Biopharmaceutical compositions and related methods

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 List This application includes a sequence listing, which is filed electronically in ASCII format and is incorporated herein by reference in its entirety.

[0002] Field of Invention The inventions described herein provide 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 intrinsic membrane receptor for its ligands, BAFF and APRIL. BCMA ligands can also bind to further receptors. TACI (Transmembrane Activator and Calcium modulator and cyclophilin ligand Interactor) binds to APRIL and BAFF, while BAFF-R (BAFF receptor or BR3) is restricted to BAFF but exhibits high affinity for it. Together, these receptors and their corresponding ligands regulate different aspects of humoral immunity, B cell development, and homeostasis.

[0004] BCMA expression is typically limited to B cell lineages and has been reported to increase during final B cell differentiation. BCMA is expressed by human plasmablasts and plasma cells derived from the tonsils, spleen, and bone marrow, but is also expressed by tonsillar memory B cells and germinal center B cells, which exhibit a TACI-BAFFR low phenotype (Darce et al., 2007). BCMA is barely present on unsensitized and memory B-25 cells (Novak et al., 2004a and Novak et al., 2004b). The BCMA antigen is expressed on the cell surface and can access antibodies, but it 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 in 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, the BCMA-APRIL signaling axis is dominant in the later stages of B cell differentiation and is suggested to be 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, such as B-cell carcinomas, including multiple myeloma (MM). MM is a clonal B-cell malignancy that arises in multiple sites within the bone marrow, either de novo or as an exacerbation from monoclonal immunoglobulinemia of unknown significance (MGUS), before spreading into the bloodstream. It is generally characterized by increased paraprotein and osteoclast activity, as well as hypercalcemia, cytopenia, renal impairment, hyperviscosity, and peripheral neuropathy. Decreased antibody levels and neutrophil counts are also common, leading to severe susceptibility to infection. BCMA is involved in the proliferation and survival of myeloma cell lines in vitro (Novak et al., 2004 and Moreaux et al., 2004). [Overview of the project]

[0006] A composition comprising an isomerization variant of an anti-BCMA antibody, wherein the isomerization variant comprises 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 , wherein the composition comprises 25% or less of the isomerization variant.

[0007] A composition comprising an oxidation variant of an anti-BCMA antibody, wherein the oxidation variant comprises 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 , wherein the composition comprises 40% or less of the oxidation variant.

[0008] A composition comprising an anti-BCMA antibody, wherein the anti-BCMA 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 , wherein the composition comprises 0.1 to 25% of isomerization at D103 of CDRH3.

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

[0010] A composition comprising an anti-BCMA antibody, The anti-BCMA antibody is at least approximately 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 comprising 0.1 to 25% isomerization of CDRH3 at D103.

[0011] A composition comprising an anti-BCMA antibody, The anti-BCMA antibody is at least approximately 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 comprising 0.1 to 40% oxidation of CDRH1 at M34.

[0012] 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] A composition comprising an anti-BCMA antibody-drug conjugate (ADC), wherein the proportion of DL0 is about 10% or less or about 5% or less. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 is a schematic diagram of a heterogeneous mixture of DL species in an ADC composition. [Figure 2] Figure 2 shows typical HIC peak characteristics for determining the DAR distribution in an ADC composition. [Figure 3] Figure 3 shows the effect of the average DAR of the ADC composition on tumor volume in a xenograft model. [Figure 4] Figure 4 shows a typical cIEF electropherogram of verantamab. [Modes for carrying out the invention]

[0015] The inventions described herein provide 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 the treatment or prevention of various BCMA-mediated diseases, including B-cell-mediated cancers such as lymphoma and multiple myeloma. The anti-BCMA antigen-binding proteins described herein can bind to human BCMA, for example, human BCMA containing the amino acid sequence of GenBank accession number Q02223.2, or human BCMA containing the amino acid sequence of a gene encoding human BCMA having at least 90% homology or at least 90% identity thereto.

[0017] As used herein, the term “antigen-binding protein” refers to BCMA, for example, antibodies, antibody fragments, and other protein constructs capable of binding to human BCMA. The antigen-binding proteins of the present invention may include heavy-chain and light-chain variable regions of the present invention that can be formatted to the structure of a native antibody or a functional fragment or equivalent thereof. Thus, the antigen-binding proteins of the present invention may include the VH region of the present invention formatted to a full-length antibody, (Fab')2 fragment, Fab fragment, or equivalent thereto (e.g., scFV, vibody, tribody, or tetrabody, Tandab, etc.) when combined with a suitable light chain. The antibody may be IgG1, IgG2, IgG3, or IgG4; IgM; IgA; IgE; IgD; or modified variants thereof. Accordingly, a constant domain of the antibody heavy chain can be selected. The light-chain constant domain may be a kappa or lambda constant domain. Furthermore, the antigen-binding proteins may include all classes of modifications, e.g., IgG dimers, Fc variants that do not bind to the Fc receptor, or Fc variants that do not regulate C1q binding. Antigen-binding proteins may also be chimeric antibodies of the type described in WO86 / 01533, comprising an antigen-binding region and a non-immunoglobulin region.

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

[0019] Chimeric antigen receptors (CARs) were developed as artificial T cell receptors to generate novel specificity in T cells without the need to bind to MHC-antigen peptide complexes. These synthetic receptors may contain a target-binding domain associated with 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, while the signaling domains contain molecular mechanisms for T cell activation and proliferation. The flexible linker, which spans the T cell membrane (i.e., forms a transmembrane domain), enables cell membrane display of the CAR's target-binding domain. CARs can successfully enable the redirection of T cells to antigens expressed on the surface of tumor cells derived from various malignancies, including lymphomas and solid tumors (Jena et al., 2010, Blood, 116(7):1035-44). In one embodiment of the present invention, the anti-BCMA antigen-binding protein may comprise a chimeric antigen receptor. In further embodiments, the CAR may comprise a binding domain, a transmembrane domain, and an intracellular effector domain.

[0020] Exemplary anti-BCMA antigen-binding proteins and methods for producing them are disclosed in International Publication No. WO2012 / 163805, which is incorporated herein by reference in its entirety. Additional exemplary anti-BCMA antigen-binding proteins are disclosed in WO2016 / 014789, WO2016 / 090320, WO2016 / 090327, W02016 / 020332, WO2016 / 079177, WO2014 / 122143, WO2014 / 122144, WO2017 / 021450, WO2016 / 014565, WO2014 / 068079, and WO2015 / 1666. This includes the documents described in 49, WO2015 / 158671, WO2015 / 052536, WO2014 / 140248, WO2013 / 072415, WO2013 / 072406, WO2014 / 089335, US2017 / 165373, WO2013 / 154760 and W02017 / 051068, each of which is incorporated herein by reference in its entirety.

[0021] In another embodiment, the anti-BCMA antigen-binding proteins described herein may inhibit the binding of BAFF and / or APRIL to the BCMA receptor. In another embodiment, the anti-BCMA antigen-binding proteins described herein may be able to bind to FcγRIIIA or have the ability to perform effector functions 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 may include monoclonal molecules, recombinant molecules, polyclonal molecules, chimeric molecules, human molecules, and humanized molecules of these types. Monoclonal antibodies may be produced by eukaryotic cell clones or prokaryotic close cells expressing the antibody. Monoclonal antibodies may also be produced by eukaryotic cell lines capable of recombinantly expressing the heavy and light chains of the antibody by having nucleic acid sequences encoding the heavy and light chains of the antibody introduced into the cell. Exemplary methods for producing antibodies from different eukaryotic cell lines, e.g., Chinese hamster ovary cells, hybridomas, or immortalized antibody cells derived from animals (e.g., humans) are well known to those skilled in the art.

[0023] Antibodies may originate from, for example, rats, mice, primates (e.g., cynomolgus monkeys, Old World monkeys, or apes), humans, or other sources (e.g., nucleic acids encoding antibody molecules produced using molecular biological techniques known in the art).

[0024] The antibody may contain a constant region, which may be any isotype or subclass. The constant region may be an IgG isotype, e.g., IgG1, IgG2, IgG3, IgG4, or a variant thereof.

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

[0026] In one embodiment, an anti-BCMA antibody has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function. The term “effector function,” as used herein, means one or more of the following: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated response, Fc-mediated phagocytosis, and / or antibody recycling via FcRn receptors. In IgG antibodies, effector functions, which may include ADCC and ADCP, can be mediated by the interaction between the heavy chain constant region and 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 antigen-bound antigen-binding proteins and Fc / Fcγ complex-forming compounds can induce a variety of effects, such as cytotoxicity, immune cell activation, phagocytosis, and / or the release of inflammatory cytokines.

[0027] In another embodiment, an anti-BCMA antibody may inhibit the binding of BAFF and / or APRIL to the BCMA receptor. In another embodiment, an anti-BCMA antibody may be able to bind to FcγRIIIA or may have the ability to perform FcγRIIIA-mediated effector functions.

[0028] In one embodiment, the composition comprises an anti-BCMA antibody containing two immunoglobulin (Ig) heavy chains ("HC") and two Ig light chains ("LC"). The basic antibody structural unit may, for example, include a tetramer of subunits. Each tetramer may contain 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 contain a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. This variable region may initially be expressed ligated to a cleavable signal peptide. A variable region without a signal peptide is sometimes called a mature variable region. Therefore, in one example, the light chain mature variable region may include a light chain variable region that does not contain a light chain signal peptide. The carboxyl-terminal portion of each chain may define a constant region. The heavy chain constant region may primarily be involved in effector function.

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

[0030] "CDR" is defined as the amino acid sequence of the complementarity-determining region of an antibody. These are the hypervariable regions of the immunoglobulin heavy and light chains. The variable portion of an immunoglobulin contains three heavy chain and three light chain CDRs (or CDR regions). Therefore, as used herein, "multiple CDRs" refers to all three heavy chain CDRs, all three light chain CDRs, all heavy chain and light chain CDRs, or at least two CDRs. In one embodiment, the composition comprises an anti-BCMA antibody containing one or more CDRs according to the invention described herein, or one or both of the heavy chain or light chain variable domains according to the invention described herein.

[0031] The terms “variant,” “antibody variant,” “CDR variant,” and “post-translational modification variant” refer to one or more amino acid changes in an antibody sequence. Variants may be the result of post-translational modifications, chemical changes, or sequence changes due to one or more deletions, substitutions, or additions. 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), while other post-translational modifications result in sequence changes, such as the conversion of one amino acid residue to another (e.g., conversion of Asn to Asp by deamidation; or lysine deletion). Further post-translational modification variants are described below. Variant antibody sequences containing sequence changes may be the result of designed sequence changes or post-translational modifications. Amino acid sequence changes may be deletions, substitutions, or additions.

[0032] In one such embodiment, the substitution is a conservative substitution. In another embodiment, the antibody variant includes one or more substitutions while retaining the standard form of the antigen-binding protein. In one embodiment, the antibody variant is an antibody that is at least about 80%, about 85%, about 90%, or about 95% identical to the primary sequence of the antibody (i.e., has sequence identity). In another embodiment, the antibody variant includes an antibody 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 protein of the present invention may have amino acid modifications that increase the affinity of the constant domain or its fragment to FcRn. Increasing the half-life (i.e., serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules has many advantages, such as reducing the dosage and / or frequency of administration of these molecules. In one embodiment, the antigen-binding protein of the present invention comprises all or part of an IgG constant domain (FcRn binding moiety) having one or more of the following amino acid modifications.

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

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

[0036] The half-life and FcRn binding can also be extended by introducing H433K and N434F mutations (commonly called "HN" mutations or "NHance" mutations) (related to IgG1) (W02006 / 130834).

[0037] WO00 / 42072 describes a polypeptide containing a variant Fc region with altered FcRn binding affinity, which includes any one or more amino acid modifications from 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 a modified IgG containing an IgG constant domain that includes one or more amino acid modifications compared to the wild-type IgG constant domain. The half-life of this modified IgG is extended compared to that of IgG with the wild-type IgG constant domain, and the one or more amino acid modifications are present at one or more of the following 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 modify residues in the Fc region of human IgG1 antibodies and then evaluated their binding to human FcRn. Sites that effectively suppressed FcRn binding when modified to alanine included I253, S254, H435, and Y436. At other sites, the decrease in binding was not as pronounced, such as E233-G236, R255, K288, L309, S415, and H433. Several amino acid sites showed improved FcRn binding when modified to alanine, and notable among these were P238, T256, E272, V305, T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Many other amino acid positions show 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, S3) in FcRn binding. (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) are shown.

[0040] The most significant improvement in FcRn binding was observed with the combination variant. At pH 6.0, the E380A / N434A variant showed more than 8 times better FcRn binding than native IgG1, compared to 2 times for E380A and 3.5 times for N434A. Adding T307A resulted in a 12-fold improvement in binding compared to native IgG1. In one embodiment, the antigen-binding protein of the present invention contains the E380A / N434A mutation, which increases FcRn binding.

[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 at 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 crossing the Fc-FcRn boundary (M252, S254, T256, H433, N434, and Y436), while the improvement is less pronounced 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, showing a 57-fold increase in affinity compared to wild-type IgG1. The in vivo behavior of such mutated human IgG1 showed an almost four-fold increase in serum half-life in cynomolgus monkeys compared to wild-type IgG1.

[0042] Therefore, the present invention provides an antigen-binding protein having 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 in the Fc region 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, 34 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, 39 It is located 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 methods for obtaining physiologically active molecules with altered half-lives, which include methods involving the introduction of FcRn-binding polypeptides into molecules (WO97 / 43316, US5869046, US5747035, WO96 / 32478, and WO91 / 14438), methods involving the fusion of molecules with antibodies to obtain antibodies that retain FcRn-binding affinity but have significantly reduced affinity for other Fc receptors (WO99 / 43713), or methods involving the fusion of molecules with the FcRn-binding domain of antibodies (WO00 / 09560, US4703039).

[0044] Fc variants that enhance FcRn affinity to improve both the cytotoxicity and half-life of antibodies were identified by screening at pH 6.0. Selected IgG variants can be constructed as low-fucosylated molecules. The resulting variants exhibit enhanced serum persistence and conserved, enhanced ADCC in hFcRn mice (Monnet et al.). Exemplary variants include (with respect 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 (where "del" indicates a deletion).

[0045] The present invention also provides a method for producing an antigen-binding protein according to the present invention, comprising the steps of: a) culturing recombinant host cells comprising an expression vector comprising an isolated nucleic acid as described herein, wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase is inactivated in the recombinant host cells; and b) recovering the antigen-binding protein. Such a method for producing an antigen-binding protein 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 a monoclonal antibody having enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity that is increased against the same monoclonal antibody produced in cells having 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 producing antigen-binding proteins, such as antibodies.

[0046] Antibodies can be recovered and purified by conventional protein purification procedures. For example, antibodies can be collected directly from the culture medium. Collection of the cell culture medium can be carried out by clarification, for example, by centrifugation and / or deep filtration. After recovering the antibodies, they are purified to ensure appropriate purity. Thus, in one embodiment, a cell culture medium containing the antibodies described herein is provided. In one embodiment, the cell culture medium contains CHO cells.

[0047] Next, the antibody can be purified from the cell culture medium. This may involve collecting the cell culture supernatant, contacting the cell culture supernatant with a purification medium (e.g., a protein A resin or protein G resin for binding antibody molecules), and eluting the antibody molecules from the purification medium to produce an eluate. Thus, in one embodiment, an eluate containing the antibody 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, the composition can be purified using affinity chromatography using a resin, such as protein A, G, or L. Alternatively, or in addition to the above, the composition can be purified using an ion exchange resin, such as a cation exchange resin.

[0049] Alternatively, the purification process may include a step using an affinity chromatography resin followed by a step using a cation exchange resin.

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

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

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

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

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

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

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

[0057] In one embodiment, the anti-BCMA antibody has a heavy chain variable region ("V") containing an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 7. H This includes ").

[0058] In one embodiment, the anti-BCMA antibody has a light chain variable region ("V") containing an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 8. L This includes ").

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

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

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

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

[0063] The "identity percentage" between a query amino acid sequence and a subject amino acid sequence is an "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 pairwise BLASTP alignment has been performed. Such pairwise BLASTP alignment between a query amino acid sequence and a subject amino acid sequence is performed with the low-complexity region filter removed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Information website. Importantly, the query sequence may be described by an amino acid sequence identified in one or more claims of this specification.

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

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

[0066] In one embodiment, the anti-BCMA antibody comprises verantamab, which includes HC having the amino acid sequence described in SEQ ID NO: 9 and LC having the amino acid sequence described 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 identifying CDRs known to those skilled in the art by numbering residues in the antibody. Other conventional methods for numbering 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 amino acids described herein are referred to numerically, the sequences may be numbered according to the Kabat numbering system or the 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. Amino acid residues in variable region sequences and full-length antibody sequences represent any antibody sequence variant position or post-translational modification variant position, e.g., isomerized variant (e.g., D103), deamidated variant (e.g., N388), or oxidized variant (e.g., M34).

[0069] References to positions within the CDRH (e.g., M34 or D103) provide positional numbers (sequential numbering) for the entire antibody sequence. Therefore, M34 in CDRH1 corresponds to the fourth residue of SEQ ID NO: 1, i.e., the underlined portion: NYW MIt is understood that H (sequence number 1) refers to the fifth residue of sequence number 3, i.e., the underlined part: GAIY D This refers to GYDVLDN (sequence number 3).

[0070] In one embodiment, the composition comprises an antibody variant having a change in one or more amino acids 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 having an amino acid change from aspartic acid (D) to asparagine (N), for example, D103N (i.e., D99N in Kabat numbering) in CDRH3.

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

[0072] In another embodiment, the anti-BCMA antibody comprises verantamab, which includes an amino acid change from aspartic acid (D) to asparagine (N), for example, 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, 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 including CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, wherein about 5% or more of the antibodies in the mixture are D103N in CDRH3, 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.

[0075] In one embodiment, the composition comprises verantamab, 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 verantamab comprises D103N in CDRH3.

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

[0077] Post-translational modification products The “post-translational modified products” of antibodies described herein are antibody compositions in which all or part of the composition comprises the “post-translational modification.” Post-translational modification may result from antibody production in host cells, upstream and downstream manufacturing, and / or storage (e.g., exposure to light, temperature, pH, water, or reaction with excipients and / or immediate container closure systems). Thus, the compositions of the present invention may be formed from the manufacturing or storage of antibodies. Exemplary post-translational modifications include changes in the antibody sequence (the “antibody variants” described above), cleavage of specific leader sequences, addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycation, deamidation, oxidation, disulfide bond scrambling and other cysteine ​​variants (e.g., free sulfhydryl, racemized disulfide, thioether and trisulfide bonds), isomerization, cleavage of the C-terminal lysine, and / or cyclization of the N-terminal glutamine.

[0078] In one example, a post-translational modification product contains "product-related impurities" that include chemical changes resulting in a decrease in function and / or activity. In another example, a post-translational modification product contains "product-related substances" that include chemical changes that do not result in a decrease in function and / or activity. Product-related impurities of antibodies described herein include isomerized variants and oxidized variants. Product-related substances of antibodies described herein include deamidated variants, glycosylated variants, C-terminal cleaved 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, and includes 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, and includes one or more functional post-translational modifications thereof.

[0080] The percentages of variants provided herein are expressed as a percentage of the total amount of antibody (e.g., a “population” of antibodies) in the composition. For example, an oxidized variant of 40% or less refers to 100% of the total antibody in the composition, of which 40% or less is oxidized. For example, an isomerized variant of 25% or less refers to 100% of the total antibody in the composition, of which 25% or less isomerized.

[0081] Glycation is a post-translational modification involving a non-enzymatic chemical reaction between a reducing sugar, such as glucose, and a free amine group of a protein, and is typically found at the epsilonamine of the lysine side chain or at the N-terminus of a protein. Glycation can occur during production and / or storage in the presence of reducing sugars.

[0082] For example, deamidation that may occur during manufacturing and / or storage can be an enzymatic or chemical reaction. Deamidation can occur by a simple chemical reaction involving intramolecular cyclization, where the amide nitrogen of an adjacent amino acid in the chain nucleophilically attacks the amide (N+1 attacks N), forming a succinimide intermediate. Deamidation may primarily convert asparagine (N) to isoaspartic acid (isoaspartinate) and aspartic acid (aspartinate) (D) in a ratio of approximately 3:1. Therefore, this deamidation reaction may be related to the isomerization of aspartic acid (D) to isoaspartic acid. Both the deamidation of asparagine and the isomerization of aspartic acid can involve the intermediate succinimide. Deamidation can occur at glutamine residues in a similar manner, but to a much lesser degree. Deamidation can occur in the CDR, Fab (non-CDR region), or Fc region. Isomerization is the conversion of aspartic acid (D) to isoaspartic acid, and involves the intermediate succinimide.

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

[0084] Disulfide bond scrambling can occur during manufacturing and / or storage conditions. Under certain circumstances, disulfide bonds may break or fail to form properly, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryl (-SH) can promote shuffling.

[0085] Thioether formation and disulfide bond racemization can occur under basic conditions during manufacturing or storage. This is due to beta-elimination of the disulfide bridge, returning to a cysteine ​​residue via dehydroalanine and persulfide intermediates. Subsequent crosslinking of dehydroalanine and cysteine ​​can form a thioether bond, or free cysteine ​​residues can reform disulfide bonds with a mixture of D-cysteine ​​and L-cysteine.

[0086] Trisulfides can arise from the insertion of a sulfur atom into a disulfide bond (Cys-SSS-Cys) and can be formed in the presence of hydrogen sulfide in production cell cultures.

[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 within the production bireactor, it can also occur 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 natural human antibodies.

[0088] C-terminal lysine cleavage is an enzymatic reaction catalyzed by carboxypeptidases and is common in recombinant and native human antibodies. Variations of this process include the removal of lysine from one or both heavy chains by cellular enzymes from recombinant host cells. Administration to human subjects / patients may result in the removal of any remaining C-terminal lysine.

[0089] The present invention comprises antibodies that have been subjected to or may have been subjected to one or more of the post-translational modifications described herein. Exemplary compositions may include mixtures or blends of the following antibodies: 1) mixtures or blends of antibodies having and not having (one or more) post-translational modifications, or 2) mixtures or blends of antibodies having two or more of the post-translational modifications described herein.

[0090] The composition may contain a mixture of antibody variants and post-translational modification variants. For example, the antibody composition may contain one or more, for example, two or three or more, of oxidized variants, deamidated variants, isomerized variants, N-terminal pyroglutamate variants, and C-terminal lysine cleavage variants.

[0091] For example, in one embodiment, the composition may comprise a mixture of antibodies, where 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 having a cleavage of the C-terminal lysine.

[0092] In another exemplary embodiment, the composition may comprise a mixture of antibodies, 10% of which comprises the amino acid sequences of SEQ ID NOs: 9 and 10, 90% of which comprises the amino acid sequences of SEQ ID NOs: 9 and 10 with a cleavage of the C-terminal lysine, and up to 100% of the total 100% of the antibody mixture having its N-terminal glutamine cyclized to pyroglutamate.

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

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

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

[0096] As used herein, “function” or “activity” is defined as one or more of the following: 1) binding to BCMA, 2) binding to FcγRIIIa, and / or 3) binding to FcRn. In one embodiment, “decreased function” or “decreased activity” means that the binding to BCMA, FcγRIIIa, or FcRn is reduced by a percentage compared to a reference standard and is significant with respect to assay variability. For example, a decrease in function or activity may 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 sequences of SEQ ID NOs: 9 and SEQ ID NOs: 10, and includes all post-translational modifications, if present.

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

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

[0100] Post-translational modifications can result in an increase or decrease in the net charge of an antibody, leading to a decrease or increase in its pI value, which can result in acidic and basic variants (collectively referred to as "charged variants") relative to the main isoform. The main isoform is the antibody population that elutes as the main peak in the chromatogram. When analyzing antibodies using IEF-based methods, acidic species are variants with low apparent pI, and basic species are variants with high apparent pI. When analyzing using chromatography-based methods, 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 later than the main peak obtained from AEX, while basic species are variants that elute later than the main peak obtained from CEX or earlier than the main peak obtained from AEX. These methods separate the main isoform of an antibody from the acidic isoform (acidic variant) and the basic isoform (basic variant). Charged 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 proportion of charged variants can be determined using capillary isoelectric focusing (cIEF). Using capillary isoelectric focusing (cIEF), the pI of dostallimab is measured and charged variants are separated (see Figure 1). Using this method, acidic and basic species can be quantified as a percentage of total area peaks. The terms “species,” “isoform,” “form,” and “peak” are used interchangeably to refer to the major isoform and charged 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 including 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 contains 1 to 70% acidic variant.

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

[0103] In one embodiment, the composition comprises a basic variant of an antibody, the basic variant comprising a heavy chain amino acid sequence including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises 1-30% of the basic variant.

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

[0105] In one embodiment, the composition comprises a major isoform of an antibody, the major isoform comprising a heavy chain amino acid sequence including 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 including 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 isoforms.

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

[0107] The proportions of acidic variants, basic variants, and major variants can be determined by isoelectric focusing, or by capillary isoelectric focusing (cIEF). It is understood that these isoform / charged variant embodiments may be used in combination with any one or a combination of the antibody variants described herein.

[0108] In one embodiment, the composition comprises a charged variant of an antibody, the charged variant comprising a heavy chain amino acid sequence including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, the composition comprising 70% or less of acidic variants and / or 30% or less of basic variants and / or 1% or more of major isoforms.

[0109] In one embodiment, the composition comprises an antibody ("isomerized" or "isomerized") or "isomerized variant" containing isomerized post-translational modification. The variant may contain amino acid residues isomerized to the heavy chain sequence and / or light chain sequence, for example, to the CDR of the heavy chain sequence and / or to the CDR of the light chain sequence. The isomerized variant may be present on one or both of the heavy chain or light chain. The isomerized post-translational modification results in isoaspartic acid and / or succinimide-aspartic acid. In one example, the isomerization of aspartic acid (Asp) may be determined using peptide mapping tandem mass spectrometry with trypsin (peptide mapping LC-MS / MS) as described herein. It is understood that embodiments of these isomerized variants can be combined with the characteristics of the antibodies described herein.

[0110] In one embodiment, the composition comprises an isomerized variant of an anti-BCMA antibody, the isomerized variant comprising a heavy chain amino acid sequence including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition contains 25% or less of the isomerized variant.

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

[0112] The composition comprises an isomerized variant of an anti-BCMA antibody, the isomerized variant comprising a heavy chain amino acid sequence including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises an isomerized variant at amino acid D103 of CDRH3 at a concentration of 25% or less.

[0113] In one embodiment, the composition comprises an isomerized variant of an anti-BCMA antibody, the isomerized variant comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, and the composition contains 25% or less of the isomerized variant.

[0114] Alternatively, the isomerized variant contains the heavy chain sequence of SEQ ID NOs. 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 isomerization to either the heavy chain sequence or the light chain sequence, for example, isomerization at amino acid D103 of CDRH3.

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

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

[0118] 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 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, a composition containing 25% or less or 23% or less of CDRH3 isomerized at D103 retains 70% or more of BCMA-specific antigen binding.

[0119] In one embodiment, the composition comprises a mixture of antibodies having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described 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 0.1-25%, 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, a composition containing 25% or less or 23% or less of CDRH3 isomerization at D103 retains 70% or more of BCMA-specific antigen binding.

[0120] In another embodiment, the composition comprises verantamab, 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 verantamab isomerized at amino acid D103 of CDRH3. In one embodiment, verantamab containing 25% or less or 23% or less of CDRH3 isomerized at D103 retains 70% or more of BCMA-specific antigen binding.

[0121] In one embodiment, the composition comprises an isomerized variant of an anti-BCMA antibody, the isomerized variant comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, and the composition contains 25% or less of the isomerized variant.

[0122] In one example, aspartic acid (Asp) isomerization can be determined using peptide mapping tandem mass spectrometry with trypsin (peptide mapping LC-MS / MS). In one example, a sample containing the composition described herein can be denatured with, for example, 6M guanidine HCl to a concentration of, for example, 4.2 μg / μL. The disulfide bond can then be reduced, for example, with 50 mM DTT for 20 minutes at room temperature. Then, for example, iodoacetic acid can be added, for example, at 100 mM, and reacted with the free cysteine ​​residues, for example, with protection from light for 30 minutes at room temperature. 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 for 15 minutes at 37°C. The obtained peptides can then be loaded onto a reverse-phase ultrahigh-performance liquid chromatography (UPLC) column and eluted using UPLC with a water-acetonitrile gradient solution, for example, a 0.1% trifluoroacetic acid solution. The peptides can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Using the extracted ion chromatograms of the unmodified and modified peptides, the level of isomerization can be calculated 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 embodiment, the composition comprises an antibody or "oxidized variant" containing oxidative post-translational modifications ("oxidized" or "oxidized"). The variant may contain oxidized amino acid residues in the heavy chain sequence and / or light chain sequence, for example, the CDR of the heavy chain sequence and / or the CDR of the light chain sequence. The oxidized variant may be present in one or both of the heavy chain or light chain. It is understood that embodiments of these oxidized variants can be combined with the features of the antibodies 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 including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition contains 40% or less of the oxidized variant.

[0125] In one embodiment, the composition comprises a population of anti-BCMA antibodies, and the population of anti-BCMA antibodies is An antibody comprising a heavy chain amino acid sequence including SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3), and a light chain amino acid sequence including SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), and Those oxidative variants Including this, less than 40% of the antibody population consists of oxidized variants.

[0126] In one embodiment, the oxidative variant comprises oxidation at one or more CDRs. In a further embodiment, the oxidative variant comprises oxidation at a methionine and / or tryptophan residue in any one of SEQ ID NOs: 1 to 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 including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises 40% or less of the oxidized variant at amino acid M34 of CDRH1.

[0128] In one embodiment, the composition comprises an oxidized variant of verantamab, the oxidized variant comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, and the composition contains 40% or less of the 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 oxidation of the heavy chain sequence, for example, oxidation at amino acids M34 (CDRH1), M256 and / or M432.

[0130] In another embodiment, the composition comprises an antibody having CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, and includes oxidation of the heavy chain sequence, for example, oxidation at amino acids M34 (CDRH1), M256 and / or M432.

[0131] In another embodiment, the anti-BCMA antibody comprises verantamab, which includes oxidation of the heavy chain sequence, for example, 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 0.1-40%, 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% of the antibodies in the mixture are oxidized at amino acid M34. In one embodiment, a composition containing oxidation of 40% or less of the heavy chain M34 retains 70% or more of the BCMA-specific antigen binding. In another embodiment, a composition containing oxidation of 37% or less of the heavy chain M34 retains 70% or more of the BCMA-specific antigen binding.

[0133] In one embodiment, the composition comprises a mixture of antibodies including CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, with approximately 40% or less of the antibodies in the mixture and approximately 35% or less. Approximately 30%, approximately 25% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, approximately 7.5% or less, approximately 5% or less, approximately 2.5% or less, approximately 2% or less, approximately 0.1-40%, approximately 0.1-35%, approximately 0.1-30%, approximately 0.1-25%, approximately 0.1-20%, approximately 0.1-15%, approximately 0.1-10%, approximately 0.1-7.5%, approximately 0.1-5%, approximately 0.1-2.5%, approximately 0.1-2%, approximately 0.5%, approximately 1%, approximately 2%, or approximately 5% are oxidized at amino acid M34. In one embodiment, a composition containing oxidation at 40% or less of heavy chain M34 retains 70% or more of BCMA-specific antigen binding. In another embodiment, a composition containing oxidation at 37% or less of heavy chain M34 retains 70% or more of BCMA-specific antigen binding.

[0134] In another embodiment, the composition comprises verantamab, 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 0.1-40%, 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% of verantamab is oxidized at amino acid M34. In one embodiment, verantamab containing 40% or less oxidation at the heavy chain M34 retains 70% or more of BCMA-specific antigen binding. In another embodiment, verantamab containing oxidation of 37% or less of the heavy chain M34 retains 70% or more of 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 including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises an oxidized variant in the Fc region of 90% or more.

[0136] In one embodiment, the antibody comprises oxidation at methionine and / or tryptophan residues in the Fc region of the heavy chain sequence and / or the Fc region of the light chain sequence. In some embodiments, the oxidized variant comprises one or a combination of oxidation 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, the oxidized variant comprising a heavy chain amino acid sequence including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and 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 verantamab, the oxidized variant comprising the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, and the composition comprises an oxidized variant in the Fc region of 90% or less.

[0139] Alternatively, the oxidized variant contains the heavy chain sequence of SEQ ID NOs: 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 0.1-90%, about 0.1-80%, about 0.1-70%, about 0.1-65%, about 0.1-50%, about 0.1-40%, about 0.1-30%, about 0.1-20%, about 0.1-10%, about 1-10%, about 1-5%, about 2-10%, about 2-4%, about 2%, about 3%, or about 4% of the antibodies in the mixture are oxidized at amino acid M256. In one embodiment, a composition containing oxidation of 90% or less or 89% or less of the heavy chain M256 retains 70% or more of the FcγRIIIA bonds. In another embodiment, a composition containing oxidation of 65% or less or 64% or less of the heavy chain M256 retains 70% or more of the FcRn bonds.

[0141] In one embodiment, the composition comprises a mixture of antibodies including CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, with approximately 90% or less of the antibodies in the mixture, approximately 80% or less, and approximately 70% or less, approximately 65% ​​or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 20% or less, approximately 10% or less, approximately 7.5% or less, approximately 5% or less, approximately 0.1-90%, approximately 0.1-80%, approximately 0.1-70%, approximately 0.1-65%, approximately 0.1-50%, approximately 0.1-40%, approximately 0.1-30%, approximately 0.1-20%, approximately 0.1-10%, approximately 1-10%, approximately 1-5%, approximately 2-10%, approximately 2-4%, approximately 2%, approximately 3%, or approximately 4% are oxidized at amino acid M256. In one embodiment, a composition containing 90% or less or 89% or less of oxidation at the heavy chain M256 retains 70% or more of the FcγRIIIA bonds. In another embodiment, a composition comprising oxidation of 65% or less or 64% or less of the heavy chain M256 retains 70% or more of the FcRn bonds.

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

[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 0.1-86%, about 0.1-70%, about 0.1-60%, about 0.1-50%, about 0.1-40%, about 0.1-30%, about 0.1-20%, about 0.1-10%, about 0.1-5%, about 0.1-3%, 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 containing oxidation of 86% or less of the heavy chain M432 retains 70% or more of the FcγRIIA bonds. In another embodiment, a composition containing oxidation of 60% or less of the heavy chain M432 retains 70% or more of the FcRn bonds.

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

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

[0146] In one example, oxidation can be determined using peptide mapping tandem mass spectrometry with trypsin (peptide mapping LC-MS / MS). In one example, a sample containing the composition described herein can be denatured with, for example, 6M guanidine HCl to a concentration of, for example, 4.2 μg / μL. The disulfide bonds can then be reduced, for example, with 50 mM DTT for 20 minutes at room temperature. Then, for example, iodoacetic acid can be added, for example, at 100 mM, and reacted with the free cysteine ​​residues, for example, with protection from light for 30 minutes at room temperature. The sample can then be buffer-exchanged using, for example, a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin for 15 minutes at 37°C. The resulting peptide can then be loaded onto a reversed-phase ultrahigh-performance liquid chromatography (UPLC) column and eluted using ULC with a solution of a water and acetonitrile gradient, for example, a 0.1% trifluoroacetic acid solution. The peptides can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Using the extracted ion chromatograms of the unmodified and modified peptides, the level of oxidation is calculated 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.

[0147] In one embodiment, the composition comprises an antibody or a "deamidation variant" comprising a deamidation post-translational modification ("deamidation" or "deamidated"). In one embodiment, the antibody comprises deamidation of an asparagine residue in the CDR of the heavy chain sequence and / or the CDR of the light chain sequence. In a further embodiment, the antibody comprises deamidation of an asparagine residue in the CDR of the heavy chain sequence. In one embodiment, the antibody comprises deamidation of an asparagine residue in the Fc region of the heavy chain sequence and / or the Fc region of the light chain sequence. The deamidation variant may be present in one or both of the heavy chain or light chain. It is understood that embodiments of these deamidation variants can be combined with the features of the antibodies 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 deamidation variant comprises a deamidation residue selected from an aspartic acid residue, a succinimide-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 of the heavy chain sequence, for example, deamidation of 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 including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises up to 100% of the deamidated variant.

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

[0152] In one embodiment, the composition comprises a deamidated variant of verantamab, the deamidated variant comprising the heavy chain sequence of SEQ ID NO: 9 and the 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 the heavy chain sequence of SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 and the light chain sequence of SEQ ID NO: 10.

[0153] In another embodiment, the composition comprises an antibody having CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, and includes deamidation of the heavy chain sequence, for example, deamidation of amino acids N388 and / or N393.

[0154] In another embodiment, the anti-BCMA antibody comprises verantamab, which includes 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 0.1-100%, about 0.1-75%, about 0.1-50%, about 0.1-40%, about 0.1-30%, about 0.1-20%, about 0.1-10%, about 0.1-5%, about 0.1-3%, 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 including CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, with approximately 100% or less, approximately 75% of the antibodies in the mixture. The following percentages are deamidated at amino acid N388: approximately 60% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 25% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, approximately 5% or less, approximately 2% or less, approximately 0.1-100%, approximately 0.1-75%, approximately 0.1-50%, approximately 0.1-40%, approximately 0.1-30%, approximately 0.1-20%, approximately 0.1-10%, approximately 0.1-5%, approximately 0.1-3%, approximately 0.5%, approximately 1%, approximately 2%, approximately 5%, or approximately 10%.

[0157] In another embodiment, the composition comprises verantamab, wherein about 100%, about 75%, about 60%, about 50%, about 40%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, about 0.1-100%, about 0.1-75%, about 0.1-50%, about 0.1-40%, about 0.1-30%, about 0.1-20%, about 0.1-10%, about 0.1-5%, about 0.1-3%, about 0.5%, about 1%, about 2%, about 5%, or about 10% of verantamab 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 0.1-100%, about 0.1-75%, about 0.1-50%, about 0.1-40%, about 0.1-30%, about 0.1-20%, about 0.1-10%, about 0.1-5%, about 0.1-3%, 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 including CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, with approximately 100% or less, approximately 85% of the antibodies in the mixture. The following percentages are deamidated at amino acid N393: approximately 70% or less, approximately 60% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 20% or less, approximately 15% or less, approximately 10% or less, approximately 5% or less, approximately 2% or less, approximately 0.1-100%, approximately 0.1-75%, approximately 0.1-50%, approximately 0.1-40%, approximately 0.1-30%, approximately 0.1-20%, approximately 0.1-10%, approximately 0.1-5%, approximately 0.1-3%, approximately 0.5%, approximately 1%, approximately 2%, approximately 5%, or approximately 10%.

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

[0161] In one example, deamidation may be determined using peptide mapping tandem mass spectrometry with trypsin (peptide mapping LC-MS / MS). In one example, a sample containing the composition described herein may be denatured with, for example, 6M guanidine HCl to a concentration of, for example, 4.2 μg / μL. The disulfide bond can then be reduced, for example, with 50 mM DTT for 20 minutes at room temperature. Then, for example, iodoacetic acid may be added at, for example, 100 mM, and reacted with the free cysteine ​​residues, for example, with protection from light for 30 minutes at room temperature. The sample can then be buffer-exchanged using, for example, a BioRad spin column (part no. 7326221), and then digested, for example, with 0.5% trypsin for 15 minutes at 37°C. The resulting peptide can then be loaded onto a reversed-phase ultrahigh-performance liquid chromatography (UPLC) column and eluted using ULC with a water and acetonitrile gradient solution, for example, a 0.1% trifluoroacetic acid solution. The peptides can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Using the extracted ion chromatograms of the unmodified and modified peptides, the level of deamidation is calculated 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.

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

[0163] In one example, the antibody variant, e.g., N103D in CDRH3, can be determined using peptide mapping tandem mass spectrometry with trypsin (peptide mapping LC-MS / MS). In one example, a sample containing the composition described herein can be denatured with, for example, 6M guanidine HCl to a concentration of, for example, 4.2 μg / μL. The disulfide bond can then be reduced, for example, with 50 mM DTT for 20 minutes at room temperature. Then, for example, iodoacetic acid can be added, for example, at 100 mM, and reacted with the free cysteine ​​residues, for example, with protection from light for 30 minutes at room temperature. 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 for 15 minutes at 37°C. The obtained peptide can then be loaded onto a reverse-phase ultrahigh-performance liquid chromatography (UPLC) column and eluted using ULC with a water-acetonitrile gradient solution, for example, a 0.1% trifluoroacetic acid solution. The peptide can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Using the extracted ion chromatograms of the unmodified and modified peptides, the level of the antibody variant, e.g., N103D in CDRH3, is calculated 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 embodiment, the composition comprises an antibody having a post-translational modification of pyroglutamic acid ("pyroglutamic acid") at the N-terminus of the heavy chain amino acid 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 which contains pyroglutamic acid at the N-terminus of the heavy chain.

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

[0166] In another embodiment, the anti-BCMA antibody comprises verantamab, with 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, 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 contain pyroglutamic acid at the N-terminus of the heavy chain amino acid sequence.

[0168] In one embodiment, the composition comprises a mixture of antibodies including CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described 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 less, 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 contain pyroglutamic acid at the N-terminus of the heavy chain amino acid sequence.

[0169] In one embodiment, the composition comprises verantamab, 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 verantamab contains pyroglutamic acid at the N-terminus of the heavy chain amino acid sequence.

[0170] In one example, the N-terminal pyroglutamic acid can be determined using peptide mapping tandem mass spectrometry with trypsin (peptide mapping LC-MS / MS). In one example, a sample containing the composition described herein can be denatured with, for example, 6M guanidine HCl to a concentration of, for example, 4.2 μg / μL. The disulfide bond can then be reduced, for example, with 50 mM DTT for 20 minutes at room temperature. Then, for example, iodoacetic acid can be added, for example, at 100 mM, and reacted with the free cysteine ​​residue, for example, with protection from light for 30 minutes at room temperature. 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 for 15 minutes at 37°C. The obtained peptide can then be loaded onto a reverse-phase ultrahigh-performance liquid chromatography (UPLC) column and eluted using ULC with a water-acetonitrile gradient solution, for example, a 0.1% trifluoroacetic acid solution. The peptide can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Using the extracted ion chromatograms of the unmodified and modified peptides, the level of pyroglutamic acid is calculated 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 embodiment, the composition comprises an antibody that includes a post-translational modification, which is a cleavage of the C-terminal lysine in the heavy chain amino acid 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 includes a cleavage of the C-terminal lysine of the heavy chain.

[0172] In another embodiment, the composition comprises an antibody having CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, and includes cleavage of the lysine at the C-terminus of the heavy chain.

[0173] In another embodiment, the anti-BCMA antibody comprises verantamab, which includes cleavage of the lysine at the C-terminus 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, 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 contain cleavage of the C-terminal lysine.

[0175] In one embodiment, the composition comprises a mixture of antibodies having the amino acid sequence described in SEQ ID NO: 1, CDRH1 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described 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 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 contain cleavage of the lysine at the C-terminus of the heavy chain.

[0176] In one embodiment, the composition comprises verantamab, 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 verantamab comprises cleavage of the lysine at the C-terminus of the heavy chain.

[0177] In one example, cleavage of the C-terminal lysine can be determined using peptide mapping tandem mass spectrometry with trypsin (peptide mapping LC-MS / MS). In one example, a sample containing the composition described herein can be denatured with, for example, 6M guanidine HCl to a concentration of, for example, 4.2 μg / μL. The disulfide bond can then be reduced, for example, with 50 mM DTT for 20 minutes at room temperature. Then, for example, iodoacetic acid can be added, for example, at 100 mM, and reacted with the free cysteine ​​residues, for example, with protection from light for 30 minutes at room temperature. 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 for 15 minutes at 37°C. The resulting peptide can then be loaded onto a reversed-phase ultrahigh-performance liquid chromatography (UPLC) column and eluted using ULC with a water and acetonitrile gradient solution, for example, a 0.1% trifluoroacetic acid solution. The peptides can then be detected using a UV detector and a mass spectrometer (e.g., Thermo Sdentific LTQ Orbitrap XL). Using the extracted ion chromatograms of the unmodified and modified peptides, the level of cleavage of the C-terminal lysine is calculated 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.

[0178] In one embodiment, the composition comprises an antibody or glycosylated variant containing a glycosylated post-translational modification ("glycosylation modification"). Exemplary glycosylation modifications include changes in the expression of G0, G1, G0-GlcNac, G2, and sialylation on the antibody. 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 contains a glycosylation modification.

[0179] In another embodiment, the composition comprises an antibody having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, and includes a glycosylated variant.

[0180] In another embodiment, the anti-BCMA antibody comprises verantamab and includes a glycosylated 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 G0 at levels 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%, or about 0-50% G1 levels of approximately 1-50%, approximately 1-40%, approximately 1-35%, or 8-31%; G0-GlcNac levels of approximately 5% or less, approximately 7.5% or less, approximately 10% or less, approximately 15% or less, approximately 20% or less, approximately 25% or less, approximately 30% or less, approximately 40% or less, approximately 50% or less, approximately 75% or less, approximately 0-100%, approximately 0.5-100%, approximately 0-50%, approximately 0.5-50%, approximately 0.5-25%, approximately 0.5-10%, approximately 0.5-7.5%, or approximately 0.9-5.3%; G2 levels of 0-100%, 1-100%, or 39-92%; and / or G0-2GlcNac levels of 0-100%, 1-100%, or 38-88%.

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

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

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

[0185] In one example, glycosylation modifications and the resulting profiles can be determined using ultra-high-performance liquid chromatography (UPLC) with hydrophilic interaction liquid chromatography (HILIC) separation and fluorescence detection. In one example, a composition described herein, for example, a composition containing verantamab, can be diluted with water to a concentration of 10 μg / μL. The glycan is then released from the composition, for example, the composition containing verantamab, by enzymatic digestion with PNGaseF using a PNGaseF kit from New England BioLabs (catalog no. P0705L). The glycan, once released by PNGaseF, can be labeled with anthranilamide (Sigma-Aidrich, catalog no. A89804). The labeled glycan can then be purified and excess labeling solution removed using a HILIC column step (a step in which the glycan can be loaded, washed with water, and eluted with acetonitrile). Next, in a Waters Acquity UPLC, labeled glycans can be separated by ammonium formate / formic acid and acetonitrile gradients using a Waters Glycan BEH Amide column (catalog number 186004742). The glycans can then be detected, for example, by using fluorescence detection with excitation at 365 nm and emission at 438 nm. The quantification of glycans can be performed, for example, by dividing the area under the curve for a given 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." The aggregated antibody may comprise a dimer or a higher-order structure formed from antibody monomers and their subunits. The aggregated variant may be, for example, a covalent or non-covalent, reducible or unreducible, and visible or subvisible aggregate of the antibody described herein. The aggregated variant or fragmented variant may be characterized based on their size and may be distinguished from the antibody. For example, the size distribution of the antibody composition may be detected using size exclusion chromatography (SEC), e.g., SE-HPLC.

[0187] In one embodiment, the composition comprises an aggregate variant of an antibody, the aggregate variant comprising a heavy chain sequence including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain sequence including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises 10% or less of the aggregate variant.

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

[0189] A fragmented variant is a variant that contains a portion of a full-length antibody. Examples of 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 embodiment, the composition comprises an antibody fragment variant, the fragment variant comprising a heavy chain sequence including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, and a light chain sequence including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, and the composition comprises 10% or less of the fragment variant.

[0191] The antibody composition may contain 10% or less of fragmented antibody, for example, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of fragmented antibody. 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 fragmented antibody. Alternatively, the composition may contain about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.5% of fragmented antibody.

[0192] The composition may comprise any one or a combination of the acidic variants, basic variants, isomerized variants, oxidized variants, deamidated variants, N-terminal pyroglutamate variants, C-terminal lysine cleavage variants, and / or glycosylated variants in any percentage, and / or aggregated variants and / or fragmented variants as described herein.

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

[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 of 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 of 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 of 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 the composition containing 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 verantamab, which is 100% activity. In one embodiment, the composition contains a variant comprising a heavy chain amino acid sequence including 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 including CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, wherein the potency of the composition is at least 70% of a composition comprising the heavy chain sequence of SEQ ID NO: 9, 11, 12, 13, or 14 and the light chain sequence of SEQ ID NO: 10, comprising any one or combination of (i) up to 23% isomerization at D103, and / or (ii) up to 37% oxidation at M34.

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

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

[0200] The binding of fetal Fc receptors (FcRn) to anti-BCMA antigen-binding proteins, such as verantamab, can be measured using surface plasmon resonance (SPR). Verantamab can be captured by FcRn immobilized on a nitrilotriacetate (NTA) sensor chip. The FcRn binding concentration of a sample can be determined by interpolating the binding reaction on a calibration curve. 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 anti-BCMA antigen-binding proteins, such as verantamab-mahodotin, to BCMA and FcγRIIIa can be measured using surface plasmon resonance (SPR). In one example, verantamab-mahodotin is injected and captured by protein A immobilized on a CM5 sensor tip. Then, BCMA is injected to bind to the captured verantamab-mahodotin. Then, FcγRIIIa is injected to bind to the captured verantamab-mahodotin. The functional concentrations of verantamab-mahodotin bound to BCMA and FcγRIIIa can be calculated from a reference standard curve and recorded as BCMA or FcγRIIIa binding concentrations, respectively. The total verantamab-mahodotin concentration of a sample can be pre-determined by absorbance at 280 nm. The specific binding activity (%) can be calculated by dividing the binding concentration of BCMA or FcγRIIIa by the absorbance, for example, the absorbance at a concentration of 280 nm.

[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, the composition comprises verantamab-mahodotin, and the average DAR or DL ​​ratio affects binding to FcRn. In yet another embodiment, the composition comprises verantamab-mahodotin, 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] The binding of fetal Fc receptors (FcRn) to anti-BCMA antigen-binding proteins, such as verantamab-mahodotin, can be measured using surface plasmon resonance (SPR). Verantamab-mahodotin can be captured by FcRn immobilized on a nitrilotriacetate (NTA) sensor chip. The FcRn binding concentration of a sample can be determined by interpolating the binding reaction on a calibration curve. Specific binding activity (%) is calculated by dividing the FcRn binding concentration by the total protein concentration.

[0204] If the anti-BCMA antigen-binding protein contains verantamab-mahodotin, the SPR method described herein may use a reference standard of verantamab or verantamab-mahodotin for specific antigen binding, FcγRIIIa, and FcRn binding. Using a verantamab or verantamab-mahodotin reference standard in the assay allows for obtaining system suitability and sample comparability data, ensuring that the method is performed correctly. The reference standard allows for the creation of a calibration curve, from which the sample concentration can be interpolated. For example, the reference standard may be a composition containing the heavy-chain amino acid sequence of SEQ ID NO: 9 and the light-chain amino acid sequence of SEQ ID NO: 10.

[0205] Antibody compositions containing the above-mentioned antibodies and antibody variants retain specific antigen binding and / or FcRn binding and / or FcγRIIIa binding and / or potency. For example, antibody compositions containing the above-mentioned antibodies, antibody variants and post-translational modification variants have greater than 0.70% BCMA-specific antigen binding and / or greater than 70% FcRn binding and / or 70% FcγRIIIa binding and / or greater than 70% potency. Therefore, variants at these levels (%) may be acceptable in antibody compositions without significantly affecting function (i.e., without causing a decrease in activity). In one embodiment, "decreased function" or "decreased activity" means that binding to BCMA, or to FcRn, or to FcγRIIIa, or potency is reduced as a percentage compared to a reference standard and is significant with respect to assay variability. For example, a decrease in function, activity, or potency may 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 the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more of cleavage of lysine at the C-terminus of the heavy chain and 100% or less of pyroglutamic acid at the N-terminus of the heavy chain. In yet another embodiment, the reference sample standard is a composition comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more of cleavage of lysine at the C-terminus of the heavy chain, 100% or less of pyroglutamic acid at the N-terminus of the heavy chain and 7% or less of isomerization of CDRH3 at amino acid D103. In a further embodiment, the reference sample standard is a composition comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more of cleavage of lysine at the C-terminus of the heavy chain, 100% or less of pyroglutamic acid at the N-terminus of the heavy chain, 7% or less of isomerization of CDRH3 at amino acid D103, and 5% or less of oxidation at amino acids M34, M256 and / or M432. In a further embodiment, the reference sample standard is a composition comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more of cleavage of lysine at the C-terminus of the heavy chain, 100% or less of pyroglutamic acid at the N-terminus of the heavy chain, 7% or less of isomerization of CDRH3 at amino acid D103, 5% or less of oxidation at amino acids M34, M256 and / or M432, and 2% or less of deamidation at amino acids N388 and / or N393. In a further embodiment, the reference sample standard is a composition comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, wherein the composition comprises 80% or more of cleavage of lysine at the C-terminus of the heavy chain, 100% of pyroglutamic acid at the N-terminus of the heavy chain, 7% or less of isomerization of CDRH3 at amino acid D103, 5% or less of oxidation at M256, 2% or less of oxidation at M34 and M432, and 2% or less of deamidation at amino acids N388 and N393.

[0207] Antibody-drug conjugate (ADC) Antibody-drug conjugates (ADCs) represent a new class of potent anticancer agents that have recently demonstrated significant clinical benefits. ADCs consist of a cytotoxic agent chemically conjugated to an antibody via a linker. It is hypothesized that 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 cell processing (e.g., mitosis), and apoptosis. ADCs combine the antigen-driven targeting properties of monoclonal antibodies with the potent antitumor effects of cytotoxic agents. For example, in 2011, ADCETRIS® (anti-CD30 antibody-MMAE ADC) received regulatory approval for the treatment of refractory Hodgkin lymphoma and systemic anaplastic lymphoma.

[0208] ADCs have been used in cancer treatment for the local delivery of cytotoxic drugs, i.e., drugs that damage or inhibit cell growth or proliferation (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; US Pat. No. 4,975,278). ADCs enable targeted delivery of the drug portion to the tumor and its intracellular accumulation there, while systemic administration of unbound drugs can result in unacceptable levels of toxicity to normal cells and tumor cells being targeted for elimination (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, such as diphtheria toxin; plant toxins, such as ricin; and low-molecular-weight toxins, such as 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 calicheamicins (Lode et al (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342) are included

[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, for example, chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioisotopes (i.e., radioconjugates).

[0210] In one embodiment, the anti-BCMA ADC has the following general structure: ABP-((Linker) n -Ctx) m [wherein ABP is an antigen-binding protein, the linker is absent, or is 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.]

[0211] ​In exemplary embodiments, enzymatically active toxins and their fragments that may be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria oficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene. See, for example, WO93 / 21232, published on October 28, 1993. Various radionuclides, for example, 211 At, 212 Bi, 131 I, 131 In, 90 Y and 186 Re can be used in the production of radioactive complex antibodies.

[0212] The anti-BCMA antibody or fragment thereof of the present invention may also be conjugated to one or more cytotoxic agents, for example, but not limited to, calicheamicin, meitansinoids, dolastatin, auristatin, trichothecene, and CC1065, or derivatives of these toxins having toxic activity. Suitable cytotoxic agents include, for example, auristatin, e.g., 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; lexitropsin; duocalmycin; taxanes, e.g., paclitaxel and docetaxel; puromycin; dolastatin; meitansinoids; and vinca alkaloids. Certain cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, drastatin-10, ethinomycin, combretatostatin, chalicheamicin, maytansin, DM-1, DM-4, ​​and netropsin. Other suitable cytotoxic agents include antitubulins (e.g., auristatin), vinca alkaloids, podophyllotoxin, taxanes, baccatin derivatives, cryptophysins, maytansinoids, combretastatin, or drastatin. Antitubulin agents include dimethylvaline-valinedrysoloiin-dlaproin-phenylalanine-p-phenylenediamine (AFP), MMAF, MMAE, auristatin E, vincristatin, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epotilon A, epotilon B, nocodazole, colchicine, colcimid, estramustine, semadolin, discodermolide, maytansine, DM-1, DM-4, ​​or eryuterobin.

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

[0214] [ka]

[0215] Cleavable linkers may be more readily cleaved under intracellular conditions. Suitable cleavable linkers include peptide linkers that can be cleaved by intracellular proteases, such as lysosomal proteases 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 that can be hydrolyzed at pH less than 5.5, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers. Examples of 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-iodoacetyl)aminobenzoate (SIAB).

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

[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 following structure.

[0218] [ka]

[0219] The anti-BCMA ADCs described herein may comprise any anti-BCMA antibody described herein together with any cytotoxic agent described herein.

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

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

[0222] In one embodiment, anti-BCMA ADC contains an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 7. H V containing an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 8. L It contains an anti-BCMA antibody that includes [specific component] and is conjugated to MMAE or MMAF.

[0223] In yet another embodiment, anti-BCMA ADC has the amino acid sequence described in SEQ ID NO: 7. H ; and V having the amino acid sequence described in Sequence ID No. 8 L It contains an anti-BCMA antibody that includes [specific component] and is conjugated to MMAF or MMAE.

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

[0225] In yet another embodiment, the anti-BCMA ADC is verantamab mahodotin comprising HC having the amino acid sequence described in SEQ ID NO: 9 and LC having the amino acid sequence described in SEQ ID NO: 10, and is conjugated to MMAF.

[0226] Preparation and Characterization of ADCs Certain natural IgG1 molecules contain 16 disulfide bonds (32 cysteine ​​or sulfhydryl groups). In certain embodiments, the antibody can be reduced to reduce only four interchain disulfide bonds, allowing it to conjugate to cytotoxic agents, enabling up to eight conjugate sites for cytotoxic agents. In other words, the drug load ("DL"), i.e., the number of cytotoxic agents per antibody molecule, can be between 0 and 8, and are described 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 conjugation to a given ADC composition, which varies in both 1) the number of drugs bound to each antibody molecule and 2) the position of cytotoxic drugs. This can result in ADC compositions 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, for example, Figure 2). The average drug-antibody ratio of the heterogeneous ADC composition is referred herein to as “average DAR” or “DAR”. For example, an ADC composition may contain a mixture of antibody species, each having its own DL (some species in the mixture are DL2, some are DL4, some are DL6, and some are DL8), and the average DAR of the composition as a whole may be approximately 4.

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

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

[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 interchain disulfides of 1 to 100 μg of antibody in 30 minutes at room temperature. However, other reducing agents and conditions may be used. Examples of reaction conditions include a pH of 5 to 8 and a temperature of 5°C to 37°C.

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

[0232] For example, DL0 does not carry any drug on the antibody. For example, DL2 has a drug load of 2. In one embodiment, the conjugation sites of DL2 are LC C214 and HC 224. For example, DL4 has a drug load of 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, DL6 has a drug load of 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, DL8 has a drug load of 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., proportions of DL0, DL2, DL4a, DL4b, DL6, and 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 a particular subspecies of DAR (e.g., the proportion of DL2a in the total DL2) is determined by collecting the specific DL species using a combination of analytical techniques that may include HIC, non-reducible separation methods, and mass spectrometry.

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

[0237] In another embodiment, the composition comprises an anti-BCMA ADC having 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, the antibody comprising CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, the cytotoxic agent being MMAE or MMAF, and the mean DAR being 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 anti-BCMA ADC, and the antibody has the amino acid sequence described in SEQ ID NO: 7. H , and V having the amino acid sequence described in Sequence ID No. 8 L The cytotoxic agents included are MMAE or MMAF, with mean DARs of approximately 2 to 6, 2.1 to 5.7, 3.4 to 4.6, or 3.8 to 4.5.

[0240] In one embodiment, the composition comprises verantamab mahodotin, with an average 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 the 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 the 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 combined species.

[0244] In one embodiment, the proportion of the 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 the 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 the 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, the 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 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, 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 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 agent 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%.

[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 are 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., an antibody that has not been conjugated with a cytotoxic agent after the conjugation process. In one embodiment, the proportion 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 proportion 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, the antibody comprising CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, the cytotoxic agent being MMAE or MMAF, and the DL0 percentage being about 10% or less or about 5% or less.

[0253] In one embodiment, the composition comprises anti-BCMA ADC, and the antibody has the amino acid sequence described in SEQ ID NO: 7. H , and V having the amino acid sequence described in Sequence ID No. 8 L The cytotoxic agents included are MMAE or MMAF, and the DL0 rate is approximately 10% or less, or approximately 5% or less.

[0254] In one embodiment, the composition contains verantamab mahodotin, and the proportion of DL0 is about 10% or less, or about 5% or less.

[0255] In one embodiment, the composition comprises verantamab mahodotin, where 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%.

[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 another embodiment, as the mean DAR or DL ​​ratio increases, cell growth inhibition is enhanced and / or tumor volume decreases. In yet another embodiment, the composition comprises verantamab mahodotin, and as the mean DAR or DL ​​ratio of the composition increases, cancer cell growth inhibition is enhanced and / or tumor volume decreases.

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

[0258] In one embodiment, the relative cell growth inhibitory potency of the composition is about 0.5 to about 1.3 or about 0.8 to about 1.1. In another embodiment, the average DAR of the composition is about 2.1 to about 5.7, and the relative cell growth inhibitory potency of the composition is about 0.5 to about 1.3. In another embodiment, the average DAR of the composition is about 3.0 to about 5.0 or about 3.5 to about 4.6, and the relative cell growth inhibitory potency of the composition is about 0.8 to about 1.1. In another embodiment, the composition comprises verantamab mahodotin with an average DAR of about 3.0 to about 5.0 or about 3.5 to about 4.6, and the relative cell growth inhibitory potency of the composition is about 0.8 to about 1.1.

[0259] In certain embodiments, the average DAR or DL ​​ratio affects ADCC activity. In another embodiment, the average DAR or DL ​​ratio does not affect ADCC activity. In yet another embodiment, the composition comprises verantamab mahodotin, and the average DAR or DL ​​ratio affects ADCC activity. In yet another embodiment, the composition comprises verantamab mahodotin, and the average DAR or DL ​​ratio does not affect ADCC activity.

[0260] The relative potency of ADCC activity can be measured, for example, by incubating verantamab mahodotin, cells (e.g., multiple myeloma cells), and NK cells (effector cells). While not bound by theory, verantamab mahodotin binds to BCMA expressed on the cell surface, and the Fc region of the antibody binds to the effector cell receptor FcγRIIIa via FcγRIIIa. The association of these receptors on the surface of effector cells leads to the synthesis and secretion of cytokines (IFNg), as well as the release of granules (perforin and granzyme) that enter the cytoplasm of target cells. Granzyme triggers signaling events within the target cells that cause apoptosis, leading to the death of these cells. The source of NK cells may be peripheral blood mononuclear cells (PBMCs) separable from human whole blood. Next, BATDA (bis-(acetoxymethyl)2,2':6',2''-terpyridine-6,6''-dicarboxylate) can be added and permeated to the target cell membrane to 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 lysed cells. ADCC activity can then be reported as the ratio of the sample's EC50 value to the reference standard's EC50 value.

[0261] In one embodiment, the relative potency of the ADCC activity of the composition is about 0.70 to about 1.30 or about 0.8 to about 1.1. In another embodiment, the average DAR of the composition is about 2.1 to about 5.7, and the relative potency of the ADCC activity of the composition is about 0.5 to about 1.3. In another embodiment, the average DAR of the composition is about 3.0 to about 5.0 or about 3.5 to about 4.6, and the relative potency of the ADCC activity of the composition is about 0.8 to about 1.1. In another embodiment, the composition contains verantamab mahodotin with an average DAR of about 3.0 to about 5.0 or about 3.5 to about 4.6, and the relative potency of the ADCC activity of the composition is 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, the composition comprises verantamab-mahodotin, and the average DAR or DL ​​ratio affects binding to BCMA. In yet another embodiment, the composition comprises verantamab-mahodotin, 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 relative BCMA-specific antigen binding of the composition is 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 verantamab mahodotin, and the relative BCMA-specific antigen binding of the composition is greater than 85% or greater than 90%. In yet another embodiment, the composition comprises verantamab mahodotin, and the average DAR of the composition is 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 relative BCMA-specific antigen binding of the composition is 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 verantamab-mahodotin, and the average DAR or DL ​​ratio affects binding to FcγRIIIa. In yet another embodiment, the composition comprises verantamab-mahodotin, 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 relative FcγRIIIa binding of the composition is 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 verantamab-mahodotin, and the relative FcγRIIIa binding of the composition is greater than 85% or greater than 90%. In yet another embodiment, the composition comprises verantamab-mahodotin, and the average DAR of the composition is 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 relative FcγRIIIa binding of the composition is greater than 85% or greater than 90%.

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

[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, the composition comprises belantamab mafodotin, and the average DAR or DL ratio affects binding to FcRn. In yet another embodiment, the 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 can weaken binding to FcRn.

[0268] Binding of the fetal Fc receptor (FcRn) to an anti-BCMA antigen binding protein, for example, 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 the sample can be determined by interpolation of the binding reaction on the calibration curve. The 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 reference standard of belantamab or belantamab mafodotin. The belantamab or belantamab mafodotin reference standard can be used in the assay to obtain system suitability and sample comparability data, ensuring that the method is performed properly. The reference standard allows a calibration curve to be generated, and the concentration of the sample is interpolated from the calibration curve. For example, the reference standard can be a composition comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10, and comprising a known DL and / or average DAR content.

[0270] Exemplary reference standards can include samples of belantamab mafodotin comprising DL species of known components / amounts and / or a known average DAR.

[0271] Pharmaceutical composition The compositions described herein may be in the form of pharmaceutical compositions. A "pharmaceutical composition" may comprise the composition described herein (i.e., the active ingredient) and one or more pharmaceutically acceptable excipients. The excipients must be compatible with the other components of the formulation, be pharmaceutical, be non-harmful to the recipient, and / or be acceptable in the sense that they do not interfere with the efficacy of the active ingredient.

[0272] As used herein, “pharmaceutically acceptable excipients” may include any solvent, diluent, carrier, dispersion medium, coating, antimicrobial agent, antifungal agent, isotonic agent and / or absorption retarder. Examples of pharmaceutically acceptable excipients include buffers, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and one or more combinations thereof. Often, isotonic agents, e.g., polyols, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride; preservatives; cosolvents; antioxidants, e.g., ascorbic acid and methionine; chelating agents, e.g., EDTA; metal complexes (e.g., Zn 2+ - Preferably, the composition contains protein complexes; biodegradable polymers; and / or salt-forming counterions such as sodium or potassium.

[0273] The exact properties of excipients or other materials may depend on the route of administration, which may be, for example, oral, rectal, nasal, topical (including cheek and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and intradural), and intratumoral. It is understood that preferred excipients may vary, for example, depending on the recipient's condition and the disease being treated.

[0274] The excipients and mixtures of their aggregates together form a “pharmaceutical formulation” (or “formulation”). The formulation may be in liquid form or lyophilized form. The composition in the liquid formulation may be filled into a container and frozen. In certain embodiments, aliquots of the frozen formulation containing the composition may be lyophilized. The lyophilized product may be reconstituted by adding water or other aqueous solution to produce a reconstituted formulation containing 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 certain embodiments, the concentration of anti-BMCA 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. The citrate buffer can be obtained, for example, by using a conjugate acid / conjugate base system (sodium citrate / citric acid) or by HCl titration of a sodium citrate solution. In certain embodiments, the concentration of the citrate buffer is about 10 mM to about 30 mM. In preferred embodiments, the concentration of the citrate buffer is 25 mM. In some embodiments, the buffer is a histidine buffer with a concentration of about 5 mM to about 35 mM.

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

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

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

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

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

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

[0283] In one embodiment, the composition contains an antibody in the formulation, the antibody comprising CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, the formulation comprising 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.

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

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

[0286] In one embodiment, the composition contains ADC in the formulation, the antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, 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 of citrate buffer, about 120 mM to about 240 mM of trehalose, about 0.01 mM to about 0.1 mM of EDTA, and about 0.01% to about 0.05% of 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 contains an ADC in the formulation, and the antibody has the amino acid sequence described in SEQ ID NO: 7. H , and V having the amino acid sequence described in Sequence ID No. 8 L The formulation contains approximately 20 mg / mL to 60 mg / mL of ADC, approximately 10 mM to 30 mM of citrate buffer, approximately 120 mM to 240 mM of trehalose, approximately 0.01 mM to 0.1 mM of EDTA, and approximately 0.01% to 0.05% of polysorbate 20 or polysorbate 80, with a pH of approximately 5.9 to 6.5.

[0288] In one embodiment, the composition contains an ADC in the formulation, the ADC being verantamab mahodotin, and the formulation contains about 20 mg / mL to about 60 mg / mL of verantamab mahodotin, about 10 mM to about 30 mM of citrate buffer, about 120 mM to about 240 mM of trehalose, about 0.01 mM to about 0.1 mM of EDTA, and about 0.01% to about 0.05% of 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 contains verantamab mahodotin in the formulation, and the formulation contains about 20 mg / mL, about 25 mg / mL, about 50 mg / mL or 60 mg / mL of verantamab mahodotin, 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 proteins within it inherently maintain their physical and / or chemical stability during manufacturing, transport, storage, and administration. Stability can be measured at a selected temperature and time. For example, for a product stored at a recommended temperature of 2°C to 8°C, the formulation is stable at room temperature, about 30°C, or 40°C for at least one month, and / or stable at about 2°C to 8°C for at least one year, preferably at least two years. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a “stable” formulation may be one in which, for example, less than about 10%, preferably less than about 5%, of the protein exists as aggregates within the formulation. Various analytical techniques for measuring protein stability are available in this field and are outlined, 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 enables the composition to maintain stability against freezing, thawing, and / or mixing.

[0292] In yet another embodiment, the present invention relates to a manufactured product, such as a kit, comprising a container for containing the composition in the formulation described herein. In one embodiment, an injection device containing the formulation is provided. The injection device may include a pen injector device or an auto-injector device. In one embodiment, the formulation is contained in a pre-filled syringe.

[0293] Methods of treatment and compositions for use The object of the present invention is 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 can be treated with anti-BCMA antigen-binding proteins. In particular, the object of the present invention is to provide a composition comprising an anti-BCMA antigen-binding protein, e.g., an anti-BCMA antibody, wherein the anti-BCMA antigen-binding protein specifically binds to BCMA (e.g., human BCMA) and modulates (i.e., inhibits or blocks) the interaction between BCMA and its ligand (e.g., BAFF and / or APRIL), in the treatment of diseases and disorders responsive to the modulation of this interaction.

[0294] Another aspect of the present invention provides a method for 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), comprising the step of administering a therapeutically effective amount of the anti-BCMA antigen-binding protein composition described herein to the subject.

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

[0296] As used herein, the terms “cancer” and “tumor” are interchangeable and refer to cells that undergo transformation, such as malignant transformation, that cause a host organism to become pathological. Primary cancer cells can be readily distinguished from non-cancerous 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 metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to the types of cancer that typically manifest as solid tumors, a “clinically detectable” tumor is a tumor that can be detected based on a mass by procedures such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound, and palpation during a physical examination, and / or a tumor that can be detected in a sample available from a patient for the expression of one or more cancer-specific antigens. Tumors can be hematopoietic (or hematologic, hematological, or blood-related) cancers, such as cancers originating from blood cells or immune cells, which are sometimes called “humoral tumors.” Specific examples of clinical conditions based on hematological tumors include leukemia, e.g., chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia; plasma cell malignancies, e.g., multiple myeloma, MGUS, and Waldenström macroglobulinemia; lymphomas, e.g., non-Hodgkin lymphoma and Hodgkin lymphoma; and so on.

[0297] Cancer can be any cancer that is characterized by an abnormal number of blast cells or undesirable cell proliferation, or a cancer diagnosed as a hematological malignancy, which includes both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic, myelogenous, or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyeloid, promyeloid, or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryoblastic (or megakaryoblastic) leukemia. These leukemias are sometimes collectively referred to as acute myeloid (or myeloid or myeloid) leukemia (AML). Myeloid malignancies include, but are not limited to, chronic myeloid (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV), as well as myeloproliferative disorders (MPDs). In particular, myeloid malignancies also include myelodysplasia (or myelodysplastic syndromes or MDS), sometimes called refractory anemia (RA), refractory anemia with blast cell proliferation (RAEB), and refractory anemia with transitional blast cell proliferation (RAEBT); as well as myelofibrosis (MFS), with or without primary myelofibrosis.

[0298] Hematopoietic carcinomas also include lymphoid malignancies, which can affect lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal sites. Lymphoid carcinomas include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin lymphoma (B-NHL). B-NHL may 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), e.g., nodular MZL, extranodal MZL, splenic MZL, and splenic MZL with hairy 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 leukemia 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 non-incisional nuclear cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHL includes immunoblastic lymphoma (or immunocytoma), primary exudative lymphoma, HIV-related (or AIDS-related) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or post-transplant lymphoma. B-cell malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenström macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocytic (LGL) leukemia, acute lymphoblastic (or lymphocytic or lymphoblastic) leukemia, and Castleman disease. In particular, NHL may also include T-cell non-Hodgkin lymphoma (T-NHL), which includes, but is not limited to, non-specific (NOS) T-cell non-Hodgkin lymphoma, peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphadenopathy (AILD), natural killer (NK) cell / T-cell lymphoma of the nose, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome.

[0299] Hematopoietic carcinomas also include Hodgkin lymphoma (or Hodgkin's disease), such as classical Hodgkin lymphoma, nodular sclerotic Hodgkin lymphoma, mixed cell type Hodgkin lymphoma, lymphocyte-dominant (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma, and lymphopenic Hodgkin lymphoma. Hematopoietic carcinomas also include plasma cell diseases or plasma cell carcinomas, such as multiple myeloma (MM) (including smoldering MM), monoclonal immunoglobulinemia of unknown significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenström macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic carcinomas may also include other cancers of further 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 tissue,” include bone marrow; peripheral blood; thymus; peripheral lymphoid tissues, such as the spleen, lymph nodes, mucosal (e.g., tonsils, Peyer's patches, and appendix) associated lymphoid tissues (e.g., gastrointestinal lymphoid tissues), and other mucosal (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 “to treat” and its derivatives include therapeutic treatment. With respect to a particular condition, to treat means: (1) to improve the condition or improve one or more of its biological signs; (2) to (a) one or more points of a biological cascade leading to the condition or causing the condition, or (b) one or more of its biological signs; (3) to alleviate one or more of the symptoms, effects, or side effects associated with the condition or its treatment; (4) to slow the progression of the condition or the progression of one or more of its biological signs; and / or (5) to cure the condition or one or more of its biological signs by eliminating or reducing one or more of its biological signs to an undetectable level for a period of time considered to be a state of remission of the biological signs of the condition, without additional treatment during a period of remission. Those skilled in the art will understand the period considered to be a state of 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 deficiency) and excessive / uncontrolled proliferation (e.g., lymphoma, leukemia). As used herein, B-cell disorders refer to both types of diseases, and methods for treating B-cell disorders by compositions described herein are provided.

[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 Waldenström macroglobulinemia.

[0304] Preventive treatment is also being considered. 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 signs, or to delay the onset of a condition or its biological signs. Preventive treatment is appropriate, for example, when a subject is considered to be at high risk of developing cancer, for example, when a subject has a strong family history of cancer or has been exposed to carcinogens.

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

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

[0307] The subject may have previously received at least one cancer treatment before being treated with the composition of the present invention. In one embodiment, the subject has previously been treated with at least one, at least two, at least three, at least four, at least five, at least six, or at least seven cancer treatments before being treated with the composition of the present invention.

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

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

[0310] In certain embodiments, the composition of the present invention is administered as a pharmaceutical composition.

[0311] As used herein, the term “administer” means the delivery of the compositions described herein to achieve a therapeutic objective. Compositions may be administered at intervals sufficient to achieve a clinical benefit. 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. Therefore, in one aspect, an injection device is provided that contains the composition, pharmaceutical composition, or formulation of the present invention. The injection device may include a pen injector device or an auto-injector device.

[0313] As used herein, the terms “therapeutic dose” or “therapeutic amount” of a composition refer to an amount effective in preventing, treating, or alleviating the symptoms of B cell-mediated disorders or disorders. The therapeutic dose and treatment plan are generally determined empirically and may depend on factors such as the patient’s age, weight, health condition, and the disease or disorder being treated. Such factors are within the control of the attending physician.

[0314] The appropriate therapeutically effective dose of a composition containing an anti-BCMA antigen-binding protein can be easily determined by those skilled in the art. The appropriate dose of the composition described herein may be calculated according to the patient's body weight, for example, an appropriate dose may be about 0.1 mg / kg to about 20 mg / kg, for example, about 1 mg / kg to about 20 mg / kg, for example, about 10 mg / kg to about 20 mg / kg, for example, about 1 mg / kg to about 15 mg / kg, or for example, about 10 mg / kg to about 15 mg / kg.

[0315] In one embodiment, the therapeutically effective dose of the composition containing the anti-BCMA antigen-binding protein is approximately 0.03 mg / kg to approximately 4.6 mg / kg. In yet another embodiment, the therapeutically effective dose of the composition containing the 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 the composition containing the 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 administered co-administered to a subject with one or more additional therapeutic agents. In other embodiments, the composition may be administered co-administered to a subject with one or more additional cancer therapeutic agents. 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 imide drugs (IMiDs) (e.g., thalidomide and its analogues).

[0317] "Simultaneous administration" means the administration of two or more different pharmaceutical compositions or treatments (e.g., radiotherapy) to a subject, either by the same pharmaceutical composition or a combination of different pharmaceutical compositions. Therefore, simultaneous 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 present invention, the present invention provides a method for treating a B-cell disease or disorder in a subject requiring such treatment by administering any of the therapeutically effective doses of compositions comprising the anti-BCMA antigen-binding protein described herein.

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

[0320] In another embodiment, the present invention provides a method for treating cancer in a subject requiring such treatment, comprising administering a therapeutically effective dose composition comprising an anti-BCMA ADC, wherein the antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, the cytotoxic agent is MMAF or MMAE, and the mean DAR is about 3.4 to about 4.6.

[0321] In another embodiment, the present invention is a method for treating cancer in a subject requiring the treatment thereof, comprising administering a therapeutically effective dose composition comprising an anti-BCMA ADC, wherein the antibody has the amino acid sequence described in SEQ ID NO: 7. H , and V having the amino acid sequence described in Sequence ID No. 8 LThe present invention provides a method comprising a cytotoxic agent which is MMAE or MMAF, and which has an average DAR of approximately 3.4 to approximately 4.6.

[0322] In another embodiment, the present invention provides a method for treating cancer in a subject requiring it, comprising administering a therapeutically effective dose of a composition comprising berantamab mahodotin having an average DAR of about 3.4 to about 4.6.

[0323] In another embodiment, the present invention provides a method for treating multiple myeloma in a subject requiring it, comprising administering a therapeutically effective dose composition comprising berantamab mahodotin having an average DAR of about 3.4 to about 4.6.

[0324] In one embodiment, the present invention provides a method for treating cancer in a subject requiring the treatment, comprising administering a therapeutically effective dose composition comprising an anti-BCMA ADC, 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%.

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

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

[0327] In yet another embodiment, the present invention provides a method for treating cancer in a subject requiring the treatment, comprising administering a therapeutically effective dose of a composition comprising verantamab mahodotin, 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%.

[0328] In yet another embodiment, the present invention provides a method for treating multiple myeloma in a subject requiring the treatment, comprising administering a therapeutically effective dose of a composition comprising verantamab mahodotin, 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%.

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

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

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

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

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

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

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

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

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

[0338] In another embodiment, the present invention provides a composition comprising the anti-BCMA ADC described herein for use in the treatment of cancer, wherein the antibody has the amino acid sequence described in SEQ ID NO: 7. H , and V having the amino acid sequence described in Sequence ID No. 8 L The present invention provides a composition comprising the above, wherein the cytotoxic agent is MMAE or MMAF, and the average DAR is approximately 3.4 to approximately 4.6.

[0339] In another embodiment, the present invention provides a composition comprising verantamab mahodotin for use in the treatment of cancer, wherein the average DAR is 3.4 to about 4.6.

[0340] In yet another embodiment, the present invention provides a composition comprising verantamab mahodotin for use in the treatment of multiple myeloma, wherein the average DAR is 3.4 to about 4.6.

[0341] In one embodiment, the present invention provides a composition for use in the treatment of cancer, comprising an anti-BCMA ADC as described herein, 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%.

[0342] In another embodiment, the present invention provides for use in the treatment of cancer, using the anti-BCMA described herein. The present invention provides a composition comprising an ADC, wherein the antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6; the cytotoxic 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%.

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

[0344] In another embodiment, the present invention provides a composition comprising verantamab mahodotin 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 26%, the proportion of DL4a is about 35% to about 38%, the proportion of DL4b is about 7% to about 10%, the proportion of DL6 is about 14% to about 20%, and / or the proportion of DL8 is about 6.0% to about 12.0%.

[0345] In yet another embodiment, the present invention provides a composition comprising verantamab mahodotin 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 embodiment, the present invention provides a composition comprising an antibody for use in the treatment of cancer, wherein the antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, and the composition comprises isomerization at D103 of heavy chain CDRH3 of 25% or less.

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

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

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

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

[0351] In yet another embodiment, the present invention provides a composition comprising verantamab mahodotin for use in the treatment of multiple myeloma, wherein the composition comprises oxidation of 40% or less of the heavy chain M34 (CDRH1).

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

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

[0354] In another embodiment, a use is provided for the manufacture of a pharmaceutical for use in the treatment of cancer, wherein the antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, the cytotoxic agent is MMAF or MMAE, and the mean DAR is about 3.4 to about 4.6.

[0355] In another embodiment, the use of a composition comprising an anti-BCMA ADC in the manufacture of a pharmaceutical for use in the treatment of cancer, wherein the antibody has the amino acid sequence described in SEQ ID NO: 7. H , and V having the amino acid sequence described in Sequence ID No. 8 L A use is provided that includes a cytotoxic agent that is MMAE or MMAF, with an average DAR of approximately 3.4 to 4.6.

[0356] In another embodiment, a use is provided for a composition comprising verantamab mahodotin in the manufacture of a pharmaceutical for use in the treatment of cancer, wherein the average DAR is about 3.4 to about 4.6.

[0357] In another embodiment, a use is provided for a composition comprising verantamab mahodotin in the manufacture of a pharmaceutical for use in the treatment of multiple myeloma, wherein the average DAR is about 3.4 to about 4.6.

[0358] In one embodiment, a use is provided for a composition comprising an anti-BCMA ADC in the manufacture of a pharmaceutical 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, in the manufacture of a pharmaceutical product for use in the treatment of cancer, anti-BCMA A composition comprising an ADC is provided, wherein the antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6; the cytotoxic 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%.

[0360] In another embodiment, the use of a composition comprising an anti-BCMA ADC in the manufacture of a pharmaceutical for use in the treatment of cancer, wherein the antibody has the amino acid sequence described in SEQ ID NO: 7.H , and V having the amino acid sequence described in Sequence ID No. 8 L A use is provided which includes; the cytotoxic 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%.

[0361] In another embodiment, a use is provided for a composition comprising an anti-BCMA ADC in the manufacture of a pharmaceutical 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, a use is provided for a composition comprising verantamab mahodotin in the manufacture of a pharmaceutical 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 a use of a composition comprising verantamab mahodotin in the manufacture of a pharmaceutical 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 embodiment, a use is provided for the manufacture of a pharmaceutical product for use in the treatment of cancer, wherein the composition comprises an antibody having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in SEQ ID NO: 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in SEQ ID NO: 4, CDRL2 having the amino acid sequence described in SEQ ID NO: 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6, and the composition comprises isomerization of 25% or less of heavy chain CDRH3 at D103.

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

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

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

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

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

[0370] All patent and document references disclosed herein are expressly and fully incorporated herein by reference.

[0371] The inventions described herein include the following: [1] A composition comprising an isomerized variant of an anti-BCMA antibody, The isomerized variant is A heavy chain amino acid sequence including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, The light chain amino acid sequence includes CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6. Includes, The composition comprising 25% or less of the isomerized variant. [2] A composition comprising an oxidized variant of an anti-BCMA antibody, The aforementioned oxidation variant A heavy chain amino acid sequence including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, The light chain amino acid sequence includes CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6. Includes, The composition comprising 40% or less of the oxidized variant. [3] A composition comprising an anti-BCMA antibody, The aforementioned anti-BCMA antibody CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in Sequence ID No. 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in Sequence ID No. 4, CDRL2 having the amino acid sequence described in Sequence ID No. 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6 Includes, The composition comprising 0.1 to 25% isomerization of CDRH3 at D103. [4] A composition comprising an anti-BCMA antibody, The aforementioned anti-BCMA antibody CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in Sequence ID No. 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in Sequence ID No. 4, CDRL2 having the amino acid sequence described in Sequence ID No. 5, and CDRL3 having the amino acid sequence described in SEQ ID NO: 6 Includes, The composition comprising 0.1 to 40% oxidation of CDRH1 at M34. [5] A composition comprising an anti-BCMA antibody, The anti-BCMA antibody is at least approximately 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 comprising 0.1 to 25% isomerization of CDRH3 at D103. [6] A composition comprising an anti-BCMA antibody, The anti-BCMA antibody is at least approximately 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 comprising 0.1 to 40% oxidation of CDRH1 at M34. [7] The composition according to any one of [1] to [6], wherein the composition comprises 65% or less of oxidation of heavy chain M256 and / or 60% or less of oxidation of heavy chain M432. [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 of the heavy chain, a change from D103 to N103 in CDRH3, cleavage of the C-terminal lysine, and conversion from 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% N103 at 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 comprises any percentage of glycoform 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 verantamab.

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

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

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

[12] , wherein the anti-BCMA antibody is verantamab mahodotin.

[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 approximately 3.4 to approximately 4.6.

[16] The composition according to any one of

[12] to

[15] , wherein the proportion of DL0 is approximately 10% or less or approximately 5% or less. A pharmaceutical composition comprising the composition described in any of

[17] [1] to

[16] and at least one pharmaceutically acceptable excipient. A preparation comprising the pharmaceutical composition described in

[18]

[17] , The aforementioned preparation comprises approximately 20 mg / mL to approximately 60 mg / mL of anti-BCMA antigen-binding protein, approximately 10 mM to approximately 30 mM of citrate buffer, approximately 120 mM to approximately 240 mM of trehalose, approximately 0.01 mM to approximately 0.1 mM of EDTA, and approximately 0.01% to approximately 0.05% of polysorbate 20 or polysorbate 80. The preparation wherein the pH of the preparation is approximately 5.9 to approximately 6.5.

[19] The preparation according to

[18] , wherein the preparation comprises approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL or approximately 60 mg / mL of verantamab mahodotin, 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 preparation is approximately 5.9 to approximately 6.5.

[20] A method for treating cancer, comprising administering a therapeutically effective amount of any of the compositions described in [1] to

[16] to a subject in need thereof.

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

[14] for use in the treatment of cancer.

[22] Use of any of the compositions described in [1] to

[16] in the manufacture of a pharmaceutical product 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 is CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in Sequence ID No. 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in Sequence ID No. 4, CDRL2 having the amino acid sequence described in Sequence ID No. 5, and CDRL3 having the amino acid sequence described 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 approximately 15% to approximately 32%, the proportion of DL4a is approximately 30% to approximately 40%, the proportion of DL4b is approximately 5% to approximately 10%, the proportion of DL6 is approximately 10% to approximately 20%, and the proportion of DL8 is approximately 4% to approximately 15%.

[26] The composition according to

[23] or

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

[27] The composition according to any one of

[23] to

[26] , wherein the mean drug-antibody ratio (DAR) is approximately 2.1 to approximately 5.7.

[28] The composition according to any one of

[23] to

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

[29] The composition according to any one of

[23] to

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

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

[31] The anti-BCMA antibody is CDRH1 having the amino acid sequence described in SEQ ID NO: 1, CDRH2 having the amino acid sequence described in Sequence ID No. 2, CDRH3 having the amino acid sequence described in SEQ ID NO: 3, CDRL1 having the amino acid sequence described in Sequence ID No. 4, CDRL2 having the amino acid sequence described in Sequence ID No. 5, and CDRL3 having the amino acid sequence described 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 approximately 5% or less.

[33] The composition according to any one of

[30] to

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

[34] The composition according to any one of

[30] to

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

[35] The composition according to any one of

[30] to

[34] , wherein the mean drug-antibody ratio (DAR) is approximately 2.1 to approximately 5.7.

[36] A composition according to any one of

[30] to

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

[37] The composition according to any one of

[30] to

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

[38] The anti-BCMA antibody having the amino acid sequence described in SEQ ID NO: 7 H , and V having the amino acid sequence described in Sequence ID No. 8 L A composition according to any one of

[23] to

[37] , comprising the above.

[39] The composition according to any one of

[23] to

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

[40] The composition according to any one of

[23] to

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

[41] The composition according to any one of

[21] to

[40] , wherein the anti-BCMA ADC is verantamab mahodotin.

[42] The composition according to any one 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 mean DAR is given by the following formula:

number

[42] , calculated from the area under the curve for each DL species using

[42] . A pharmaceutical composition comprising the composition described in any of

[44] ,

[23] , or

[43] , and at least one pharmaceutically acceptable excipient. A preparation comprising the pharmaceutical composition described in

[45] and

[44] , The aforementioned preparation comprises approximately 20 mg / mL to approximately 60 mg / mL of anti-BCMA antigen-binding protein, approximately 10 mM to approximately 30 mM of citrate buffer, approximately 120 mM to approximately 240 mM of trehalose, approximately 0.01 mM to approximately 0.1 mM of EDTA, and approximately 0.01% to approximately 0.05% of polysorbate 20 or polysorbate 80. The preparation wherein the pH of the preparation is approximately 5.9 to approximately 6.5.

[46] The preparation according to

[45] , wherein the preparation comprises approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL or approximately 60 mg / mL of verantamab mahodotin, 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 preparation is approximately 5.9 to approximately 6.5.

[47] A method for treating cancer, comprising administering a therapeutically effective amount of any of the compositions described in

[23] to

[43] to a subject in need thereof.

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

[23] to

[43] for use in the treatment of cancer.

[49] Use of any of the compositions described in

[23] to

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

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

[51] A composition comprising an acidic variant of an antibody, The aforementioned acidic variant CDRH1 of sequence number 1, CDRH2 of sequence number 2, CDRH3, which is sequence number 3, The light chain amino acid sequence includes CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6. Includes, The composition comprising 70% or less of the acidic variant.

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

[54] A composition comprising the main isoform of an antibody, The aforementioned main isoforms are, A heavy chain amino acid sequence including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, The light chain amino acid sequence includes CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6. Includes, The composition comprising 1 to 90% of the main isoform.

[55] A composition comprising major isoforms of an antibody, The aforementioned main isoforms are, A heavy chain amino acid sequence including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, The light chain amino acid sequence includes CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6. Includes, The composition comprising 1% or more of the main isoform.

[56] A composition comprising an antibody charge variant, The aforementioned charge variant is A heavy chain amino acid sequence including CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 of SEQ ID NO: 3, The light chain amino acid sequence includes CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6. Includes, The composition comprising 70% or less of an acidic variant and / or 30% or less of a basic variant and / or 1% or more of a major isoform. [Examples]

[0372] Example 1: Determination of the proportion of DL species and average DAR in the ADC composition For Examples 2-9, the proportion of DLs and the average DAR were calculated as follows: The proportion of a specific DL species (e.g., proportion of DL0, DL2, DL4a, DL4b, DL6, and 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 average DAR for each sample was calculated from the area under the curve for each DL species using the following formula.

[0374]

number

[0375] The 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 average DAR on cell growth inhibition The cell growth inhibition of verantamab-mahodotin was determined by measuring the cell viability of NCI-H929 cells, a human multiple myeloma cell line, after 48-hour incubation with verantamab-mahodotin. Cell viability was measured using Promega's CellTiter Glo technology. Increasing verantamab-mahodotin concentration corresponded relatively to a decrease in the CellTiter Glo luminescence signal. Dose-response (EC50) was generated using SoftMaxPro with a 4-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. The results are summarized in Table 2.

[0378] [Table 2]

[0379] Example 3: Effect of average DAR on ADCC activity Verantamab mahodotin, multiple myeloma cells, and NK cells (effector cells) were incubated. While not constrained by theory, verantamab mahodotin bound to BCMA expressed on the surface of multiple myeloma cells, and the antibody's Fc domain bound to the effector cell receptor FcγRIIIa via FcγRIIIa. The association of these receptors on the effector cell surface resulted in the synthesis and secretion of cytokines (IFNg) and the release of granules (perforin and granzyme) that entered the cytoplasm of target cells. Granzyme triggered intracellular signaling events in the target cells, leading to apoptosis and subsequent death of these cells. The source of NK cells was peripheral blood mononuclear cells (PBMCs) isolated from human whole blood. Next, 10 μL of BATDA (bis-(acetoxymethyl)2,2':6',2''-terpyridine-6,6''-dicarboxylate) (Perkin-Elmer catalog number C136-100), a fluorescence-enhancing ligand that permeates the cell membrane, was added to 1 mL of NCI-H929 cells (human multiple myeloma cell line). The ester bond of BATDA is hydrolyzed to form a hydrophilic ligand (TDA), which can no longer pass through the cell membrane. Labeled cells were added to various amounts of verantamab mahodotin and effector cells (PBMCs). The cells lysed and released TDA. After cell lysis, this 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 fluorescence plate reader, the measured fluorescence directly correlates with the amount of lysed cells. The ADCC activity of verantamab mahodotin was reported as the ratio of the sample's EC50 value to the EC50 value of 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 verantamab-mahodotin to BCMA and FcγRIIIa. Verantamab-mahodotin was diluted to 10 μg / mL with PBST, injected, and captured by protein A immobilized on a CM5 sensor tip. BCMA was then injected to bind to the captured verantamab-mahodotin. FcγRIIIa was then injected to bind to the captured verantamab-mahodotin. The functional concentrations of verantamab-mahodotin bound to BCMA and FcγRIIIa were calculated from the reference standard curve (reference standard #132371424) and reported as BCMA or FcγRIIIa binding concentrations, respectively. The total verantamab-mahodotin concentration of the sample was pre-determined by absorbance at 280 nm. The specific binding activity (%) was calculated by dividing the binding concentration of BCMA or FcγRIIIa by the absorbance at 280 nm. 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 transplanted 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 longest and shortest diameters of each mouse tumor were recorded to calculate the tumor volume (volume = longest diameter × shortest diameter). 2 ) × 0.5). The average tumor volume is approximately 200 mm². 3 Upon reaching a certain stage, mice were randomized into multiple groups and administered verantamab mahodotin, containing one of the mean DAR samples, twice weekly for two weeks. All tumors were measured in this manner, and the mean tumor size was 2.0 mm. 3 Each mouse was euthanized when it reached either day 1 or day 60, whichever came first. The study design is summarized 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 Samples of verantamab mahodotin containing specific DL species were prepared by collecting separate peaks in HIC chromatograms. Binding of verantamab mahodotin 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 receptors (FcRn) to verantamab mahodotin. Samples were diluted, and verantamab mahodotin was captured by FcRn immobilized on a nitrilotriacetate (NTA) sensor tip. The FcRn binding concentration of each sample was determined by interpolation of binding reactions on a calibration curve. Specific binding activity (%) was calculated by dividing the FcRn binding 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 verantamab mahodotin 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 mahodotin was conjugated to BCMA expressed on the surface of multiple myeloma cells. The Fc region of belantamab mahodotin was conjugated to FcγRIIIa (CD16a) on Jurkat T effector cells (Promega, catalog number G7102, BioCat#140011) designed to stably express 1) a human FcγRIIIa receptor V158 high-affinity variant and 2) a luciferase reporter gene fused to a promoter downstream of the NFAT activating sequence. When the antibody simultaneously binds to H929 and effector cells, activation of the NFAT pathway results in gene transcription from the luciferase reporter gene and expression of firefly luciferase in effector cells. A luminescent substrate (Bio-Glo® Luciferase Assay System, Promega, catalog number G7940) was added, and after cell lysis occurred, the luciferase produced by NFAT activation was measured as relative luminescence units (RLU) using a plate reader. In this assay, verantamab mahodotin was added in a dose-dependent manner. Therefore, a nonlinear logistic regression model was used to generate a dose-response (median effect 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: Average DAR and DL species percentages of several lots of verantamab mahodotin Several batches (19) of verantamab mahodotin were prepared. As described in Example 1, the average DAR and DL species percentages for each lot were calculated. The results are summarized in Tables 9-12.

[0394] [Table 9]

[0395] [Table 10]

[0396] [Table 11]

[0397] [Table 12]

[0398] Example 10: Test design for forced degradation of verantamab mahodotin Table 13 shows an overview of the test designs for Examples 11 to 18.

[0399] [Table 13]

[0400] Example 11: Oxidation conditions To prepare oxidative stress samples, verantamab mahodotin samples were diluted to 10 mg / mL, and hydrogen peroxide was added to achieve a molar ratio of 500:1 between hydrogen peroxide and verantamab mahodotin. The samples were quenched with methionine, and the buffer was replaced using a 3 kDa molecular weight cutoff filter (MWCO).

[0401] Deamidation and oxidation were determined using trypsin peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). The sample was denatured with 6M guanidine HCl solution to a concentration of 4.2 μg / μL. The disulfide bond was reduced by 50 mM DTT at room temperature for 20 minutes. 100 mM iodoacetic acid was added and reacted with the free cysteine ​​residue at room temperature for 30 minutes, then protected from light. The sample was buffer-changed using a BioRad spin column (part number 7326221). The peptide was then digested with 0.5% trypsin using Worthington trypsin (part number TRTPCK) at 37°C for 15 minutes. The resulting peptide was loaded onto a Waters reversed-phase ultrahigh performance liquid chromatography (UPLC) column (part number 186003687) and eluted with a water and acetonitrile gradient solution (0.1% trifluoroacetic acid solution) using a Waters Acquity UPLC. Peptides were detected using a UV detector and a mass spectrometer, such as the Thermo Scientific LTQ OrbitrapXL. Using extracted ion chromatograms of both the unmodified and modified peptides, the level of either deamidation or oxidation was calculated 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] As described in Example 4, surface plasmon resonance (SPR) was used to measure the binding of bellantamab mahodotin to BCMA and FcγRIIIa. As described in Example 6, surface plasmon resonance (SPR) was used to measure the binding of fetal Fc receptor (FcRn) to bellantamab mahodotin.

[0403] Reference standard #182407660A was used. The results are summarized in Tables 14 and 15.

[0404] [Table 14]

[0405] [Table 15]

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

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

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

[0409] Example 12: Chemical reaction: High pH (base treatment) To prepare a high-pH sample, the verantamab mahodotin sample was diluted with 4 mM Tris buffer to adjust the pH to 9. The sample was further diluted to 10 mg / mL and incubated at 25°C / 50% RH for up to 21 days.

[0410] As described in Example 11, deamidation, isomerization, and oxidation were determined using trypsin peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). Using extracted ion chromatograms of the unmodified and modified peptides, the level of deamidation, isomerization, or oxidation was calculated 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.

[0411] As described in Example 4, surface plasmon resonance (SPR) was used to measure the binding of BCMA and FcγRIIIa by verantamab mahodotin. As described in Example 6, surface plasmon resonance (SPR) was used to measure the binding of the embryonic Fc receptor (FcRn) to verantamab mahodotin.

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

[0413] [Table 16]

[0414] [Table 17]

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

[0416] Example 13: Chemical reaction: Low pH (acid treatment) To prepare a low-pH sample, the verantamab mahodotin sample was diluted in citrate buffer to adjust the pH to 5. The sample was further diluted to 10 mg / mL and incubated at 25°C / 50% RH for up to 21 days.

[0417] As described in Example 11, isomerization, deamidation, and oxidation were determined using trypsin peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). Using extracted ion chromatograms of the unmodified and modified peptides, the level of isomerization, deamidation, or oxidation was calculated 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.

[0418] As described in Example 4, surface plasmon resonance (SPR) was used to measure the binding of BCMA and FcγRIIIa by verantamab mahodotin. As described in Example 6, surface plasmon resonance (SPR) was used to measure the binding of the embryonic Fc receptor (FcRn) to verantamab mahodotin.

[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 prepare heat stress samples, verantamab mahodotin samples were diluted to 10 mg / mL with pharmaceutical buffer and incubated at 40°C / 75% RH for up to 28 days.

[0423] As described in Example 11, isomerization, deamidation, and oxidation were determined using trypsin peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). Using extracted ion chromatograms of the unmodified and modified peptides, the level of isomerization, deamidation, or oxidation was calculated 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.

[0424] As described in Example 4, surface plasmon resonance (SPR) was used to measure the binding of BCMA and FcγRIIIa by verantamab mahodotin. As described in Example 6, surface plasmon resonance (SPR) was used to measure the binding of the embryonic Fc receptor (FcRn) to verantamab mahodotin.

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

[0426] [Table 20]

[0427] [Table 21]

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

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

[0430] As described in Example 11, oxidation, deamidation, and isomerization were determined using trypsin peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). Using extracted ion chromatograms of the unmodified and modified peptides, the level of oxidation, deamidation, or isomerization was calculated 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.

[0431] As described in Example 4, surface plasmon resonance (SPR) was used to measure the binding of BCMA and FcγRIIIa by verantamab mahodotin. As described in Example 6, surface plasmon resonance (SPR) was used to measure the binding of the embryonic Fc receptor (FcRn) to verantamab mahodotin.

[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 cleavage and N-terminal pyroglutamate As described in Example 11, several lots of verantamab were analyzed for the levels of N-terminal pyroglutamate and C-terminal cleavage using trypsin peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS). Using extracted ion chromatograms of the unmodified and modified peptides, the levels of either N-terminal pyroglutamate or C-terminal cleavage were calculated 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.

[0436] As described in Example 4, surface plasmon resonance (SPR) was used to measure the binding of belantamab to BCMA and FcγRIIIa. As described in Example 6, surface plasmon resonance (SPR) was used to measure the binding of fetal Fc receptor (FcRn) to belantamab.

[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 The glycosylation patterns of several lots of verantamab were analyzed. The profiles were determined using ultra-high-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 verantamab by enzymatic digestion using PNGaseF with the New England BioLabs PNGaseF kit (catalog number P0705L). The glycans released by PNGaseF were labeled with anthranilamide (Sigma-Aidrich, catalog number A89804). The labeled glycans were then purified using a HILIC column step (a step in which the glycans are loaded, washed with water, and eluted with acetonitrile) to remove excess labeling solution. The labeled glycans were then separated using an ammonium formate / formic acid and acetonitrile gradient with a Waters Glycan BEH Amide column (catalog number 186004742) in a Waters Acquity UPLC. 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 given 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: Glycotechnology The effects of verantamab glucose-enriched samples on ADCC activity and binding were determined. ADCC activity was measured as in Example 3. BCMA and FcγRIIIa binding were determined as in Example 4.

[0445] For the galactosylation experiment, glycosylation was measured by reduced LC-MS. The sample was diluted to 1 mg / mL, 50 μL of 1 M DTT was added, and the mixture was reacted at 25°C or 37°C for 30 minutes before analysis by mass spectrometer (Micromass Q-tof). Heavy and light chains were separated using size exclusion chromatography with isocratic flow of water, acetonitrile, and trifluoroacetic acid. The spectra of each heavy and light chain were summed and deconvolved using Waters MaxEnt software. The major glycoform was detected, and its relative amount was evaluated from the signal count or area under the curve. The results are summarized in Tables 28-29.

[0446] [Table 28]

[0447] [Table 29]

[0448] Example 19: Tolerance The acceptable range (70–130% activity) was determined using the data from Tables 11–18 at the initial and final time points (day 21 or day 28, depending on the conditions). Binding data was plotted against the relative percentage of relevant post-translational modifications, and the slope of the relationship was determined. Using this information, the predicted level of each post-translational modification was calculated 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 verantamab or verantamab mahodotin.

[0449] [Table 30]

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

[0451] [Table 31]

[0452] Example 21 Table 32 shows an overview of the test designs for forced degradation of verantamab in Examples 21-26. The methods were largely the same as those described in Examples 11-18 above for verantamab mahodotin (except where otherwise noted).

[0453] [Table 32]

[0454] Example 22: Oxidation conditions for verantamab After 24 hours, oxidation of HC M256 increased from approximately 2% to approximately 98%, and M432 increased from approximately 1% to approximately 98%. SPR reduced specific binding to FcγRIIIa by 21–25% and FcRn binding by 10–18%. Oxidation of Fc may alter the binding activity of verantamab to FcγRIIIa and FcRn. After 24 hours, oxidation of HC M34 of CDR1 increased from approximately 0.3% to 47.7–48.5%, with no change in antigen binding, and within the range of assay variability. Oxidation levels of cysteine ​​and tryptophan 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% for HC N31, from approximately 2.0% to approximately 10% for HC N388, and from approximately 1.7% to approximately 18.5% for HC N393. Furthermore, oxidation of HC M256 was also observed to increase from approximately 2.2% to approximately 3.7% in verantamab 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 range of variability in the antigen, FcγRIIIa, and FcRn specific binding assays. Therefore, binding by SPR is equivalent to that of verantamab exposed to pH 9.0 stress on day 28.

[0456] [Table 33]

[0457] Example 24: Acid treatment of verantamab Fragmentation increased from 0.8% to 2.3-2.7% after 28 days. Succinimide formation at 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 also increased from approximately 2.2% to approximately 3.7% in verantamab 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 changes observed in antigen, FcγRIIIa, and FcRn specific binding were within the range of assay variability. Therefore, binding by SPR is equivalent to that of verantamab exposed to pH 3.5 stress at 28 days.

[0458] [Table 34]

[0459] Example 25: Heat-treated verantamab Fragmentation increased from 0.8% to 2.2-2.3% after 28 days. Aggregation remained unchanged. Succinimide formation at 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, in verantamab exposed to heat stress for 28 days, HC M256 oxidation also increased from approximately 2.2% to approximately 5.3%, and HC M432 oxidation increased from approximately 1% to 2%. 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% at 28 days, consistent with increased HC D103 isomerization, which has been shown to affect antigen binding. The changes observed in FcγRIIIa and FcRn-specific binding were within the range of assay variability.

[0460] Example 26: Phototreated verantamab Fragmentation increased from 0.8% to 1.5% in 1.5×ICH, and aggregation increased from approximately 1% to 6.5–7.5%. In 1.5×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 changes observed in antigen, FcγRIIIa, and FcRn specific binding were within the range of assay variability. Therefore, binding by SPR is comparable to verantamab exposed to pH 3.5 stress at 28 days.

[0461] [Table 35]

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

[0463] Although cIEF data for verantamab mahodotin is not shown because the number of drug-carrying molecules contributes to the charge profile, in the case of verantamab, cIEF effectively separates acidic and basic variants from the major species (see Figure 4).

[0464] Sequence List Sequence ID 1:CDRH1 NYWMH

[0465] Sequence ID 2: CDRH2 ATYRGHSDTYYNQKFKG

[0466] Sequence ID 3: CDRH3 GAIYDGYDVLDN

[0467] Sequence ID 4: CDRL1 SASQDISNYLN

[0468] Sequence ID 5: CDRL2 YTSNLHS

[0469] Sequence ID 6: CDRL3 QQYRKLPWT

[0470] Sequence ID 7: Heavy chain variable region (CDR is underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDNWGQGTLVTVSS

[0471] Sequence ID 8: Light chain variable region (CDR is underlined) DIQMTQSPSSLSASVGDRVTITC SASQDISNYLN WYQQKPGKAPKLLIY YTSNLHS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYRKLPWT FGQGTKLEIKR

[0472] Sequence ID 9: Heavy chain region (CDR is underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0473] Sequence ID 10: Light chain region (CDR is underlined) DIQMTQSPSSLSASVGDRVTITC SASQDISNYLN WYQQKPGKAPKLLIY YTSNLHS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0474] Sequence ID 11: Heavy chain region containing D103N (CDR is underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYNGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0475] Sequence ID 12: Heavy chain region containing N388D (CDR is underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0476] Sequence ID 13: Heavy chain region containing N393D (CDR is underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0477] Sequence ID 14: Heavy chain region containing N388D and N393D (CDR is underlined) VQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESDGQPEDNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A composition comprising an antibody-drug conjugate (ADC), The ADC comprises an anti-B cell maturation antigen (BCMA) antibody. The anti-BCMA antibody comprises the heavy chain amino acid sequence described in SEQ ID NO: 9 and the light chain amino acid sequence described in SEQ ID NO:

10. 0.1% to 23% of the total amount of anti-BCMA antibody in the composition contains isomerized aspartic acid at amino acid residue 103 of SEQ ID NO:

9. (a) 0.1% to 37% of the total amount of anti-BCMA antibody in the composition contains oxidation at M34 of SEQ ID NO: 9, (b) 0.1% to 64% of the total amount of anti-BCMA antibody in the composition contains oxidation at M256 of SEQ ID NO: 9, and / or (c) 0.1% to 61% of the total amount of anti-BCMA antibody in the composition contains oxidation at M432 of SEQ ID NO: 9 The anti-BCMA antibody may include (a) deamidation at N388 and / or N393 of SEQ ID NO: 9, (b) cleavage of the C-terminal lysine of SEQ ID NO: 9, and / or (c) conversion of the N-terminal glutamine of SEQ ID NO: 9 to pyroglutamic acid. The aforementioned anti-BCMA antibody is conjugated with a cytotoxic agent. The composition wherein the cytotoxic agent is selected from calicheamicin, meitansinoids, drastatin, auristatin, trichothecene, DNA minor groove binders, DNA minor groove alkylating agents, enediyne, lexitropsin, duocalmycin, taxane, puromycin, vinca alkaloids, antitubulin agents, podophyllotoxin, baccatin derivatives, cryptophycin, and combretastatin.

2. The composition according to claim 1, wherein the percentage of anti-BCMA antibody in the composition, comprising isomerized aspartic acid at amino acid residue 103 of SEQ ID NO: 9, oxidation at M34 of SEQ ID NO: 9, oxidation at M256 of SEQ ID NO: 9, and oxidation at M432 of SEQ ID NO: 9, is determined by peptide mapping tandem mass spectrometry (peptide mapping LC-MS / MS) using trypsin.

3. The composition according to claim 1, wherein the cytotoxic agent is selected from drastatin and auristatin.

4. The composition according to claim 2, wherein the cytotoxic agent is selected from drastatin and auristatin.

5. The composition according to claim 3, wherein the cytotoxic agent is selected from monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

6. The composition according to claim 4, wherein the cytotoxic agent is selected from monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

7. The composition according to claim 5, wherein the cytotoxic agent is an MMAF.

8. The composition according to claim 6, wherein the cytotoxic agent is an MMAF.

9. The composition according to claim 1, wherein the anti-BCMA antibody is conjugated to the cytotoxic agent via a linker.

10. The composition according to claim 2, wherein the anti-BCMA antibody is conjugated to the cytotoxic agent via a linker.

11. The composition according to claim 7, wherein the anti-BCMA antibody is conjugated to the MMAF via a linker.

12. The composition according to claim 8, wherein the anti-BCMA antibody is conjugated to the MMAF via a linker.

13. The composition according to claim 9, wherein the linker is a severable linker.

14. The composition according to claim 10, wherein the linker is a severable linker.

15. The composition according to claim 11, wherein the linker is a severable linker.

16. The composition according to claim 12, wherein the linker is a severable linker.

17. The composition according to claim 9, wherein the linker is an inseparable linker.

18. The composition according to claim 10, wherein the linker is an incuttable linker.

19. The composition according to claim 11, wherein the linker is an inseparable linker.

20. The composition according to claim 12, wherein the linker is an incuttable linker.

21. The composition according to claim 9, wherein the linker is selected from 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-iodoacetyl)aminobenzoate (SIAB).

22. The composition according to claim 10, wherein the linker is selected from 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-iodoacetyl)aminobenzoate (SIAB).

23. The composition according to claim 11, wherein the linker is selected from 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-iodoacetyl)aminobenzoate (SIAB).

24. The composition according to claim 12, wherein the linker is selected from 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-iodoacetyl)aminobenzoate (SIAB).

25. The composition according to claim 21, wherein the linker is MC.

26. The composition according to claim 22, wherein the linker is MC.

27. ​​The composition according to claim 23, wherein the linker is MC.

28. The composition according to claim 24, wherein the linker is MC.

29. The composition according to claim 27, wherein the anti-BCMA antibody is not fucosylated.

30. The composition according to claim 28, wherein the anti-BCMA antibody is not fucosylated.

31. A formulation comprising (a) the composition according to claim 9 in a concentration of 20 mg / mL to 60 mg / mL, (b) 10 mM to 30 mM citrate buffer, (c) 120 mM to 240 mM trehalose, (d) 0.01 mM to 0.1 mM EDTA, and (e) 0.01% to 0.05% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

32. A formulation comprising (a) the composition according to claim 10 in a concentration of 20 mg / mL to 60 mg / mL, (b) 10 mM to 30 mM citrate buffer, (c) 120 mM to 240 mM trehalose, (d) 0.01 mM to 0.1 mM EDTA, and (e) 0.01% to 0.05% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

33. A formulation comprising (a) the composition according to claim 11 in a concentration of 20 mg / mL to 60 mg / mL, (b) 10 mM to 30 mM citrate buffer, (c) 120 mM to 240 mM trehalose, (d) 0.01 mM to 0.1 mM EDTA, and (e) 0.01% to 0.05% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

34. A formulation comprising (a) the composition according to claim 12 in a concentration of 20 mg / mL to 60 mg / mL, (b) 10 mM to 30 mM citrate buffer, (c) 120 mM to 240 mM trehalose, (d) 0.01 mM to 0.1 mM EDTA, and (e) 0.01% to 0.05% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

35. A formulation comprising (a) the composition according to claim 29 in a concentration of 20 mg / mL to 60 mg / mL, (b) 10 mM to 30 mM citrate buffer, (c) 120 mM to 240 mM trehalose, (d) 0.01 mM to 0.1 mM EDTA, and (e) 0.01% to 0.05% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

36. A formulation comprising (a) the composition according to claim 30 in a concentration of 20 mg / mL to 60 mg / mL, (b) 10 mM to 30 mM citrate buffer, (c) 120 mM to 240 mM trehalose, (d) 0.01 mM to 0.1 mM EDTA, and (e) 0.01% to 0.05% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

37. A formulation comprising (a) 50 mg / mL of the composition according to claim 9, (b) 25 mM citrate buffer, (c) 200 mM trehalose, (d) 0.05 mM EDTA, and (e) 0.02% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

38. A formulation comprising (a) 50 mg / mL of the composition according to claim 10, (b) 25 mM citrate buffer, (c) 200 mM trehalose, (d) 0.05 mM EDTA, and (e) 0.02% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

39. A formulation comprising (a) 50 mg / mL of the composition according to claim 11, (b) 25 mM citrate buffer, (c) 200 mM trehalose, (d) 0.05 mM EDTA, and (e) 0.02% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

40. A formulation comprising (a) the composition according to claim 12 in a concentration of 50 mg / mL, (b) 25 mM citrate buffer, (c) 200 mM trehalose, (d) 0.05 mM EDTA, and (e) 0.02% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

41. A formulation comprising (a) the composition according to claim 29 in a concentration of 50 mg / mL, (b) 25 mM citrate buffer, (c) 200 mM trehalose, (d) 0.05 mM EDTA, and (e) 0.02% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.

5.

42. A formulation comprising (a) the composition according to claim 30 in a concentration of 50 mg / mL, (b) 25 mM citrate buffer, (c) 200 mM trehalose, (d) 0.05 mM EDTA, and (e) 0.02% polysorbate 20 or polysorbate 80, The preparation wherein the pH of the preparation is 5.9 to 6.5.

Images