Biological pharmaceutical composition and related method

Abstract

To provide a composition for treating BCMA mediated disease or disorder.SOLUTION: Provided is a composition including an isomerization variant of an anti BCMA antibody, where the isomerization variant includes a heavy chain amino acid sequence including CDRH1, CDRH2 and CDRH3 of specific sequences and a light chain amino acid sequence including CDRL1, CDRL2 and CDRL3 of specific sequences, and the composition contains 25% or less of the isomerization variant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.

[0002] FIELD OF THE INVENTION The invention described herein provides compositions comprising anti-BCMA antigen binding proteins and related methods for treating BCMA-mediated diseases or disorders. [Background technology]

[0003] BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily. It is a non-glycosylated integral membrane receptor for the ligands BAFF and APRIL. BCMA ligands can also bind to additional receptors. TACI (Transmembrane Activator and Calcium Modulator and Cyclophilin Ligand Interactor) binds to APRIL and BAFF, and BAFF-R (BAFF Receptor or BR3) is restricted to, but exhibits high affinity for, BAFF. Together, these receptors and their corresponding ligands regulate different aspects of humoral immunity, B cell development, and homeostasis.

[0004] BCMA expression is typically restricted to the B cell lineage and has been reported to increase during terminal B cell differentiation. BCMA is expressed by human plasmablasts and plasma cells derived from tonsils, spleen, and bone marrow, but is also expressed by tonsillar memory B cells and germinal center B cells, which have a TACI-BAFFR-low phenotype (Darce et al., 2007). BCMA is nearly absent on naive and memory B-25 cells (Novak et al., 2004a and Novak et al., 2004b). BCMA antigen is expressed on the cell surface, making it accessible to antibodies, but is also expressed in the Golgi apparatus. As suggested by its expression profile, BCMA signaling is typically associated with B cell survival and proliferation and is important during the later stages of B cell differentiation, but is also important for the survival of long-lived bone marrow plasma cells (O'Connor et al., 2004) and plasmablasts (Avery et al., 2003). Furthermore, because BCMA binds to APRIL with high affinity, it has been suggested that the BCMA-APRIL signaling axis is dominant at later stages of B cell differentiation and is perhaps the most physiologically important interaction.

[0005] BCMA expression (both transcript and protein) has been reported to correlate with disease progression in various B-cell disorders, including B-cell cancers such as multiple myeloma (MM). MM is a clonal B-cell malignancy that develops at multiple sites within the bone marrow, either de novo or as a progression from monoclonal gammopathy of undetermined significance (MGUS), before spreading to the circulation. It is generally characterized by increased paraproteins and osteoclast activity, as well as hypercalcemia, cytopenia, nephropathy, hyperviscosity, and peripheral neuropathy. Decreases in both normal antibody levels and neutrophil counts are also common, resulting in severe susceptibility to infection. BCMA has been implicated in the proliferation and survival of myeloma cell lines in vitro (Novak et al., 2004 and Moreaux et al., 2004). Summary of the Invention

[0006] 1. A composition comprising an isomerization variant of an anti-BCMA antibody, The isomerization variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises 25% or less of the isomerization variant.

[0007] 1. A composition comprising an oxidized variant of an anti-BCMA antibody, The oxidation variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises no more than 40% of the oxidation variant.

[0008] 1. A composition comprising an anti-BCMA antibody, the anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2; CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4; CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 Including, The composition, wherein the composition contains 0.1 to 25% isomerization at D103 of CDRH3.

[0009] 1. A composition comprising an anti-BCMA antibody, the anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2; CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4; CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 Including, The composition, wherein the composition comprises 0.1 to 40% oxidation at M34 of CDRH1.

[0010] 1. A composition comprising an anti-BCMA antibody, the anti-BCMA antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10; The composition, wherein the composition contains 0.1 to 25% isomerization at D103 of CDRH3.

[0011] 1. A composition comprising an anti-BCMA antibody, the anti-BCMA antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10; The composition, wherein the composition comprises 0.1 to 40% oxidation at M34 of CDRH1.

[0012] 1. A composition comprising an anti-BCMA antibody-drug conjugate (ADC), wherein the proportion of DL2 is at least about 30%, about 15% to about 27%, or about 15% to about 32%; the proportion of DL4a is at least about 30%, about 35% to about 38%, or about 30% to about 40%; the proportion of DL4b is at least about 5%, about 7% to about 9%, or about 5% to about 10%; the proportion of DL6 is at least about 10%, about 14% to about 20%, or about 10% to about 20%; and / or the proportion of DL8 is at least about 1%, about 6.0% to about 12.0%, or about 4% to about 15%.

[0013] 1. A composition comprising an anti-BCMA antibody-drug conjugate (ADC), wherein the percentage of DL0 is about 10% or less, or about 5% or less. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows a schematic representation of a heterogeneous mixture of DL species in an ADC composition. [Figure 2] FIG. 2 shows representative HIC peak characteristics for determining DAR distribution in ADC compositions. [Figure 3] Figure 3 shows the effect of the mean DAR of ADC compositions on tumor volume in a xenograft model. [Figure 4] FIG. 4 shows a representative cIEF electropherogram of belantamab. DETAILED DESCRIPTION OF THE INVENTION

[0015] The invention described herein provides compositions comprising anti-BCMA antigen binding proteins and related methods for treating BCMA-mediated diseases or disorders. A composition comprising an anti-BCMA antibody as described herein may also refer to a population of anti-BCMA antibodies as described herein, and these terms are interchangeable.

[0016] Anti-BCMA antigen-binding protein The anti-BCMA antigen binding proteins in the compositions described herein may be useful for treating or preventing a variety of BCMA-mediated diseases, including B-cell mediated cancers, e.g., lymphoma and multiple myeloma. The anti-BCMA antigen binding proteins described herein may bind to human BCMA, for example, human BCMA comprising the amino acid sequence of GenBank Accession No. Q02223.2, or a human BCMA comprising the amino acid sequence of a gene encoding human BCMA that has at least 90% homology or at least 90% identity thereto.

[0017] The term "antigen-binding protein" as used herein refers to antibodies, antibody fragments, and other protein constructs capable of binding to BCMA, e.g., human BCMA. Antigen-binding proteins of the present invention may comprise heavy and light chain variable regions of the present invention, which may be formatted into the structure of a natural antibody or a functional fragment or equivalent thereof. Thus, antigen-binding proteins of the present invention may comprise VH regions of the present invention formatted into full-length antibodies, (Fab')2 fragments, Fab fragments, or equivalents thereof (e.g., scFVs, bibodies, tribodies, or tetrabodies, Tandabs, etc.) when combined with an appropriate light chain. The antibody may be IgG1, IgG2, IgG3, or IgG4; IgM; IgA; IgE; IgD; or engineered variants thereof. The constant domain of the antibody heavy chain can be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. Furthermore, antigen-binding proteins may comprise all classes of modifications, such as IgG dimers, Fc variants that do not bind to Fc receptors, or Fc variants that do not modulate C1q binding. The antigen binding protein may also be a chimeric antibody of the type described in WO86 / 01533, comprising an antigen binding region and a non-immunoglobulin region.

[0018] In another embodiment of the invention, the antigen-binding protein may be a dAb, Fab, Fab', F(ab')2, Fv, diabody, triabody, tetrabody, miniantibody or minibody. In one embodiment of the invention, the antigen-binding protein may be a fully human antibody, a humanized antibody or a chimeric antibody. In a further embodiment, the antigen-binding protein is a humanized antibody. In one embodiment of the invention, the antigen-binding protein is a monoclonal antibody.

[0019] Chimeric antigen receptors (CARs) have been developed as artificial T cell receptors to generate novel specificities in T cells without the need for binding to MHC-antigen peptide complexes. These synthetic receptors can contain a target-binding domain linked to one or more signaling domains via a flexible linker within a single fusion molecule. The target-binding domain can be used to target T cells to specific targets present on the surface of pathological cells, and the signaling domain contains the molecular machinery for T cell activation and proliferation. A flexible linker that penetrates the T cell membrane (i.e., forms a transmembrane domain) enables cell membrane display of the CAR's target-binding domain. CARs can successfully redirect T cells to antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors (Jena et al., 2010, Blood, 116(7):1035-44). In one embodiment of the present invention, an anti-BCMA antigen-binding protein can comprise a chimeric antigen receptor. In a further embodiment, the CAR can comprise a binding domain, a transmembrane domain, and an intracellular effector domain.

[0020] Exemplary anti-BCMA antigen binding proteins and methods of making the same are disclosed in International Publication No. WO2012 / 163805, which is incorporated by reference in its entirety. Additional exemplary anti-BCMA antigen binding proteins include WO2016 / 014789, WO2016 / 090320, WO2016 / 090327, WO2016 / 020332, WO2016 / 079177, WO2014 / 122143, WO2014 / 122144, WO2017 / 021450, WO2016 / 014565, WO2014 / 068079, WO2015 / 1666 49, WO2015 / 158671, WO2015 / 052536, WO2014 / 140248, WO2013 / 072415, WO2013 / 072406, WO2014 / 089335, US2017 / 165373, WO2013 / 154760, and WO2017 / 051068, each of which is incorporated herein by reference in its entirety.

[0021] In another embodiment, the anti-BCMA antigen binding proteins described herein are capable of inhibiting the binding of BAFF and / or APRIL to the BCMA receptor. In another embodiment, the anti-BCMA antigen binding proteins described herein are capable of binding to FcγRIIIA or are capable of effector function mediated by FcγRIIIA.

[0022] In one embodiment, the anti-BCMA antigen binding protein comprises an antibody ("anti-BCMA antibody"). In another embodiment, the anti-BCMA antigen binding protein comprises a monoclonal antibody. As used herein, the term "antibody" refers to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE) and can include monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of these types. Monoclonal antibodies can be produced by eukaryotic cell clones or prokaryotic close cells expressing antibodies. Monoclonal antibodies can also be produced by eukaryotic cell lines capable of recombinantly expressing antibody heavy and light chains by having nucleic acid sequences encoding the antibody heavy and light chains introduced into the cells. Exemplary methods for producing antibodies from different eukaryotic cell lines, such as Chinese hamster ovary cells, hybridomas, or immortalized antibody cells derived from animals (e.g., humans), are well known to those of skill in the art.

[0023] Antibodies may be derived from, for example, rat, mouse, primate (e.g., cynomolgus monkey, Old World monkey or ape), human, or other sources (e.g., nucleic acids encoding antibody molecules generated using molecular biology techniques known in the art).

[0024] The antibody may comprise a constant region and may be of any isotype or subclass. The constant region may be of an IgG isotype, such as IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0025] The antigen binding protein may comprise one or more modifications including, for example, a mutated constant domain, so as to enhance antibody effector function / ADCC and / or complement activation, if the antigen binding protein is an antibody.

[0026] In one embodiment, the anti-BCMA antibody has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function. The term "effector function," as used herein, refers to one or more of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated responses, Fc-mediated phagocytosis, and / or antibody recycling via the FcRn receptor. In IgG antibodies, effector functions, which may include ADCC and ADCP, may be mediated by interaction of the heavy chain constant region with the Fcγ receptor family present on the surface of immune cells. In humans, these may include FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). The interaction between the antigen-bound antigen-binding protein and the Fc / Fcγ complex formation may induce various effects, such as cytotoxicity, immune cell activation, phagocytosis, and / or release of inflammatory cytokines.

[0027] In another embodiment, the anti-BCMA antibody may inhibit the binding of BAFF and / or APRIL to the BCMA receptor, hi another embodiment, the anti-BCMA antibody may bind to FcγRIIIA or be capable of FcγRIIIA-mediated effector function.

[0028] In one embodiment, the composition comprises an anti-BCMA antibody comprising two immunoglobulin (Ig) heavy chains ("HC") and two Ig light chains ("LC"). The basic antibody structural unit may comprise, for example, a tetramer of subunits. Each tetramer may comprise two pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain may comprise a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. This variable region may be initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, in one example, a light chain mature variable region may comprise a light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain may define a constant region. The heavy chain constant region may be primarily responsible for effector function.

[0029] The mature variable regions of each light / heavy chain pair can form an antibody binding site (also called an antigen-binding site). An "antigen-binding site" refers to the site of an antibody that can specifically bind to an antigen, which can be a single variable domain or a pair of V domains as found in a standard antibody. H / V L The domains may be FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Thus, an intact antibody may have, for example, two binding sites. Except in the case of bifunctional or bispecific antibodies, the two binding sites may be identical. All chains may exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or "CDRs." The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. Thus, in one example, from the N-terminus to the C-terminus, both the light chain and the heavy chain comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0030] "CDRs" are defined as the amino acid sequences of the complementarity determining regions 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, as used herein, "multiple CDRs" refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs. In one embodiment, the composition comprises an anti-BCMA antibody comprising one or more CDRs according to the invention described herein, or one or both of the heavy or light chain variable domains according to the invention described herein.

[0031] The terms "variant," "antibody variant," "CDR variant," and "post-translationally modified variant" refer to one or more amino acid changes in an antibody sequence. Variants can be the result of post-translational modification, chemical change, or sequence change due to one or more deletions, substitutions, or additions. While some post-translational modifications result in chemical changes that do not alter the sequence (e.g., Met and oxidized Met; or Asp; or isomerization / iso-Asp; or aggregation), other post-translational modifications result in sequence changes, such as conversion of one amino acid residue to another (e.g., conversion of Asn to Asp by deamidation; or loss of lysine). Additional post-translationally modified variants are described below. Variant antibody sequences containing sequence changes can be the result of engineered sequence changes or post-translational modifications. The amino acid sequence change can be a deletion, substitution, or addition.

[0032] In one such embodiment, the substitutions are conservative substitutions. In another embodiment, antibody variants contain one or more substitutions while retaining the canonical form of the antigen-binding protein. In one embodiment, an antibody variant is an antibody that is at least about 80%, about 85%, about 90%, or about 95% identical (i.e., has sequence identity) to the primary sequence of the antibody. In another embodiment, antibody variants include antibodies comprising a heavy chain amino acid sequence that is at least about 80%, about 85%, about 90%, or about 95% identical to the amino acid sequence of SEQ ID NO:9 and / or a heavy chain amino acid sequence that is at least about 80%, about 85%, about 90%, or about 95% identical to the amino acid sequence of SEQ ID NO:10.

[0033] The antigen-binding proteins of the present invention may have amino acid modifications that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life (i.e., serum half-life) of therapeutic and diagnostic IgG antibodies and other biologically active molecules has many advantages, for example, reducing the dosage and / or frequency of administration of these molecules. In one embodiment, the antigen-binding proteins of the present invention comprise all or a portion of an IgG constant domain (FcRn-binding portion) with one or more of the following amino acid modifications:

[0034] For example, for IgG1, M252Y / S254T / T256E (commonly referred to as the “YTE” mutation) and M428L / N434S (commonly referred to as the “LS” mutation) increase FcRn binding at pH 6.0 (Wang et al. 2018).

[0035] Half-life can also be extended by T250Q / M428L, V259I / V308F / M428L, N434A and T307A / E380A / N434A mutations (relative to IgG1 and Kabat numbering) (Monnet et al.).

[0036] Half-life and FcRn binding can also be extended by introducing H433K and N434F mutations (commonly referred to as "HN" or "NHance" mutations) (for IgG1) (WO2006 / 130834).

[0037] WO00 / 42072 describes polypeptides comprising variant Fc regions with altered FcRn binding affinity, which contain amino acid modifications at any one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and 447 (EU index numbering) of the Fc region.

[0038] WO02 / 060919 describes modified IgGs comprising an IgG constant domain which comprises one or more amino acid modifications compared to the wild-type IgG constant domain, the half-life of which is extended compared to the half-life of an IgG having the wild-type IgG constant domain, and the one or more amino acid modifications are at one or more of positions 251, 253, 255, 285-290, 308-314, 385-389 and 428-435.

[0039] Shields et al. (2001, J Biol Chem; 276:6591-604) used alanine-scanning mutagenesis to alter residues in the Fc region of a human IgG1 antibody and then evaluated binding to human FcRn. Positions that effectively inhibit FcRn binding when altered to alanine include I253, S254, H435, and Y436. Other positions, such as E233-G236, R255, K288, L309, S415, and H433, showed less pronounced decreases in binding. Several amino acid positions showed improved FcRn binding when altered to alanine, notably P238, T256, E272, V305, T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Many other amino acid positions showed either a slight improvement (D265, N286, V303, K360, Q362, and A378) or no change (S239, K246, K248, D249, M252, E258, T260, S267, H268, S269, D270, K274, N276, Y278, D280, V282, E283, H285, T289, K290, R292, E293, E294, Q295, Y296, N297, S298, R301, N315, E318, K320, K322, S330, R301, N315, E318, K320, K322, S330, R301, N316, E318, K322, K322, S330, R301, N317, E318, K320, K322, S330, R301, N318, E319, K331, K332, S333, R301, N319, E323, K334, K335, S336, R301, N319, E324, K336, S337, R301, N325, E326, K338, K339, S340, R301, N326, E327, K341, K342, S343, R301, N327, E328, K343, K344, S345, R301, N328, E329, K350, K351, K352, S353, 24, K326, A327, P329, P331, E333, K334, T335, S337, K338, K340, Q342, R344, E345, Q345, Q347, R356, M358, T359, K360, N361, Y373, S375, S383, N384, Q386, E388, N389, N390, K392, L398, S400, D401, K414, R416, Q418, Q419, N421, V422, E430, T437, K439, S440, S442, S444 and K447).

[0040] The most significant effect in terms of improved FcRn binding was found for the combination variant. At pH 6.0, the E380A / N434A variant showed over 8-fold better FcRn binding than native IgG1, compared to 2-fold for E380A and 3.5-fold for N434A. The addition of T307A to this resulted in a 12-fold improvement in binding compared to native IgG1. In one embodiment, the antigen-binding protein of the invention comprises the E380A / N434A mutations and has increased binding to FcRn.

[0041] Dall'Acqua et al. (2002, J Immunol.;169:5171-80) described random mutagenesis and screening of a human IgG1 hinge-Fc fragment phage display library against mouse FcRn. They described random mutagenesis of positions 251, 252, 254-256, 308, 309, 311, 312, 314, 385-387, 389, 428, 433, 434, and 436. The main improvement in the stability of the IgG1-human FcRn complex occurs when substituting residues located in the band across the Fc-FcRn interface (M252, S254, T256, H433, N434, and Y436), with lesser improvements occurring when substituting peripheral residues, such as V308, L309, Q311, G385, Q386, P387, and N389. The variant with the highest affinity for human FcRn was obtained by combining the M252Y / S254T / T256E ("YTE") and H433K / N434F / Y436H mutations, and showed a 57-fold increase in affinity compared to wild-type IgG1. The in vivo behavior of this mutated human IgG1 showed an approximately four-fold increase in serum half-life in cynomolgus monkeys compared to wild-type IgG1.

[0042] Thus, the present invention provides antigen binding proteins with optimized binding to FcRn. In a preferred embodiment, the antigen binding protein comprises one or more amino acid modifications in the Fc region of the antigen binding protein, the modifications being selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 0, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399 and at an amino acid position selected from the group consisting of 9, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446, and 447.

[0043] Furthermore, various publications describe how to obtain physiologically active molecules with altered half-lives, either by introducing an FcRn-binding polypeptide into the molecule (WO97 / 43316, US5869046, US5747035, WO96 / 32478 and WO91 / 14438) or by fusing the molecule to an antibody, thereby obtaining an antibody that retains its FcRn-binding affinity but has a significantly reduced affinity for other Fc receptors (WO99 / 43713) or by fusing the molecule to the FcRn-binding domain of an antibody (WO00 / 09560, US4703039).

[0044] FcRn affinity-enhancing Fc variants for improving both antibody cytotoxicity and half-life were identified in screens at pH 6.0. Selected IgG variants can be produced as hypofucosylated molecules. The resulting variants exhibit enhanced serum persistence in hFcRn mice and preserved, enhanced ADCC (Monnet et al.). Exemplary variants include (relative to IgG1 and Kabat numbering): P230T / V303A / K322R / N389T / F404L / N434S; P228R / N434S; Q311R / K334R / Q342E / N434Y; C226G / Q386R / N434Y; T307P / N389T / N434Y; P230S / N434S; P230T / V305A / T307A / A378V / L398P / N434S; P230T / P387S / N434S; P230Q / E269D / N434S; N276S / A378V / N434S; T307A / N315D / A330V / 382V / N389T / N434Y; T256N / A378V / S383N / N434Y; N315D / A330V / N361D / A387V / N434Y; V259I / N315D / M428L / N434Y; P230S / N315D / M428L / N434Y; F241L / V264E / T307P / A378V / H433R; T250A / N389K / N434Y; V305A / N315D / A330V / P395A / N434Y; V264E / Q386R / P396L / N434S / K439R; E294del / T307P / N434Y ("del" indicates deletion).

[0045] The present invention also provides methods for producing antigen-binding proteins according to the present invention, comprising the steps of: a) culturing a recombinant host cell containing an expression vector comprising an isolated nucleic acid described herein, wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase is inactivated in the recombinant host cell; and b) recovering the antigen-binding protein. Such methods for producing antigen-binding proteins can be carried out, for example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity relative to the same monoclonal antibodies produced in cells with a functional FUT8 gene. Embodiments of the POTELLIGENT technology system are described in US7214775, US6946292, WO0061739, and WO0231240, all of which are incorporated herein by reference. Those skilled in the art will also recognize other suitable systems and methods for generating antigen binding proteins, eg, antibodies.

[0046] The antibody can be recovered and purified by conventional protein purification procedures. For example, the antibody can be harvested directly from the culture medium. Harvesting of the cell culture medium can be accomplished via clarification, for example, by centrifugation and / or depth filtration. After the antibody is harvested, it is purified to ensure appropriate purity. Thus, in one aspect, a cell culture medium comprising an antibody described herein is provided. In one embodiment, the cell culture medium comprises CHO cells.

[0047] The antibody can then be purified from the cell culture medium. This may include harvesting the cell culture supernatant, contacting the cell culture supernatant with a purification medium (e.g., a Protein A resin or a Protein G resin to bind the antibody molecules), and eluting the antibody molecules from the purification medium to produce an eluate. Thus, in one embodiment, an eluate comprising the antibodies described herein is provided.

[0048] One or more chromatography steps may be used for purification, for example, one or more chromatography resins and / or one or more filtration steps may be used for purification. For example, affinity chromatography using a resin such as Protein A, G, or L may be used to purify the composition. Alternatively, or in addition, an ion exchange resin, for example, a cation exchange resin, may be used to purify the composition.

[0049] Alternatively, the purification step comprises an affinity chromatography step followed by a cation exchange resin step.

[0050] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region CDR1 ("CDRH1") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the heavy chain variable region CDR1 ("CDRH1") comprises an amino acid sequence having one amino acid mutation ("variant") in the amino acid sequence set forth in SEQ ID NO: 1.

[0051] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region CDR2 ("CDRH2") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the heavy chain variable region CDR2 ("CDRH2") comprises an amino acid sequence having a single amino acid mutation ("variant") in the amino acid sequence set forth in SEQ ID NO: 2.

[0052] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region CDR3 ("CDRH3") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the heavy chain variable region CDR3 ("CDRH3") comprises an amino acid sequence having a single amino acid mutation ("variant") in the amino acid sequence set forth in SEQ ID NO: 3.

[0053] In one embodiment, the anti-BCMA antibody comprises a light chain variable region CDR1 ("CDRL1") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the light chain variable region CDR1 ("CDRL1") comprises an amino acid sequence having one amino acid mutation ("variant") in the amino acid sequence set forth in SEQ ID NO: 4.

[0054] In one embodiment, the anti-BCMA antibody comprises a light chain variable region CDR2 ("CDRL2") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In one embodiment, the light chain variable region CDR2 ("CDRL2") comprises an amino acid sequence having a single amino acid mutation ("variant") in the amino acid sequence set forth in SEQ ID NO: 5.

[0055] In one embodiment, the anti-BCMA antibody comprises a light chain variable region CDR3 ("CDRL3") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the light chain variable region CDR3 ("CDRL3") comprises an amino acid sequence having a single amino acid mutation ("variant") in the amino acid sequence set forth in SEQ ID NO: 6.

[0056] In one embodiment, the anti-BCMA antibody comprises a CDRH1 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; a CDRH2 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; or a CDRH2 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. CDRH3; CDRL1 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; CDRL2 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; and / or CDRL3 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

[0057] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region (“V”) comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7. H ").

[0058] In one embodiment, the anti-BCMA antibody comprises a light chain variable region (“V”) comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. L ").

[0059] In one embodiment, the anti-BCMA antibody 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 set forth in SEQ ID NO:7. H and V comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. L Includes.

[0060] In one embodiment, the anti-BCMA antibody comprises a heavy chain region ("HC") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9.

[0061] In one embodiment, the anti-BCMA antibody comprises a light chain region ("LC") comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0062] In one embodiment, the anti-BCMA antibody comprises an HC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; and an LC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:10.

[0063] The "percent identity" between a query amino acid sequence and a subject amino acid sequence is the "identity" value, expressed as a percentage, calculated by the BLASTP algorithm when the subject amino acid sequence has 100% query coverage with the query amino acid sequence after a pairwise BLASTP alignment is performed. Such a pairwise BLASTP alignment between a query amino acid sequence and a subject amino acid sequence is performed using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Information website, with the filter for low complexity regions removed. Importantly, the query sequence can be described by an amino acid sequence identified in one or more claims herein.

[0064] In one embodiment, the anti-BCMA antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2; a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4; a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5; and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0065] In one embodiment, the anti-BCMA antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8 L Includes.

[0066] In one embodiment, the anti-BCMA antibody comprises belantamab, which comprises an HC having the amino acid sequence set forth in SEQ ID NO:9 and an LC having the amino acid sequence set forth in SEQ ID NO:10.

[0067] Antibody sequences can be identified by the Kabat numbering system (Kabat et al. Sequences of proteins of Immunological Interest NIH, 1987). Alternatively, they can be identified using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273,927-948), the contact definition method (MacCallum RM, and Martin ACR and Thornton JM, (1996), Journal of Molecular Biology, 262 (5), 732-745), or any other established method for numbering residues in antibodies and identifying CDRs known to those skilled in the art. Other numbering conventions for antibody sequences available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University of London) methods. Finally, antibody sequences can be numbered sequentially.

[0068] When referring to amino acids described herein numerically, the sequences may be numbered according to the Kabat or sequential numbering method. Unless otherwise specified, numerical references to specific amino acid numbers are described herein using the sequential numbering system. Throughout this specification, the terms "CDR," "CDRL1," "CDRL2," "CDRL3," "CDRH1," "CDRH2," and "CDRH3" follow the Kabat numbering system. Amino acid residues in variable region sequences and full-length antibody sequences indicate any antibody sequence variant positions or post-translational modification variant positions, such as isomerization variants (e.g., D103), deamidation variants (e.g., N388), or oxidation variants (e.g., M34).

[0069] Reference to a position within a CDR (e.g., M34 or D103) provides the position number (consecutive numbering) relative to the entire antibody sequence. Thus, M34 in CDRH1 corresponds to the fourth residue of SEQ ID NO: 1, i.e., the underlined: NYW MH (SEQ ID NO: 1). Similarly, D103 in CDRH3 refers to the fifth residue of SEQ ID NO: 3, i.e., the underlined: GAIY. D GYDVLDN (SEQ ID NO: 3).

[0070] In one aspect, the composition comprises an antibody variant comprising one or more amino acid changes in the primary sequence. 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: 9 and / or the light chain sequence of SEQ ID NO: 10, and that has an aspartic acid (D) to asparagine (N) amino acid change, e.g., D103N in CDRH3 (i.e., D99N in Kabat numbering).

[0071] In another embodiment, the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and comprising an amino acid change from aspartic acid (D) to asparagine (N), e.g., D103N in CDRH3.

[0072] In another embodiment, the anti-BCMA antibody comprises belantamab and comprises an aspartic acid (D) to asparagine (N) amino acid change, e.g., D103N in CDRH3.

[0073] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, and wherein about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 50% or more, about 75% or more, or about 90% or more of the antibodies in the mixture contain D103N in CDRH3.

[0074] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, or at least about 90% of the antibodies in the mixture contain D103N in CDRH3.

[0075] In one embodiment, the composition comprises belantamab, wherein about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 50% or more, about 75% or more, or about 90% or more of the belantamab comprises D103N in CDRH3.

[0076] In one embodiment, the composition comprises belantamab comprising at least one antibody variant using the Kabat numbering system selected from the group consisting of G27Y, S30T, A93T, A24G, K73T, M48I, V67A, F71Y, D99N, M4L, and K45E.

[0077] Post-translational modification products A "post-translationally modified product" of an antibody described herein is an antibody composition in which all or a portion of the composition comprises a "post-translation modification." The post-translation modification can be the result of antibody production in a host cell, upstream and downstream manufacturing, and / or storage (e.g., the effects of exposure to light, temperature, pH, water, or reaction with excipients and / or immediate container closure systems). Thus, a composition of the invention can be formed from antibody manufacturing or storage. Exemplary post-translational modifications include alterations in the antibody sequence ("antibody variants" above), cleavage of specific leader sequences, addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycosylation, deamidation, oxidation, disulfide bond scrambling and other cysteine ​​variants (e.g., free sulfhydryls, racemic disulfides, thioether and trisulfide bonds), isomerization, truncation of a C-terminal lysine, and / or cyclization of an N-terminal glutamine.

[0078] In one example, post-translationally modified products include "product-related impurities" that include chemical changes that result in a decrease in function and / or activity. In another example, post-translationally modified products include "product-related substances" that include chemical changes that do not result in a decrease in function and / or activity. Product-related impurities of the antibodies described herein include isomerized variants and oxidized variants. Product-related substances of the antibodies described herein include deamidated variants, glycosylated variants, C-terminal truncated variants, and N-terminal pyroglutamate variants.

[0079] In one embodiment, the composition comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, including one or more functional post-translational modifications thereof. In another embodiment, the composition comprises the heavy chain sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 and the light chain sequence of SEQ ID NO: 10, including one or more functional post-translational modifications thereof.

[0080] The percentage of variants provided herein is expressed as a percentage of the total amount of antibody (e.g., antibody "population") in the composition. For example, 40% or less oxidation variants refer to 100% of the total amount of antibody in the composition, of which 40% or less is oxidized. For example, 25% or less isomerization variants refer to 100% of the total amount of antibody in the composition, of which 25% or less isomerized.

[0081] Glycation is a post-translational modification involving a non-enzymatic chemical reaction between reducing sugars, e.g., glucose, and free amine groups of proteins, typically found at the epsilon amine of lysine side chains or the N-terminus of proteins. Glycation can occur during production and / or storage in the presence of reducing sugars.

[0082] For example, deamidation, which may occur during manufacturing and / or storage, can be an enzymatic or chemical reaction. Deamidation can occur through a simple chemical reaction involving intramolecular cyclization, in which the amide nitrogen of the adjacent amino acid in the chain undergoes a nucleophilic attack on the amide (N+1 attacks N), forming a succinimide intermediate. Deamidation may primarily convert asparagine (N) to isoaspartic acid (isoasparaginate) and aspartic acid (asparaginate) (D) in a ratio of approximately 3:1. Therefore, this deamidation reaction may be associated with the isomerization of aspartic acid (D) to isoaspartic acid. Both the deamidation of asparagine and the isomerization of aspartic acid may involve the intermediate succinimide. Deamidation can occur at glutamine residues in a similar manner, albeit to a much lesser extent. Deamidation can occur in the CDR, Fab (non-CDR region), or Fc region. Isomerization is the conversion of aspartic acid (D) to isoaspartic acid, involving the intermediate succinimide.

[0083] Oxidation can occur during manufacture and / or storage (i.e., in the presence of oxidizing conditions) and results in the covalent modification of proteins induced directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation occurs primarily at methionine residues, but can also occur at tryptophan and free cysteine ​​residues. Oxidation can occur in the CDRs, Fab (non-CDR) regions, or Fc regions.

[0084] Disulfide bond scrambling can occur during manufacturing and / or storage conditions. Under certain circumstances, disulfide bonds can break or not form properly, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryls (-SH) can facilitate shuffling.

[0085] Thioether formation and disulfide bond racemization can occur under basic conditions during manufacturing or storage. This leads to beta-elimination of the disulfide bridge back to a cysteine ​​residue via a dehydroalanine and persulfide intermediate. Subsequent cross-linking of dehydroalanine and cysteine ​​can form a thioether bond, or a free cysteine ​​residue can reform the disulfide bond with a mixture of D- and L-cysteine.

[0086] The trisulfide can result from the insertion of a sulfur atom into a disulfide bond (Cys-SSS-Cys) and can be formed by the presence of hydrogen sulfide in the production cell culture.

[0087] The N-terminal glutamine (Q) and glutamate (glutamic acid) (E) of the heavy and / or light chains can form pyroglutamate (pGlu) via cyclization. While pGlu formation can occur in the production bioreactor, it can also be formed non-enzymatically, depending, for example, on the pH and temperature of processing and storage conditions. Cyclization of the N-terminal Q or E is commonly observed in native human antibodies.

[0088] Cleavage of the C-terminal lysine is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in recombinant and native human antibodies. A variation of this process involves the removal of lysines from one or both heavy chains by cellular enzymes from the recombinant host cell. Administration to a human subject / patient may result in the removal of the remaining C-terminal lysine.

[0089] The present invention encompasses antibodies that have been or may have been subjected to one or more of the post-translational modifications described herein. Exemplary compositions may include mixtures or blends of antibodies: 1) mixtures or blends of antibodies with and without the post-translational modification(s), or 2) mixtures or blends of antibodies with two or more of the post-translational modifications described herein.

[0090] The composition may comprise a mixture of antibody variants and post-translationally modified variants, for example, the antibody composition may comprise one or more, e.g., two or more, of oxidized variants, deamidated variants, isomerized variants, N-terminal pyroglutamate variants, and C-terminal lysine truncated variants.

[0091] For example, in one embodiment, a composition may comprise a mixture of antibodies, wherein 10% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10, and 90% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10 with C-terminal lysine truncations.

[0092] In another exemplary embodiment, a composition may comprise a mixture of antibodies, wherein 10% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10, 90% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10 with C-terminal lysine truncation, and up to 100% of the N-terminal glutamines of a total 100% antibody mixture are cyclized to pyroglutamate.

[0093] In another exemplary embodiment, a composition may comprise a mixture of antibodies, wherein 10% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10, 90% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10 with C-terminal lysine truncation, and of the total 100% antibody mixture, up to 100% are N-terminal pyroglutamate and up to 23% are isomerized at D103 in CDRH3.

[0094] In yet another exemplary embodiment, a composition comprises a mixture of antibodies, wherein 20% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10, 80% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10 with a variant at N103 in CDRH3, and of the total 100% antibody mixture, up to 37% of the antibodies are oxidized at amino acid M34 in CDRH1.

[0095] In one embodiment, the post-translational modifications described herein do not result in significant changes in antigen binding affinity, biological activity, pharmacokinetics (PK) / pharmacodynamics (PD), aggregation, immunogenicity, and / or binding to Fc receptors, except as designated and listed as product-related impurities.

[0096] As used herein, "function" or "activity" is defined as one or more of: 1) binding to BCMA, 2) binding to FcγRIIIa, and / or 3) binding to FcRn. In one embodiment, "reduced function" or "reduced activity" means a percentage decrease in binding to BCMA, FcγRIIIa, or FcRn compared to a reference standard that is significant relative to assay variability. For example, a decrease in function or activity can be described as a decrease of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more.

[0097] In one embodiment, the anti-BCMA antibody comprises an antibody that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and SEQ ID NO:10, including all post-translational modifications, if present.

[0098] In another embodiment, the anti-BCMA antibody comprises belantamab, including all post-translational modifications, if present.

[0099] Antibody variants are commonly observed when the composition of an antibody is analyzed by charge-based separation techniques, such as gel electrophoresis, which is isoelectric focusing (IEF), gel electrophoresis, which is capillary isoelectric focusing (cIEF), cation exchange chromatography (CEX), and anion exchange chromatography (AEX).

[0100] Post-translational modifications can increase or decrease the net charge of an antibody, resulting in a decrease or increase in the pI value, thereby resulting in acidic and basic variants (collectively referred to as "charge variants") of the main isoform. The main isoform is the antibody population that elutes as the main peak in a chromatogram. When analyzing antibodies using an IEF-based method, acidic species are variants with a low apparent pI, and basic species are variants with a high apparent pI. When analyzing by a chromatography-based method, acidic and basic species are defined based on their retention time relative to the main peak. Acidic species are variants that elute earlier than the main peak obtained from CEX or variants that elute later than the main peak obtained from AEX, and basic species are variants that elute later than the main peak obtained from CEX or variants that elute earlier than the main peak obtained from AEX. These methods separate the main isoforms of antibodies from acidic and basic isoforms (acidic variants) and basic variants (basic variants). Charge variants can be detected by various methods, such as ion exchange chromatography (e.g., WCX-10 HPLC (weak cation exchange chromatography)) or IEF (isoelectric focusing). The percentage of charge variants can be determined using capillary isoelectric focusing (cIEF). Capillary isoelectric focusing (cIEF) is used to measure the pI of dostarlimab and separate the charge variants (see Figure 1). Using this method, acidic and basic species can be quantified as a percentage of the total area peak. The terms "species," "isoform," "form," and "peak" are used interchangeably to refer to the major isoforms and charge variants (acidic and basic variants).

[0101] In one embodiment, the composition comprises an acidic variant of an antibody, the acidic variant comprising a light chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises 1 to 70% acidic variant.

[0102] In one aspect, the composition comprises 70% or less of an acidic variant. In one embodiment, the composition comprises 60% or less, 50% or less, 40% or less, 35% or less, or 30% or less of an acidic variant. Alternatively, the composition comprises 10-70%, 10-60%, 10-50%, 10-40%, 10-35%, or 10-30% of an acidic variant. Alternatively, the composition comprises 20-70%, 20-60%, 20-50%, 20-40%, 20-35%, or 20-30% of an acidic variant. Alternatively, the composition comprises about 60%, about 50%, about 40%, about 35%, about 30%, about 25%, or about 20% of an acidic variant.

[0103] In one aspect, the composition comprises a basic variant of an antibody, the basic variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises 1 to 30% basic variant.

[0104] In one aspect, the composition contains 30% or less of a basic variant. In one embodiment, the composition contains 25% or less, 20% or less, 15% or less, 10% or less, 7.5% or less, or 5% or less of a basic variant. In one embodiment, the composition contains 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, or 1-5% of a basic variant. Alternatively, the composition contains about 15%, about 10%, or about 5% of a basic variant.

[0105] In one aspect, the composition comprises a major isoform of an antibody, the major isoform comprising a heavy chain amino acid sequence comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises 1 to 90% of the major isoform.

[0106] In one aspect, the composition comprises 1% or more of a major isoform. In one embodiment, the composition comprises 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 90% or more of a major isoform. In one embodiment, the composition comprises 10-90%, 20-90%, 30-90%, 40-90%, 50-90%, or 60-90% of a major isoform. In one embodiment, the composition comprises 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, or 60-80% of a major isoform. Alternatively, the composition comprises about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, or about 55% of a major isoform.

[0107] The percentage of acidic variants, the percentage of basic variants, and the percentage of major variants can be determined using isoelectric focusing, such as capillary isoelectric focusing (cIEF). It is understood that these isoform / charge variant embodiments can be used with any one or combination of antibody variants described herein.

[0108] In one aspect, the composition comprises charge variants of an antibody, wherein the charge variants comprise a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises no more than 70% acidic variants and / or no more than 30% basic variants and / or 1% or more of the major isoforms.

[0109] In one aspect, the composition includes an antibody comprising an isomerization post-translational modification ("isomerization" or "isomerized") or "isomerization variant." The variant may include isomerized amino acid residues in the heavy chain sequence and / or the light chain sequence, e.g., in the CDR of the heavy chain sequence and / or the CDR of the light chain sequence. The isomerization variant may be present in one or both of the heavy and light chains. The isomerization post-translational modification results in isoaspartic acid and / or succinimide-aspartic acid. In one example, the isomerization of aspartic acid (Asp) can be determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described herein. It is understood that these isomerization variant embodiments can be combined with the antibody features described herein.

[0110] In one embodiment, the composition comprises an isomerization variant of an anti-BCMA antibody, wherein the isomerization variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises no more than 25% isomerization variant.

[0111] In one embodiment, the composition comprises a population of anti-BCMA antibodies, the population of anti-BCMA antibodies comprising: An antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:1 (CDRH1), SEQ ID NO:2 (CDRH2), and SEQ ID NO:3 (CDRH3), and a light chain amino acid sequence comprising SEQ ID NO:4 (CDRL1), SEQ ID NO:5 (CDRL2), and SEQ ID NO:6 (CDRL3), and Their isomerized variants , and 25% or less of the antibody population is made up of isomerization variants.

[0112] The composition comprises isomerization variants of an anti-BCMA antibody, the isomerization variants comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises no more than 25% isomerization variants at amino acid D103 of CDRH3.

[0113] In one embodiment, the composition comprises an isomerization variant of an anti-BCMA antibody, wherein the isomerization variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and wherein the composition comprises no more than 25% isomerization variant.

[0114] Alternatively, the isomerization variant comprises the heavy chain sequence of SEQ ID NO: 11, 12, 13 or 14.

[0115] 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: 9 and / or the light chain sequence of SEQ ID NO: 10, and includes an isomerization in either the heavy chain or light chain sequence, e.g., an isomerization at amino acid D103 of CDRH3.

[0116] In another embodiment, the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and comprising an isomerization in at least one of the six CDR regions, for example, an isomerization at amino acid D103 of CDRH3.

[0117] In another embodiment, the anti-BCMA antibody comprises belantamab and comprises an isomerization in either the heavy or light chain sequence, for example, an isomerization at amino acid D103 of CDRH3.

[0118] In one embodiment, a composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 and / or the light chain sequence of SEQ ID NO:10, wherein about 25% or less, about 23% or less, about 20% or less, about 15% or less, about 10% or less, about 8% or less, about 7% or less, about 0.1-25%, about 0.1-20%, about 0.1-15%, about 0.1-10%, about 0.1-8%, about 0.1-7%, about 1-6%, about 2-6%, about 3-6%, about 4%, about 5%, or about 6% of the antibodies in the mixture are isomerized at amino acid D103 of CDRH3. In one embodiment, compositions comprising 25% or less or 23% or less isomerization at D103 of CDRH3 retain 70% or more BCMA-specific antigen binding.

[0119] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein about 25% or less, about 23% or less, about 20% or less, about 15% or less, about 10% or less, about 8% or less, about 7% or less, about 0.1 to 25%, about 0.1 to 20%, about 0.1 to 15%, about 0.1 to 10%, about 0.1 to 8%, about 0.1 to 7%, about 1 to 6%, about 2 to 6%, about 3 to 6%, about 4%, about 5%, or about 6% of the antibodies in the mixture are isomerized at amino acid D103 of CDRH3. In one embodiment, a composition comprising 25% or less, or 23% or less isomerization at D103 of CDRH3 retains 70% or more BCMA-specific antigen binding.

[0120] In another embodiment, the composition comprises belantamab, wherein about 25% or less, about 23% or less, about 20% or less, about 15% or less, about 10% or less, about 8% or less, about 7% or less, about 0.1-25%, about 0.1-20%, about 0.1-15%, about 0.1-10%, about 0.1-8%, about 0.1-7%, about 1-6%, about 2-6%, about 3-6%, about 4%, about 5%, or about 6% of the belantamab isomerized at amino acid D103 of CDRH3. In one embodiment, belantamab with 25% or less or 23% or less isomerization at D103 of CDRH3 retains 70% or more BCMA-specific antigen binding.

[0121] In one embodiment, the composition comprises an isomerization variant of an anti-BCMA antibody, wherein the isomerization variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and wherein the composition comprises no more than 25% isomerization variant.

[0122] In one example, aspartic acid (Asp) isomerization can be determined using trypsin-based peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). In one example, a sample containing a composition described herein can be denatured, for example, with 6 M guanidine HCl to a concentration of, for example, 4.2 μg / μL. Disulfide bonds can then be reduced, for example, with 50 mM DTT at room temperature for 20 minutes. Iodoacetic acid, for example, can then be added, for example, at 100 mM, and reacted with free cysteine ​​residues, for example, at room temperature for 30 minutes, protected from light. The sample can then be buffer-exchanged, for example, using a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin at 37° C. for 15 minutes. The resulting peptides can then be loaded onto a reverse-phase ultra-performance liquid chromatography (UPLC) column and eluted using a gradient of water and acetonitrile, e.g., 0.1% trifluoroacetic acid. The peptides can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Extracted ion chromatograms of the unmodified and modified peptides can be used to calculate the level of isomerization by dividing the area under the curve of the modified peptide by the total area under the curve of both the modified and unmodified peptides.

[0123] In one aspect, the composition comprises an antibody comprising an oxidative post-translational modification ("oxidation" or "oxidized") or "oxidation variant." The variant may comprise oxidized amino acid residues in the heavy and / or light chain sequences, e.g., in the CDRs of the heavy chain sequence and / or the CDRs of the light chain sequence. The oxidation variant may be present in one or both of the heavy and light chains. It is understood that these oxidation variant embodiments can be combined with the antibody features described herein.

[0124] In one embodiment, the composition comprises an oxidized variant of an anti-BCMA antibody, the oxidized variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises no more than 40% oxidized variant.

[0125] In one embodiment, the composition comprises a population of anti-BCMA antibodies, the population of anti-BCMA antibodies comprising: An antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:1 (CDRH1), SEQ ID NO:2 (CDRH2), and SEQ ID NO:3 (CDRH3), and a light chain amino acid sequence comprising SEQ ID NO:4 (CDRL1), SEQ ID NO:5 (CDRL2), and SEQ ID NO:6 (CDRL3), and Their oxidized variants , with up to 40% of the antibody population being made up of oxidized variants.

[0126] In one embodiment, the oxidation variant comprises oxidation in one or more CDRs. In a further embodiment, the oxidation variant comprises oxidation in methionine and / or tryptophan residues in any one of SEQ ID NOs: 1-6.

[0127] In another embodiment, the composition comprises an oxidized variant of an anti-BCMA antibody, the oxidized variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises no more than 40% oxidized variant at amino acid M34 of CDRH1.

[0128] In one embodiment, the composition comprises an oxidized variant of belantamab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and the composition comprises no more than 40% oxidized variant.

[0129] 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: 9 and / or the light chain sequence of SEQ ID NO: 10, and includes an oxidation in the heavy chain sequence, e.g., an oxidation at amino acids M34 (CDRH1), M256, and / or M432.

[0130] In another embodiment, a composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and comprising oxidations in the heavy chain sequence, e.g., oxidations at amino acids M34 (CDRH1), M256, and / or M432.

[0131] In another embodiment, the anti-BCMA antibody comprises belantamab and comprises an oxidation in the heavy chain sequence, for example, an oxidation at amino acids M34 (CDRH1), M256 and / or M432.

[0132] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, wherein about 40% or less, about 35% or less, about 30%, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 7.5% or less, about 5% or less, about 2.5% or less, about 2% or less, about 0.1 to 40%, about 0.1 to 35%, about 0.1 to 30%, about 0.1 to 25%, about 0.1 to 20%, about 0.1 to 15%, about 0.1 to 10%, about 0.1 to 7.5%, about 0.1 to 5%, about 0.1 to 2.5%, about 0.1 to 2%, about 0.5%, about 1%, about 2%, or about 5% of the antibodies in the mixture are oxidized at amino acid M34. In one embodiment, compositions comprising 40% or less heavy chain M34 oxidation retain 70% or more BCMA-specific antigen binding, hi another embodiment, compositions comprising 37% or less heavy chain M34 oxidation retain 70% or more BCMA-specific antigen binding.

[0133] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein the antibodies in the mixture comprise no more than about 40%, no more than about 35% of the antibodies. , about 30%, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 7.5% or less, about 5% or less, about 2.5% or less, about 2% or less, about 0.1-40%, about 0.1-35%, about 0.1-30%, about 0.1-25%, about 0.1-20%, about 0.1-15%, about 0.1-10%, about 0.1-7.5%, about 0.1-5%, about 0.1-2.5%, about 0.1-2%, about 0.5%, about 1%, about 2%, or about 5% are oxidized at amino acid M34. In one embodiment, compositions comprising 40% or less heavy chain M34 oxidation retain 70% or more BCMA-specific antigen binding. In another embodiment, compositions comprising 37% or less heavy chain M34 oxidation retain 70% or more BCMA-specific antigen binding.

[0134] In another embodiment, the composition comprises belantamab, wherein about 40% or less, about 35% or less, about 30%, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 7.5% or less, about 5% or less, about 2.5% or less, about 2% or less, about 0.1 to 40%, about 0.1 to 35%, about 0.1 to 30%, about 0.1 to 25%, about 0.1 to 20%, about 0.1 to 15%, about 0.1 to 10%, about 0.1 to 7.5%, about 0.1 to 5%, about 0.1 to 2.5%, about 0.1 to 2%, about 0.5%, about 1%, about 2%, or about 5% of the belantamab is oxidized at amino acid M34. In one embodiment, belantamab comprising 40% or less heavy chain oxidation at M34 retains 70% or more BCMA-specific antigen binding. In another embodiment, belantamab containing 37% or less oxidation in heavy chain M34 retains 70% or more BCMA-specific antigen binding.

[0135] In one embodiment, the composition comprises an oxidized variant of an anti-BCMA antibody, the oxidized variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises an oxidized variant in at least 90% of the Fc region.

[0136] In one embodiment, the antibody comprises oxidation of methionine and / or tryptophan residues in the Fc region of the heavy chain sequence and / or the Fc region of the light chain sequence, hi some embodiments, the oxidation variant comprises one or a combination of oxidations at M256 and / or M432 in the Fc region of the heavy chain sequence.

[0137] In another embodiment, the composition comprises an oxidized variant of an anti-BCMA antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises 90% or less of the oxidized variant at M256 and / or M432.

[0138] In one embodiment, the composition comprises an oxidized variant of belantamab, wherein the oxidized variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and the composition comprises oxidized variants in up to 90% of the Fc region.

[0139] Alternatively, the oxidation variant comprises the heavy chain sequence of SEQ ID NO: 11, 12, 13 or 14.

[0140] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, wherein about 90% or less, about 80% or less, about 70% or less, about 65% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 7.5% or less, about 5% or less, about 0.1 to 90%, about 0.1 to 80%, about 0.1 to 70%, about 0.1 to 65%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 1 to 10%, about 1 to 5%, about 2 to 10%, about 2 to 4%, about 2%, about 3%, or about 4% of the antibodies in the mixture are oxidized at amino acid M256. In one embodiment, compositions comprising up to 90% or up to 89% heavy chain M256 oxidation retain 70% or more FcγRIIIA binding, hi another embodiment, compositions comprising up to 65% or up to 64% heavy chain M256 oxidation retain 70% or more FcRn binding.

[0141] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein up to about 90%, up to about 80%, up to about 70% or less, about 65% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 7.5% or less, about 5% or less, about 0.1 to 90%, about 0.1 to 80%, about 0.1 to 70%, about 0.1 to 65%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 1 to 10%, about 1 to 5%, about 2 to 10%, about 2 to 4%, about 2%, about 3%, or about 4% are oxidized at amino acid M256. In one embodiment, a composition comprising 90% or less or 89% or less heavy chain M256 oxidation retains 70% or more FcγRIIIA binding. In another embodiment, compositions comprising 65% or less or 64% or less oxidation at heavy chain M256 retain 70% or more FcRn binding.

[0142] In another embodiment, the composition comprises belantamab, and about 90% or less, about 80% or less, about 70% or less, about 65% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 7.5% or less, about 5% or less, about 0.1 to 90%, about 0.1 to 80%, about 0.1 to 70%, about 0.1 to 65%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 1 to 10%, about 1 to 5%, about 2 to 10%, about 2 to 4%, about 2%, about 3%, or about 4% of the belantamab is oxidized at amino acid M256. In one embodiment, belantamab containing 90% or less or 89% or less heavy chain M256 oxidation retains 70% or more FcγRIIIA binding, hi another embodiment, belantamab containing 65% or less or 64% or less heavy chain M256 oxidation retains 70% or more FcRn binding.

[0143] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, wherein about 86% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 7.5% or less, about 5% or less, about 2.5% or less, about 2% or less, about 0.1 to 86%, about 0.1 to 70%, about 0.1 to 60%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 3%, about 0.5%, about 1%, about 2%, or about 3% of the antibodies in the mixture are oxidized at amino acid M432. In one embodiment, a composition comprising 86% or less heavy chain M432 oxidation retains 70% or more FcγRIIA binding, hi another embodiment, a composition comprising 60% or less heavy chain M432 oxidation retains 70% or more FcRn binding.

[0144] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein no more than about 86%, more than about 70% of the antibodies in the mixture About 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 7.5% or less, about 5% or less, about 2.5% or less, about 2% or less, about 0.1 to 86%, about 0.1 to 70%, about 0.1 to 60%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 3%, about 0.5%, about 1%, about 2%, or about 3% are oxidized at amino acid M432. Compositions containing 86% or less heavy chain M432 oxidation retain 70% or more FcγRIIIA binding. In another embodiment, compositions containing 60% or less heavy chain M432 oxidation retain 70% or more FcRn binding.

[0145] In another embodiment, the composition comprises belantamab, wherein about 86% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 7.5% or less, about 5% or less, about 2.5% or less, about 2% or less, about 0.1 to 86%, about 0.1 to 70%, about 0.1 to 60%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 3%, about 0.5%, about 1%, about 2%, or about 3% of the belantamab is oxidized at amino acid M432. In one embodiment, belantamab with 86% or less heavy chain oxidation at M432 retains 70% or more FcγRIIIA binding. In another embodiment, belantamab containing 60% or less oxidation at heavy chain M432 retains 70% or more FcRn binding.

[0146] In one example, oxidation can be determined using trypsin-based peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). In one example, a sample containing a composition described herein can be denatured, for example, with 6 M guanidine HCl to a concentration of, for example, 4.2 μg / μL. Disulfide bonds can then be reduced, for example, with 50 mM DTT at room temperature for 20 minutes. Iodoacetic acid, for example, can then be added at 100 mM and reacted with free cysteine ​​residues, for example, at room temperature for 30 minutes, protected from light. The sample can then be buffer exchanged, for example, using a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin at 37°C for 15 minutes. The resulting peptides can then be loaded onto a reverse-phase ultra-performance liquid chromatography (UPLC) column and eluted using UPLC with a gradient of water and acetonitrile, for example, with 0.1% trifluoroacetic acid. The peptides can then be detected with a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Extracted ion chromatograms of the unmodified and modified peptides are used to calculate the level of oxidation by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0147] In one aspect, the composition comprises an antibody or "deamidation variant" comprising a deamidation post-translational modification ("deamidation" or "deamidated"). In one embodiment, the antibody comprises deamidation of asparagine residues in the CDRs of the heavy chain sequence and / or the CDRs of the light chain sequence. In a further embodiment, the antibody comprises deamidation of asparagine residues in the CDRs of the heavy chain sequence. In one embodiment, the antibody comprises deamidation of asparagine residues in the Fc region of the heavy chain sequence and / or the Fc region of the light chain sequence. The deamidation variant can be present in one or both of the heavy and light chains. It is understood that these deamidation variant embodiments can be combined with the antibody features described herein. In some embodiments, the deamidation variant comprises one or a combination of deamidation at N388 and / or N393 in the Fc region of the heavy chain sequence.

[0148] In one embodiment, the deamidated variant comprises a deamidated residue selected from an aspartic acid residue, a succinimido-aspartic acid residue, or an isoaspartic acid residue.

[0149] 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: 9 and / or the light chain sequence of SEQ ID NO: 10, and includes deamidation in the heavy chain sequence, e.g., deamidation at amino acids N388 and / or N393.

[0150] In one embodiment, the composition comprises a deamidated variant of an anti-BCMA antibody, the deamidated variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises up to 100% deamidated variant.

[0151] In another embodiment, the composition comprises a deamidated variant of an anti-BCMA antibody, wherein the oxidized variant comprises a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises up to 100% deamidated variants at N388 and / or N393.

[0152] In one embodiment, the composition comprises a deamidated variant of belantamab, wherein the deamidated variant comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10, and the composition comprises up to 100% of the deamidated variant. In another embodiment, the composition comprises a deamidated variant comprising a heavy chain sequence of SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and a light chain sequence of SEQ ID NO: 10.

[0153] In another embodiment, a composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and comprising deamidation in the heavy chain sequence, e.g., deamidation at amino acid N388 and / or N393.

[0154] In another embodiment, the anti-BCMA antibody comprises belantamab and comprises a deamidation in the heavy chain sequence, for example, deamidation at amino acids N388 and / or N393.

[0155] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, wherein about 100% or less, about 75% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, about 0.1 to 100%, about 0.1 to 75%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 3%, about 0.5%, about 1%, about 2%, about 5%, or about 10% of the antibodies in the mixture are deamidated at amino acid N388.

[0156] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein about 100% or less, about 75% or less of the antibodies in the mixture are CDRH1, CDRH2, CDRH3, and CDRL3. less than about 60%; less than about 50%; less than about 40%; less than about 30%; less than about 25%; less than about 20%; less than about 15%; less than about 10%; less than about 5%; less than about 2%; about 0.1 to 100%; about 0.1 to 75%; about 0.1 to 50%; about 0.1 to 40%; about 0.1 to 30%; about 0.1 to 20%; about 0.1 to 10%; about 0.1 to 5%; about 0.1 to 3%; about 0.5%; about 1%; about 2%; about 5%; or about 10% is deamidated at amino acid N388.

[0157] In another embodiment, the composition comprises belantamab, wherein about 100% or less, about 75% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, about 0.1 to 100%, about 0.1 to 75%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 3%, about 0.5%, about 1%, about 2%, about 5%, or about 10% of the belantamab is deamidated at amino acid N388.

[0158] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, wherein about 100% or less, about 85% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, about 0.1 to 100%, about 0.1 to 75%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 3%, about 0.5%, about 1%, about 2%, about 5%, or about 10% of the antibodies in the mixture are deamidated at amino acid N393.

[0159] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein up to about 100%, about 85%, or less of the antibodies in the mixture are CDRH1, CDRH2, CDRH3, and CDRL3. less than about 70%; less than about 60%; less than about 50%; less than about 40%; less than about 30%; less than about 20%; less than about 15%; less than about 10%; less than about 5%; less than about 2%; about 0.1 to 100%; about 0.1 to 75%; about 0.1 to 50%; about 0.1 to 40%; about 0.1 to 30%; about 0.1 to 20%; about 0.1 to 10%; about 0.1 to 5%; about 0.1 to 3%; about 0.5%; about 1%; about 2%; about 5%; or about 10% is deamidated at amino acid N393.

[0160] In another embodiment, the composition comprises belantamab, wherein about 100% or less, about 85% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, about 0.1 to 100%, about 0.1 to 75%, about 0.1 to 50%, about 0.1 to 40%, about 0.1 to 30%, about 0.1 to 20%, about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 3%, about 0.5%, about 1%, about 2%, about 5%, or about 10% of the belantamab is deamidated at amino acid N393.

[0161] In one example, deamidation can be determined using trypsin-mediated peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). In one example, a sample containing a composition described herein can be denatured, for example, with 6 M guanidine HCl to a concentration of, for example, 4.2 μg / μL. Disulfide bonds can then be reduced, for example, with 50 mM DTT at room temperature for 20 minutes. Iodoacetic acid, for example, can then be added at 100 mM and reacted with free cysteine ​​residues, for example, at room temperature for 30 minutes, protected from light. The sample can then be buffer exchanged, for example, using a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin at 37°C for 15 minutes. The resulting peptides can then be loaded onto a reverse-phase ultra-performance liquid chromatography (UPLC) column and eluted using UPLC with a gradient of water and acetonitrile, for example, with 0.1% trifluoroacetic acid. The peptides can then be detected with a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Extracted ion chromatograms of the unmodified and modified peptides are used to calculate the level of deamidation by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0162] In one embodiment, the post-translational modification is an antibody sequence variant. Exemplary post-translational modified antibody sequence variants include an asparagine (N) to aspartic acid (D) switch, truncation of N-terminal pyroglutamic acid and / or C-terminal lysine.

[0163] In one example, antibody variants, such as N103D in CDRH3, can be determined using trypsin-based peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). In one example, a sample containing a composition described herein can be denatured, for example, with 6 M guanidine HCl to a concentration of, for example, 4.2 μg / μL. Disulfide bonds can then be reduced, for example, with 50 mM DTT at room temperature for 20 minutes. Iodoacetic acid, for example, can then be added, for example, at 100 mM, and reacted with free cysteine ​​residues, for example, at room temperature for 30 minutes, protected from light. The sample can then be buffer-exchanged, for example, using a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin at 37° C. for 15 minutes. The resulting peptides can then be loaded onto a reverse-phase ultra-performance liquid chromatography (UPLC) column and eluted with a gradient of water and acetonitrile, e.g., 0.1% trifluoroacetic acid. The peptides can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Extracted ion chromatograms of the unmodified and modified peptides can be used to calculate the level of the antibody variant, e.g., N103D in CDRH3, by dividing the area under the curve of the modified peptide by the total area under the curve of both the modified and unmodified peptides.

[0164] In one aspect, a composition comprises an antibody comprising an N-terminal pyroglutamic acid ("pyroglutamic acid") post-translational modification in its heavy chain amino acid sequence. In one embodiment, a composition comprises an antibody that is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 and / or the light chain sequence of SEQ ID NO:10, and comprises pyroglutamic acid at the N-terminus of the heavy chain.

[0165] In another embodiment, the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and comprising pyroglutamic acid at the N-terminus of the heavy chain.

[0166] In another embodiment, the anti-BCMA antibody comprises belantamab and comprises a pyroglutamic acid at the N-terminus of the heavy chain.

[0167] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, and about 25% or more, about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, or about 50% or less of the antibodies in the mixture comprise a pyroglutamic acid at the N-terminus of their heavy chain amino acid sequence.

[0168] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein about 25% or more, about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, or about 50% or less of the antibodies in the mixture comprise pyroglutamic acid at the N-terminus of their heavy chain amino acid sequences.

[0169] In one embodiment, the composition comprises belantamab, wherein about 25% or more, about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, or about 50% or less of the belantamab comprises pyroglutamic acid at the N-terminus of its heavy chain amino acid sequence.

[0170] In one example, N-terminal pyroglutamic acid can be determined using trypsin-based peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). In one example, a sample containing a composition described herein can be denatured, for example, with 6 M guanidine HCl to a concentration of, for example, 4.2 μg / μL. Disulfide bonds can then be reduced, for example, with 50 mM DTT at room temperature for 20 minutes. Iodoacetic acid, for example, can then be added at 100 mM and reacted with free cysteine ​​residues, for example, at room temperature for 30 minutes, protected from light. The sample can then be buffer-exchanged, for example, using a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin at 37° C. for 15 minutes. The resulting peptides can then be loaded onto a reverse-phase ultra-performance liquid chromatography (UPLC) column and eluted with a gradient of water and acetonitrile, e.g., 0.1% trifluoroacetic acid solution, using UPLC. The peptides can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Extracted ion chromatograms of the unmodified and modified peptides can be used to calculate the level of pyroglutamic acid by dividing the area under the curve of the modified peptide by the total area under the curve of both the modified and unmodified peptides.

[0171] In one aspect, the composition comprises an antibody comprising a post-translational modification in the heavy chain amino acid sequence that is a C-terminal lysine truncation. 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: 9 and / or the light chain sequence of SEQ ID NO: 10, and comprises a C-terminal lysine truncation in the heavy chain.

[0172] In another embodiment, the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and comprising a truncation of the C-terminal lysine of the heavy chain.

[0173] In another embodiment, the anti-BCMA antibody comprises belantamab and includes a truncation of the C-terminal lysine of the heavy chain.

[0174] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, and wherein about 25% or more, about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, or about 50% or less of the antibodies in the mixture comprise C-terminal lysine truncations.

[0175] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein about 25% or more, about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, or about 50% or less of the antibodies in the mixture comprise truncation of the C-terminal lysine of their heavy chains.

[0176] In one embodiment, the composition comprises belantamab, wherein about 25% or more, about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, or about 50% or less of the belantamab comprises cleavage of the C-terminal lysine of the heavy chain.

[0177] In one example, C-terminal lysine cleavage can be determined using trypsin-mediated peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). In one example, a sample containing a composition described herein can be denatured, for example, with 6 M guanidine HCl to a concentration of, for example, 4.2 μg / μL. Disulfide bonds can then be reduced, for example, with 50 mM DTT at room temperature for 20 minutes. Iodoacetic acid, for example, can then be added at 100 mM and reacted with free cysteine ​​residues, for example, at room temperature for 30 minutes, protected from light. The sample can then be buffer exchanged, for example, using a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin at 37°C for 15 minutes. The resulting peptides can then be loaded onto a reverse-phase ultra-performance liquid chromatography (UPLC) column and eluted using UPLC with a gradient of water and acetonitrile, for example, with 0.1% trifluoroacetic acid. The peptides can then be detected with a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Extracted ion chromatograms of the unmodified and modified peptides are used to calculate the level of C-terminal lysine cleavage by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0178] In one aspect, a composition comprises an antibody or glycosylation variant comprising a glycosylation post-translational modification ("glycosylation modification"). Exemplary glycosylation modifications include altered expression of G0, G1, G0-GlcNac, G2, and sialylation on the antibody. In one embodiment, a composition comprises an antibody that is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 and / or the light chain sequence of SEQ ID NO:10, and comprises a glycosylation modification.

[0179] In another embodiment, the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, including glycosylation variants.

[0180] In another embodiment, the anti-BCMA antibody comprises belantamab and includes a glycosylation variant.

[0181] In one embodiment, the composition comprises a mixture of antibodies that are at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10, and the composition comprises a GO level of about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 0-100%, about 1-100%, about 30-100%, about 40-90%, about 50-80%, or 55-80%; about 2.5% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 50% or more, about 0-100%, about 1-100%, about 0-50%. %, about 1-50%, about 1-40%, about 1-35% or 8-31%; G1 at a level of about 5% or less, about 7.5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 40% or less, about 50% or less, about 75% or less, about 0-100%, about 0.5-100%, about 0-50%, about 0.5-50%, about 0.5-25%, about 0.5-10%, about 0.5-7.5% or about 0.9-5.3%; G2 at a level of 0-100%, 1-100% or 39-92%; and / or G0-2GlcNac at a level of 0-100%, 1-100% or 38-88%.

[0182] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein the composition has a GO level of about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 0-100%, about 1-100%, about 30-100%, about 40-90%, about 50-80%, or 55-80%; about 2.5% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 0-100%, about 1-100%, about 30-100%, about 40-90%, about 50-80%, or 55-80%; % or more, about 20% or more, about 25% or more, about 30% or more, about 50% or more, about 0-100%, about 1-100%, about 0-50%, about 1-50%, about 1-40%, about 1-35%, or 8-31% of G1; about 5% or less, about 7.5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 40% or less, about 50% or less, about 75% or less, G0-GlcNac at a level of about 0-100%, about 0.5-100%, about 0-50%, about 0.5-50%, about 0.5-25%, about 0.5-10%, about 0.5-7.5% or about 0.9-5.3%; G2 at a level of 0-100%, 1-100% or 39-92%; and / or G0-2GlcNac at a level of 0-100%, 1-100% or 38-88%.

[0183] In one embodiment, the composition comprises belantamab, and the composition has a level of G0 of about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 0-100%, about 1-100%, about 30-100%, about 40-90%, about 50-80%, or 55-80%; about 2.5% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 50% or more, about 0-100%, about 1-100%, about 0-50%, about 1-50%, about 1-40%, about 1-35%, or 8%. G1 at a level of about 31% or less; G0-GlcNac at a level of about 5% or less, about 7.5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 40% or less, about 50% or less, about 75% or less, about 0-100%, about 0.5-100%, about 0-50%, about 0.5-50%, about 0.5-25%, about 0.5-10%, about 0.5-7.5% or about 0.9-5.3%; G2 at a level of 0-100%, 1-100% or 39-92%; and / or G0-2GlcNac at a level of 0-100%, 1-100% or 38-88%.

[0184] In one embodiment, the composition comprises a mixture of antibodies that are 100% non-fucosylated, hi another embodiment, the composition comprises a mixture of antibodies that are 0% fucosylated.

[0185] In one example, glycosylation modifications and resulting profiles can be determined using ultra-performance liquid chromatography (UPLC) with hydrophilic interaction liquid chromatography (HILIC) separation and fluorescence detection. In one example, a composition described herein, such as a composition described herein containing belantamab, can be diluted with water to a concentration of 10 μg / μL. Glycans can then be released from the composition, such as a composition containing belantamab, by enzymatic digestion with PNGaseF using a PNGaseF kit (catalog number P0705L) from New England BioLabs. The glycans can be released with PNGaseF and labeled with anthranilamide (Sigma-Aidrich, catalog number A89804). The labeled glycans can then be purified to remove excess labeling solution using a HILIC column step (where the glycans can be loaded, washed with water, and eluted with acetonitrile). The labeled glycans can then be separated using a Waters Acquity UPLC with a Waters Glycan BEH Amide column (catalog number 186004742) using an ammonium formate / formic acid and acetonitrile gradient. The glycans can then be detected, for example, by using fluorescence detection with excitation at 365 nm and emission at 438 nm. Quantitation of the glycans can be performed, for example, by dividing the area under the curve for a glycan by the total area under the curve for all detected glycans.

[0186] In one embodiment, the composition comprises an antibody that is an aggregated antibody (high molecular weight (HMW) species), also referred to herein as an "aggregated variant." Aggregated antibodies can include dimers or higher-order structures formed from antibody monomers and their subunits. Aggregated variants can be, for example, covalent or non-covalent, reducible or non-reducible, and visible or subvisible aggregates of the antibodies described herein. Aggregated or fragmented variants can be characterized based on their size and can be distinguished from antibodies. For example, the size distribution of an antibody composition can be detected using size exclusion chromatography (SEC), e.g., SE-HPLC.

[0187] In one aspect, the composition comprises aggregate variants of an antibody, wherein the aggregate variants comprise a heavy chain sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises no more than 10% aggregate variants.

[0188] The antibody composition may contain 10% or less aggregate variants, for example, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less aggregate variants. In other embodiments, the composition may contain 1-10%, 1-5%, 1-4%, 1-3%, or 1-2% aggregate variants. Alternatively, the composition may contain more than 1% but less than 10% aggregate variants. Alternatively, the composition may contain about 7.5%, about 5%, about 4%, about 3%, about 2%, or about 1% aggregate variants.

[0189] Fragmented variants ("fragment variants") are variants that contain a portion of a full-length antibody. For example, such fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and immunoglobulin single variable domains.

[0190] In one aspect, the composition comprises a fragment variant of an antibody, wherein the fragment variant comprises a heavy chain sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2 and a CDRH3 of SEQ ID NO: 3, and a light chain sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5 and a CDRL3 of SEQ ID NO: 6, wherein the composition comprises no more than 10% fragment variant.

[0191] The antibody composition may contain 10% or less of antibody fragments, for example, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of antibody fragments. In another embodiment, the composition may contain 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, 0.5-1.5%, or 0.5-1% of antibody fragments. Alternatively, the composition may contain about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% of antibody fragments.

[0192] The composition may comprise any one or combination of acidic variants, basic variants, isomerized variants, oxidized variants, deamidated variants, N-terminal pyroglutamic acid variants, C-terminal lysine truncated variants, and / or any percentage of glycosylation modified variants, and / or aggregated and / or fragmented variants described herein.

[0193] In one embodiment, the composition has BCMA-specific antigen binding of 70% or greater, FcγRIIIa binding of 70% or greater, and / or FcRn binding of 70% or greater.

[0194] In another embodiment, the composition has about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more BCMA-specific antigen binding. In another embodiment, the composition has about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more FcγRIIIa binding. In another embodiment, the composition has about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more FcRn binding.

[0195] In another embodiment, the composition has about 70-130% specific antigen binding, about 70-130% FcγRIIIa binding, and / or about 70-130% FcRn binding.

[0196] In some embodiments, the composition has about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 70%, about 80%, about 90%, 100%, about 110%, about 120%, or about 130% specific antigen binding. In some embodiments, the composition has about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 90%, about 95%, about 100%, about 105%, or about 110% FcγRIIIa binding. In some embodiments, the composition has about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 90%, about 95%, about 100%, about 105%, or about 110% FcRn binding.

[0197] In another embodiment, the activity of a composition comprising the variant is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of belantamab, which is 100% active. In one aspect, a composition comprises a variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6, wherein the potency of the composition is at least 70% of a composition comprising a heavy chain sequence of SEQ ID NO: 9, 11, 12, 13, or 14 and a light chain sequence of SEQ ID NO: 10, wherein the potency of the composition comprises any one or combination of: (i) up to 23% isomerization at D103, and / or (ii) up to 37% oxidation at M34.

[0198] In another aspect, the composition comprises a variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6, wherein the potency of the composition is at least 70% of a composition comprising a heavy chain sequence of SEQ ID NO: 9, 11, 12, 13, or 14 and a light chain sequence of SEQ ID NO: 10, wherein the potency of the composition is at least 70% of a composition comprising any one or combination of: (i) up to 23% isomerization at D103; (ii) up to 37% oxidation at M34; (iii) up to 64% oxidation at M256; (iv) up to 61% oxidation at M432; (v) up to 100% deamidation at N388; and / or (vi) up to 100% deamidation at N393. In yet another aspect, the composition comprises a variant comprising a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3, and a light chain amino acid sequence comprising a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6, wherein the potency of the composition is a composition comprising a heavy chain sequence of SEQ ID NO: 9, 11, 12, 13, or 14 and a light chain sequence of SEQ ID NO: 10, wherein the composition exhibits (i) up to 23% isomerization at D103, (ii) up to 23% isomerization at D103, (iii) up to 23% isomerization at D103, and (iv) up to 23% isomerization at D103. At least 70% of the composition comprises any one or combination of: (i) up to 37% oxidation at M34; (ii) up to 64% oxidation at M256; (iii) up to 64% oxidation at M256; (iv) up to 61% oxidation at M432; (v) up to 100% deamidation at N388; (vi) up to 100% deamidation at N393; (vii) up to 100% truncation of lysine at the C-terminus of HC; and / or (viii) up to 100% pyroglutamic acid at the N-terminus of HC.

[0199] In one example, the binding of belantamab mafodotin to BCMA and FcγRIIIa is measured using surface plasmon resonance (SPR). Belantamab mafodotin can be diluted to 10 μg / mL in PBST, injected, and captured by Protein A immobilized on a CM5 sensor chip. BCMA is then injected and allowed to bind to the captured belantamab mafodotin. FcγRIIIa is then injected and allowed to bind to the captured belantamab mafodotin. The functional concentrations of belantamab mafodotin bound to BCMA and FcγRIIIa can be calculated from a reference standard curve and reported as BCMA or FcγRIIIa binding concentrations, respectively. The total belantamab mafodotin concentration of the sample is previously determined by absorbance at 280 nm. Specific binding activity (%) can be calculated by dividing the bound BCMA or FcγRIIIa concentration by the absorbance at 280 nm.

[0200] The binding of fetal Fc receptor (FcRn) to anti-BCMA antigen-binding proteins, such as belantamab, can be measured using surface plasmon resonance (SPR). Belantamab can be captured by FcRn immobilized on a nitrilotriacetic acid (NTA) sensor chip. The FcRn binding concentration of the sample can be determined by interpolating the binding response on a standard curve. The specific binding activity (%) is calculated by dividing the FcRn binding concentration by the total protein concentration.

[0201] Among other methods known to those skilled in the art, the binding of BCMA and FcγRIIIa by anti-BCMA antigen-binding proteins, such as belantamab mafodotin, can be measured using surface plasmon resonance (SPR). In one example, belantamab mafodotin is injected and captured by Protein A immobilized on a CM5 sensor chip. BCMA is then injected and allowed to bind to the captured belantamab mafodotin. FcγRIIIa is then injected and allowed to bind to the captured belantamab mafodotin. The functional concentrations of belantamab mafodotin bound to BCMA and FcγRIIIa can be calculated from a reference standard curve and reported as BCMA or FcγRIIIa-bound concentrations, respectively. The total belantamab mafodotin concentration of the sample can be previously determined by absorbance at 280 nm. Specific binding activity (%) can be calculated by dividing the bound concentration of BCMA or FcγRIIIa by the absorbance, for example, the absorbance at 280 nm at that concentration.

[0202] In certain embodiments, the average DAR or DL ​​ratio affects binding to FcRn. In another embodiment, the average DAR or DL ​​ratio does not affect binding to FcRn. In yet another embodiment, a composition comprises belantamab mafodotin, and the average DAR or DL ​​ratio affects binding to FcRn. In yet another embodiment, a composition comprises belantamab mafodotin, and the average DAR or DL ​​ratio does not affect binding to FcRn. In one embodiment, the average DAR or DL ​​ratio may weaken binding to FcRn.

[0203] Binding of fetal Fc receptor (FcRn) to anti-BCMA antigen-binding proteins, such as belantamab mafodotin, can be measured using surface plasmon resonance (SPR). Belantamab mafodotin can be captured by FcRn immobilized on a nitrilotriacetic acid (NTA) sensor chip. The FcRn binding concentration of a sample can be determined by interpolation of the binding response on a standard curve. Specific binding activity (%) is calculated by dividing the FcRn binding concentration by the total protein concentration.

[0204] When the anti-BCMA antigen-binding protein comprises belantamab mafodotin, the SPR methods described herein for specific antigen binding, FcγRIIIa, and FcRn binding can use a belantamab or belantamab mafodotin reference standard. The belantamab or belantamab mafodotin reference standard can be used in the assay to obtain system suitability and sample comparability data and ensure that the method is performing properly. The reference standard can generate a calibration curve from which sample concentrations are interpolated. For example, the reference standard can be a composition comprising a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10.

[0205] Antibody compositions comprising the above-described antibodies and antibody variants retain specific antigen binding and / or FcRn binding and / or FcγRIIIa binding and / or potency. For example, an antibody composition comprising the above-described antibodies and antibody variants and post-translationally modified variants has BCMA-specific antigen binding of greater than 0.70, and / or greater than 70% FcRn binding, and / or 70% FcγRIIIa binding, and / or greater than 70% potency. Thus, variants at these levels (%) can be tolerated in antibody compositions without significantly affecting function (i.e., without reducing activity). In one embodiment, "reduced function" or "reduced activity" refers to a percentage reduction in BCMA binding, FcRn binding, FcγRIIIa binding, or potency compared to a reference standard that is significant relative to assay variability. For example, a decrease in function or activity or efficacy can be described as a decrease of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more.

[0206] In another embodiment, the reference sample standard is a composition comprising a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more heavy chain C-terminal lysine truncation and 100% or less heavy chain N-terminal pyroglutamic acid. In a further embodiment, the reference sample standard is a composition comprising a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more heavy chain C-terminal lysine truncation, 100% or less heavy chain N-terminal pyroglutamic acid, and 7% or less isomerization at amino acid D103 of CDRH3. In a further embodiment, the reference sample standard is a composition comprising a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more heavy chain C-terminal lysine truncation, 100% or less heavy chain N-terminal pyroglutamic acid, 7% or less isomerization at amino acid D103 of CDRH3, and 5% or less oxidation at amino acids M34, M256, and / or M432. In a further embodiment, the reference sample standard is a composition comprising a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more heavy chain C-terminal lysine truncation, 100% or less heavy chain N-terminal pyroglutamic acid, 7% or less isomerization at amino acid D103 of CDRH3, 5% or less oxidation at amino acids M34, M256, and / or M432, and 2% or less deamidation at amino acids N388 and / or N393. In a further embodiment, the reference sample standard is a composition comprising a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, which composition comprises 80% or more cleavage of heavy chain C-terminal lysine, 100% pyroglutamic acid at the heavy chain N-terminus, 7% or less isomerization at amino acid D103 of CDRH3, 5% or less oxidation at M256, 2% or less oxidation at M34 and M432, and 2% or less deamidation at amino acids N388 and N393.

[0207] Antibody-drug conjugates (ADCs) Antibody-drug conjugates (ADCs) are a new class of potent anticancer agents that have recently demonstrated remarkable clinical benefits. ADCs consist of a cytotoxic drug chemically attached to an antibody via a linker. Presumably, ADCs can destroy cancer cells overexpressing cell surface proteins through a series of events including antigen binding on the cell surface, endocytosis, transport to lysosomes, ADC degradation, payload release, disruption of cellular processing (e.g., mitosis), and apoptosis. ADCs combine the antigen-driven targeting properties of monoclonal antibodies with the potent antitumor effects of cytotoxic drugs. For example, in 2011, ADCETRIS® (an anti-CD30 antibody-MMAE ADC) received regulatory approval for the treatment of refractory Hodgkin's lymphoma and systemic anaplastic lymphoma.

[0208] ADCs have been used for the local delivery of cytotoxic drugs, i.e., drugs that damage or inhibit cell growth or proliferation, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278). ADCs allow targeted delivery of drug moieties to tumors and their intracellular accumulation there; systemic administration of unconjugated drugs can result in unacceptable levels of toxicity to normal cells and the tumor cells being eliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (A. Pinchera et al., eds) pp. 475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother. 21:183-87). Toxins used in antibody-toxin conjugates include bacterial toxins, e.g., diphtheria toxin, plant toxins, e.g., ricin, small molecule toxins, e.g., geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342).

[0209] In one embodiment, the BCMA antigen binding protein is an antibody-drug conjugate ("anti-BCMA ADC") comprising an antibody or antibody fragment conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0210] In one embodiment, the anti-BCMA ADC has the following general structure: ABP-((linker) n -Ctx) m [In the formula, ABP is an antigen-binding protein, the linker is absent or any cleavable or non-cleavable linker; Ctx is any cytotoxic agent described herein; n is 0, 1, 2 or 3, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. It has.

[0211] In exemplary embodiments, enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria oficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. See, e.g., WO 93 / 21232, published October 28, 1993. Various radionuclides, e.g. 211 At, 212 Bi, 131 I, 131 In, 90 Y and 186 Re is available for the production of radioconjugated antibodies.

[0212] The anti-BCMA antibodies of the invention, or fragments thereof, may also be conjugated to one or more cytotoxic agents, including, but not limited to, calicheamicin, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, or derivatives of these toxins that have toxic activity. Suitable cytotoxic agents include, for example, auristatins such as dovaline-valine-dolaisoleunine-dolaproinephenylalanine (MMAF) and monomethyl auristatin E (MMAE) and ester forms of MMAE; DNA minor groove binders; DNA minor groove alkylating agents; enediynes; lexitropsins; duocarmycins; taxanes such as paclitaxel and docetaxel; puromycins; dolastatins; maytansinoids; and vinca alkaloids. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatostatin, calicheamicin, maytansine, DM-1, DM-4, ​​and netropsin. Other suitable cytotoxic agents include antitubulin agents (e.g., auristatins), vinca alkaloids, podophyllotoxins, taxanes, baccatin derivatives, cryptophysins, maytansinoids, combretastatins, or dolastatins. Antitubulin agents include dimethylvaline-valinedolaisoloiin-dolaproine-phenylalanine-p-phenylenediamine (AFP), MMAF, MMAE, auristatin E, vincristatine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-1, DM-4, ​​or eleutherobin.

[0213] In one embodiment, the anti-BCMA ADC comprises an anti-BCMA antibody linked to MMAE or MMAF.

[0214] [ka]

[0215] The cleavable linker may be susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include peptide linkers cleavable by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit) or phenylalanine-lysine (phe-lys) linker. Other suitable linkers include, for example, linkers hydrolyzable at pH below 5.5, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers. Exemplary linkers include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

[0216] In one embodiment, the linker may comprise a thiol-reactive maleimide, a caproyl spacer, the dipeptide valine-5 citrulline, p-aminobenzyloxycarbonyl, a self-immolative fragmenting group, or a protease-resistant maleimidocaproyl.

[0217] In another embodiment, the anti-BCMA ADC comprises an anti-BCMA antibody linked to MMAE or MMAF by an MC linker, as shown in the structure below:

[0218] [ka]

[0219] The anti-BCMA ADCs described herein may comprise any of the anti-BCMA antibodies described herein together with any of the cytotoxic agents described herein.

[0220] In one embodiment, the anti-BCMA ADC comprises a CDRH1 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; a CDRH2 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; a CDRH4 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and / or CDRL3 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6, wherein the CDRL1 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 set forth in SEQ ID NO:5; CDRL2 comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6, wherein the CDRL3 is conjugated to MMAE or MMAF.

[0221] In yet another embodiment, an anti-BCMA ADC comprises an anti-BCMA antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, conjugated to MMAF or MMAE.

[0222] In one embodiment, the anti-BCMA ADC 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 set forth in SEQ ID NO:7. H and / or V comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. L and an anti-BCMA antibody comprising the compound conjugated to MMAE or MMAF.

[0223] In yet another embodiment, the anti-BCMA ADC has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8 L and an anti-BCMA antibody comprising the compound conjugated to MMAF or MMAE.

[0224] In one embodiment, an anti-BCMA ADC comprises an anti-BCMA antibody comprising an HC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; and / or an LC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:10, conjugated to MMAF or MMAE.

[0225] In yet another embodiment, the anti-BCMA ADC is belantamab mafodotin comprising an HC having the amino acid sequence set forth in SEQ ID NO: 9; and an LC having the amino acid sequence set forth in SEQ ID NO: 10, conjugated to MMAF.

[0226] Preparation and characterization of ADCs A particular native IgG1 molecule contains 16 disulfide bonds (32 cysteine ​​or sulfhydryl groups). In certain embodiments, the antibody can be reduced to reduce only four interchain disulfide bonds and conjugate to a cytotoxic drug, allowing for up to eight attachment sites for the cytotoxic drug. In other words, the drug load ("DL"), i.e., the number of cytotoxic drugs per antibody molecule, can range from 0 to 8, and are referred to herein as DL0, DL2 (including DL2a and DL2b), DL4 (including DL4a, DL4b, and DL4c), DL6 (including DL6a and DL6b), and DL8.

[0227] The conjugation process can result in heterogeneity of drug-antibody linkages for a given ADC composition, varying both in 1) the number of drugs attached to each antibody molecule and in 2) the location of the cytotoxic drug. This can result in an ADC composition with various DL species, as illustrated in Figure 1. As used herein, the term "ADC composition" refers to a composition containing a heterogeneous mixture of antibody species with various drug loading numbers ("DL") (see, e.g., Figure 2). The average drug-antibody ratio across a heterogeneous ADC composition is referred to herein as the "average DAR" or "DAR." For example, an ADC composition may contain a mixture of antibody species, each with its own DL (some species in the mixture are DL2, some species in the mixture are DL4, some species in the mixture are DL6, and some species in the mixture are DL8), and the average DAR across the composition may be about 4.

[0228] In another embodiment, the term "proportion of DL" may be used to describe the proportion of a particular DL species in a heterogeneous ADC composition (e.g., the proportion of DL2 is about 10% to about 30% of the total heterogeneous ADC composition).

[0229] In certain embodiments of the present invention, a drug can be conjugated to an antibody via a sulfhydryl group on the antibody. The sulfhydryl group can be a sulfhydryl group of a cysteine ​​side chain. The cysteine ​​residue can be naturally present in the antibody (e.g., an interchain disulfide) or can be introduced by other means, such as mutagenesis. Methods for conjugating a drug to a sulfhydryl group on an antibody are well known in the art (see, for example, U.S. Pat. Nos. 7,659,241 and 7,498,298, and International Publication Nos. WO2011 / 130613, WO2014 / 152199, WO2015 / 077605, and Bioconjugate Chem. 2005, 16, 1282-1290). The antibody is usually reduced prior to conjugation to make the sulfhydryl group available for conjugation. The antibody can be reduced using conditions known in the art. Reducing conditions are conditions that generally do not cause substantial denaturation of the antibody and generally do not affect the antigen binding affinity of the antibody.

[0230] In one embodiment of the present invention, the reducing agent used in the reduction step is TCEP (tris(2-carboxyethyl)phosphine), which is added in excess for, for example, 30 minutes at room temperature. For example, 250 μL of a 10 mM TCEP solution at pH 7.4 readily reduces the interchain disulfides of 1 to 100 μg of antibody in 30 minutes at room temperature. However, other reducing agents and conditions may also be used. Example reaction conditions include a pH of 5 to 8 and a temperature of 5°C to 37°C.

[0231] Various methods exist for calculating the proportion and / or average DAR of DL species in an ADC composition and are known to those skilled in the art. For example, the heterogeneity of cysteine-linked ADCs is typically measured by hydrophobic interaction chromatography (HIC). This chromatography separates DL species based on the number of drugs loaded. LC-MS assays have also been developed to assess DL distribution. An exemplary method for calculating the drug loading number distribution in an ADC composition can be found, for example, in Journal of Chromatography B 1060 (2017) 182-189.

[0232] For example, DL0 does not carry a drug on the antibody. For example, the number of drugs carried by DL2 is 2. In one embodiment, the conjugation sites of DL2 are LC C214 and HC 224. For example, the number of drugs carried by DL4 is 4. In one embodiment, the conjugation sites of DL4a are LC C214, HC 224, LC C214, and HC 224. In one embodiment, the conjugation sites of DL4b are LC C230, HC 233, LC C230, and HC 233. For example, the number of drugs carried by DL6 is 6. In one embodiment, the conjugation sites of DL6 are LC C214, HC 224, LC C230, HC 233, LC C230, and HC 233. For example, the number of drugs carried by DL8 is 8. In one embodiment, the conjugation sites of DL8 are LC C214, HC 224, LC C214, HC 224, LC C230, HC 233, LC C230 and HC 233.

[0233] In one embodiment, the proportion of a particular DL species (e.g., proportion DL0, proportion DL2, proportion DL4a, proportion DL4b, proportion DL6, proportion DL8) can be determined by separating the individual DL species using hydrophobic interaction chromatography (HIC), calculating the area under the curve for each DL peak, and dividing each DL peak by the total area under the curve for all DL species combined. In one embodiment, the average DAR can be calculated from the area under the curve for each DL species using the following formula:

[0234]

number

[0235] In one embodiment, the proportion of DAR of a particular subspecies (e.g., the proportion of DL2a in total DL2) is determined by collecting the particular DL species using a combination of analytical techniques, which may include HIC, non-reducing separation methods, and mass spectrometry techniques.

[0236] In one embodiment, the average DAR of the anti-BCMA ADC composition is from about 2 to about 7, from about 2 to about 6, from about 2.1 to about 5.7, from about 2.1 to about 5.0, from about 2.1 to about 4.6, from about 2.1 to about 4.1, from about 2.1 to about 3.5, from about 2.1 to about 3.0, from about 3.0 to about 5.7, from about 3.0 to about 5.0, from about 3.0 to about 4.6, from about 3.0 to about 4.1, from about 3.0 to about 3.5, or from about 3.5 to about 5.7, about 3.5 to about 5.0, about 3.5 to about 4.6, about 3.5 to about 4.1, about 3.8 to about 4.5, about 4.1 to about 5.7, about 4.1 to about 5.0, about 4.1 to about 4.6, about 4.6 to about 5.7, about 4.6 to about 5.0, about 5.0 to about 5.7, about 2.1, about 3.0, about 3.5, about 4.1, about 4.6, about 5.0 or about 5.7.

[0237] In another embodiment, the composition comprises an anti-BCMA ADC with an average DAR of about 2.1 to about 5.7, about 3.4 to about 4.6, about 3.8 to about 4.5, or about 4.

[0238] In one embodiment, the composition comprises an anti-BCMA ADC, wherein the antibody comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; the cytotoxic agent is MMAE or MMAF; and the average DAR is about 2 to about 6, about 2.1 to about 5.7, about 3.4 to about 4.6, or about 3.8 to about 4.5.

[0239] In one embodiment, the composition comprises an anti-BCMA ADC, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L wherein the cytotoxic agent is MMAE or MMAF, and the average DAR is about 2 to about 6, about 2.1 to about 5.7, about 3.4 to about 4.6, or about 3.8 to about 4.5.

[0240] In one embodiment, the composition comprises belantamab mafodotin and has a mean DAR of about 2 to about 6, about 2.1 to about 5.7, about 3.4 to about 4.6, or about 3.8 to about 4.5.

[0241] In one embodiment, the proportion of DL0 species in the anti-BCMA ADC composition is about 10% or less, about 5% or less, about 1% to about 10%, about 1% to about 5%, or about 2.8% to about 4.7%.

[0242] In one embodiment, the proportion of DL2 species in the anti-BCMA ADC composition is at least about 10%, at least about 15%, about 15.8% to about 26.3%, about 15% to about 27%, about 15% to about 32%, or about 10% to about 40%.

[0243] In one embodiment, the proportion of DL4a species in the anti-BCMA ADC composition is at least about 30%, at least about 35%, about 35.5% to about 37.9%, about 35% to about 38%, about 30% to about 40%, or about 20% to about 50%. In another embodiment, the proportion of DL4a species is the predominant species in the anti-BCMA ADC composition and is about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more of all species combined.

[0244] In one embodiment, the proportion of DL4b species in the anti-BCMA ADC composition is at least about 5%, at least about 7%, about 7.1% to about 8.5%, about 7% to about 9%, about 5% to about 10%, or about 1% to about 15%.

[0245] In one embodiment, the proportion of DL6 species in the anti-BCMA ADC composition is at least about 10%, at least about 14%, about 14.0% to about 19.1%, about 14% to about 20%, about 10% to about 20%, or about 5% to about 30%.

[0246] In one embodiment, the proportion of DL8 species in the anti-BCMA ADC composition is at least about 1%, at least about 6%, about 6.0% to about 12.0%, about 4% to about 15%, or about 1% to about 20%.

[0247] In one embodiment, a composition comprises an anti-BCMA ADC, wherein the proportion of DL2 is about 15% to about 27% or about 15% to about 32%, the proportion of DL4a is about 35% to about 38% or about 30% to about 40%, the proportion of DL4b is about 7% to about 9% or about 5% to about 10%, the proportion of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0248] In one embodiment, the composition comprises an anti-BCMA ADC, wherein the antibodies include a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; and the cytotoxic drug is MMAE or MMAF, wherein the proportion of DL2 is about 15% to about 27% or about 15% to about 32%, the proportion of DL4a is about 35% to about 38% or about 30% to about 40%, the proportion of DL4b is about 7% to about 9% or about 5% to about 10%, the proportion of DL6 is about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0249] In one embodiment, the composition comprises an anti-BCMA ADC, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L wherein the cytotoxic drug is MMAE or MMAF, wherein the DL2 proportion is about 15% to about 27% or about 15% to about 32%, the DL4a proportion is about 35% to about 38% or about 30% to about 40%, the DL4b proportion is about 7% to about 9% or about 5% to about 10%, the DL6 proportion is about 14% to about 20% or about 10% to about 20%, and / or the DL8 proportion is about 6.0% to about 12.0% or about 4% to about 15%.

[0250] In one embodiment, the composition comprises belantamab mafodotin, wherein the proportion of DL2 is about 15% to about 27% or about 15% to about 32%, the proportion of DL4a is about 35% to about 38% or about 30% to about 40%, the proportion of DL4b is about 7% to about 9% or about 5% to about 10%, the proportion of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0251] As used herein, the term "undesirable DAR species" refers to any DAR species that is undesirable in the final composition and may adversely affect certain properties of the final therapeutic product (e.g., target binding, efficacy, safety, etc.). In one embodiment, the undesirable DAR species is DL0, i.e., antibody that is not conjugated to a cytotoxic drug after the conjugation process. In one embodiment, the percentage of DL0 in the ADC composition is about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less. In another embodiment, the percentage of DL0 in the ADC composition is about 1% to about 10%, about 2% to about 5%, or about 2.0% to about 4.8%.

[0252] In one embodiment, the composition comprises an anti-BCMA ADC, wherein the antibodies comprise CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; the cytotoxic agent is MMAE or MMAF; and the DL0 rate is about 10% or less or about 5% or less.

[0253] In one embodiment, the composition comprises an anti-BCMA ADC, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L wherein the cytotoxic agent is MMAE or MMAF, and the DL0 rate is about 10% or less, or about 5% or less.

[0254] In one embodiment, the composition comprises belantamab mafodotin and the percent DL0 is about 10% or less, or about 5% or less.

[0255] In one embodiment, the composition comprises belantamab mafodotin, wherein the percentage of DL0 is about 10% or less or about 5% or less, the percentage of DL2 is about 15% to about 27% or about 15% to about 32%, the percentage of DL4a is about 35% to about 38% or about 30% to about 40%, the percentage of DL4b is about 7% to about 9% or about 5% to about 10%, the percentage of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the percentage of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0256] In certain embodiments, the mean DAR or DL ​​ratio affects cell growth inhibition and / or tumor volume. In certain embodiments, the mean DAR does not affect cell growth inhibition and / or tumor volume. In other embodiments, as the mean DAR or DL ​​ratio increases, cell growth inhibition is enhanced and / or tumor volume is reduced. In yet another embodiment, a composition comprises belantamab mafodotin, and as the mean DAR or DL ​​ratio of the composition increases, cancer cell growth inhibition is enhanced and / or tumor volume is reduced.

[0257] The relative potency of cell growth inhibition can be determined by measuring cell viability of a cell line (e.g., a multiple myeloma cell line) after incubation with a composition described herein (e.g., belantamab mafodotin). Cell viability can be measured using cell viability assays known to those skilled in the art. A dose-response (half maximal effective concentration or EC50) can be generated using a nonlinear logistic regression model. The ratio of the EC50 of the reference standard to the EC50 of the sample containing the composition can be calculated to determine relative potency.

[0258] In one embodiment, the composition has a relative potency of cell growth inhibition of about 0.5 to about 1.3 or about 0.8 to about 1.1. In another embodiment, the composition has an average DAR of about 2.1 to about 5.7, and the composition has a relative potency of cell growth inhibition of about 0.5 to about 1.3. In another embodiment, the composition has an average DAR of about 3.0 to about 5.0 or about 3.5 to about 4.6, and the composition has a relative potency of cell growth inhibition of about 0.8 to about 1.1. In another embodiment, the composition comprises belantamab mafodotin with an average DAR of about 3.0 to about 5.0 or about 3.5 to about 4.6, and the composition has a relative potency of cell growth inhibition of about 0.8 to about 1.1.

[0259] In certain embodiments, the mean DAR or DL ​​ratio affects ADCC activity. In other embodiments, the mean DAR or DL ​​ratio does not affect ADCC activity. In yet other embodiments, the composition comprises belantamab mafodotin, and the mean DAR or DL ​​ratio affects ADCC activity. In yet other embodiments, the composition comprises belantamab mafodotin, and the mean DAR or DL ​​ratio does not affect ADCC activity.

[0260] The relative potency of ADCC activity can be measured, for example, by incubating belantamab mafodotin, cells (e.g., multiple myeloma cells), and NK cells (effector cells). Without being bound by theory, belantamab mafodotin binds to BCMA expressed on the cell surface, and the Fc region of the antibody binds to effector cell FcγRIIIa via the effector cell receptor FcγRIIIa. Engagement of these receptors on the surface of effector cells results in the synthesis and secretion of cytokines (IFNg) and the release of granules (perforin and granzymes) that enter the cytoplasm of target cells. Granzymes initiate signaling events within the target cells that result in the death of these cells by apoptosis. NK cells can also be sourced from peripheral blood mononuclear cells (PBMCs), which can be isolated from human whole blood. BATDA (bis-(acetoxymethyl) 2,2':6',2''-terpyridine-6,6''-dicarboxylate) can then be added to permeabilize the target cell membrane and label the cells. After cell lysis, this ligand can be mixed with DELFIA Europium Solution to form a highly fluorescent and stable chelate (EuTDA). The measured signal directly correlates with the amount of cells lysed. ADCC activity can then be reported as the ratio of the EC50 value of the sample to the EC50 value of the reference standard.

[0261] In one embodiment, the composition has a relative potency of ADCC activity of about 0.70 to about 1.30 or about 0.8 to about 1.1. In another embodiment, the composition has a mean DAR of about 2.1 to about 5.7, and the composition has a relative potency of ADCC activity of about 0.5 to about 1.3. In another embodiment, the composition has a mean DAR of about 3.0 to about 5.0 or about 3.5 to about 4.6, and the composition has a relative potency of ADCC activity of about 0.8 to about 1.1. In another embodiment, the composition comprises belantamab mafodotin having a mean DAR of about 3.0 to about 5.0 or about 3.5 to about 4.6, and the composition has a relative potency of ADCC activity of about 0.8 to about 1.1.

[0262] In certain embodiments, the average DAR or DL ​​ratio affects binding to BCMA. In another embodiment, the average DAR or DL ​​ratio does not affect binding to BCMA. In yet another embodiment, a composition includes belantamab mafodotin, and the average DAR or DL ​​ratio affects binding to BCMA. In yet another embodiment, a composition includes belantamab mafodotin, and the average DAR or DL ​​ratio does not affect binding to BCMA. In one embodiment, the average DAR or DL ​​ratio may weaken binding to BCMA.

[0263] In one embodiment, the composition has a relative BCMA-specific antigen binding of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. In another embodiment, the composition comprises belantamab mafodotin, and the composition has a relative BCMA-specific antigen binding of greater than 85% or greater than 90%. In another embodiment, the composition comprises belantamab mafodotin, and the composition has a mean DAR of about 2.1 to about 5.7, or about 3.0 to about 5.0, or about 3.5 to about 4.6, and the composition has a relative BCMA-specific antigen binding of greater than 85% or greater than 90%.

[0264] In certain embodiments, the average DAR or DL ​​ratio affects binding to FcγRIIIa. In another embodiment, the average DAR or DL ​​ratio does not affect binding to FcγRIIIa. In yet another embodiment, the composition comprises belantamab mafodotin, and the average DAR or DL ​​ratio affects binding to FcγRIIIa. In yet another embodiment, the composition comprises belantamab mafodotin, and the average DAR or DL ​​ratio does not affect binding to FcγRIIIa. In one embodiment, the average DAR or DL ​​ratio may weaken binding to FcγRIIIa.

[0265] In one embodiment, the composition has a relative FcγRIIIa binding of 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater. In another embodiment, the composition comprises belantamab mafodotin, and the composition has a relative FcγRIIIa binding of greater than 85% or greater than 90%. In another embodiment, the composition comprises belantamab mafodotin, and the composition has a mean DAR of about 2.1 to about 5.7, or about 3.0 to about 5.0, or about 3.5 to about 4.6, and the composition has a relative FcγRIIIa binding of greater than 85% or greater than 90%.

[0266] Among other methods known to those skilled in the art, the binding of BCMA and FcγRIIIa by anti-BCMA antigen-binding proteins, such as belantamab mafodotin, can be measured using surface plasmon resonance (SPR). In one example, belantamab mafodotin is injected and captured by Protein A immobilized on a CM5 sensor chip. BCMA is then injected and allowed to bind to the captured belantamab mafodotin. FcγRIIIa is then injected and allowed to bind to the captured belantamab mafodotin. The functional concentrations of belantamab mafodotin bound to BCMA and FcγRIIIa can be calculated from a reference standard curve and reported as BCMA or FcγRIIIa-bound concentrations, respectively. The total belantamab mafodotin concentration of the sample can be previously determined by absorbance at 280 nm. Specific binding activity (%) can be calculated by dividing the bound concentration of BCMA or FcγRIIIa by the absorbance, for example, the absorbance at 280 nm at that concentration.

[0267] In certain embodiments, the average DAR or DL ​​ratio affects binding to FcRn. In another embodiment, the average DAR or DL ​​ratio does not affect binding to FcRn. In yet another embodiment, a composition comprises belantamab mafodotin, and the average DAR or DL ​​ratio affects binding to FcRn. In yet another embodiment, a composition comprises belantamab mafodotin, and the average DAR or DL ​​ratio does not affect binding to FcRn. In one embodiment, the average DAR or DL ​​ratio may weaken binding to FcRn.

[0268] Binding of fetal Fc receptor (FcRn) to anti-BCMA antigen-binding proteins, such as belantamab mafodotin, can be measured using surface plasmon resonance (SPR). Belantamab mafodotin can be captured by FcRn immobilized on a nitrilotriacetic acid (NTA) sensor chip. The FcRn binding concentration of a sample can be determined by interpolation of the binding response on a standard curve. Specific binding activity (%) is calculated by dividing the FcRn binding concentration by the total protein concentration.

[0269] When the anti-BCMA antigen binding protein comprises belantamab mafodotin, the SPR methods described herein for specific antigen binding, FcγRIIIa, and FcRn binding may use a belantamab or belantamab mafodotin reference standard. The belantamab or belantamab mafodotin reference standard can be used in the assay to obtain system suitability and sample comparability data and ensure that the method is performing properly. The reference standard allows for the generation of a calibration curve, from which sample concentrations are interpolated. For example, the reference standard can be a composition comprising a heavy chain amino acid sequence of SEQ ID NO: 9 and a light chain amino acid sequence of SEQ ID NO: 10, with a known DL and / or average DAR content.

[0270] An exemplary reference standard may include a sample of belantamab mafodotin containing known components / amounts of DL species and / or average DAR.

[0271] Pharmaceutical Composition The compositions described herein may be in the form of a pharmaceutical composition. A "pharmaceutical composition" may comprise a composition described herein (i.e., an active ingredient) and one or more pharmaceutically acceptable excipients. An excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of being formulated pharmaceuticals, not harmful to the recipient thereof, and / or not interfering with the efficacy of the active ingredient.

[0272] As used herein, "pharmaceutically acceptable excipients" can include any and all solvents, diluents, carriers, dispersion media, coatings, antibacterial agents, antifungal agents, isotonicity agents, and / or absorption delaying agents. Examples of pharmaceutically acceptable excipients include one or more of buffers, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, isotonicity agents, such as polyols, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride; preservatives; cosolvents; antioxidants, such as ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn 2+ -protein complex); a biodegradable polymer; and / or a salt-forming counterion such as sodium or potassium is preferably included in the composition.

[0273] The precise nature of the excipient or other material may depend on the route of administration, which may be, for example, oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and intrathecal), and intratumoral. It will be appreciated that preferred excipients may vary with, for example, the condition of the recipient and the disease being treated.

[0274] The mixture of excipients and each of the aggregates together form a "pharmaceutical formulation" (or "formulation"). The formulation may be in liquid or lyophilized form. The composition in a liquid formulation may be filled into a container and frozen. In certain embodiments, an aliquot of the frozen formulation comprising the composition may be lyophilized. The lyophilizate may be reconstituted by adding water or other aqueous solution to produce a reconstituted formulation comprising the composition.

[0275] In some embodiments, the anti-BMCA antigen binding protein is present in the formulation at a concentration of at least about 10 mg / mL or at least about 20 mg / mL. In some embodiments, the anti-BMCA antigen binding protein is present in the formulation at a concentration of about 20 mg / mL to about 100 mg / mL, or about 20 mg / mL to about 60 mg / mL. In specific embodiments, the concentration of the anti-BCMA antigen binding protein present in the formulation is about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 60 mg / mL, or about 100 mg / mL. In one embodiment, the anti-BMCA antigen binding protein is present in the liquid formulation at a concentration of about 20 mg / mL or about 25 mg / mL. In another embodiment, the anti-BMCA antigen binding protein is present in the lyophilized formulation at a concentration of about 50 mg / mL or about 60 mg / mL. In yet another embodiment, the anti-BMCA antigen binding protein is present in the reconstituted formulation at a concentration of about 50 mg / mL.

[0276] In certain embodiments, the buffer is a citrate buffer. Citrate buffering can be achieved, for example, by using a conjugate acid / conjugate base system (sodium citrate / citric acid) or by titrating a sodium citrate solution with HCl. In certain embodiments, the citrate buffer has a concentration of about 10 mM to about 30 mM. In a preferred embodiment, the citrate buffer has a concentration of 25 mM. In some embodiments, the buffer is a histidine buffer at a concentration of about 5 mM to about 35 mM.

[0277] Buffering agents can be used to help maintain a preferred pH range, hi certain embodiments, the pH of the formulation is from about 5.5 to about 7, or from about 5.9 to about 6.5, preferably pH 6.2.

[0278] In some embodiments, the formulation comprises a polyol. In some embodiments, the polyol is a sugar, preferably a non-reducing sugar. In some embodiments, the non-reducing sugar is trehalose. In some embodiments, the formulation comprises about 120 mM to about 240 mM trehalose. In yet another embodiment, the formulation comprises about 200 mM trehalose.

[0279] In one embodiment, the formulation includes a chelating agent. In another embodiment, the chelating agent is EDTA. In a specific embodiment, the formulation includes EDTA at a concentration of 0.01 mM to about 0.1 mM. In yet another embodiment, the formulation includes EDTA at a concentration of 0.05 mM.

[0280] In some embodiments, the formulation contains a surfactant. A "surfactant" is a surface active agent that can exert its effect at solid-solid, solid-liquid, liquid-liquid, and liquid-air interfaces due to its chemical composition containing both hydrophilic and hydrophobic groups. Surfactants can reduce the concentration of proteins in dilute solutions at air-water and / or water-solid interfaces, where proteins may adsorb and potentially aggregate. Surfactants can bind to hydrophobic interfaces in protein formulations. Some parentally acceptable nonionic surfactants contain either polysorbate or polyether groups. Polysorbates 20 and 80 are suitable surfactant stabilizers in the formulations of the present invention. In some embodiments, the formulation contains about 0.01% to about 0.05% polysorbate 20 or polysorbate 80. In yet another embodiment, the formulation contains about 0.02% polysorbate 20 or polysorbate 80. In a preferred embodiment, the formulation contains about 0.02% polysorbate 80.

[0281] One aspect of the invention focuses on a formulation comprising about 20 mg / mL to about 100 mg / mL of an anti-BCMA antigen binding protein, about 10 mM to about 25 mM of a buffering agent, and about 120 mM to about 240 mM of a polyol, wherein the formulation has a pH of 5.5 to 6.5.

[0282] In one embodiment, the formulation comprises about 20 mg / mL to about 60 mg / mL of anti-BCMA antigen binding protein, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0283] In one embodiment, the composition comprises an antibody in a formulation, the antibody comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; the formulation comprises about 20 mg / mL to about 60 mg / mL of the antibody, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0284] In one embodiment, the composition comprises an antibody in a formulation, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L The formulation comprises about 20 mg / mL to about 60 mg / mL of antibody, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0285] In one embodiment, the composition comprises an antibody in a formulation, wherein the antibody is belantamab, and the formulation comprises about 20 mg / mL to about 60 mg / mL of belantamab, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0286] In one embodiment, the composition comprises an ADC in a formulation, the antibodies comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, the cytotoxin is MMAF or MMAE, the formulation comprises about 20 mg / mL to about 60 mg / mL of the ADC, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0287] In one embodiment, the composition comprises an ADC in a formulation, wherein the antibody is a V antibody having the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L wherein the cytotoxin is MMAF or MMAE, the formulation comprises about 20 mg / mL to about 60 mg / mL of ADC, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0288] In one embodiment, the composition comprises an ADC in a formulation, wherein the ADC is belantamab mafodotin, and the formulation comprises about 20 mg / mL to about 60 mg / mL of belantamab mafodotin, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0289] In one embodiment, the composition comprises belantamab mafodotin in a formulation comprising about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, or 60 mg / mL of belantamab mafodotin, 25 mM citrate buffer, 200 mM trehalose, 0.05 mM EDTA disodium salt, and 0.02% polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0290] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability during manufacture, transportation, storage, and administration. Stability can be measured at a selected temperature for a selected time. For example, for a product stored at a recommended temperature of 2°C to 8°C, the formulation may be stable at room temperature, about 30°C, or 40°C for at least one month, and / or at about 2-8°C for at least one year, preferably at least two years. For example, the degree of aggregation during storage is used as an indicator of protein stability. Thus, a "stable" formulation can be, for example, one in which less than about 10%, preferably less than about 5%, of the protein is present as aggregates in the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).

[0291] In certain embodiments of the present invention, the formulation allows the composition to remain stable upon freezing, thawing and / or mixing.

[0292] In yet another aspect, the present invention relates to an article of manufacture, e.g., a kit, comprising a container containing the composition in the formulation described herein. In one aspect, an injection device containing the formulation is provided. The injection device may include a pen injector device or an autoinjector device. In one embodiment, the formulation is contained in a pre-filled syringe.

[0293] Methods of Treatment and Compositions for Use It is an object of the present invention to provide a therapeutic approach to the treatment of B cell related disorders or diseases (e.g. antibody mediated diseases, plasma cell mediated diseases or plasma cell malignancies (e.g. cancer, e.g. multiple myeloma)), or other diseases that may be treated with anti-BCMA antigen binding proteins. In particular, it is an object of the present invention to provide compositions comprising an anti-BCMA antigen binding protein, e.g. an anti-BCMA antibody, which specifically binds to BCMA (e.g. human BCMA) and modulates (i.e. inhibits or blocks) the interaction of BCMA with its ligands (e.g. BAFF and / or APRIL) in the treatment of diseases and disorders responsive to modulation of that interaction.

[0294] In another aspect of the invention, there is provided a method of treating a subject (e.g., a human patient) suffering from a B-cell related disorder or disease, such as an antibody-mediated disease, a plasma cell-mediated disease or a plasma cell malignancy (e.g., cancer, e.g., multiple myeloma), the method comprising administering to the subject a therapeutically effective amount of an anti-BCMA antigen binding protein composition as described herein.

[0295] In yet another embodiment, the invention provides a method of treating a patient with cancer, the method comprising administering to the patient a therapeutically effective amount of an anti-BCMA antigen binding protein composition described herein.

[0296] As used herein, the terms "cancer" and "tumor" are used interchangeably and refer to cells that have undergone transformation, e.g., malignant transformation, in either the singular or plural form, resulting in a pathological condition in the host organism. Primary cancer cells can be readily distinguished from noncancerous cells by established techniques, particularly histological examination. The definition of cancer cells as used herein includes not only primary cancer cells but also any cells that originate from cancer cells. This includes metastasized cancer cells as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically present as solid tumors, a "clinically detectable" tumor is one that is detectable based on mass, for example, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during a physical examination, and / or is detectable in samples obtained from a patient due to the expression of one or more cancer-specific antigens. The tumor may be a hematopoietic (or hematologic or hematological or blood-related) cancer, such as a cancer derived from blood cells or immune cells, which are sometimes referred to as "liquid tumors." Specific examples of clinical conditions based on hematological tumors include leukemias, such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia; plasma cell malignancies, such as multiple myeloma, MGUS, and Waldenstrom's macroglobulinemia; lymphomas, such as non-Hodgkin's lymphoma and Hodgkin's lymphoma; and the like.

[0297] The cancer may be either one in which there is an abnormal number of blasts or unwanted cell proliferation, or one diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyelocytic (or promyelocytic or promyelocytic or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryocytic (or megakaryoblastic) leukemia. These leukemias are sometimes collectively referred to as acute myeloid (or myeloid or myeloid) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPDs), including, but not limited to, chronic myeloid (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV). In particular, myeloid malignancies also include myelodysplasia (or myelodysplastic syndromes or MDS), which are sometimes referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transition (RAEBT); and myelofibrosis with or without primary myelofibrosis (MFS).

[0298] Hematopoietic cancers also include lymphoid malignancies, which can affect lymph nodes, spleen, bone marrow, peripheral blood, and / or extra-lymphatic sites. Lymphoid cancers include B-cell malignancies, including, but not limited to, B-cell non-Hodgkin's lymphoma (B-NHL). B-NHL can be indolent (or low-grade), intermediate-grade (or aggressive), or high-grade (highly aggressive). Indolent B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL), such as nodal MZL, extranodal MZL, splenic MZL, and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHL includes mantle cell lymphoma (MCL) with or without leukemic involvement; diffuse large cell lymphoma (DLBCL); follicular large cell (or grade 3 or grade 3B) lymphoma; and primary mediastinal lymphoma (PML). High-grade B-NHL includes Burkitt lymphoma (BL), Burkitt-like lymphoma, small noncleaved cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV-associated (or AIDS-associated) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or post-transplant lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocytic (LGL) leukemia, acute lymphocytic (or lymphocytic or lymphoblastic) leukemia, and Castleman's disease. In particular, NHL may also include T-cell non-Hodgkin's lymphoma (T-NHL), including, but not limited to, T-cell non-Hodgkin's lymphoma, not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphadenopathy (AILD), nasal natural killer (NK)-cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome.

[0299] Hematopoietic cancers also include Hodgkin's lymphoma (or Hodgkin's disease), such as classical Hodgkin's lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed cytology Hodgkin's lymphoma, lymphocyte-predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocytopenic Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell dyscrasias or plasma cell cancers, such as multiple myeloma (MM) (including smoldering MM), monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers can also include other cancers of additional hematopoietic cells, such as polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes, and natural killer cells. Tissues containing hematopoietic cells, referred to herein as "hematopoietic cell tissues," include bone marrow; peripheral blood; thymus; peripheral lymphoid tissues, such as the spleen, lymph nodes, mucosa- (e.g., tonsils, Peyer's patches, and appendix)-associated lymphoid tissues (e.g., gut-associated lymphoid tissues), and other mucosa- (e.g., bronchial lining)-associated lymphoid tissues.

[0300] In one embodiment, the cancer is selected from the group consisting of colorectal cancer (CRC), gastric cancer, esophageal cancer, cervical cancer, bladder cancer, breast cancer, head and neck cancer, ovarian cancer, melanoma, renal cell carcinoma (RCC), EC squamous cell carcinoma, non-small cell lung cancer, mesothelioma, pancreatic cancer, and prostate cancer.

[0301] As used herein, the term "treating" and its derivatives are intended to include therapeutic therapy. With respect to a particular condition, treating means: (1) improving the condition or ameliorating one or more of the biological symptoms of the condition; (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more of the biological symptoms of the condition; (3) alleviating one or more symptoms, effects, or side effects associated with the condition or its treatment; (4) slowing the progression of the condition or the progression of one or more of the biological symptoms of the condition; and / or (5) curing the condition or one or more of the biological symptoms of the condition by eliminating or reducing one or more of the biological symptoms of the condition to undetectable levels, a period of time that is considered to be in remission of the biological symptoms of the condition without further treatment during the remission period. Those of skill in the art will understand the period of time that is considered to be in remission of a particular disease or condition.

[0302] B cell disorders can be divided into defects in B cell development / immunoglobulin production (e.g., immune deficiencies) and excessive / uncontrolled proliferation (e.g., lymphoma, leukemia). As used herein, B cell disorders refer to both types of disease, and methods for treating B cell disorders are provided with the compositions described herein.

[0303] In certain embodiments, the disease or disorder is multiple myeloma (MM), chronic lymphocytic leukemia (CLL), solitary plasmacytoma (bone, extramedullary), amyloidosis (AL), smoldering multiple myeloma (SMM), solitary plasmacytoma (bone, extramedullary), or Waldenstrom's macroglobulinemia.

[0304] Preventive treatment is also contemplated. Those skilled in the art will recognize that "prevention" is not an absolute term. In medicine, "prevention" is understood to mean the prophylactic administration of a drug to substantially reduce the likelihood or severity of a condition or its biological manifestations, or to delay the onset of a condition or its biological manifestations. Preventive treatment is appropriate, for example, when a subject is considered to be at high risk of developing cancer, for example, when the subject has a strong family history of cancer or when the subject has been exposed to a carcinogen.

[0305] "Subject" or "patient" are used interchangeably herein and are broadly defined to include any person in need of treatment, for example, a person in need of cancer treatment. A subject may include a mammal. In one embodiment, a subject is a human patient. Subjects in need of cancer treatment may include patients at various stages of disease, for example, newly diagnosed patients, relapsed patients, refractory patients, patients with progressive disease, patients in remission, and others. Subjects in need of cancer treatment may also include patients who have undergone stem cell transplantation or who are deemed transplant ineligible.

[0306] Subjects can be pre-screened to be selected for treatment with the compositions described herein, hi one embodiment, a sample from the subject is tested for BCMA expression prior to treatment with the compositions described herein.

[0307] The subject may have previously undergone at least one cancer treatment prior to being treated with a composition of the invention, hi one embodiment, the subject has previously been treated with at least one, at least two, at least three, at least four, at least five, at least six, or at least seven cancer treatments prior to being treated with a composition of the invention.

[0308] In another embodiment, the subject is newly diagnosed with cancer and has not received any previous treatment before being treated with the compositions of the invention.

[0309] The compositions of the present invention can be administered by any suitable route. For some compositions, routes of administration include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and intrathecal), and intratumoral. It is understood that the preferred route may vary depending, for example, on the condition of the recipient and the cancer being treated.

[0310] In certain embodiments, the compositions of the invention are administered as pharmaceutical compositions.

[0311] As used herein, the term "administering" refers to the delivery of the compositions described herein to achieve a therapeutic purpose. The compositions may be administered at intervals sufficient for a period of time to achieve a clinical benefit. The compositions may be administered to a subject in a manner that targets the therapy to a specific site.

[0312] In some embodiments, the composition is administered by injection. Thus, in one aspect, an injection device is provided comprising the composition, pharmaceutical composition, or formulation of the invention. The injection device may comprise a pen injector device or an autoinjector device.

[0313] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" of a composition refers to an amount effective to prevent or treat or alleviate the symptoms of a B cell-mediated disorder or disorder. Therapeutically effective amounts and treatment regimens are generally determined empirically and may depend on factors such as the patient's age, the patient's weight, the patient's health, and the disease or disorder being treated. Such factors are within the control of the attending physician.

[0314] An appropriate therapeutically effective dose of a composition comprising an anti-BCMA antigen-binding protein can be readily determined by one of skill in the art. The appropriate dose of the compositions described herein can be calculated based on the patient's body weight; for example, the appropriate dose can be about 0.1 mg / kg to about 20 mg / kg, e.g., about 1 mg / kg to about 20 mg / kg, e.g., about 10 mg / kg to about 20 mg / kg, e.g., about 1 mg / kg to about 15 mg / kg, or e.g., about 10 mg / kg to about 15 mg / kg.

[0315] In one embodiment, the therapeutically effective dose of a composition comprising an anti-BCMA antigen binding protein is about 0.03 mg / kg to about 4.6 mg / kg. In yet another embodiment, the therapeutically effective dose of a composition comprising an anti-BCMA antigen binding protein is 0.03 mg / kg, 0.06 mg / kg, 0.12 mg / kg, 0.24 mg / kg, 0.48 mg / kg, 0.96 mg / kg, 1.92 mg / kg, 3.4 mg / kg, or 4.6 mg / kg. In yet another embodiment, the therapeutically effective dose of a composition comprising an anti-BCMA antigen binding protein is 1.9 mg / kg, 2.5 mg / kg, or 3.4 mg / kg.

[0316] In certain embodiments, the composition may be co-administered to a subject with one or more additional therapeutic agents. In another embodiment, the composition may be co-administered to a subject with one or more additional cancer therapeutic agents. The additional cancer therapeutic agents may include, but are not limited to, other immunomodulatory agents, therapeutic antibodies (e.g., anti-CD38 antibodies, e.g., daratumumab), CAR-T therapeutics, BiTEs, HDAC inhibitors, proteasome inhibitors (e.g., bortezomib), anti-inflammatory compounds, and immunomodulatory imid drugs (IMiDs) (e.g., thalidomide and its analogs).

[0317] "Co-administered" refers to the administration of two or more different pharmaceutical compositions or treatments (e.g., radiation therapy) administered to a subject in the same pharmaceutical composition or in a combination of separate pharmaceutical compositions. Thus, co-administration includes the simultaneous administration of a single pharmaceutical composition containing two or more pharmaceutical agents, or the administration of two or more different compositions to the same subject at the same or different times.

[0318] In one aspect of the invention, the invention provides a method of treating a B-cell disease or disorder in a subject in need thereof by administering a therapeutically effective dose of any of the compositions comprising an anti-BCMA antigen binding protein described herein.

[0319] In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising an anti-BCMA ADC having a mean DAR of about 3.4 to about 4.6.

[0320] In another embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising an anti-BCMA ADC, wherein the antibodies comprise CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; the cytotoxic agent is MMAF or MMAE, and the average DAR is about 3.4 to about 4.6.

[0321] In another embodiment, the invention provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective dose of a composition comprising an anti-BCMA ADC, wherein the antibody is a V having the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. Lwherein the cytotoxic agent is MMAE or MMAF, and the average DAR is about 3.4 to about 4.6.

[0322] In another embodiment, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising belantamab mafodotin, having a mean DAR of about 3.4 to about 4.6.

[0323] In another embodiment, the present invention provides a method of treating multiple myeloma in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising belantamab mafodotin, having a mean DAR of about 3.4 to about 4.6.

[0324] In one embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising an anti-BCMA ADC, wherein the DL0 rate is about 10% or less or about 5% or less, the DL2 rate is about 15% to about 27%, or about 15% to about 32%, the DL4a rate is about 35% to about 38%, or about 30% to about 40%, the DL4b rate is about 7% to about 9%, or about 5% to about 10%, the DL6 rate is about 14% to about 20%, or about 10% to about 20%, and / or the DL8 rate is about 6.0% to about 12.0%, or about 4% to about 15%.

[0325] In another embodiment, the invention provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective dose of a composition comprising an anti-BCMA ADC, wherein the antibody comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; and a cytotoxin. The method is provided in which the sexually active agent is MMAF or MMAE; the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27% or about 15% to about 32%, the proportion of DL4a is about 35% to about 38% or about 30% to about 40%, the proportion of DL4b is about 7% to about 9% or about 5% to about 10%, the proportion of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0326] In another embodiment, the invention provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective dose of a composition comprising an anti-BCMA ADC, wherein the antibody is a V having the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L the cytotoxic drug is MMAF or MMAE; the DL0 rate is about 10% or less or about 5% or less, the DL2 rate is about 15% to about 27% or about 15% to about 32%, the DL4a rate is about 35% to about 38% or about 30% to about 40%, the DL4b rate is about 7% to about 9% or about 5% to about 10%, the DL6 rate is about 14% to about 20% or about 10% to about 20%, and / or the DL8 rate is about 6.0% to about 12.0% or about 4% to about 15%.

[0327] In yet another embodiment, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising belantamab mafodotin, wherein the DL0 rate is about 10% or less or about 5% or less, the DL2 rate is about 15% to about 27% or about 15% to about 32%, the DL4a rate is about 35% to about 38% or about 30% to about 40%, the DL4b rate is about 7% to about 9% or about 5% to about 10%, the DL6 rate is about 14% to about 20% or about 10% to about 20%, and / or the DL8 rate is about 6.0% to about 12.0% or about 4% to about 15%.

[0328] In yet another embodiment, the present invention provides a method of treating multiple myeloma in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising belantamab mafodotin, wherein the DL0 rate is about 10% or less or about 5% or less, the DL2 rate is about 15% to about 27%, or about 15% to about 32%, the DL4a rate is about 35% to about 38%, or about 30% to about 40%, the DL4b rate is about 7% to about 9%, or about 5% to about 10%, the DL6 rate is about 14% to about 20%, or about 10% to about 20%, and / or the DL8 rate is about 6.0% to about 12.0%, or about 4% to about 15%.

[0329] In one aspect, the invention provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective dose of a composition comprising an antibody, wherein the antibody comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and the composition comprises 25% or less isomerization at D103 of the heavy chain CDRH3.

[0330] In one embodiment, the invention provides a method of treating multiple myeloma in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising an antibody, wherein the antibody comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and the composition comprises 25% or less isomerization at D103 of the heavy chain CDRH3.

[0331] In one embodiment, the present invention provides a method for treating multiple myeloma in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising belantamab, wherein the composition comprises 25% or less isomerization at D103 of heavy chain CDRH3.

[0332] In one aspect, the invention provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective dose of a composition comprising an antibody, wherein the antibody comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO:1, CDRH2 having the amino acid sequence set forth in SEQ ID NO:2, CDRH3 having the amino acid sequence set forth in SEQ ID NO:3, CDRL1 having the amino acid sequence set forth in SEQ ID NO:4, CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6, and the composition comprises 40% or less oxidation in heavy chain M34(CDRH1).

[0333] In one embodiment, the invention provides a method of treating multiple myeloma in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising an antibody, wherein the antibody comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO:1, CDRH2 having the amino acid sequence set forth in SEQ ID NO:2, CDRH3 having the amino acid sequence set forth in SEQ ID NO:3, CDRL1 having the amino acid sequence set forth in SEQ ID NO:4, CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6, and the composition comprises 40% or less oxidation in heavy chain M34(CDRH1).

[0334] In one embodiment, the present invention provides a method of treating multiple myeloma in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising belantamab, wherein the composition comprises 40% or less oxidation in heavy chain M34 (CDRH1).

[0335] In one aspect of the invention, the invention provides a composition comprising an anti-BCMA antigen binding protein as described herein for use in the treatment of a B-cell disease or disorder.

[0336] In one embodiment, the invention provides a composition comprising an anti-BCMA ADC described herein for use in treating cancer, wherein the composition has an average DAR of about 3.4 to about 4.6.

[0337] In another embodiment, the invention provides a composition comprising an anti-BCMA ADC described herein for use in treating cancer, wherein the antibodies comprise CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; the cytotoxic agent is MMAF or MMAE, and the composition has an average DAR of about 3.4 to about 4.6.

[0338] In another embodiment, the invention provides a composition comprising an anti-BCMA ADC described herein for use in treating cancer, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L wherein the cytotoxic drug is MMAE or MMAF, and the average DAR is about 3.4 to about 4.6.

[0339] In another embodiment, the present invention provides a composition comprising belantamab mafodotin for use in treating cancer, the composition having a mean DAR of 3.4 to about 4.6.

[0340] In yet another embodiment, the present invention provides a composition comprising belantamab mafodotin for use in the treatment of multiple myeloma, the composition having a mean DAR of 3.4 to about 4.6.

[0341] In one embodiment, the invention provides a composition comprising an anti-BCMA ADC described herein for use in treating cancer, wherein the percentage of DL0 is about 10% or less or about 5% or less, the percentage of DL2 is about 15% to about 27%, or about 15% to about 32%, the percentage of DL4a is about 35% to about 38%, or about 30% to about 40%, the percentage of DL4b is about 7% to about 9%, or about 5% to about 10%, the percentage of DL6 is about 14% to about 20%, or about 10% to about 20%, and / or the percentage of DL8 is about 6.0% to about 12.0%, or about 4% to about 15%.

[0342] In another embodiment, the present invention provides an anti-BCMA antibody as described herein for use in the treatment of cancer. The present invention provides a composition comprising an ADC, wherein the antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; the cytotoxic drug is MMAF or MMAE; and the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27%, or about 15% to about 32%, the proportion of DL4a is about 35% to about 38%, or about 30% to about 40%, the proportion of DL4b is about 7% to about 9%, or about 5% to about 10%, the proportion of DL6 is about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0%, or about 4% to about 15%.

[0343] In another embodiment, the invention provides a composition comprising an anti-BCMA ADC described herein for use in treating cancer, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L the cytotoxic drug is MMAF or MMAE; the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27% or about 15% to about 32%, the proportion of DL4a is about 35% to about 38% or about 30% to about 40%, the proportion of DL4b is about 7% to about 9% or about 5% to about 10%, the proportion of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0344] In another embodiment, the present invention provides a composition comprising belantamab mafodotin for use in treating cancer, wherein the percentage of DL0 is about 10% or less or about 5% or less, the percentage of DL2 is about 15% to about 26%, the percentage of DL4a is about 35% to about 38%, the percentage of DL4b is about 7% to about 10%, the percentage of DL6 is about 14% to about 20%, and / or the percentage of DL8 is about 6.0% to about 12.0%.

[0345] In yet another embodiment, the present invention provides a composition comprising belantamab mafodotin for use in the treatment of multiple myeloma, wherein the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27%, or about 15% to about 32%, the proportion of DL4a is about 35% to about 38%, or about 30% to about 40%, the proportion of DL4b is about 7% to about 9%, or about 5% to about 10%, the proportion of DL6 is about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0%, or about 4% to about 15%.

[0346] In one aspect, the invention provides a composition comprising an antibody for use in treating cancer, wherein the antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and the composition comprises 25% or less isomerization at D103 of the heavy chain CDRH3.

[0347] In one aspect, the invention provides a composition comprising an antibody for use in treating multiple myeloma, wherein the antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and the composition comprises 25% or less isomerization at D103 of the heavy chain CDRH3.

[0348] In yet another embodiment, the present invention provides a composition comprising belantamab mafodotin for use in treating multiple myeloma, the composition comprising 25% or less isomerization at D103 of heavy chain CDRH3.

[0349] In one aspect, the invention provides a composition comprising an antibody for use in treating cancer, wherein the antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and the composition comprises 40% or less oxidation in heavy chain M34(CDRH1).

[0350] In one aspect, the invention provides a composition comprising an antibody for use in treating multiple myeloma, wherein the antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO:1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO:2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO:3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO:4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:6, and the composition comprises 40% or less oxidation in heavy chain M34(CDRH1).

[0351] In yet another embodiment, the present invention provides a composition comprising belantamab mafodotin for use in treating multiple myeloma, the composition comprising 40% or less oxidation in heavy chain M34 (CDRH1).

[0352] In one aspect of the invention there is provided the use of a composition in the manufacture of a medicament for use in the treatment of a B cell disease or disorder.

[0353] In one embodiment, there is provided use of a composition comprising an anti-BCMA ADC in the manufacture of a medicament for use in the treatment of cancer, wherein the composition has an average DAR of about 3.4 to about 4.6.

[0354] In another embodiment, there is provided use of a composition comprising an anti-BCMA ADC in the manufacture of a medicament for use in treating cancer, wherein the antibodies comprise CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, and the cytotoxic agent is MMAF or MMAE, and the average DAR is about 3.4 to about 4.6.

[0355] In another embodiment, there is provided use of a composition comprising an anti-BCMA ADC in the manufacture of a medicament for use in treating cancer, wherein the antibody is a V having the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L wherein the cytotoxic agent is MMAE or MMAF, and the average DAR is about 3.4 to about 4.6.

[0356] In another embodiment, there is provided use of a composition comprising belantamab mafodotin in the manufacture of a medicament for use in treating cancer, wherein the composition has a mean DAR of about 3.4 to about 4.6.

[0357] In another embodiment, there is provided a use of a composition comprising belantamab mafodotin in the manufacture of a medicament for use in the treatment of multiple myeloma, wherein the composition has a mean DAR of about 3.4 to about 4.6.

[0358] In one embodiment, there is provided use of a composition comprising an anti-BCMA ADC in the manufacture of a medicament for use in the treatment of cancer, wherein the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27%, or about 15% to about 32%, the proportion of DL4a is about 35% to about 38%, or about 30% to about 40%, the proportion of DL4b is about 7% to about 9%, or about 5% to about 10%, the proportion of DL6 is about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0%, or about 4% to about 15%.

[0359] In another embodiment, an anti-BCMA Provided is a use of a composition comprising an ADC, wherein the antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6; the cytotoxic drug is MMAF or MMAE; and the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27%, or about 15% to about 32%, the proportion of DL4a is about 35% to about 38%, or about 30% to about 40%, the proportion of DL4b is about 7% to about 9%, or about 5% to about 10%, the proportion of DL6 is about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0%, or about 4% to about 15%.

[0360] In another embodiment, there is provided use of a composition comprising an anti-BCMA ADC in the manufacture of a medicament for use in treating cancer, wherein the antibody is a V having the amino acid sequence set forth in SEQ ID NO:7.H and V having the amino acid sequence set forth in SEQ ID NO:8. L the cytotoxic drug is MMAF or MMAE; the DL0 rate is about 10% or less or about 5% or less, the DL2 rate is about 15% to about 27% or about 15% to about 32%, the DL4a rate is about 35% to about 38% or about 30% to about 40%, the DL4b rate is about 7% to about 9% or about 5% to about 10%, the DL6 rate is about 14% to about 20% or about 10% to about 20%, and / or the DL8 rate is about 6.0% to about 12.0% or about 4% to about 15%.

[0361] In another embodiment, there is provided use of a composition comprising an anti-BCMA ADC in the manufacture of a medicament for use in the treatment of cancer, wherein the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27%, or about 15% to about 32%, the proportion of DL4a is about 35% to about 38%, or about 30% to about 40%, the proportion of DL4b is about 7% to about 9%, or about 5% to about 10%, the proportion of DL6 is about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0%, or about 4% to about 15%.

[0362] In another embodiment, there is provided use of a composition comprising belantamab mafodotin in the manufacture of a medicament for use in the treatment of cancer, wherein the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27% or about 15% to about 32%, the proportion of DL4a is about 35% to about 38% or about 30% to about 40%, the proportion of DL4b is about 7% to about 9% or about 5% to about 10%, the proportion of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0363] In yet another embodiment, there is provided use of a composition comprising belantamab mafodotin in the manufacture of a medicament for use in the treatment of multiple myeloma, wherein the proportion of DL0 is about 10% or less or about 5% or less, the proportion of DL2 is about 15% to about 27% or about 15% to about 32%, the proportion of DL4a is about 35% to about 38% or about 30% to about 40%, the proportion of DL4b is about 7% to about 9% or about 5% to about 10%, the proportion of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0364] In one aspect, there is provided use of a composition comprising an anti-BCMA antigen binding protein in the manufacture of a medicament for use in the treatment of cancer, wherein the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein the composition comprises 25% or less isomerization at D103 of the heavy chain CDRH3.

[0365] In one embodiment, there is provided use of a composition comprising an anti-BCMA antigen binding protein in the manufacture of a medicament for use in multiple myeloma, wherein the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein the composition comprises 25% or less isomerization at D103 of the heavy chain CDRH3.

[0366] In yet another embodiment, there is provided a use of a composition comprising belantamab in the manufacture of a medicament for use in the treatment of multiple myeloma, wherein the composition comprises 25% or less isomerization at D103 of heavy chain CDRH3.

[0367] In one aspect, there is provided use of a composition comprising an anti-BCMA antigen binding protein in the manufacture of a medicament for use in the treatment of cancer, wherein the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein the composition comprises 40% or less oxidation in heavy chain M34(CDRH1).

[0368] In one embodiment, there is provided use of a composition comprising an anti-BCMA antigen binding protein in the manufacture of a medicament for use in treating multiple myeloma, wherein the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO: 6, wherein the composition comprises 40% or less oxidation in heavy chain M34(CDRH1).

[0369] In yet another embodiment, there is provided a use of a composition comprising belantamab in the manufacture of a medicament for use in the treatment of multiple myeloma, wherein the composition comprises 40% or less oxidation in heavy chain M34 (CDRH1).

[0370] All patent and literature references disclosed herein are expressly and entirely incorporated by reference.

[0371] The inventions described herein include the following: [1] A composition comprising an isomerization variant of an anti-BCMA antibody, The isomerization variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises 25% or less of the isomerization variant. [2] A composition comprising an oxidized variant of an anti-BCMA antibody, The oxidation variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises no more than 40% of the oxidation variant. [3] A composition comprising an anti-BCMA antibody, the anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2; CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4; CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 Including, The composition, wherein the composition contains 0.1 to 25% isomerization at D103 of CDRH3. [4] A composition comprising an anti-BCMA antibody, the anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2; CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4; CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 Including, The composition, wherein the composition comprises 0.1 to 40% oxidation at M34 of CDRH1. [5] A composition comprising an anti-BCMA antibody, the anti-BCMA antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10; The composition, wherein the composition contains 0.1 to 25% isomerization at D103 of CDRH3. [6] A composition comprising an anti-BCMA antibody, the anti-BCMA antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10; The composition, wherein the composition comprises 0.1 to 40% oxidation at M34 of CDRH1. [7] The composition according to any one of [1] to [6], wherein the composition contains 65% or less oxidation at M256 of the heavy chain and / or 60% or less oxidation at M432 of the heavy chain. [8] The composition comprises an antibody variant; The composition according to any one of [1] to [7], wherein the antibody variant comprises one or more selected from the group consisting of deamidation at N388 and / or N393 in the heavy chain, a change from D103 to N103 in CDRH3, truncation of C-terminal lysine, and conversion of N-terminal glutamine to pyroglutamic acid. [9] The composition according to any one of [1] to [8], wherein the composition comprises one or more selected from the group consisting of up to 100% deamidation at N388 and / or N393, up to 100% cleavage of N103 in CDRH3, up to 100% cleavage of C-terminal lysine, and up to 100% conversion of N-terminal glutamine to pyroglutamic acid.

[10] The composition according to any one of [1] to [9], wherein the composition contains any percentage of glycoforms G0, G1, G2, G0-GlcNac, or G0-2GlcNac.

[11] The composition according to any one of [1] to

[10] , wherein the anti-BCMA antibody is belantamab.

[12] The composition according to any one of [1] to

[11] , wherein the anti-BCMA antibody is conjugated to a cytotoxic drug to form an antibody-drug complex.

[13] The composition according to any one of [1] to

[12] , wherein the anti-BCMA antibody is belantamab mafodotin.

[14] The composition according to

[12] or

[13] , wherein the proportion of DL2 is at least about 30%, about 15% to about 27%, or about 15% to about 32%, the proportion of DL4a is at least about 30%, about 35% to about 38%, or about 30% to about 40%, the proportion of DL4b is at least about 5%, about 7% to about 9%, or about 5% to about 10%, the proportion of DL6 is at least about 10%, about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is at least about 1%, about 6.0% to about 12.0%, or about 4% to about 15%.

[15] The composition according to any one of

[12] to

[14] , wherein the average DAR is about 3.4 to about 4.6.

[16] The composition according to any one of

[12] to

[15] , wherein the proportion of DL0 is about 10% or less or about 5% or less.

[17] A pharmaceutical composition comprising the composition according to any one of [1] to

[16] and at least one pharmaceutically acceptable excipient.

[18] A formulation comprising the pharmaceutical composition according to

[17] , the formulation comprises about 20 mg / mL to about 60 mg / mL of an anti-BCMA antigen-binding protein, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80; The formulation has a pH of about 5.9 to about 6.5.

[19] The formulation of

[18] , wherein the formulation comprises about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, or about 60 mg / mL of belantamab mafodotin, 25 mM citrate buffer, 200 mM trehalose, 0.05 mM disodium EDTA, and 0.02% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[20] A method for treating cancer, comprising administering a therapeutically effective amount of the composition according to any one of [1] to

[16] to a subject in need thereof.

[21] The composition according to any one of [1] to

[14] for use in the treatment of cancer.

[22] Use of the composition according to any one of [1] to

[16] in the manufacture of a medicament for use in the treatment of cancer.

[23] A composition comprising an anti-BCMA antibody-drug conjugate (ADC), wherein the proportion of DL2 is at least about 30%, about 15% to about 27%, or about 15% to about 32%; the proportion of DL4a is at least about 30%, about 35% to about 38%, or about 30% to about 40%; the proportion of DL4b is at least about 5%, about 7% to about 9%, or about 5% to about 10%; the proportion of DL6 is at least about 10%, about 14% to about 20%, or about 10% to about 20%; and / or the proportion of DL8 is at least about 1%, about 6.0% to about 12.0%, or about 4% to about 15%.

[24] The anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2; CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4; CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 The composition according to

[23] , comprising:

[25] The composition according to

[23] or

[24] , wherein the proportion of DL2 is about 15% to about 32%, the proportion of DL4a is about 30% to about 40%, the proportion of DL4b is about 5% to about 10%, the proportion of DL6 is about 10% to about 20%, and the proportion of DL8 is about 4% to about 15%.

[26] The composition according to

[23] or

[24] , wherein the proportion of DL2 is about 15% to about 27%, the proportion of DL4a is about 35% to about 38%, the proportion of DL4b is about 7% to about 9%, the proportion of DL6 is about 14% to about 20%, and the proportion of DL8 is about 6.0% to about 12.0%.

[27] The composition according to any one of

[23] to

[26] , having an average drug-antibody ratio (DAR) of about 2.1 to about 5.7.

[28] The composition according to any one of

[23] to

[26] , wherein the average DAR is about 3.4 to about 4.6.

[29] The composition according to any one of

[23] to

[26] , wherein the average DAR is about 3.8 to about 4.5.

[30] A composition comprising an anti-BCMA antibody-drug conjugate (ADC), wherein the percentage of DL0 is about 10% or less, or about 5% or less.

[31] The anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO: 2; CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4; CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 The composition according to

[30] , comprising:

[32] The composition according to

[30] or

[31] , wherein the proportion of DL0 is about 5% or less.

[33] The composition according to any one of

[30] to

[32] , wherein the proportion of DL2 is about 15% to about 32%, the proportion of DL4a is about 30% to about 40%, the proportion of DL4b is about 5% to about 10%, the proportion of DL6 is about 10% to about 20%, and the proportion of DL8 is about 4% to about 15%.

[34] The composition according to any one of

[30] to

[32] , wherein the proportion of DL2 is about 15% to about 27%, the proportion of DL4a is about 35% to about 38%, the proportion of DL4b is about 7% to about 9%, the proportion of DL6 is about 14% to about 20%, and the proportion of DL8 is about 6.0% to about 12.0%.

[35] The composition according to any one of

[30] to

[34] , having an average drug-antibody ratio (DAR) of about 2.1 to about 5.7.

[36] The composition according to any one of

[30] to

[34] , wherein the average DAR is about 3.4 to about 4.6.

[37] The composition according to any one of

[30] to

[34] , wherein the average DAR is about 3.8 to about 4.5.

[38] The anti-BCMA antibody has the amino acid sequence set forth in SEQ ID NO: 7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L The composition according to any one of

[23] to

[37] , comprising:

[39] The composition according to any one of

[23] to

[38] , wherein the anti-BCMA antibody is belantamab.

[40] The composition according to any one of

[23] to

[37] , wherein the cytotoxic drug is MMAE or MMAF.

[41] The composition according to any one of

[21] to

[40] , wherein the anti-BCMA ADC is belantamab mafodotin.

[42] The composition of any of

[23] to

[41] , wherein the proportion of DL is determined by separating individual DL species using hydrophobic interaction chromatography (HIC), calculating the area under the curve for each DL peak, and dividing each DL peak by the total area under the curve for all DL species combined.

[43] The average DAR is calculated using the following formula:

number

[42] is calculated from the area under the curve of each DL species using the formula:

[44] A pharmaceutical composition comprising the composition according to any one of

[23] to

[43] and at least one pharmaceutically acceptable excipient.

[45] A formulation comprising the pharmaceutical composition according to

[44] , the formulation comprises about 20 mg / mL to about 60 mg / mL of an anti-BCMA antigen-binding protein, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80; The formulation has a pH of about 5.9 to about 6.5.

[46] The formulation of

[45] , wherein the formulation comprises about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, or about 60 mg / mL of belantamab mafodotin, 25 mM citrate buffer, 200 mM trehalose, 0.05 mM disodium EDTA, and 0.02% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[47] A method for treating cancer, comprising administering a therapeutically effective amount of the composition according to any one of

[23] to

[43] to a subject in need thereof.

[48] ​​The composition according to any one of

[23] to

[43] for use in the treatment of cancer.

[49] Use of the composition according to any one of

[23] to

[43] in the manufacture of a medicament for use in the treatment of cancer.

[50] A composition comprising an acidic variant of an antibody, The acidic variant is CDRH1 of SEQ ID NO: 1; CDRH2 of SEQ ID NO: 2; CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises 1 to 70% of the acidic variant.

[51] A composition comprising an acidic variant of an antibody, The acidic variant is CDRH1 of SEQ ID NO: 1; CDRH2 of SEQ ID NO: 2; CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises no more than 70% of the acidic variant.

[52] A composition comprising a basic variant of an antibody, The basic variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises 1 to 30% of the basic variant.

[53] A composition comprising a basic variant of an antibody, The basic variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises no more than 30% of the basic variant.

[54] A composition comprising a major isoform of an antibody, The major isoform is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises 1 to 90% of the major isoform.

[55] A composition comprising a major isoform of an antibody, The major isoform is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition, wherein the composition comprises 1% or more of the major isoform.

[56] A composition comprising a charge variant of an antibody, The charge variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6; Including, The composition comprises no more than 70% acidic variants and / or no more than 30% basic variants and / or 1% or more of the major isoform. [Example]

[0372] Example 1: Determination of the Proportion and Mean DAR of DL Species in ADC Compositions For Examples 2-9, the percentage of DL and average DAR were calculated as follows: The percentage of a specific DL species (e.g., percentage DL0, percentage DL2, percentage DL4a, percentage DL4b, percentage DL6, and percentage DL8) was determined by separating the individual DL species using hydrophobic interaction chromatography (HIC) (illustrated in Figure 2), calculating the area under the curve for each DL peak, and dividing each DL peak by the total area under the curve for all DL species combined.

[0373] The mean DAR for each sample was calculated from the area under the curve for each DL species using the following formula:

[0374]

number

[0375] Reference standards used in Examples 2-8 and which may also be used in other experiments include the samples in Table 1.

[0376] [Table 1]

[0377] Example 2: Effect of mean DAR on cell growth inhibition The cell growth inhibition of belantamab mafodotin was determined by measuring cell viability of NCI-H929 cells, a human multiple myeloma cell line, after 48 hours of incubation with belantamab mafodotin. Cell viability was measured using Promega's CellTiter Glo technology. Increasing concentrations of belantamab mafodotin corresponded relatively to decreases in the CellTiter Glo luminescence signal. Dose-response (EC50) curves were generated using SoftMaxPro using a four-parameter nonlinear logistic regression model. Relative potency was determined by calculating the ratio of the EC50 of the reference standard #132371424 to the EC50 of the sample. Results are summarized in Table 2.

[0378] [Table 2]

[0379] Example 3: Effect of mean DAR on ADCC activity Belantamab mafodotin was incubated with multiple myeloma cells and NK cells (effector cells). Without being bound by theory, belantamab mafodotin bound to BCMA expressed on the surface of multiple myeloma cells, and the Fc region of the antibody bound to the effector cell receptor FcγRIIIa via the effector cell receptor FcγRIIIa. Engagement of these receptors on the surface of effector cells resulted in the synthesis and secretion of cytokines (IFNg) and the release of granules (perforin and granzymes) that entered the cytoplasm of target cells. Granzymes initiated signaling events within the target cells that led to the death of these cells by apoptosis. The source of NK cells was peripheral blood mononuclear cells (PBMCs) isolated from human whole blood. Next, 1 mL of NCI-H929 cells (a human multiple myeloma cell line) was loaded with 10 μL of BATDA (bis-(acetoxymethyl) 2,2':6',2''-terpyridine-6,6''-dicarboxylate) (Perkin-Elmer, catalog number C136-100), an acetoxymethyl ester of the fluorescence-enhancing ligand that permeates the cell membrane. The ester bond of BATDA was hydrolyzed to form the hydrophilic ligand (TDA), which can no longer cross the cell membrane. The labeled cells were then added to various amounts of belantamab mafodotin and effector cells (PBMCs). The cells were lysed, releasing TDA. After cell lysis, the ligand was mixed with 200 μL of DELFIA Europium Solution (Perkin-Elmer, catalog number C135-100) to form a highly fluorescent and stable chelate (EuTDA). When measured with a fluorescent plate reader, the measured fluorescence directly correlates with the amount of cells lysed. The ADCC activity of belantamab mafodotin was reported as the ratio of the EC50 value of the sample to the EC50 value of the reference standard #132371424. The results are summarized in Table 3.

[0380] [Table 3]

[0381] Example 4: Effect of mean DAR on BCMA binding and FcγRIIIa binding Surface plasmon resonance (SPR) was used to measure the binding of belantamab mafodotin to BCMA and FcγRIIIa. Belantamab mafodotin was diluted to 10 μg / mL in PBST, injected, and captured by Protein A immobilized on a CM5 sensor chip. BCMA was then injected and allowed to bind to the captured belantamab mafodotin. FcγRIIIa was then injected and allowed to bind to the captured belantamab mafodotin. The functional concentrations of belantamab mafodotin bound to BCMA and FcγRIIIa were calculated from a reference standard curve (Reference Standard #132371424) and reported as BCMA or FcγRIIIa-bound concentrations, respectively. The total belantamab mafodotin concentration of the samples was previously determined by absorbance at 280 nm. Specific binding activity (%) was calculated by dividing the bound concentration of BCMA or FcγRIIIa by the absorbance at 280 nm at that concentration. The results are summarized in Table 4.

[0382] [Table 4]

[0383] Example 5: Effect of mean DAR on tumor volume Multiple myeloma cell lines were subcutaneously implanted into the flanks of severe combined immunodeficiency (SCID) mice. Starting around day 15, all tumors were measured three times a week using a caliper system, and the long and short diameters of each mouse tumor were recorded to calculate tumor volume (volume = long diameter × short diameter). 2 ) × 0.5). The average tumor volume was approximately 200 mm 3 Once tumors reached the mean DAR, mice were randomized into groups and administered belantamab mafodotin containing one of the mean DAR samples twice weekly for two weeks. All tumors were measured in this manner, and tumors reached a mean tumor measurement of 2.0 mm. 3 Individual mice were euthanized upon reaching 0.05 mg / kg or day 60, whichever came first. The study design is outlined in Table 5, and the results are shown in Figure 3.

[0384] [Table 5]

[0385] Example 6: Effect of DL species on BCMA binding, FcγRIIIa binding, and FcRn binding. Belantamab mafodotin samples containing specific DL species were prepared by collecting distinct peaks in HIC chromatograms. Binding of belantamab mafodotin to BCMA or FcγRIIIa was measured using surface plasmon resonance (SPR) as described in Example 4.

[0386] Surface plasmon resonance (SPR) was used to measure the binding of fetal Fc receptor (FcRn) to belantamab mafodotin. Samples were diluted, and belantamab mafodotin was captured by FcRn immobilized on a nitrilotriacetic acid (NTA) sensor chip. The FcRn-bound concentration of the sample was determined by interpolation of the binding response on a standard curve. Specific binding activity (%) was calculated by dividing the FcRn-bound concentration by the total protein concentration. The results are summarized in Table 6.

[0387] Reference standard #162397940 was used.

[0388] [Table 6]

[0389] Example 7: Effect of DL species on cell growth inhibition Specific DL species samples of belantamab mafodotin were prepared as in Example 7. Cell growth inhibition was determined as in Example 2. Reference standard #162397940 was used. The results are summarized in Table 7.

[0390] [Table 7]

[0391] Example 8: Effect of DL species on ADCC activity Belantamab mafodotin was bound to BCMA expressed on the surface of multiple myeloma cells. The Fc region of belantamab mafodotin was bound to FcγRIIIa (CD16a) on Jurkat T effector cells (Promega, Catalog No. G7102, BioCat #140011) engineered to stably express 1) the high-affinity variant of the human FcγRIIIa receptor V158 and 2) a luciferase reporter gene fused to a promoter downstream of an NFAT activation sequence. When the antibody simultaneously bound to H929 and effector cells, activation of the NFAT pathway resulted in gene transcription from the luciferase reporter gene and expression of firefly luciferase in the effector cells. A luminescent substrate (Bio-Glo™ Luciferase Assay System, Promega, Cat. No. G7940) was added, and after cell lysis, luciferase produced by NFAT activation was measured using a plate reader as relative luminescence units (RLU). In this assay, belantamab mafodotin was added in a dose-dependent manner. Therefore, a nonlinear logistic regression model was used to generate a dose-response (half maximal effective concentration or EC50). Relative potency was determined by calculating the ratio of the EC50 of the reference standard to the EC50 of the sample. Reference standard #162397940 was used. The results are summarized in Table 8.

[0392] [Table 8]

[0393] Example 9: Mean DAR and percentage of DL species for several lots of belantamab mafodotin Several (19) batches of belantamab mafodotin were manufactured. The mean DAR and percentage of DL species for each batch were calculated as described in Example 1. The results are summarized in Tables 9-12.

[0394] [Table 9]

[0395] [Table 10]

[0396] [Table 11]

[0397] [Table 12]

[0398] Example 10: Belantamab mafodotin forced degradation study design An overview of the study design for Examples 11 to 18 is shown in Table 13.

[0399] [Table 13]

[0400] Example 11: Oxidation Conditions To generate oxidative stress samples, belantamab mafodotin samples were diluted to 10 mg / mL and hydrogen peroxide was added to a molar ratio of hydrogen peroxide to belantamab mafodotin of 500:1. Samples were quenched with methionine and buffer exchanged using a 3 kDa molecular weight cutoff filter (MWCO).

[0401] Deamidation and oxidation were determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). Samples were denatured with 6 M guanidine HCl solution to a concentration of 4.2 μg / μL. Disulfide bonds were reduced with 50 mM DTT for 20 minutes at room temperature. Iodoacetic acid was added at 100 mM and reacted with free cysteine ​​residues at room temperature for 30 minutes, protected from light. Samples were buffer exchanged using a BioRad spin column (part number 7326221). Samples were then digested with 0.5% trypsin using Worthington trypsin (part number TRTPCK) at 37°C for 15 minutes. The resulting peptides were loaded onto a Waters reverse-phase ultra-performance liquid chromatography (UPLC) column (part number 186003687) and eluted with a gradient of water and acetonitrile (0.1% trifluoroacetic acid) using a Waters Acquity UPLC. Peptides were detected by a UV detector and a mass spectrometer, such as a Thermo Scientific LTQ OrbitrapXL. Extracted ion chromatograms of the unmodified and modified peptides were used to calculate the level of either deamidation or oxidation by dividing the area under the curve of the modified peptide by the total area under the curve of both the modified and unmodified peptides.

[0402] Binding of BCMA and FcγRIIIa by belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 4. Binding of fetal Fc receptor (FcRn) to belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 6.

[0403] Reference standard #182407660A was used. A summary of the results is shown in Tables 14 and 15.

[0404] [Table 14]

[0405] [Table 15]

[0406] Extrapolating from the data for oxidation with HC Met34, up to 37% oxidation results in at least 70% antigen-specific binding activity. This was calculated using the linear slopes of the M34-oxidized samples at time 0 (0.2-0.4%) and 24 hours (45.1-45.5%), which have antigen-specific binding activities of 88-99% and 62-88%, respectively.

[0407] Extrapolating from the data for oxidation at HC Met256, up to 89% oxidation results in at least 70% FcγRIIIa-specific binding activity. This is calculated using the linear slopes of the M256-oxidized samples at time 0 (2.9-3.5%) and 24 hours (98.3-98.7%), which have FcγRIIIa-specific binding activity of 89-99% and 66-70%, respectively. Extrapolating from the data for oxidation at HC Met256, up to 64% oxidation results in at least 70% FcRn-specific binding activity. This is calculated using the linear slopes of the M256-oxidized samples at time 0 (2.9-3.5%) and 24 hours (98.3-98.7%), which have FcRn-specific binding activity of 93-97% and 52-57%, respectively.

[0408] Extrapolating from the data for oxidation with HC Met432, up to 86% oxidation results in at least 70% FcγRIIIa-specific binding activity. This is calculated using the linear slopes of the M432-oxidized samples at time 0 (0.4-0.6%) and 24 hours (94.8-95.2%), which have FcγRIIIa-specific binding activity of 88-99% and 66-70%, respectively. Extrapolating from the data for oxidation with HC Met432, up to 61% oxidation results in at least 70% FcRn-specific binding activity. This is calculated using the linear slopes of the M432-oxidized samples at time 0 (0.4-0.6%) and 24 hours (94.8-95.2%), which have FcRn-specific binding activity of 93-97% and 52-57%, respectively.

[0409] Example 12: Chemistry: High pH (Base Treatment) To generate high pH samples, belantamab mafodotin samples were diluted with 4 mM Tris buffer to adjust the pH to 9. Samples were further diluted to 10 mg / mL and incubated at 25°C / 50% RH for up to 21 days.

[0410] Deamidation, isomerization, and oxidation were determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described in Example 11. Extracted ion chromatograms of the unmodified and modified peptides were used to calculate the level of either deamidation, isomerization, or oxidation by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0411] Binding of BCMA and FcγRIIIa by belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 4. Binding of fetal Fc receptor (FcRn) to belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 6.

[0412] Reference standard #182407660A was used. The results are summarized in Tables 16 and 17.

[0413] [Table 16]

[0414] [Table 17]

[0415] Deamidation at HC Asn388 and HC Asn393 is expected to be higher than the reported levels of 10.3% and 14.5%, respectively, without affecting antigen-, FcγRIIIa-, and FcRn-specific binding.

[0416] Example 13: Chemistry: Low pH (Acid Treatment) To generate low pH samples, belantamab mafodotin samples were diluted with citrate buffer to adjust the pH to 5. Samples were further diluted to 10 mg / mL and incubated at 25°C / 50% RH for up to 21 days.

[0417] Isomerization, deamidation, and oxidation were determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described in Example 11. Extracted ion chromatograms of the unmodified and modified peptides were used to calculate the level of either isomerization, deamidation, or oxidation by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0418] Binding of BCMA and FcγRIIIa by belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 4. Binding of fetal Fc receptor (FcRn) to belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 6.

[0419] Reference standard #182407660A was used. The results are summarized in Tables 18 and 19.

[0420] [Table 18]

[0421] [Table 19]

[0422] Example 14: Heat: High Temperature To generate heat-stressed samples, belantamab mafodotin samples were diluted to 10 mg / mL in formulation buffer and incubated at 40°C / 75% RH for up to 28 days.

[0423] Isomerization, deamidation, and oxidation were determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described in Example 11. Extracted ion chromatograms of the unmodified and modified peptides were used to calculate the level of either isomerization, deamidation, or oxidation by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0424] Binding of BCMA and FcγRIIIa by belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 4. Binding of fetal Fc receptor (FcRn) to belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 6.

[0425] Reference standard #182407660A was used. The results are summarized in Tables 20 and 21.

[0426] [Table 20]

[0427] [Table 21]

[0428] Extrapolating from the data for isomerization at HC Asp130, up to 23% isomerization results in at least 70% antigen-specific binding activity. This was calculated using the linear slopes of the D103 isomerized samples at time 0 (4.1-4.4%) and 28 days (28.7-29.3%), which have antigen-specific binding activities of 88-99% and 55-62%, respectively.

[0429] Example 15: Exposure To prepare the exposed samples, belantamab mafodotin samples were diluted to 10 mg / mL, filled into glass vials, and transferred to a Caron Photostability Chamber at 25° C. for various degrees of exposure as shown in Table 10.

[0430] Oxidation, deamidation, and isomerization were determined using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described in Example 11. Extracted ion chromatograms of the unmodified and modified peptides were used to calculate the level of either oxidation, deamidation, or isomerization by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0431] Binding of BCMA and FcγRIIIa by belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 4. Binding of fetal Fc receptor (FcRn) to belantamab mafodotin was measured using surface plasmon resonance (SPR) as described in Example 6.

[0432] Reference standard #182407660A was used. The results are summarized in Tables 22 and 23.

[0433] [Table 22]

[0434] [Table 23]

[0435] Example 16: C-terminal truncation and N-terminal pyroglutamic acid Several lots of belantamab were analyzed for levels of N-terminal pyroglutamic acid and C-terminal truncations using tryptic peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) as described in Example 11. Extracted ion chromatograms of the unmodified and modified peptides were used to calculate the levels of either N-terminal pyroglutamic acid or C-terminal truncations by dividing the area under the curve for the modified peptide by the total area under the curve for both the modified and unmodified peptides.

[0436] Binding of BCMA and FcγRIIIa by belantamab was measured using surface plasmon resonance (SPR) as described in Example 4. Binding of fetal Fc receptor (FcRn) to belantamab was measured using surface plasmon resonance (SPR) as described in Example 6.

[0437] The reference standards used were #122368059 and #172405900. The results are summarized in Tables 24-25.

[0438] [Table 24]

[0439] [Table 25]

[0440] Example 17: Glycosylation Several lots of belantamab were analyzed for glycosylation patterns. The profiles were determined using ultra-performance liquid chromatography (UPLC) with hydrophilic interaction liquid chromatography (HILIC) separation and fluorescence detection. Samples were diluted with water to a concentration of 10 μg / μL. Glycans were released from belantamab by enzymatic digestion with PNGaseF using the PNGaseF kit from New England BioLabs (catalog no. P0705L). The glycans released by PNGaseF were labeled with anthranilamide (Sigma-Aidrich, catalog no. A89804). The labeled glycans were then purified to remove excess labeling solution using a HILIC column step (a step in which the glycans can be loaded, washed with water, and eluted with acetonitrile). The labeled glycans were then separated on a Waters Acquity UPLC using a Waters Glycan BEH Amide column (catalog no. 186004742) with an ammonium formate / formic acid and acetonitrile gradient. Glycans were detected using fluorescence detection with excitation at 365 nm and emission at 438 nm. Glycan quantification was performed by dividing the area under the curve for a glycan by the total area under the curve for all detected glycans.

[0441] The reference standard used was #122368059. The results are summarized in Tables 26-27.

[0442] [Table 26]

[0443] [Table 27]

[0444] Example 18: Glycoengineering The effect of glyco-enriched samples of belantamab on ADCC activity and binding was determined. ADCC activity was measured as in Example 3. BCMA and FcγRIIIa binding was determined as in Example 4.

[0445] For galactosylation experiments, glycosylation was measured by reduced LC-MS. Samples were diluted to 1 mg / mL, 50 μL of 1 M DTT was added, and the mixture was incubated at 25°C or 37°C for 30 minutes before analysis by mass spectrometry (Micromass Q-tof). Heavy and light chains were separated using size-exclusion chromatography with an isocratic flow of water, acetonitrile, and trifluoroacetic acid. Spectra for each heavy and light chain were summed and deconvoluted using Waters MaxEnt software. Major glycoforms were detected, and relative abundances were assessed from signal counts or area under the curve. The results are summarized in Tables 28-29.

[0446] [Table 28]

[0447] [Table 29]

[0448] Example 19: Tolerance The data in Tables 11-18 for the first and last (day 21 or day 28, depending on the condition) time points were used to determine the acceptable range (70-130% activity). Binding data were plotted against the relative percentage of the relevant post-translational modification to determine the slope of the relationship. This information was used to calculate the predicted level of each post-translational modification for at least 70% binding measurements. The results of this extrapolation are summarized in Table 30. The reported trends broadly reflect the observations seen with either belantamab or belantamab mafodotin.

[0449] [Table 30]

[0450] Example 20: Summary of degradation products The incidence of degradation products observed under various forced degradation conditions that do not affect belantamab mafodotin activity is summarized below (Table 31).

[0451] [Table 31]

[0452] Example 21 A summary of the study design for belantamab forced degradation in Examples 21-26 is shown in Table 32. Methods were generally similar to those described above for belantamab mafodotin in Examples 11-18 (except where noted).

[0453] [Table 32]

[0454] Example 22: Oxidation conditions for belantamab After 24 hours, oxidation of HC M256 increased from approximately 2% to approximately 98%, and oxidation of M432 increased from approximately 1% to approximately 98%. SPR revealed a 21-25% decrease in specific binding to FcγRIIIa and a 10-18% decrease in FcRn binding. Fc oxidation may alter the binding activity of belantamab to FcγRIIIa and FcRn. After 24 hours, oxidation of CDR1 HC M34 increased from approximately 0.3% to 47.7-48.5%, with no change in antigen binding and within the assay variability. Cysteine ​​and tryptophan oxidation levels were low throughout this study, and no other significant post-translational modifications were detected.

[0455] Example 23: Base treatment of belantamab HC D103 isomerization was observed to increase from approximately 3.5% to approximately 6.5% after 28 days. After 28 days, deamidation increased from 0.1% to approximately 2.5% in HC N31, from approximately 2.0% to approximately 10% in HC N388, and from approximately 1.7% to approximately 18.5% in HC N393. Furthermore, oxidation of HC M256 was also observed to increase from approximately 2.2% to approximately 3.7% in belantamab exposed to pH 9 stress for 28 days. cIEF analysis showed that acidic variants increased from approximately 25% to approximately 62%, and basic variants decreased from approximately 9% to approximately 4.5% (see Table 33 below). All observed changes were within the variability of the antigen-, FcγRIIIa-, and FcRn-specific binding assays. Thus, binding by SPR is comparable to belantamab stressed at pH 9.0 at 28 days.

[0456] [Table 33]

[0457] Example 24: Acid treatment of belantamab Fragments increased from 0.8% to 2.3-2.7% after 28 days. Succinimide formation in HC D103 increased from 0.2% to 4.0%, and HC D103 isomerization increased from approximately 3.5% to approximately 5.8% after 28 days. In addition to aspartic acid isomerization, HC M256 oxidation was also observed to increase from approximately 2.2% to approximately 3.7% in belantamab exposed to pH 3.5 stress for 28 days. cIEF analysis showed that acidic variants increased from approximately 25% to approximately 28%, and basic variants increased from approximately 9% to approximately 13% (see Table 34 below). All observed changes in antigen, FcγRIIIa, and FcRn-specific binding were within the range of assay variability. Thus, binding by SPR is comparable to belantamab stressed at pH 3.5 after 28 days.

[0458] [Table 34]

[0459] Example 25: Heat-treated belantamab Fragmentation increased from 0.8% to 2.2-2.3% after 28 days. % aggregation remained unchanged. Succinimide formation in HC D103 increased from 0.2% to 2.2%, and HC D103 isomerization increased from approximately 3.5% to approximately 29% after 28 days. In addition to aspartic acid isomerization, HC M256 oxidation increased from approximately 2.2% to approximately 5.3%, and HC M432 oxidation increased from approximately 1% to 2% in belantamab exposed to heat stress for 28 days. After 28 days, HC N329 deamidation increased from approximately 0% to approximately 7%, HC N388 deamidation increased from approximately 2.0% to approximately 2.5%, and HC N393 deamidation increased from approximately 1.7% to approximately 2.6%. Antigen-specific binding decreased to 63-67% by day 28, consistent with increased HC D103 isomerization, which has been shown to affect antigen binding. The observed changes in FcγRIIIa and FcRn-specific binding were within the variability of the assay.

[0460] Example 26: Phototreated Belantamab Fragmentation increased from 0.8% to 1.5% at 1.5x ICH, and aggregation increased from approximately 1% to 6.5-7.5%. At 1.5x ICH, HC M34 oxidation increased from approximately 0.3% to 1.6-2.1%, HC M256 oxidation increased from approximately 2.2% to 18.4-251%, and HC M432 oxidation increased from approximately 1% to 13.7-19.5%. cIEF analysis showed that acidic variants increased from approximately 25% to approximately 34%, while basic variants remained unchanged (see Table 35 below). All observed changes in antigen, FcγRIIIa, and FcRn-specific binding were within the variability of the assay. Therefore, binding by SPR is equivalent for belantamab exposed to pH 3.5 stress after 28 days.

[0461] [Table 35]

[0462] The belantamab forced degradation study showed results consistent with the belantamab mafodotin forced degradation study described above, except that oxidation up to 48.5% in HC M34 did not result in changes in antigen binding and was within the range of assay variability.

[0463] Note that cIEF data for belantamab mafodotin is not shown because the drug loading contributes to the charge profile, but in the case of belantamab, cIEF effectively separates the acidic and basic variants from the main species (see Figure 4).

[0464] Sequence Listing SEQ ID NO: 1: CDRH1 NYWMH

[0465] SEQ ID NO: 2: CDRH2 ATYRGHSDTYYNQKFKG

[0466] SEQ ID NO: 3: CDRH3 GAIYDGYDVLDN

[0467] SEQ ID NO: 4: CDRL1 SASQDISNYLN

[0468] SEQ ID NO: 5: CDRL2 YTSNLHS

[0469] SEQ ID NO: 6: CDRL3 QQYRKLPWT

[0470] SEQ ID NO: 7: Heavy chain variable region (CDRs are underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDNWGQGTLVTVSS

[0471] SEQ ID NO: 8: Light chain variable region (CDRs are underlined) DIQMTQSPSSLSASVGDRVTITC SASQDISNYLN WYQQKPGKAPKLLIY YTSNLHS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYRKLPWT FGQGTKLEIKR

[0472] SEQ ID NO: 9: Heavy chain region (CDRs are underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0473] SEQ ID NO: 10: Light chain region (CDRs are underlined) DIQMTQSPSSLSASVGDRVTITC SASQDISNYLN WYQQKPGKAPKLLIY YTSNLHS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0474] SEQ ID NO: 11: Heavy chain region with D103N (CDRs are underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYNGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0475] SEQ ID NO: 12: Heavy chain region with N388D (CDRs are underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0476] SEQ ID NO: 13: Heavy chain region with N393D (CDRs are underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0477] SEQ ID NO: 14: Heavy chain region with N388D and N393D (CDRs are underlined) VQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESDGQPEDNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. 1. A composition comprising an isomerization variant of an anti-BCMA antibody, The isomerization variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises 25% or less of the isomerization variant.

2. 1. A composition comprising an oxidized variant of an anti-BCMA antibody, The oxidation variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises no more than 40% of the oxidation variant.

3. 1. A composition comprising an anti-BCMA antibody, The anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 Including, The composition, wherein the composition comprises 0.1 to 25% isomerization at D103 of CDRH3.

4. 1. A composition comprising an anti-BCMA antibody, The anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 Including, The composition, wherein the composition comprises 0.1 to 40% oxidation at M34 of CDRH1.

5. 1. A composition comprising an anti-BCMA antibody, the anti-BCMA antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 and the light chain amino acid sequence of SEQ ID NO:10; The composition, wherein the composition comprises 0.1 to 25% isomerization at D103 of CDRH3.

6. 1. A composition comprising an anti-BCMA antibody, the anti-BCMA antibody is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 and the light chain amino acid sequence of SEQ ID NO:10; The composition, wherein the composition comprises 0.1 to 40% oxidation at M34 of CDRH1.

7. The composition of any one of claims 1 to 6, wherein the composition comprises no more than 65% oxidation at heavy chain M256 and / or no more than 60% oxidation at heavy chain M432.

8. the composition comprises an antibody variant; The composition of any one of claims 1 to 7, wherein the antibody variant comprises one or more selected from the group consisting of deamidation at N388 and / or N393 of the heavy chain, a change from D103 to N103 in CDRH3, truncation of C-terminal lysine, and conversion of N-terminal glutamine to pyroglutamic acid.

9. 9. The composition of claim 1, wherein the composition comprises one or more selected from the group consisting of up to 100% deamidation at N388 and / or N393, up to 100% deamidation at N103 in CDRH3, up to 100% cleavage of C-terminal lysine, and up to 100% conversion of N-terminal glutamine to pyroglutamic acid.

10. 10. The composition of any one of claims 1 to 9, wherein the composition comprises any percentage of glycoforms G0, G1, G2, G0-GlcNac or G0-2GlcNac.

11. The composition of any one of claims 1 to 10, wherein the anti-BCMA antibody is belantamab.

12. The composition of any one of claims 1 to 11, wherein the anti-BCMA antibody is conjugated to a cytotoxic drug to form an antibody-drug conjugate.

13. The composition of any one of claims 1 to 12, wherein the anti-BCMA antibody is belantamab mafodotin.

14. The composition of claim 12 or 13, wherein the proportion of DL2 is at least about 30%, about 15% to about 27%, or about 15% to about 32%, the proportion of DL4a is at least about 30%, about 35% to about 38%, or about 30% to about 40%, the proportion of DL4b is at least about 5%, about 7% to about 9%, or about 5% to about 10%, the proportion of DL6 is at least about 10%, about 14% to about 20%, or about 10% to about 20%, and / or the proportion of DL8 is at least about 1%, about 6.0% to about 12.0%, or about 4% to about 15%.

15. 15. The composition of any one of claims 12 to 14, having an average DAR of from about 3.4 to about 4.

6.

16. The composition according to any one of claims 12 to 15, wherein the proportion of DL0 is about 10% or less or about 5% or less.

17. A pharmaceutical composition comprising the composition of any one of claims 1 to 16 and at least one pharmaceutically acceptable excipient.

18. 18. A formulation comprising the pharmaceutical composition of claim 17, the formulation comprises about 20 mg / mL to about 60 mg / mL of anti-BCMA antigen binding protein, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80; The formulation, wherein the pH of the formulation is from about 5.9 to about 6.

5.

19. 19. The formulation of claim 18, wherein the formulation comprises about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, or about 60 mg / mL belantamab mafodotin, 25 mM citrate buffer, 200 mM trehalose, 0.05 mM disodium EDTA, and 0.02% polysorbate 20 or polysorbate 80, and wherein the pH of the formulation is about 5.9 to about 6.

5.

20. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1 to 16.

21. A composition according to any one of claims 1 to 14 for use in the treatment of cancer.

22. 20. Use of a composition according to any one of claims 1 to 16 in the manufacture of a medicament for use in the treatment of cancer.

23. 1. A composition comprising an anti-BCMA antibody-drug conjugate (ADC), wherein the DL2 percentage is at least about 30%, about 15% to about 27%, or about 15% to about 32%; the DL4a percentage is at least about 30%, about 35% to about 38%, or about 30% to about 40%; the DL4b percentage is at least about 5%, about 7% to about 9%, or about 5% to about 10%; the DL6 percentage is at least about 10%, about 14% to about 20%, or about 10% to about 20%; and / or the DL8 percentage is at least about 1%, about 6.0% to about 12.0%, or about 4% to about 15%.

24. The anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 24. The composition of claim 23, comprising:

25. The composition of claim 23 or 24, wherein the proportion of DL2 is about 15% to about 32%, the proportion of DL4a is about 30% to about 40%, the proportion of DL4b is about 5% to about 10%, the proportion of DL6 is about 10% to about 20%, and the proportion of DL8 is about 4% to about 15%.

26. The composition of claim 23 or 24, wherein the proportion of DL2 is about 15% to about 27%, the proportion of DL4a is about 35% to about 38%, the proportion of DL4b is about 7% to about 9%, the proportion of DL6 is about 14% to about 20%, and the proportion of DL8 is about 6.0% to about 12.0%.

27. 27. The composition of any one of claims 23 to 26, wherein the average drug-to-antibody ratio (DAR) is from about 2.1 to about 5.

7.

28. 27. The composition of any one of claims 23 to 26, having an average DAR of from about 3.4 to about 4.

6.

29. 27. The composition of any one of claims 23 to 26, having an average DAR of from about 3.8 to about 4.

5.

30. A composition comprising an anti-BCMA antibody-drug conjugate (ADC), wherein the DL0 rate is about 10% or less, or about 5% or less.

31. The anti-BCMA antibody CDRH1 having the amino acid sequence set forth in SEQ ID NO: 1; CDRH2 having the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 having the amino acid sequence set forth in SEQ ID NO:6 31. The composition of claim 30, comprising:

32. 32. The composition of claim 30 or 31, wherein the proportion of DL0 is about 5% or less.

33. The composition of any one of claims 30 to 32, wherein the proportion of DL2 is about 15% to about 32%, the proportion of DL4a is about 30% to about 40%, the proportion of DL4b is about 5% to about 10%, the proportion of DL6 is about 10% to about 20%, and the proportion of DL8 is about 4% to about 15%.

34. The composition of any one of claims 30 to 32, wherein the proportion of DL2 is about 15% to about 27%, the proportion of DL4a is about 35% to about 38%, the proportion of DL4b is about 7% to about 9%, the proportion of DL6 is about 14% to about 20%, and the proportion of DL8 is about 6.0% to about 12.0%.

35. 35. The composition of any one of claims 30 to 34, wherein the average drug-to-antibody ratio (DAR) is from about 2.1 to about 5.

7.

36. 35. The composition of any one of claims 30 to 34, having an average DAR of from about 3.4 to about 4.

6.

37. 35. The composition of any one of claims 30 to 34, having an average DAR of from about 3.8 to about 4.

5.

38. The anti-BCMA antibody has the amino acid sequence set forth in SEQ ID NO:7 Ｈ and V having the amino acid sequence set forth in SEQ ID NO:

8. Ｌ The composition of any one of claims 23 to 37, comprising:

39. The composition of any one of claims 23 to 38, wherein the anti-BCMA antibody is belantamab.

40. The composition of any one of claims 23 to 37, wherein the cytotoxic agent is MMAE or MMAF.

41. 41. The composition of any one of claims 21 to 40, wherein the anti-BCMA ADC is belantamab mafodotin.

42. 42. The composition of any one of claims 23 to 41, wherein the percent DL is determined by separating individual DL species using hydrophobic interaction chromatography (HIC), calculating the area under the curve for each DL peak, and dividing each DL peak by the total area under the curve for all DL species combined.

43. The average DAR is calculated using the following formula: [Equation 1] 43. The composition of claim 42, calculated from the area under the curve of each DL species using:

44. A pharmaceutical composition comprising the composition of any one of claims 23 to 43 and at least one pharmaceutically acceptable excipient.

45. 45. A formulation comprising the pharmaceutical composition of claim 44, the formulation comprises about 20 mg / mL to about 60 mg / mL of anti-BCMA antigen binding protein, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80; The formulation, wherein the pH of the formulation is from about 5.9 to about 6.

5.

46. 46. ​​The formulation of claim 45, wherein the formulation comprises about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, or about 60 mg / mL belantamab mafodotin, 25 mM citrate buffer, 200 mM trehalose, 0.05 mM disodium EDTA, and 0.02% polysorbate 20 or polysorbate 80, and wherein the pH of the formulation is about 5.9 to about 6.

5.

47. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 23 to 43.

48. A composition according to any one of claims 23 to 43 for use in the treatment of cancer.

49. 44. Use of a composition according to any one of claims 23 to 43 in the manufacture of a medicament for use in the treatment of cancer.

50. 1. A composition comprising an acidic variant of an antibody, The acidic variant is CDRH1 of SEQ ID NO: 1; CDRH2 of SEQ ID NO: 2; and a CDRH3 of SEQ ID NO: 3; and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises 1-70% of the acidic variant.

51. 1. A composition comprising an acidic variant of an antibody, The acidic variant is CDRH1 of SEQ ID NO: 1; CDRH2 of SEQ ID NO: 2; and a CDRH3 of SEQ ID NO: 3; and a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises no more than 70% of the acidic variant.

52. A composition comprising a basic variant of an antibody, The basic variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises 1 to 30% of the basic variant.

53. A composition comprising a basic variant of an antibody, The basic variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises no more than 30% of the basic variant.

54. 1. A composition comprising a major isoform of an antibody, The major isoform is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises 1 to 90% of the major isoform.

55. 1. A composition comprising a major isoform of an antibody, The major isoform is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition, wherein the composition comprises 1% or more of the major isoform.

56. 1. A composition comprising a charge variant of an antibody, The charge variant is a heavy chain amino acid sequence comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and a CDRH3 of SEQ ID NO: 3; a light chain amino acid sequence comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:6; Including, The composition comprises no more than 70% acidic variants and / or no more than 30% basic variants and / or 1% or more of the major isoform.

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