in a therapeutically effective amount.

[0334] As used herein, the "therapeutically effective amount" or "therapeutically effective dose" of a composition refers to the amount of an agent (such as an antibody or pharmaceutical composition) that provides a therapeutic benefit in the treatment or control of one or more symptoms of a disorder to be treated.

[0335] Therapeutically effective amounts and regimens are usually determined empirically and can depend on a variety of factors such as the age, weight, and health status of the patient and the disease or condition to be treated. These factors are within the purview of the treating physician.

[0336] Any type of range provided herein includes all values within the specified range and values at the endpoints of the specific range.

[0337] In some embodiments, the therapeutically effective dose is a fixed dose of about 10 mg - 500 mg (e.g., a fixed dose of about 10 mg, a fixed dose of about 25 mg, a fixed dose of about 50 mg, a fixed dose of about 75 mg, a fixed dose of about 100 mg, a fixed dose of about 200 mg, a fixed dose of about 300 mg, a fixed dose of about 400 mg, or a fixed dose of about 500 mg). In some embodiments, the therapeutically effective dose is about 1 mg / kg. In some embodiments, the therapeutically effective dose is about 3 mg / kg. In some embodiments, the therapeutically effective dose is about 10 mg / kg. In some embodiments, the therapeutically effective dose is about 15 mg / kg. In some embodiments, the therapeutically effective dose is a fixed dose of about 50 mg. In some embodiments, the therapeutically effective dose is about 75 mg. In some embodiments, the therapeutically effective dose is about 100 mg.

[0338] In one embodiment, the composition is administered once every 2 - 6 weeks (e.g., 2, 3, or 4 weeks, particularly 3 weeks).

[0339] In one embodiment, the composition is administered once every 3 weeks for 2 - 6 dosing cycles (e.g., the first 3, 4, or 5 dosing cycles, particularly the first 4 dosing cycles).

[0340] In some embodiments, the first dose and the second dose are different. In some embodiments, the first dose is about 75 mg and the second dose is about 100 mg.

[0341] In some embodiments, the composition is administered at an administration interval (or treatment cycle) of once every 3 weeks (Q3W), once every 4 weeks (Q4W), once every 5 weeks (Q5W), or once every 6 weeks (Q6W). In some embodiments, the composition is administered at an administration interval (or treatment cycle) of once every 3 weeks (Q3W). In some embodiments, the composition is administered at an administration interval (or treatment cycle) of once every 4 weeks (Q4W). In some embodiments, the composition is administered at an administration interval (or treatment cycle) of once every 5 weeks (Q5W). In some embodiments, the composition is administered at an administration interval (or treatment cycle) of once every 6 weeks (Q6W). In some embodiments, the composition is administered for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or longer. In some embodiments, the composition is administered on the first day of the treatment cycle or within 1, 2, or 3 days after the first day of the treatment cycle.

[0342] In some embodiments, the composition is administered at a dose of 75 mg every 3 weeks. In another embodiment, the composition is administered at a dose of 75 mg every 3 weeks for 4 cycles. In another embodiment, the composition is administered at a dose of 75 mg every 3 weeks for 4 cycles, followed by an additional 75 mg dose at week 16.

[0343] In some embodiments, the composition is administered as a single dose of 300 mg. In another embodiment, the composition is administered as a single dose of 300 mg in combination with 1500 mg of durvalumab on the first day of the treatment cycle, wherein durvalumab monotherapy continues every 4 weeks.

[0344] In some embodiments, the composition is administered according to Table 1.

[0345] Table 1. Recommended Dosage

[0346]

[0347]

[0348] a Patients weighing 40 kg or less must receive weight-based dosing equivalent to 1 mg / kg of tremelimumab and 20 mg / kg of durvalumab until their weight improves to greater than 30 kg.

[0349] b Tremelimumab is administered first; durvalumab is then administered, followed by chemotherapy on the day of dosing, if applicable.

[0350] cWhen tremelimumab is administered in combination with durvalumab and chemotherapy, for dosing information, refer to the prescribing information for durvalumab.

[0351] d When tremelimumab is administered in combination with durvalumab and platinum-based chemotherapy, for dosing information, refer to the prescribing information for albumin-bound paclitaxel, gemcitabine, pemetrexed, and carboplatin or cisplatin.

[0352] In some embodiments, the compositions described herein are administered according to a dosing regimen that demonstrates 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 an embodiment, the clinical benefit is partial response ("PR").

[0353] In an embodiment, the clinical benefit is complete response ("CR"). In some embodiments, PR or CR is determined according to the Response Evaluation Criteria In Solid Tumors (RECIST). In some embodiments, the composition is administered for a longer period of time to maintain the clinical benefit.

[0354] All patents and references disclosed herein are hereby expressly and fully incorporated by reference.

[0355] Examples

[0356] Example 1: Trametinib Impurities

[0357] Tremelimumab is evaluated to determine product-related impurities and process-related impurities. Test methods for evaluating impurities are described herein.

[0358] Methods

[0359] Amino Acid Analysis

[0360] Amino acid analysis is used to determine the amino acid composition of a protein. The sample is acid hydrolyzed, labeled, and analyzed by liquid chromatography. The quantification of each amino acid is determined relative to an external standard curve.

[0361] Analytical Ultracentrifugation

[0362] Analytical ultracentrifugation is used to measure purity, fragmentation, and aggregation. Substances of different sizes are separated based on their sedimentation behavior in a strong centrifugal force field. Substances that dissolve at sedimentation coefficients below the monomer peak are recorded as fragments. Substances that dissolve at sedimentation coefficients above the monomer peak are recorded as aggregates.

[0363] Competitive Binding ELISA

[0364] The competitive enzyme-linked immunosorbent assay (ELISA) was used as the binding assay. This assay measures the ability of tremelimumab to block the binding of CTLA-4 to the B7.2 fusion protein. The binding activity was evaluated as a dose response normalized to a reference standard, and a potency value relative to the tremelimumab reference standard was generated.

[0365] Capillary Isoelectric Focusing (cIEF)

[0366] The charge heterogeneity of tremelimumab was determined by imaging capillary electrophoresis (ICe). The tremelimumab sample was mixed with carrier ampholytes and injected into the capillary. Separation of the charge isomers was achieved when a potential was applied and the carrier ampholytes formed a pH gradient within the capillary; the tremelimumab charge isomers concentrated at the point of neutral net charge (pI) within the capillary.

[0367] Reducing Capillary Gel Electrophoresis

[0368] The protein was subjected to treatment with the denaturing agent sodium dodecyl sulfate (SDS) under reducing conditions. When placed in an electric field within the capillary, the SDS-coated protein migrated towards the anode through a replaceable SDS polymer sieving matrix and was separated according to the apparent molecular weight.

[0369] Non-Reducing Capillary Gel Electrophoresis

[0370] In non-reducing capillary gel electrophoresis, the protein was subjected to treatment with the denaturing agent SDS under non-reducing conditions. When placed in an electric field within the capillary, the SDS-coated protein migrated towards the anode through a replaceable SDS polymer sieving matrix and was separated according to the apparent molecular weight.

[0371] FcRn Binding Assay by SPR

[0372] A surface plasmon resonance binding assay was used to measure FcRn binding. This method is based on the ability of the test sample to bind to recombinant FcRn that has been immobilized on the surface of the SPR sensor. A reference flow cell was created using the same standard amine coupling procedure without the addition of FcRn.

[0373] High Performance Size Exclusion Chromatography

[0374] The purity (monomer) and product-related impurities (aggregates) of tremelimumab were determined by high performance size exclusion chromatography (HPSEC).

[0375] The HPSEC method separates size variants based on the relative molecular size and shape of the size variants, using differential exclusion from the pores of the stationary phase in the column. When the mobile phase carries the sample through the chromatographic column, the solute diffuses into the pores at a rate inversely proportional to its hydrodynamic volume, which depends on the size of the solute and the pore exclusion limit. The separated variants are detected and quantified by a UV detector.

[0376] Ion Exchange Chromatography (IEC)

[0377] Ion exchange chromatography is used to evaluate charge heterogeneity. The sample is injected onto an ion exchange column and eluted with a salt gradient. The eluted protein is detected using ultraviolet absorbance at 220 (or 280) nm. The results are reported as the percentages of the 0-Lys, 1-Lys, and 2-Lys peaks. To evaluate charge heterogeneity in the absence of C-terminal lysine, the sample is digested with carboxypeptidase B (CBP) before analysis. The results are reported as the percentages of the areas of the pre-peak, main peak, and post-peak.

[0378] Ligand Binding Assay by SPR

[0379] This assay is performed using surface plasmon resonance (SPR) to measure the association (ka) and dissociation (kd) of the interaction between tremelimumab and CTLA-4. The ratio of kd / ka provides information on the equilibrium dissociation constant (K D ) of the interaction between tremelimumab and CTLA-4. The binding affinity of the test sample is calculated relative to a reference standard. D

[0380] Methionine Oxidation by Lys-C Mapping

[0381] The methionine oxidation of the tremelimumab sample is quantitatively determined by peptide mapping. The resulting peptide fragments are separated by reversed-phase HPLC and detected using a UV detector. A methionine-containing peptide fragment and its corresponding oxidized form are monitored. The percentage of oxidation of the fragment containing methionine-256 is reported.

[0382] N-Terminal Sequencing

[0383] N-terminal sequencing by Edman degradation is used to determine the amino acid sequences at the N-termini of the heavy and light chains of tremelimumab. The results are compared with the theoretical sequences of the light and heavy chains.

[0384] Peptide Mapping by Mass Spectrometry

[0385] Peptide mapping was used to verify the primary sequence and evaluate post-translational modifications. The samples were denatured, reduced, alkylated, and digested with trypsin. Peptides were separated by reversed-phase liquid chromatography and measured using a UV detector and a mass spectrometer. The results of the peptide fragmentation pattern of the test samples compared to the reference standard were reported.

[0386] Q-TOF Mass Spectrometry

[0387] Q-TOF mass spectrometry (MS) was used to confirm the primary structure of the molecule based on the differences in the molecular mass-to-charge ratio (m / z). The measured mass of the molecule was obtained by deconvoluting the m / z data and compared with the theoretical mass of the molecule based on the amino acid composition. The results were reported in units of molecular weight (Da).

[0388] Reporter Gene Bioassay

[0389] CTLA4 is a cell surface receptor on activated T cells. The reporter gene bioassay measures the ability (potency) 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. Different concentrations of tremelimumab were incubated with cell lines in the presence of the superantigen SEE. The amount of luminescence produced was proportional to T cell activity and was quantified in a luminometer after reaction with the luciferase substrate. The potency of the test samples was determined relative to a reference standard.

[0390] Reverse Phase HPLC with Mass Spectrometry

[0391] The RP-HPLC / MS method was used only for peak identification. The samples were deglycosylated using PNGase F. Both non-reduced and reduced samples were separated by RP-HPLC and analyzed by Q-TOF mass spectrometry. Mass spectrometry data were collected and analyzed. Each peak in the RP-HPLC chromatogram was identified and reported.

[0392] Product-Related Impurities

[0393] Product-related impurities such as aggregates and fragments are commonly found in monoclonal antibody products due to degradation or modification. The important quality attributes of the identified tremelimumab include the following (Table 2).

[0394] Table 2.

[0395] Aggregates CDR Deamidation (Light Chain Asn-30) Fragments CDR Oxidation (Heavy Chain Trp-52) Fe Oxidation (Met-256 and Met-432)

[0396] Aggregation

[0397] Aggregation is one of the major degradation pathways of therapeutic antibodies. Multiple aggregation mechanisms have been observed in protein solutions, including the association of native monomers, the aggregation of conformationally altered monomers, the aggregation of chemically modified monomers, nucleation-controlled aggregation, and surface-induced aggregation. Aggregates can be broadly classified into two categories, covalent and non-covalent. Non-covalent aggregates can be further classified as reversible and irreversible.

[0398] Trastuzumab aggregates, including dimers and higher molecular weight (BMW) aggregates, were measured by high-performance size-exclusion chromatography (HPSEC).

[0399] Characterization of Aggregates

[0400] To evaluate the impact on biological activity, trastuzumab aggregates were induced by a combination of low pH (2.5 to 3.0) and high temperature (45 °C) treatment with shear force (oscillation). The forced-treated material was fractionated by preparative SEC to generate enriched aggregates. Subsequently, the aggregates and monomer fractions were characterized by physicochemical and biological assays (Table 4). The results showed that, surprisingly, the aggregates had only a moderate impact on biological activity, as a reduction in relative potency was observed only in the case of the high aggregate fraction.

[0401] Table 4. Effect of Aggregates on Potency

[0402]

[0403] Summary

[0404] The results showed that trastuzumab retained activity at relatively high levels of aggregation.

[0405] Aggregation increased after the material was subjected to forced conditions (i.e., low pH, high temperature, and shear force). The impact of the aggregates on biological activity was moderate. A reduced relative potency was observed when the material had >70% aggregates.

[0406] CDR Deamidation (Light Chain Asn-30)

[0407] Asparagine deamidation is another degradation pathway for therapeutic antibodies. When deamidation occurs in the CDR (complementary determining region), its impact on biological activity varies. The effect of deamidation on activity can vary from product to product. It has been reported that CDR deamidation at Asn-33, Asn-55, or Asn-102 in IgG has been found to reduce antigen binding and biological activity. In addition, it has been reported that the formation of succinimide (a deamidation intermediate) at Asn-55 in the CDR2 region of IgG1 reduces biological activity by 70% and ligand binding affinity by 50%. Subsequent hydrolysis of this succinimide results in a further decrease in potency. The effect of deamidation on protein activity cannot be predicted with the certainty required for pharmaceutical compositions.

[0408] Trastuzumab (IgG2) has a CDR deamidation site at Asn-30 on the light chain. As measured by peptide mapping, the LC Asn-30 deamidation of the tested samples ranged from 6.5% - 6.9% (Table 5).

[0409] Table 5. Deamidation of Trametinib Samples Measured by Peptide Mapping and cIEF

[0410]

[0411] Characterization of CDR Deamidation (LC Asn-30)

[0412] When analyzing trastuzumab samples that were force-treated at 40 °C during stability studies, a significant increase in deamidation at LC Asn-30 was observed by peptide mapping.

[0413] These data are consistent with the increase in acidic peaks measured by cIEF (Table 6).

[0414] Table 6. Deamidation of 40 °C Heat-Stressed Samples Measured by Peptide Mapping and cIEF

[0415]

[0416] To evaluate the effect of deamidation on biological activity, trastuzumab was incubated at pH 9 for 7 days and then fractionated by ion exchange chromatography (IEC, Figure 1 ) to enrich for LC Asn-30 deamidated species. Subsequently, the main peak (M) fraction (fraction B) and two acidic fractions containing LC Asn-30 deamidation (fractions D, E) were characterized by physicochemical and biological assays. The cIEF analysis of the IEC fractions is shown as Figure 2 follows.

[0417] Table 7 summarizes the levels of LC Asn-30 deamidation in the IEC fractions measured by peptide mapping and cIEF, and its effects on bioactivity (reporter gene bioassay), CTLA-4 binding, and FcRn binding activity. IEC fraction D contained 41.2% Asn-30 deamidation and eluted mainly as acidic peak 1 (A1) in cIEF. IEC fraction D showed reduced bioactivity (78%) and reduced CTLA-4 binding (62%). IEC fraction E had lower CTLA-4 binding (54%), had higher LC Asn-30 deamidation, and eluted mainly as acidic peak 2 (A2) in cIEF.

[0418] Table 7. Effect of Deamidation on Relative Potency, Relative CTLA-4 Binding, and Relative FcRn Binding Activity

[0419]

[0420] A1 = acidic peak 1; A2 = acidic peak 2; A3 = acidic peak 3; A4 = acidic peak 4; M = main peak; NT = not tested

[0421] Trastuzumab was further characterized after incubation in PBS and in human serum at 37 °C to evaluate changes in the deamidation levels under physiological conditions. Deamidation at LC Asn-30 was the major degradation product detected in these studies (Table 8).

[0422] Table 8. LC in Trametinib Incubated with Human Serum and PBS Asn-30 Deamidation

[0423]

[0424] The correlation between bioactivity and the level of LC Asn-30 deamidation (measured by cIEF acidic peaks and by peptide mapping) is shown in Figure 3 . The effect of LC Asn-30 deamidation on bioactivity was moderate. When LC Asn-30 deamidation > 80% (measured by cIEF acidic peaks) or > 40% (measured by peptide mapping), more than 75% of the relative potency was maintained.

[0425] Summary

[0426] Deamidation increased under heat stress conditions at 40 °C (measured by peptide mapping as 37.7% after 3 months) and increased during incubation in serum or PBS at 37 °C. The effect of CDR deamidation at LC Asn-30 on bioactivity was moderate. More than 75% of the relative potency was retained in materials with > 80% cIEF acidic peaks.

[0427] Fragmentation

[0428] 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 radical impurities, or residual host cell proteolytic enzymes. The hinge region of IgG2 is less prone to cleavage than that of IgG1. Under similar conditions, the total fragmentation of IgG2 is generally less than that of IgG1.

[0429] Characterization of Fragments

[0430] Trastuzumab is resistant to fragmentation, even over an extended period of time under forced conditions. As shown in Table 10, the fragments in trastuzumab forced at 40 °C for up to 6 months were measured to be less than or equal to 6.3% by non-reducing gel electrophoresis and less than or equal to 5.0% by reducing gel electrophoresis. The low level of fragments is consistent with the literature reports that IgG2 is less prone to fragmentation due to the lack of peptide bond hydrolysis in its hinge region.

[0431] Table 10. Fragments in Heat-Stressed Trametinib Samples Determined by Gel Electrophoresis

[0432]

[0433]

[0434] To identify the fragments observed by gel electrophoresis, control and heat-forced (40 °C for 6 months) samples were analyzed by RP-LC / MS. Fragments were detected in the heat-forced samples compared to the undetectable fragment levels in the control samples. The major fragments were identified as heavy chain fragments (HC 1-99, HC330-450), light chain fragments (LC1-212, LC1-213), and light chain fragments containing succinimide. Succinimide is a deamidation or isomerization intermediate. Succinimide intermediates with increased levels of deamidation or isomerization were identified in heat-forced IgG. Trace amounts of heavy chain fragments HC 239-450 and HC 241-450 were also detected. A schematic diagram of trastuzumab with labeled fragmentation sites is shown in Figure 4 Figure.

[0435] As summarized in Table 11, orthogonal physicochemical methods fully characterized the trastuzumab fragmentation pattern. The expected fragments and other product-related impurities detected by RP-LC / MS in heat-forced trastuzumab are listed. Gel electrophoresis peak identification was based on the results from RP-LC / MS, deglycosylation studies, and partial reduction studies. In addition, peptide mapping was performed to verify the fragments and non-glycosylated species.

[0436] Table 11. Trametinib Fragments Measured by RP-LC / MS and Gel Electrophoresis

[0437]

[0438] HC: Heavy chain; LC: Light chain; ND: Not detected; NR: Non-reducing; R: Reducing; Su-LC: LC containing succinimide

[0439] To evaluate the effect of fragmentation on bioactivity, the relative potency (reporter gene assay) of trastuzumab samples forced at 40 °C for 0, 2, and 3 months was determined (Table 12). The results showed that in the presence of low levels of fragments, >95% relative potency was maintained after 3 months of incubation at 40 °C.

[0440] Table 12. Effect of Fragments on Potency

[0441] Sample Description Fragments Measured by Non-Reducing Gel Electrophoresis (%) Relative Potency (%) 40 °C for 0 months 1.7 95 40 °C for 2 months 2.6 97 40 °C for 3 months 2.7 97

[0442] Summary

[0443] The fragmentation pattern was characterized by RP-LC / MS. The major fragment species were the heavy chain N-terminal fragment (HC 1-99), the heavy chain C-terminal fragment (HC 330-450), and the light chain C-terminal fragments (LC 1-212 and LC 1-213).

[0444] Hinge region fragments were not detected. Trastuzumab fragmentation occurred very slowly under forced conditions (40 °C). The presence of low levels of fragments (2.7% after 3 months at 40 °C) had little effect on bioactivity. Fragmentation was not the major degradation pathway of trastuzumab.

[0445] Oxidation

[0446] It is known that amino acid oxidation can affect the structure, activity, and degradation rate of proteins. Met and Trp oxidation are chemical modifications that occur during monoclonal antibody purification, formulation, and storage. Oxidation of other residues (Cys and Tyr) has also been observed. It has been reported that Fe oxidation (Met-252 and Met-428; EU numbering) reduces FcRn binding and results in a shorter serum half-life. Loss of bioactivity has been shown to be associated with oxidation in the CDR region.

[0447] As a characterization test, trastuzumab oxidation at Trp-52 in the CDR and at Met-256, Met-362, Met-401, and Met-432 in the Fe region was measured by tryptic peptide mapping. As the most readily oxidized site, Met-256 oxidation was monitored by Lys-C peptide mapping.

[0448] Characterization of Oxidative Heat Stress

[0449] After 3 months of heat stress treatment at 40 °C, a slight increase in the oxidation of tryptophan and methionine residues was observed (Table 14).

[0450] Table 14. Oxidation of Heat-Stressed Trametinib Determined by Peptide Mapping

[0451]

[0452] Chemical Oxidation

[0453] Before peptide mapping and bioanalysis, the tremelimumab samples were chemically oxidized by treatment with hydrogen peroxide (100 ppm) at room temperature (Table 15). Met-256 and Met-401 were more easily oxidized than Met-432 and Met-362. Up to 79.8% oxidation of Met-256 had no effect on potency. However, at oxidation levels above 35.5%, FcRn binding decreased.

[0454] Table 15. Effect of Chemical Oxidation on Potency and FcRn Binding

[0455]

[0456] Photooxidation

[0457] In addition to chemical oxidation, photo-stress studies were conducted at exposure levels of IX and 3X as outlined in the ICH guideline QIB; that is, for UV-A, the light radiation exposure exceeded 200 watt-hours per square meter, and for cold white light (CWL), the light radiation exposure exceeded 12 million lux-hours. Another study was conducted on tremelimumab samples exposed to a CWL intensity of approximately 2000 lux for 14 days (a total of 672,000 lux-hours).

[0458] Table 16 summarizes the oxidation levels in the photo-stress treated samples measured by peptide mapping and their effects on potency and FcRn binding. No major effects on potency or FcRn binding were observed at exposure according to the IX ICH guideline or 14-day exposure to CWL. A decrease in potency and FcRn binding occurred after exposure according to the 3X ICH guideline. Oxidation on Trp-52, Met-256, and Met-432 was observed at exposure according to the IX ICH guideline and 14-day exposure to CWL, and increased significantly after exposure according to the 3X ICH guideline. Trp-52 is in the heavy chain CDR region, and oxidation at this site can lead to loss of potency at excessive exposure (3X ICH). The study showed that 36.7% photo-stress oxidation of tryptophan in the CDR had an effect on bioactivity.

[0459] Table 16. Oxidation, Potency, and FcRn Binding of Photo-Stressed Trametinib Samples

[0460]

[0461] Summary

[0462] Chemical oxidation studies and photo-oxidation studies demonstrated that up to 35.5% oxidation of Met-256 did not affect potency or FcRn binding. Oxidation of 36.7% of Trp-52 reduced potency and FcRn binding.

[0463] Host Cell Protein (HCP)

[0464] HCP LC-MS characterization was performed on tremelimumab and pre-purification samples. The results are summarized in Tables 17 to 21. Twenty or fewer HCPs were identified in the tremelimumab samples, while 569 - 660 HCPs were detected in three different pre-purification samples (3 - 5), respectively. This method enabled high sensitivity HCP identification by using native digestion to deplete most of the mAb molecules. The concentration of each identified HCP was estimated by a label-free quantification method. The results confirmed that HCPs were cleared during the purification process, although a small amount of low-abundance HCPs remained at reduced levels.

[0465] Table 17.

[0466]

[0467] *Semi-quantitative

[0468] Table 18.

[0469]

[0470]

[0471] Table 19.

[0472]

[0473] ppm: parts per million of active substance, ng / mg protein

[0474] A useful upper limit was obtained using a tolerance interval based on approximately five times the standard deviation of the data set, i.e., an upper limit of approximately 34 ng / mg was generated. The useful upper limit for host cell proteins is much lower than the currently permitted range for monoclonal antibodies (≤100 ng / mg protein) (Champion K. et al., Bioprocess International; 2005; 3(8):52 - 7).

[0475] A useful upper limit for each HCP can also be established based on the same method. For example, the upper limit for the endoplasmic reticulum chaperone BiP is approximately 14 ng / mg.

[0476] Table 21. Overview of Quality Attributes

[0477]

[0478]

Claims

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; 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 one or more deamidated variants of the anti-CTLA-4 antibody, wherein the composition comprises ≤45%, ≤40%, ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3% of the deamidated variant.

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

3. The composition according to claim 1 or claim 2, wherein the amount of the deamidated variant of the anti-CTLA antibody is: (i) Any one selected from the following ranges: 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％、0.01％-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) 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 according to claim 3, wherein the amount of the deamidated variant of the anti-CTLA antibody is 0.01% - 45%.

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

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

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

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

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

1.

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

11.

11. The composition according to any one of the preceding claims, 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% of deamidation at N30 of the anti-CTLA- 4 antibody.

12. The composition according to any one of the preceding claims, 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 of deamidation at N30 of the anti-CTLA-4 antibody.

13. The composition according to any one of the preceding claims, 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% of deamidation at N388 of the anti-CTLA-4 antibody.

14. The composition according to any one of the preceding claims, 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 of deamidation at N388 of the anti-CTLA-4 antibody.

15. The composition according to any one of the preceding claims, 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 at N393 of the anti-CTLA-4 antibody.

16. The composition according to any one of the preceding claims, 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 according to any one of the preceding claims, wherein 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) deamidation at N393 of the anti-CTLA-4 antibody.

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

19. The composition according to any one of the preceding claims, 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. The composition according to 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. The composition according to any one of the preceding claims, 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. The composition according to 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. The composition according to 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 according to any one of the preceding claims, 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. The composition according to any one of the preceding claims, wherein the method for identifying the deamidated variant or deamidation at the residue of the anti-CTLA-4 antibody is capillary isoelectric focusing (cIEF).

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

27. 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; 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 one or more oxidized variants of the anti-CTLA antibody, wherein the composition comprises ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3% of the oxidized variant.

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

29. The composition according to claim 27 or claim 28, wherein the amount of the oxidized variant of the anti-CTLA antibody is: (i) Any one selected from the following ranges: 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％、0.05％-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) Approximately 35%, approximately 30%, approximately 25%, approximately 20%, approximately 10%, approximately 5%, approximately 4%, approximately 3%, approximately 2% or approximately 1%.

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

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

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

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

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

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

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

37. The composition according to any one of claims 24 to 36, wherein the composition comprises oxidation at W52 in CDRH2 of the anti-CTLA antibody in an amount of: (i) Any one selected from the following ranges: 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％-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%; or (ii) about 20%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

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

41. The composition according to 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 the method for identifying 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 according to any one of claims 24 to 41, wherein the composition comprises oxidation at M256 of the heavy chain of the anti-CTLA antibody in an amount of: (i) Any one selected from the following ranges: 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％、0.05％-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) About 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

43. The composition according to 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 the method for identifying 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 according to any one of claims 24 to 43, wherein the composition comprises oxidation at M401 of the heavy chain of the anti-CTLA antibody in an amount of: (i) Any one selected from the following ranges: 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) About 16%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

45. The composition according to 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 the method for identifying 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 according to any one of claims 24 to 45, wherein the composition comprises oxidation at M362 of the heavy chain of the anti-CTLA antibody in an amount of: (i) Any one selected from the following ranges: 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％、0.1％-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%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

47. The composition according to 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 the method for identifying 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 according to any one of claims 24 to 47, wherein the composition comprises oxidation at M432 of the heavy chain of the anti-CTLA antibody, in an amount of: (i) any one selected from the following ranges: 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％、0.05％-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) about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

49. The composition according to 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 the method for identifying 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 according to any one of claims 26 to 49, wherein the composition comprises oxidation at M256 of the oxidized variant of the anti-CTLA-4 antibody, in an amount of: (i) any one selected from the following ranges: 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％、0.05％-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) about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

51. The composition according to 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 the method for identifying 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 according to any one of claims 26 to 51, wherein the composition comprises oxidation at M401 of the anti-CTLA-4 antibody in an amount of: (i) any one selected from the following ranges: 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) about 16%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

53. The composition according to 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 the method for identifying oxidation at M362 of the anti-CTLA-4 antibody and 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2% or 1%.

54. The composition according to any one of claims 26 to 53, wherein the composition comprises oxidation at M362 of the anti-CTLA-4 antibody in an amount of: (i) any one selected from the following ranges: 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％、0.1％-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%; or (ii) about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

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

56. A composition according to any one of claims 26 to 55, wherein the composition comprises oxidation at M432 of the anti-CTLA-4 antibody in an amount of: (i) any one selected from the following ranges: 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％、0.05％-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) about 35%, about 30%, about 25%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

57. A composition according to any one of claims 26 to 56, wherein the method for identifying the oxidation variant or oxidation at the residue of the anti-CTLA-4 antibody is capillary isoelectric focusing (cIEF).

58. 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; and one or more aggregation variants of the anti-CTLA-4 antibody, wherein the composition comprises ≤26%, ≤25%, ≤20%, ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of the aggregation variants.

59. A composition according to claim 58, wherein the amount of the aggregation variant of the anti-CTLA-4 antibody is between the lower limit of detection of the method for identifying the aggregation variant of the anti-CTLA-4 antibody and 26%, 25%, 20%, 10%, 5%, 4%, 3%, 2% or 1%.

60. A composition according to claim 58 or claim 59, wherein the composition comprises the aggregation variant of the anti-CTLA-4 antibody in an amount of: (i) Any one selected from the following ranges: 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%, 0.5% - 2% and 0.5% - 1%; or (ii) Approximately 10%, approximately 5%, approximately 4%, approximately 3%, approximately 2% or approximately 1%.

61. The composition according to any one of claims 58 to 60, wherein the method for identifying the aggregated variant of the anti-CTLA-4 antibody is size exclusion chromatography.

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

63. 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; and a fragmented variant of the CTLA-4 antibody, wherein the composition comprises ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of the fragmented variant.

64. The composition according to claim 63, wherein the amount of the fragmented variant of the anti-CTLA-4 antibody is between the lower limit of detection of the method for identifying the aggregated variant of the anti-CTLA-4 antibody and 10%, 5%, 4%, 3%, 2% or 1%.

65. The composition according to claim 63 or claim 64, wherein the composition comprises the fragmented variant of the anti-CTLA-4 antibody in an amount of: (i) Any one selected from the following ranges: 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) About 10%, about 5%, about 4%, about 3%, about 2% or about 1%.

66. The composition according to any one of claims 63 to 65, wherein the fragmented variant comprises a heavy chain N-terminal fragment (HC 1-99), a heavy chain C-terminal fragment (HC 330-450) and a light chain C-terminal fragment (LC 1-212 and LC 1-213).

67. The composition according to 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. The composition according to any one of claims 63 to 67, wherein the method for identifying the aggregated variant of the anti- CTLA-4 antibody is reduced or non-reduced capillary gel electrophoresis (CGE).

69. A composition, the composition comprises an anti-CTLA antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain, the light chain variable domain comprises 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, the heavy chain variable domain comprises 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% of the heavy chain N-terminal pyroglutamic acid variant and / or up to 100% of the heavy chain C-terminal lysine cleavage variant of the CTLA-4 antibody.

70. A composition, the composition comprising an anti-CTLA antibody, wherein the anti-CTLA-4 antibody comprises a light chain variable domain, the 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, the 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; the composition further comprises ≤34 ng / mg of host cell protein (HCP).

71. The composition according to claim 70, the composition further comprising ≤34 ng / mg of HCP.

72. The composition according to claim 70 or claim 71, wherein the HCP is selected from elongation factor 1-alpha 1, glyceraldehyde-3-phosphate dehydrogenase, Mus musculus RuvB-like protein 2, 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, lipid droplet coating protein-3, mitotic checkpoint protein BUB3, methylosome protein 50, aminoacyl tRNA synthetase complex-interacting multifunctional protein 2, fatty acid synthase, hypoxia upregulated protein 1, endoplasmic reticulum chaperone BiP, protein containing staphylococcal nuclease domain 1, cytoplasmic dynein 1 light intermediate chain 1, HEP AC AM family member 2, and pyrroline-5-carboxylate reductase 2.

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

74. The composition according to any one of the preceding claims, 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 according to any one of the preceding claims, 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. A composition according to any one of the preceding claims, wherein the acidic variant and / or the oxidized 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. The composition according to claim 76, wherein the acidic variant and / or the oxidized 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 according to claim 75, wherein the anti-CTLA-4 antibody comprises oxidation at one or more of M256, M362, and / or M432 of the amino acid sequence of SEQ ID NO:

10.

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

10.

80. 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) ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3% of the oxidized variant of the anti-CTLA-4 antibody; (ii) ≤26%, ≤25%, ≤20%, ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% of the aggregated variant of the anti-CTLA-4 antibody; (iii) Deamidated variants of the anti-CTLA-4 antibody that are ≤45%, ≤40%, ≤35%, ≤30%, ≤20%, ≤15%, ≤10%, ≤5%, ≤4% or ≤3%; (iv) Fragmented variants of the anti-CTLA-4 antibody that are ≤10%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1%.

81. 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 further comprises: (i) Variants oxidized at Trp-52 of the heavy chain of the anti-CTLA-4 antibody that are ≤30%; (ii) Variants deamidated at Met256 and / or Met-432 of the heavy chain of the anti-CTLA-4 antibody that are ≤35%; (iii) ≤34 ng / mg of host cell protein; (iv) Aggregated variants of the anti-CTLA-4 antibody that are ≤4%; and / or (iv) Fragmented variants of the anti-CTLA-4 antibody that are ≤10%.

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

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

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

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

86. A formulation comprising the pharmaceutical composition according to claim 84, wherein the formulation comprises from about 5 mg / mL to about 125 mg / mL of the anti-CTLA-4 antibody and a buffer having a pH of from about 5.0 to about 6.

5.

87. The formulation according to claim 86, wherein the buffer is a histidine buffer.

88. The formulation according to claim 87, wherein the buffer is a histidine buffer having a pH of about 5.

5.

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

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

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

92. A formulation comprising the pharmaceutical composition according to claim 85, wherein the formulation comprises: (a) About 20 mg / mL of the anti-CTLA-4 antibody; (b) About 20 mM of a histidine buffer; (c) About 222 mM of trehalose dihydrate; (d) About 0.27 mM of disodium ethylenediaminetetraacetate dihydrate; and (e) About 0.02% w / v of polysorbate 80, having a pH of about 5.

5.

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

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

95. An eluent, the eluent comprising the composition according to any one of claims 1 to 84.

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

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

98. The composition according to any one of claims 1 to 84, the pharmaceutical composition according to claim 85, or the preparation according to any one of claims 86 to 92, for use in therapy.

99. The composition according to any one of claims 1 to 84, the pharmaceutical composition according to claim 85, or the preparation according to any one of claims 86 to 92, for use in treating cancer.

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

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