Biopharmaceutical composition and stable isotope-labeled peptide mapping method

Abstract

Disclosed herein is a stable isotope labeled (SIL) peptide mapping method for accurate and sensitive quantification of conjugation sites. Also disclosed herein are compositions comprising antibody drug conjugates (ADCs) that target BCMA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 228,951, filed August 3, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. The XML file is named 054624_09_5031_Sequence_Listing.xml, was created on July 25, 2022, and is 15,855 bytes in size.

[0003] Field Disclosed herein are methods for determining site-specific conjugation levels of cysteine-conjugated antibody drug conjugates bearing small molecule cytotoxic payloads (e.g., MMAF or MMAE) using stable isotope labeling (SIL) peptide mapping. [Background technology]

[0004] background Antibody-drug conjugates (ADCs) are a growing class of biopharmaceuticals for targeted tumor therapy that combine the specificity of monoclonal antibodies (mAbs) with the potency of cytotoxic small molecule drugs (Hafeez et al., Antibody-Drug Conjugates for Cancer Therapeutics., Molecules., 2020, vol. 25, pp. 4764 and Boni et al., The Resurgence of Antibody Drug Conjugates in Cancer Therapeutics: Novel Targets and Payloads., Am Soc Clin Oncol Educ Book., 2020, vol. 40, pp. 1-17). Small molecule payloads are typically conjugated via cysteine ​​or lysine protein residues, resulting in a heterogeneous mixture of different drug-loaded (DL) species (Ponziani et al., Antibody-Drug Conjugates: The New Frontier of Chemotherapy., Int. J. Mol. Sci., 2020, vol. 21, pp. 5510). The drug-to-antibody ratio (DAR) of ADCs has been identified as a critical quality attribute (CQA) because it impacts drug potency and efficacy (Li et al., Impact of Physiologically Based Pharmacokinetics, Population Pharmacokinetics and Pharmacokinetics / Pharmacodynamics in the Development of Antibody-Drug Conjugates., The Journal of Clinical Pharmacology., 2020, 60, 105-119). As a result, ADCs pose analytical challenges in characterizing these drug-loaded species in addition to the protein sequence and post-translational modifications (PTMs) typically characterized for mAb biopharmaceuticals.

[0005] A wide range of DARs for ADCs can be determined by several analytical methods: hydrophobic interaction chromatography (HIC) (Bobaly et al., Optimization of non-linear gradient in hydrophobic interaction chromatography for the analytical characterization of antibody-drug conjugates., Journal of Chromatography A., 2017, Vol. 1481, pp. 82-91), hydrophilic interaction chromatography (HILIC) (D'Atri et al., Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry., Journal of Chromatography B., 2018, Vol. 1080, pp. 37-41), and capillary gel electrophoresis (CGE) (Lechner et al., Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016-2018., Journal of Chromatography B., 2018, Vol. 1080, pp. 37-41). B., 2019, vol. 1122-1123, pp. 1-17), and native and subunit liquid chromatography mass spectrometry (LC-MS) (Zhu et al., Current LC-MS-based strategies for characterization and quantification of antibody-drug conjugates., Journal of Pharmaceutical Analysis., 2020, vol. 10, pp. 209-220). These methods can also provide qualitative information on the location of the conjugation sites, but it has been difficult to assess site-specific conjugation levels.

[0006] Liquid chromatography tandem mass spectrometry (LC-MS / MS) of enzymatic digests (peptide mapping) is a widely used analytical method for characterizing protein sequences and quantifying post-translational modifications (PTMs) of biopharmaceuticals by performing relative quantification of native and modified peptides. MMAF-conjugated peptides complicate this approach because of the relatively large differences in mass and retention time between native and conjugated peptides due to the addition of hydrophobic drug payloads. These differences result in peptide pairs with significantly different ionization efficiencies, making them unsuitable for relative quantification between peptides.

[0007] As a result, most ADC peptide mapping applications are qualitative in nature, only confirming the location of conjugation sites, and few attempts have been described to quantify the level of conjugation at these sites. One method described by Q. Luo et al. (Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate., Analytical Chemistry., 2016, vol. 88, pp. 695-702) involved the analysis of an unconjugated mAb intermediate sample along with the ADC sample. The unconjugated peak area detected in the mAb sample was compared to the area detected in the ADC sample, and area loss was attributed to conjugation at that peptide site. However, this method did not normalize for sample preparation variability between mAb and ADC samples, and did not account for multiple conjugation sites on a single peptide, such as the heavy chain hinge peptide of a cysteine-conjugated ADC.

[0008] Another method described by L. Chen et al. (In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate., mAbs., 2016, vol. 8, pp. 1210-1223) attempted to correct for sample preparation variability by normalizing the peak area of ​​the conjugated peptide to the peak area when a known amount of leucine enkephalin peptide was added. However, this method only provided relative conjugation quantification between samples, but not the true site occupancy of a particular conjugation site.

[0009] A third method described by H. Sang et al. (Conjugation site analysis of antibody-drug-conjugates (ADCs) by signature ion fingerprinting and normalized area quantitation approach using nano-liquid chromatography coupled to high resolution mass spectrometry., Analytica Chimica Acta., 2017, vol. 955, pp. 67-78) attempted to account for ionization differences by normalizing the peak area of ​​the conjugated peptide by the peak area of ​​the respective unconjugated peptide. This ratio was then multiplied by a relative ionization intensity coefficient calculated by dividing the slope of the calibration curve for the unconjugated and conjugated peptides to obtain a normalized ratio. The conjugation level of the site was then calculated as a function of this normalized ratio. However, this method required the analysis of standards to generate a calibration curve for the peptide pairs of all conjugation sites. Furthermore, this formula for conjugation level did not take into account the presence of multiple conjugation sites on a single peptide.

[0010] Stable isotope labeling (SIL) is the process of incorporating heavy isotope atoms into an analyte of interest, resulting in a mass change that can be detected by mass spectrometry. SIL peptide mapping is a common method in the field of proteomics to provide relative quantification of proteins in differentially labeled samples (Liu et al., Advances and applications of stable isotope labeling-based methods for proteome relative quantitation., Trends in Anal.Chem., 2020, vol. 124, pp. 115815), but it has also been applied to protein PTM characterization. Liu et al. (Accurate Determination of Protein Methionine Oxidation by Stable Isotope Labeling and LC-MS Analysis., Anal.Chem., 2013, vol. 85, pp. 11705-11709) investigated the oxidation level of methionines by reacting mAb samples with oxygen-18 hydrogen peroxide to completely oxidize the methionines of interest. The isotopic peak areas were then used to perform relative quantification of the native (+16 Da) and SIL (+18 Da) forms of the oxidized peptides. Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there is a need in the art to provide improved analytical methods for ADCs. [Means for solving the problem]

[0012] overview According to a first aspect of the present disclosure, (i) preparing an isotopically labeled antibody drug conjugate (ADC) sample by conjugation of an unoccupied cysteine ​​site of a cysteine-conjugated ADC using an isotopically labeled cytotoxin containing a carbonyl group and a reducing agent; and (ii) performing peptide mapping of said sample; A method (e.g., an analytical method) is provided that includes:

[0013] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody, CDRH1 comprising the amino acid sequence of SEQ ID NO:1; CDRH2 comprising the amino acid sequence of SEQ ID NO:2; CDRH3 comprising the amino acid sequence of SEQ ID NO:3; CDRL1 comprising the amino acid sequence of SEQ ID NO:4, CDRL2 comprising the amino acid sequence of SEQ ID NO:5, and CDRL3 comprising the amino acid sequence of SEQ ID NO:6 and the cytotoxic agent is MMAF or MMAE, and Compositions are provided having a drug loading percentage at LC C214 of about 56% to about 80%, and / or a drug loading percentage at HC C224 of about 58% to about 81%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 15% to about 46%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 11% to about 15%.

[0014] According to a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising the composition described herein and at least one pharma- ceutically acceptable excipient.

[0015] According to a further embodiment of the present disclosure, there is provided a formulation comprising the pharmaceutical composition described herein, which comprises about 20 mg / mL to about 60 mg / mL of ADC, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, wherein the pH of the formulation is about 5.9 to about 6.5.

[0016] According to a further aspect of the present disclosure, there is provided a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a composition or formulation disclosed herein.

[0017] According to a further aspect of the present disclosure, there is provided a composition or formulation as disclosed herein for use in the treatment of cancer.

[0018] According to a further aspect of the present disclosure, there is provided a use of a composition disclosed herein in the manufacture of a medicament for use in the treatment of cancer.

[0019] According to a further aspect of the present disclosure, there is provided a method for determining a conjugation level of a cysteine-conjugated antibody drug conjugate, comprising: reducing the antibody-drug conjugate to form a reduced antibody-drug conjugate; conjugating the reduced antibody drug conjugate with an isotopically labeled cytotoxin to form an isotopically labeled antibody drug conjugate; generating an isotopically labeled conjugate peptide from the isotopically labeled antibody-drug conjugate and performing peptide mapping on the isotopically labeled conjugate peptide; detecting the mass-to-charge ratio of the isotopically labeled conjugated peptide; and comparing the mass-to-charge ratio of the isotopically labeled conjugated peptide to the mass-to-charge ratio of a non-isotopically labeled conjugated peptide to determine the conjugation level of the cysteine-conjugated antibody drug complex. In one embodiment, the cytotoxin is MMAF or MMAE. In another embodiment, the cysteine-conjugated antibody-drug conjugate is first reduced with a reducing agent and then conjugated with an isotopically labeled cytotoxin. In one embodiment, the reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In another embodiment, excess reducing agent is removed by eluting the sample from a size-exclusion chromatography column prior to peptide mapping. In yet another embodiment, conjugation occurs by reacting the cysteine-conjugated antibody-drug conjugate with an isotopically labeled cytotoxin. In another embodiment, excess isotopically labeled cytotoxin is removed by eluting the sample from a size-exclusion chromatography column prior to peptide mapping. In yet another embodiment, peptide mapping comprises the use of liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis. In some embodiments, peptide mapping comprises denaturing the sample, reducing residual disulfide bonds, and alkylating the resulting free sulfhydryls. In another embodiment, the peptide mapping comprises enzymatic digestion of the sample to generate isotopically labeled conjugated peptides, and optionally quenching the enzymatic digestion by the addition of strong acid. In some embodiments, the method comprises reacting the cytotoxin with isotopically labeled water to generate an isotopically labeled cytotoxin. In another embodiment, the cytotoxin is reacted with isotopically labeled water in acetonitrile. In yet another embodiment, the cysteine-conjugated antibody drug conjugate is belantamab mafodotin. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows the UV chromatogram and MS spectrum of stable isotope-labeled MMAF. [Diagram 2] FIG. 2 shows reduced LC-MS spectra of belantamab mafodotin light and heavy chains before and after stable isotope labeling. [Diagram 3] FIG. 3 shows a schematic diagram of the drug loading species heterogeneous mixture in belantamab mafodotin. [Figure 4] FIG. 4 compares the XICs of the light chain, heavy chain fab and heavy chain hinge peaks detected from standard peptide mapping and stable isotope labeled peptide mapping. [Diagram 5] Figure 5 shows representative MS spectra of labeled and unlabeled conjugated light chain peptides. The natural isotope ratios used for the calculation of isotopomer contributions are shown. [Figure 6] Figure 6 shows representative MS spectra of labeled and unlabeled conjugated heavy chain Fab peptides. Natural isotope ratios used for calculation of isotopomer contributions are indicated. [Figure 7] Figure 7 shows representative MS spectra of labeled and unlabeled conjugated heavy chain hinge peptides. Natural isotope ratios used for calculation of isotopomer contributions are indicated. [Figure 8] FIG. 8 compares the linear response curves between standard and stable isotope-labeled peptide mapping for all conjugation sites. [Figure 9] FIG. 9 compares the calculated and theoretical DARs for standard and SIL peptide mapping for belantamab mafodotin linear samples. [Figure 10] FIG. 10 shows the conjugation values ​​of belantamab mafodotin samples with different DAR. [Figure 11] FIG. 11 compares the calculated DARs from SIL peptide mapping with the theoretical HIC DARs for belantamab mafodotin samples with different DARs. [Figure 12] FIG. 12 shows analytical and preparative-scale hydrophobic interaction chromatography traces used to collect drug-loaded fractions. [Figure 13] FIG. 13 shows representative NR-CGE electropherograms of purified DL0, DL2, DL4a, DL4b, DL6 and DL8. [Figure 14] FIG. 14 shows intact mass spectra of purified DL0, DL2, DL4a, DL4b, DL6 and DL8 drug-loaded variants. [Figure 15] FIG. 15 shows reduced mass spectra of the heavy (A) and light (B) chains of purified DL0, DL2, DL4a, DL4b, DL6 and DL8 drug-loaded variants. [Figure 16] FIG. 16 shows capillary differential scanning calorimetry (DSC) traces of purified DL0, DL2, DL4a, DL4b, DL6 and DL8 drug loaded variants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Stable isotope labeling (SIL) peptide mapping method The term "about" or "approximately" may mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range depends in part on how the value is measured or determined, e.g., the limitations of the measurement system.

[0022] For example, "about" may mean plus or minus 10%, according to the practice of those skilled in the art. Alternatively, "about" may mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within 10-fold, 5-fold, or 2-fold of a value. When a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable range of error for the particular value, unless otherwise indicated. Also, when a range and / or subrange of values ​​is given, the range and / or subrange may include the endpoints of the range and / or subrange.

[0023] The present disclosure provides methods for determining site-specific conjugation levels of cysteine-conjugated antibody-drug conjugates bearing small molecule payloads (e.g., cytotoxins) using stable isotope-labeled peptide mapping LC-MS / MS analysis.

[0024] According to a first aspect of the present disclosure, (i) preparing an isotopically labeled antibody drug conjugate (ADC) sample by conjugation of an unoccupied cysteine ​​site of a cysteine-conjugated ADC using an isotopically labeled cytotoxin containing a carbonyl group and a reducing agent; and (ii) performing peptide mapping of said sample; An analytical method is provided, comprising:

[0025] In one embodiment, the cytotoxin contains a carbonyl group (eg, a carbonyl oxygen bearing a double bond).

[0026] In one embodiment, the cytotoxin is monomethylauristatin F (MMAF) or monomethylauristatin E (MMAE).

[0027] Liquid chromatography tandem mass spectrometry (LC-MS / MS) of enzymatic digests (peptide mapping) is an analytical method well known to those skilled in the art for identifying protein sequences of biopharmaceuticals and quantifying PTMs by performing relative quantification between native and modified peptides. The analytical method described herein reduces (including eliminates) the problem of different ionization efficiencies associated with prior art methods by conjugating unoccupied cysteine ​​sites with stable isotope-labeled cytotoxins to generate peptide pairs with identical retention times and minimal differences in mass and hydrophobicity, allowing for parallel relative quantification of other monitored post-translational modifications (PTMs) in a single multi-attribute analytical method (MAM).

[0028] Stable isotope labeling (SIL) is the process of incorporating heavy isotope atoms (e.g., carbon-13, nitrogen-15, oxygen-18) into an analyte of interest, resulting in a mass change detectable by mass spectrometry. The peptide mapping method described herein mitigates (including eliminates) the problem of different peptide ionization efficiencies by labeling available conjugation sites with isotopically labeled cytotoxins to create conjugation site-peptide pairs with identical retention times and minimal mass differences. This method allows for accurate quantification of site-specific conjugation levels and provides the first known example of "bottom-up" DAR characterization in parallel with protein sequence and PTM characterization in one multi-attribute analytical method (MAM).

[0029] In one embodiment, the analytical methods described herein involve isotopically labeling cytotoxins with isotopically labeled water (e.g., H2 18O) to produce an isotopically labeled cytotoxin. In one embodiment, the isotopically labeled water undergoes solvent exchange with a cytotoxin containing a carbonyl group (oxygen with a double bond, e.g., a ketone) to produce an isotopically labeled cytotoxin. This reaction can occur at room temperature or at 37° C. The reaction time can range from 2 days to several weeks. In one embodiment, the reaction time is 7 to 14 days.

[0030] In one embodiment, the cytotoxin is dissolved in an organic solvent, such as acetonitrile (ACN), prior to reaction with isotopically labeled water. 18 In another embodiment, MMAF or MMAE in ACN is reacted with H2O to produce an isotopically labeled cytotoxin. 18 O to generate isotopically labeled MMAF or MMAE. In certain embodiments, cytotoxin is reacted with isotopically labeled water under strong acidic conditions. Acid includes TFA or formic acid. In certain embodiments, the isotopic purity of the labeled molecule can be evaluated by ionizing the isotopically labeled cytotoxin and detecting the mass-to-charge ratio associated with the isotopically labeled cytotoxin.

[0031] In one embodiment, the unoccupied cysteine ​​site of a cysteine-conjugated antibody drug conjugate (ADC) is conjugated to an isotope-labeled cytotoxin.

[0032] In certain embodiments, a reducing agent is used to reduce the ADC interchain disulfide bond to generate free sulfhydryl groups (e.g., unoccupied cysteine ​​sites) prior to conjugation with an isotopically labeled cytotoxin. The free sulfhydryl groups are then available for conjugation with an isotopically labeled cytotoxin. Thus, in one embodiment, the ADC is first reduced with a reducing agent and then conjugated with an isotopically labeled cytotoxin.

[0033] In one embodiment, the reducing agent is any compound or reagent capable of reducing interchain disulfide bonds. In certain embodiments, the reducing agent is dithiothreitol (DTT), 2-mercaptoethanol, and / or tris(2-carboxyethyl)phosphine (TCEP). In further embodiments, the reducing agent is DTT. In another embodiment, the reducing agent is TCEP. Other reducing agents can be used in the methods disclosed herein and are known to those of skill in the art.

[0034] The reducing agent is applied to reduce the interchain disulfide bonds. In one embodiment, the reducing agent is applied in excess to ensure complete reduction of the interchain disulfide bonds, thereby ensuring subsequent labeling at most, if not all, disulfide bond sites. Methods for optimizing the amount of reducing agent are known to those skilled in the art. A concentration of reducing agent can be added in increasing amounts until the intrachain disulfide bonds are reduced, which can be detected by measuring the separated heavy and light chains after the reduction reaction. If complete reduction of the interchain disulfide bonds does not occur, some disulfide bonds will not be labeled with the cytotoxin (natural or isotopically labeled cytotoxin), which may result in an artificially higher quantification of the natural cytotoxin.

[0035] In one embodiment, excess reducing agent is removed prior to conjugation. In certain embodiments, excess reducing agent needs to be removed prior to conjugation because it may interfere with subsequent conjugation steps. For example, in one embodiment, excess reducing agent is removed by eluting the sample from a size exclusion chromatography column. In another embodiment, excess reducing agent is removed by a molecular weight cut-off (MWCO) filter. In a further embodiment, excess reducing agent is removed prior to conjugation with an isotope-labeled cytotoxin.

[0036] In one embodiment, conjugation occurs by reacting the ADC with an isotopically labeled cytotoxin. The reaction can occur, for example, by mixing the ADC and the isotopically labeled cytotoxin at room temperature or about 37° C. The reaction time can be optimized. In one embodiment, the reaction time is 5 minutes to about 60 minutes. In one embodiment, the isotopically labeled cytotoxin is an isotopically labeled MMAF or MMAE. In one embodiment, the ratio of ADC to labeled cytotoxin is optimized to ensure that there are no disulfide bonds without the cytotoxin (natural cytotoxin or isotopically labeled cytotoxin), e.g., all resulting ADCs should have a drug loading of 8 (DL8). Various methods for testing the drug loading of an ADC are known to those skilled in the art.

[0037] In one embodiment, excess isotopically labeled cytotoxin (not conjugated to an antibody) is removed prior to peptide mapping. In one embodiment, excess isotopically labeled cytotoxin (not conjugated to an antibody) is removed prior to peptide mapping by eluting the sample from a size exclusion chromatography column.

[0038] In one embodiment, after conjugation, an ADC sample is obtained having a mixture of isotopically labeled and non-isotopically labeled cytotoxins (eg, "native cytotoxins").

[0039] In another embodiment, after conjugation with an isotope-labeled cytotoxin, peptide mapping of the ADC sample (e.g., peptide mapping by LC-MS / MS analysis) is performed. This step involves denaturation of the isotope-labeled antibody-drug conjugate, reduction of all remaining disulfide bonds, alkylation of the resulting free sulfhydryls, enzymatic digestion of the sample, and analysis of the sample by mass spectrometry. Various peptide mapping methods are well known to those skilled in the art and are described, for example, in Analytical Biochemistry, vol. 266, pp. 31-47 (1999). In one embodiment, the denaturant may include, for example, guanidine HCl, urea, or any denaturant that opens up all inter- and intrachain disulfide bonds for subsequent reduction. Examples of reducing agents include TCEP and DTT. After reduction, an alkylating agent can be applied to the sample to ensure that disulfide bonds are not reformed. An exemplary alkylating agent includes sodium iodoacetate. In certain embodiments, the denaturant remaining before enzymatic digestion can be removed, for example, by size exclusion chromatography, before adding enzyme. In certain embodiments, the peptides of the sample are enzymatically digested. Exemplary enzymes include trypsin or Lys-C. The enzymatic digestion of the sample can be quenched by adding a strong acid, such as HCl or TFA. In some embodiments, the resulting peptides are then ionized, and the mass-to-charge ratios associated with natural (not isotope-labeled) and isotope-labeled conjugate peptides are detected and compared.

[0040] Antibody-drug conjugates The analytical methods described herein are particularly well suited for analyzing ADCs that contain a cytotoxin that contains a carbonyl group (e.g., MMAF or MMAE). In one embodiment, the ADC is an anti-BCMA ADC. In a further embodiment, the anti-BCMA ADC is belantamab mafodotin. Belantamab mafodotin comprises an anti-BCMA antibody linked to an MMAF cytotoxic agent by a maleimidocaproyl (MC) linker.

[0041] The present disclosure also provides compositions comprising anti-BCMA antibody drug conjugates (ADCs) and related methods for treating BCMA-mediated diseases or disorders. Compositions comprising the anti-BCMA ADCs described herein may also be referred to as populations of the anti-BCMA ADCs described herein, and these terms are understood to be interchangeable.

[0042] In one embodiment, the anti-BCMA antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and / or a CDRL3 comprising the amino acid sequence of SEQ ID NO:6.

[0043] In another embodiment, the anti-BCMA antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7; and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:8.

[0044] In yet another embodiment, the anti-BCMA antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:9; and / or a light chain (LC) comprising the amino acid sequence of SEQ ID NO:10.

[0045] In one aspect of the disclosure, the analytical method described herein can determine the location of specific amino acid residues of a cytotoxic agent on an antibody (e.g., belantamab) as well as the quantitative amount of cytotoxin at each amino acid residue. In one embodiment, the cytotoxin is conjugated to a cysteine-containing amino acid, e.g., light chain (LC) C214, heavy chain (HC) C224, heavy chain hinge region (HC hinge) C230, and / or heavy chain hinge region (HC hinge) C233. In one embodiment, the heavy chain hinge region contains two cytotoxin molecules at both C230 and C233. This may be referred to herein as "HC hinge DL2". In another embodiment, the heavy chain hinge region contains one cytotoxin molecule at either C230 or C233. This may be referred to herein as "HC hinge DL1". The method described herein can distinguish between HC hinge DL2 and HC hinge DL1 isoforms. If the HC-hinge-DL1 isoform is detected, the methods described herein can determine the presence or absence of the HC-hinge-DL1 isoform.

[0046] In one embodiment, the anti-BCMA antibody is belantamab comprising the heavy chain sequence of SEQ ID NO:9 (CDRs are underlined; HC C224, HC C230 and HC C233 are bolded / underlined).

[0047] [ka]

[0048] In one embodiment, the anti-BCMA antibody is belantamab comprising the light chain sequence of SEQ ID NO: 10 (CDRs underlined; LC C214 in bold / underlined).

[0049] [ka]

[0050] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 2.1; and Compositions are provided having a drug loading percentage at LC C214 of about 38% to about 44%, and / or a drug loading percentage at HC C224 of about 40% to about 46%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 5% to about 9%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 3% to about 7%.

[0051] Thus, according to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody comprises a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10; and the cytotoxic agent is MMAF or MMAE (particularly MMAF), and an average drug-antibody ratio (DAR) of about 2.1; and Compositions are provided in which the drug loading percentage at LC C214 is about 41%, and / or the drug loading percentage at HC C224 is about 43%, and / or the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 7%, and / or the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 5%.

[0052] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3.0; and Compositions are provided having a drug loading percentage at LC C214 of about 53% to about 59%, and / or a drug loading percentage at HC C224 of about 55% to about 61%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 13% to about 19%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 8% to about 14%.

[0053] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody comprises a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10; and the cytotoxic agent is MMAF or MMAE (particularly MMAF), and an average drug-antibody ratio (DAR) of about 3.0; and Compositions are provided in which the drug loading percentage at LC C214 is about 56%, and / or the drug loading percentage at HC C224 is about 58%, and / or the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 16%, and / or the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 11%.

[0054] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3.5; and Compositions are provided having a drug loading percentage at LC C214 of about 60% to about 66%, and / or a drug loading percentage at HC C224 of about 62% to about 68%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 20% to about 26%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 8% to about 14%.

[0055] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody comprises a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10; and the cytotoxic agent is MMAF or MMAE (particularly MMAF), and an average drug-antibody ratio (DAR) of about 3.5; and Compositions are provided in which the drug loading percentage at LC C214 is about 63%, and / or the drug loading percentage at HC C224 is about 65%, and / or the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 23%, and / or the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 11%.

[0056] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 4.0; and Compositions are provided having a drug loading percentage at LC C214 of about 65% to about 71%, and / or a drug loading percentage at HC C224 of about 68% to about 74%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 24% to about 30%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 12% to about 18%.

[0057] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody comprises a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10; and the cytotoxic agent is MMAF or MMAE (particularly MMAF), and an average drug-antibody ratio (DAR) of about 4.0; and Compositions are provided in which the drug loading percentage at LC C214 is about 68%, and / or the drug loading percentage at HC C224 is about 71%, and / or the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 27%, and / or the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 15%.

[0058] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-to-antibody ratio (DAR) of about 4.6; and Compositions are provided having a drug loading percentage at LC C214 of about 72% to about 78%, and / or a drug loading percentage at HC C224 of about 73% to about 79%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 37% to about 43%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 13% to about 19%.

[0059] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody comprises a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10; and the cytotoxic agent is MMAF or MMAE (particularly MMAF), and an average drug-to-antibody ratio (DAR) of about 4.6; and Compositions are provided in which the drug loading percentage at LC C214 is about 75%, and / or the drug loading percentage at HC C224 is about 76%, and / or the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 40%, and / or the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 16%.

[0060] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody (e.g., belantamab) comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 5.0; and Compositions are provided having a drug loading percentage at LC C214 of about 75% to about 81%, and / or a drug loading percentage at HC C224 of about 77% to about 83%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 43% to about 49%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 11% to about 17%.

[0061] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody comprises a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10; and the cytotoxic agent is MMAF or MMAE (particularly MMAF), and an average drug-antibody ratio (DAR) of about 5.0; and Compositions are provided in which the drug loading percentage at LC C214 is about 78%, and / or the drug loading percentage at HC C224 is about 80%, and / or the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 46%, and / or the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 14%.

[0062] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 5.7; and Compositions are provided having a drug loading percentage at LC C214 of about 81% to about 87%, and / or a drug loading percentage at HC C224 of about 82% to about 88%, and / or a drug loading percentage at HC hinge DL2 at HC C230 and HC C233 of about 56% to about 61%, and / or a drug loading percentage at HC hinge DL1 at HC C230 or HC C233 of about 10% to about 16%.

[0063] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: The antibody comprises a heavy chain amino acid sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:10; and the cytotoxic agent is MMAF or MMAE (particularly MMAF), and an average drug-antibody ratio (DAR) of about 5.7; and Compositions are provided in which the drug loading percentage at LC C214 is about 84%, and / or the drug loading percentage at HC C224 is about 85%, and / or the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 58%, and / or the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 13%.

[0064] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided having a drug loading percentage in LC C214 of about 56% to about 80%.

[0065] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided having a drug loading percentage in HC C224 of about 58% to about 81%.

[0066] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided in which the percentage of drug loading of the HC hinge DL2 in HC C230 and HC C233 is about 15% to about 46%.

[0067] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided in which the percentage of drug loading of HC hinge DL1 in HC C230 or HC C233 is about 11% to about 15%.

[0068] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided in which the drug loading percentage at LC C214 is about 56% to about 80%, the drug loading percentage at HC C224 is about 58% to about 81%, the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 15% to about 46%, and the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 11% to about 15%.

[0069] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3 to about 5; and Compositions are provided having a drug loading percentage in LC C214 of about 56% to about 80%.

[0070] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3 to about 5; and Compositions are provided having a drug loading percentage in HC C224 of about 58% to about 81%.

[0071] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3 to about 5; and Compositions are provided in which the percentage of drug loading of the HC hinge DL2 in HC C230 and HC C233 is about 15% to about 46%.

[0072] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3 to about 5; and Compositions are provided in which the percentage of drug loading of HC hinge DL1 in HC C230 or HC C233 is about 11% to about 15%.

[0073] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3 to about 5; and Compositions are provided in which the drug loading percentage at LC C214 is about 56% to about 80%, the drug loading percentage at HC C224 is about 58% to about 81%, the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 15% to about 46%, and the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 11% to about 15%.

[0074] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided having a drug loading percentage in LC C214 of about 63% to about 76%.

[0075] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided having a drug loading percentage in HC C224 of about 65% to about 78%.

[0076] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided in which the percentage of drug loading of the HC hinge DL2 in HC C230 and HC C233 is about 22% to about 40%.

[0077] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided, wherein the percentage of drug loading of HC hinge DL1 in HC C230 or HC C233 is about 11% to about 16%.

[0078] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and Compositions are provided in which the drug loading percentage at LC C214 is about 63% to about 76%, the drug loading percentage at HC C224 is about 65% to about 78%, the drug loading percentage at C-hinge DL2 at HC C230 and HC C233 is about 22% to about 40%, and the drug loading percentage at HC-hinge DL1 at HC C230 or HC C233 is about 11% to about 16%.

[0079] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3.5 to about 4.6; and Compositions are provided having a drug loading percentage in LC C214 of about 63% to about 76%.

[0080] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3.5 to about 4.6; and Compositions are provided having a drug loading percentage in HC C224 of about 65% to about 78%.

[0081] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3.5 to about 4.6; and Compositions are provided in which the percentage of drug loading of the HC hinge DL2 in HC C230 and HC C233 is about 22% to about 40%.

[0082] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3.5 to about 4.6; and Compositions are provided, wherein the percentage of drug loading of HC hinge DL1 in HC C230 or HC C233 is about 11% to about 16%.

[0083] According to a further aspect of the present disclosure, there is provided a composition comprising an anti-BCMA antibody (e.g., belantamab) conjugated to a cytotoxic agent, forming an antibody drug conjugate (ADC), comprising: the antibody comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:1; a CDRH2 comprising the amino acid sequence of SEQ ID NO:2; a CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a CDRL1 comprising the amino acid sequence of SEQ ID NO:4; a CDRL2 comprising the amino acid sequence of SEQ ID NO:5; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:6; and the cytotoxic agent is MMAF or MMAE, and an average drug-antibody ratio (DAR) of about 3.5 to about 4.6; and Compositions are provided in which the drug loading percentage at LC C214 is about 63% to about 76%, the drug loading percentage at HC C224 is about 65% to about 78%, the drug loading percentage at HC hinge DL2 at HC C230 and HC C233 is about 22% to about 40%, and the drug loading percentage at HC hinge DL1 at HC C230 or HC C233 is about 11% to about 16%.

[0084] The anti-BCMA ADCs in the compositions described herein may be useful for the treatment and / or prevention of a variety of BCMA-mediated diseases (including, for example, B-cell mediated cancers such as lymphoma and multiple myeloma). The anti-BCMA ADCs described herein may bind to human BCMA (e.g., human BCMA comprising the amino acid sequence of GenBank Accession No. Q02223.2) or a BCMA protein having at least 90% amino acid sequence homology or at least 90% amino acid sequence identity thereto.

[0085] Anti-BCMA ADCs include anti-BCMA antigen binding proteins. The term "antigen binding protein" as used herein refers to antibodies, antibody fragments and other protein constructs capable of binding to antigens, such as anti-BCMA antigen binding proteins capable of binding to BCMA (e.g., human BCMA). Antigen binding proteins can include heavy and light chain variable regions of the present disclosure that can be formatted into the structure of a natural antibody or a functional fragment or equivalent thereof. Thus, antigen binding proteins include the VFVs of the present disclosure that, when combined with an appropriate light chain, are formatted into a full-length antibody, (Fab')2 fragment, Fab fragment or equivalent thereof (e.g., scFV, bibody, tribody or tetrabody, Tandab, etc.). H The antibody may be an IgG1, IgG2, IgG3 or IgG4, IgM, IgA, IgE or IgD or modified variants thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. Furthermore, the antigen binding protein may include all classes of modifications, such as IgG dimers, Fc variants that no longer bind Fc receptors or Fc variants that no longer mediate C1q binding. The antigen binding protein may also be a chimeric antibody of the type described in WO 86 / 01533, which includes an antigen binding region and a non-immunoglobulin region.

[0086] The antigen binding protein can be either a dAb, Fab, Fab', F(ab')2, Fv, diabody, triabody, tetrabody, miniantibody or minibody. The antigen binding protein can be either a fully human antibody, a humanized antibody or a chimeric antibody. The antigen binding protein can be a humanized antibody. The antigen binding protein can be a monoclonal antibody.

[0087] Exemplary anti-BCMA antigen binding proteins and methods of making the same are disclosed in WO 2012 / 163805, which is incorporated by reference in its entirety. Further exemplary anti-BCMA antigen binding proteins include those disclosed in WO 2016 / 014789, WO 2016 / 090320, WO 2016 / 090327, WO 2016 / 020332, WO 2016 / 079177, WO 2014 / 122143, WO 2014 / 122144, WO 2017 / 021450, WO 2016 / 014565, WO 2014 / 068079, WO 2015 / 166649 Nos. 2015 / 158671, 2015 / 052536, 2014 / 140248, 2013 / 072415, 2013 / 072406, 2014 / 089335, U.S. Patent Publication No. 2017 / 165373, WO 2013 / 154760, and WO 2017 / 051068, each of which is incorporated by reference in its entirety.

[0088] In another embodiment, the anti-BCMA antigen binding proteins described herein may inhibit binding of BAFF and / or APRIL to the BCMA receptor. In another embodiment, the anti-BCMA antigen binding proteins described herein may bind to FcγRIIIA or exhibit effector functions mediated by FcγRIIIA.

[0089] Anti-BCMA antigen binding proteins may include antibodies ("anti-BCMA antibodies"). The term "antibody" as used herein refers to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD or IgE) and may include monoclonal, recombinant, polyclonal, chimeric, human and humanized molecules of this type. Monoclonal antibodies may be produced by eukaryotic or prokaryotic close cells expressing the antibody. Monoclonal antibodies may also be produced by eukaryotic cell lines that may recombinantly express the antibody heavy and light chains by having nucleic acid sequences encoding the antibody heavy and light chains introduced into the cell. Exemplary methods for producing antibodies from various 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 of skill in the art.

[0090] Antibodies may be derived, for example, from rat, mouse, primate (e.g., cynomolgus monkey, Old World monkey or great ape), human, or from other sources (e.g., nucleic acids encoding the antibody molecule generated using molecular biology techniques well known to those of skill in the art).

[0091] The antibody may comprise a constant region and may be of any isotype or subclass. The constant region may be of an IgG isotype, such as IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0092] The antigen binding protein may contain one or more modifications, e.g., where the antigen binding protein is an antibody, constant domains mutated to enhance antibody effector function / ADCC and / or complement activation.

[0093] Anti-BCMA antibodies have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function. The term "effector function" as used herein is meant to refer to 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 the FcRn receptor. In the case of IgG antibodies, effector functions that may include ADCC and ADCP may be mediated by interaction of the heavy chain constant region with the family of Fcγ receptors present on the surface of immune cells. In humans, these may include FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Interaction between antigen-bound antigen-binding proteins and Fc / Fcγ complex formation may induce various effects, such as cytotoxicity, immune cell activation, phagocytosis and / or release of inflammatory cytokines.

[0094] The anti-BCMA antibody may inhibit the binding of BAFF and / or APRIL to the BCMA receptor. The anti-BCMA antibody may bind to FcγRIIIA or exhibit FcγRIIIA-mediated effector function.

[0095] An anti-BCMA antibody may comprise two immunoglobulin (Ig) heavy chains ("HC") and two Ig light chains ("LC"). The basic antibody structural unit may comprise, for example, a tetramer of subunits. Each tetramer may comprise two pairs of polypeptide chains, each pair may comprise one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain may comprise a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. This variable region may be expressed initially linked to a cleavable signal peptide. The variable region without the signal peptide is referred to as the mature variable region. Thus, in one example, the light chain mature variable region may comprise the light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain may define a constant region. The heavy chain constant region may be primarily responsible for effector function.

[0096] The mature variable regions of each light / heavy chain pair can form an antibody binding site (also called an antigen-binding site). An "antigen-binding site" refers to the site on an antibody that can specifically bind to an antigen, which can be a single variable domain or a paired V domain as found in a standard antibody. H / V L The domains may be FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Thus, an intact antibody may have, for example, two binding sites. Except in the case of bifunctional or bispecific antibodies, these two binding sites may be identical. These chains may all exhibit the same general structure of relatively conserved framework regions (FR) bound by three hypervariable regions, also called complementarity determining regions or "CDRs". The CDRs from the two chains of each pair are aligned by the framework regions, allowing binding to a specific epitope. Thus, in one example, both light and heavy chains comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0097] "CDR" is defined as the amino acid sequence of the complementarity determining region of an antibody. These are the hypervariable regions of the immunoglobulin heavy and light chains. There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs. In one embodiment, the composition comprises an anti-BCMA antibody comprising one or more CDRs as described herein, or one or both of the heavy chain variable domains or light chain variable domains as described herein.

[0098] The terms "variant", "antibody variant", "CDR variant" and "post-translationally modified variant" refer to at least one amino acid change in the antibody sequence. The variant may be the result of a post-translational modification, a chemical change or a sequence change by at least one deletion, substitution or addition. Some post-translational modifications result in a chemical change that does not change the sequence (e.g., Met to oxidized Met, or Asp to isomerization / iso-Asp, or aggregation), while other post-translational modifications result in a sequence change, for example, conversion of one amino acid residue to another amino acid residue (e.g., conversion of Asn to Asp by deamidation, or deletion of lysine). Further post-translationally modified variants are described below. The variant antibody sequence containing a sequence change may be the result of a designed sequence change or a post-translational modification. The sequence change of an amino acid may be a deletion, substitution or addition.

[0099] In one such embodiment, the substitution is a conservative substitution. In another embodiment, the antibody variant may contain at least one substitution and retain the canonical structure of the antigen-binding protein. In one embodiment, the antibody variant is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of the parent antibody (i.e., has amino acid sequence identity with the amino acid sequence of the parent antibody). In another embodiment, the antibody variant comprises a heavy chain amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least 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%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO:10.

[0100] The antigen binding protein may have amino acid modifications (e.g., amino acid substitutions) that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life (e.g., serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules has many benefits, including reducing the dosage and / or frequency of administration of these molecules. In one embodiment, the antigen binding protein of the present disclosure comprises all or a portion of an IgG constant domain (FcRn binding portion) with one or more of the following amino acid modifications:

[0101] For example, with respect to IgG1, the M252Y / S254T / T256E (commonly referred to as “YTE”) and / or M428L / N434S (commonly referred to as “LS”) modifications increase FcRn binding at pH 6.0 ( Wang et al., 2018 ).

[0102] Half-life can also be extended by the T250Q / M428L, V259I / V308F / M428L, N434A and T307A / E380A / N434A modifications (see IgG1 and Kabat numbering) (Monnet et al.).

[0103] Half-life and FcRn binding can also be extended by introducing H433K and N434F modifications (commonly referred to as "HN" or "NHance") (see IgG1) (WO 2006 / 130834).

[0104] WO 00 / 42072 discloses polypeptides comprising a variant Fc region with altered FcRn binding affinity, the polypeptides comprising any one or more amino acid modifications at 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.

[0105] WO 02 / 060919 discloses modified IgGs comprising an IgG constant domain which comprises one or more amino acid modifications compared to a wild-type IgG constant domain, which modified IgGs exhibit an increased half-life compared to the half-life of an IgG having a wild-type IgG constant domain, and wherein the one or more amino acid modifications are present at one or more of 251, 253, 255, 285-290, 308-314, 385-389 and 428-435.

[0106] Shields et al. (2001, J Biol Chem, vol. 276, pp. 6591-604) used alanine scanning mutagenesis to alter residues in the Fc region of a human IgG1 antibody and then evaluated binding to human FcRn. Positions that effectively inhibit binding to FcRn when changed to alanine include I253, S254, H435, and Y436. The following other positions did not show a significant decrease in binding: E233-G236, R255, K288, L309, S415, and H433. Several amino acid positions showed improved FcRn binding when changed to alanine, notable among these are P238, T256, E272, V305, T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Many other amino acid positions showed either slight improvement in FcRn binding (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, K321, K322, K323, K324, K325, K326, K327, K328, K329, K330, K331, K332, K333, K334, K335, K336, K337, K338, K339, K340, K341, K342, K343, K344, K345, K346, K347, K348, K349, K350, K351, K352, K353, K354, K355, K356, K357, K358, K360, K361, K362, K363, K364, K365, K366, K367, K368, K369, K370, K371, K372, K373, K374, K375, K376, K377, K378, K379, K380, K382, K383, K 22, S324, 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).

[0107] The most significant effect in terms of improved FcRn binding was seen in the combination variants. At pH 6.0, the E380A / N434A variant bound to FcRn more than 8-fold better than native IgG1, compared to 2-fold for E380A and 3.5-fold for N434A. Addition of T307A to this improved binding by 12-fold compared to native IgG1. In one embodiment, an antigen binding protein of the present disclosure comprises E380A / N434A substitutions and exhibits increased binding to FcRn.

[0108] Dall'Acqua et al. (2002, J Immunol. 169:5171-80) describe random mutagenesis and screening of a phage display library of human IgG1 hinge Fc fragments against mouse FcRn. The authors disclose random mutagenesis of positions 251, 252, 254-256, 308, 309, 311, 312, 314, 385-387, 389, 428, 433, 434 and 436. The main improvement in IgG1-human FcRn complex stability occurred when substituting residues located in a band along the Fc-FcRn interface (M252, S254, T256, H433, N434 and Y436), with lesser improvements observed for substitutions of periphery residues, e.g., 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, which showed 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.

[0109] The antigen binding protein may exhibit optimal binding to FcRn. Thus, the antigen binding protein may comprise at least one amino acid modification in the Fc region of said antigen binding protein, said modification comprising 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344 70, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 3 45, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 4 and 447 (numbering according to the EU index as in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991))).

[0110] Furthermore, various publications describe methods to obtain bioactive molecules with altered half-lives, either by introducing FcRn-binding polypeptides into the molecules (WO 97 / 43316, US Patent Publication Nos. 5,869,046, 5,747,035, WO 96 / 32478 and 91 / 14438) or by fusing them to antibodies with preserved FcRn-binding affinity but significantly reduced affinity for other Fc receptors (WO 99 / 43713) or with the FcRn-binding domain of an antibody (WO 00 / 09560, US Patent Publication No. 4,703,039).

[0111] Fc variants with enhanced FcRn affinity to improve both antibody cytotoxicity and half-life were identified in a screen at pH 6.0. Selected IgG variants can be made as hypofucosylated molecules. The resulting variants show enhanced serum persistence in hFcRn mice as well as preserved and enhanced ADCC (Monnet et al.). Exemplary variants include (see IgG1 and numbering according to the EU index of Kabat et al.): P230T / V303A / K322R / N389T / F404L / N434S; P228R / N434S; Q311R / K334R / Q342E / N434Y; C226G / Q386R / N434Y; T307P / N389T / N434Y; P230S / N434S; P230T / V305A / T307A / A378V / L398P / N434S; P230T / P387S / N434S; P230Q / E269D / N434S; N276S / A378V / N434S; T307A / N315D / A330V / 382V / N389T / N434Y; T256N / A378V / S383N / N434Y; N315D / A330V / N361D / A387V / N434Y; V259I / N315D / M428L / N434Y; P230S / N315D / M428L / N434Y; F241L / V264E / T307P / A378V / H433R; T250A / N389K / N434Y; V305A / N315D / A330V / P395A / N434Y; V264E / Q386R / P396L / N434S / K439R; E294del / T307P / N434Y ("del" indicates deletion).

[0112] Also described are methods for producing the antigen binding proteins described herein, comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising an isolated nucleic acid described herein, wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase is inactivated in the recombinant host cell; and b) recovering the antigen binding protein. Such methods for producing antigen binding proteins can be performed, for example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity over the same monoclonal antibodies produced in cells with a functional FUT8 gene. Embodiments of the POTELLIGENT technology system are described in U.S. Patent Publication Nos. 7,214,775, 6,946,292, WO 0061,739, and WO 0231,240, all of which are incorporated herein by reference. Those of skill in the art will also recognize other suitable systems and methods for generating antigen binding proteins, e.g., antibodies.

[0113] The antibody can be collected and purified by conventional protein purification procedures. For example, the antibody can be collected directly from the culture medium. The cell culture medium can be collected by clarification, for example, by centrifugation and / or depth filtration. After collecting the antibody, purification is performed to ensure sufficient purity. Thus, a cell culture medium comprising the antibody described herein is also described. In one embodiment, the cell culture medium comprises CHO cells.

[0114] The antibody can then be purified from the cell culture medium. This can include harvesting the cell culture supernatant, contacting the cell culture supernatant with a purification medium (e.g., a protein A resin or a protein G resin for binding the antibody molecules), and eluting the antibody molecules from the purification medium to produce an eluate. Thus, in one embodiment, an eluate is provided that comprises the antibody described herein.

[0115] Purification may involve one or more chromatography steps, for example, one or more chromatography resins and / or one or more filtration steps. For example, affinity chromatography using a resin, for example, Protein A, G, or L, may be used to purify the composition. Alternatively, or in addition, a cation exchange resin, for example, a cation exchanger, may be used to purify the composition.

[0116] Alternatively, the purification steps include an affinity chromatography step followed by a cation exchange resin step.

[0117] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region CDR1 ("CDRH1") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the heavy chain variable region CDR1 ("CDRH1") comprises an amino acid sequence having one amino acid mutation ("variant") relative to the amino acid sequence set forth in SEQ ID NO: 1.

[0118] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region CDR2 ("CDRH2") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the heavy chain variable region CDR2 ("CDRH2") comprises an amino acid sequence having one amino acid mutation ("variant") relative to the amino acid sequence set forth in SEQ ID NO:2.

[0119] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region CDR3 ("CDRH3") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the heavy chain variable region CDR3 ("CDRH3") comprises an amino acid sequence having one amino acid mutation ("variant") relative to the amino acid sequence set forth in SEQ ID NO:3.

[0120] In one embodiment, the anti-BCMA antibody comprises a light chain variable region CDR1 ("CDRL1") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the light chain variable region CDL1 ("CDR1") comprises an amino acid sequence having one amino acid mutation ("variant") relative to the amino acid sequence set forth in SEQ ID NO: 4.

[0121] In one embodiment, the anti-BCMA antibody comprises a light chain variable region CDR2 ("CDRL2") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In one embodiment, the light chain variable region CDL2 ("CDR2") comprises an amino acid sequence having one amino acid mutation ("variant") relative to the amino acid sequence set forth in SEQ ID NO:5.

[0122] In one embodiment, the anti-BCMA antibody comprises a light chain variable region CDR3 ("CDRL3") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the light chain variable region CDL3 ("CDR3") comprises an amino acid sequence having one amino acid mutation ("variant") relative to the amino acid sequence set forth in SEQ ID NO: 6.

[0123] In one embodiment, the anti-BCMA antibody is a CDRH1 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; CDRH2 comprising an amino acid sequence having 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; CDRH3 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; RH3; CDRL1 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; a CDRL2 comprising an amino acid sequence having at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6; and / or a CDRL3 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

[0124] In one embodiment, the anti-BCMA antibody comprises a heavy chain variable region ("V") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7. H ").

[0125] In one embodiment, the anti-BCMA antibody comprises a light chain variable region ("V") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. L ").

[0126] In one embodiment, the anti-BCMA antibody comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7. H and V comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. L and the anti-BCMA antibody retains binding to BCMA.

[0127] In one embodiment, the anti-BCMA antibody comprises a heavy chain region ("HC") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9.

[0128] In one embodiment, the anti-BCMA antibody comprises a light chain region ("LC") comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:10.

[0129] In one embodiment, the anti-BCMA antibody comprises a HC comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; and a LC comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:10.

[0130] The "sequence identity" between a query amino acid sequence and a subject amino acid sequence is the "identity" value expressed as a percentage calculated by the BLASTP algorithm when the subject amino acid sequence has 100% query coverage with the query amino acid sequence after a pairwise BLASTP alignment is performed. Such pairwise BLASTP alignment between a query amino acid sequence and a subject amino acid sequence is performed using the default settings of the BLASTP algorithm available at the National Center for Biotechnology Institute website with the low complexity region filter turned off. Importantly, the query sequence may be described by an amino acid sequence identified in one or more claims herein.

[0131] In one embodiment, the anti-BCMA antibody comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO:1; a CDRH2 having the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 having the amino acid sequence set forth in SEQ ID NO:3; a CDRL1 having the amino acid sequence set forth in SEQ ID NO:4; a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5; and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:6.

[0132] In one embodiment, the anti-BCMA antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L Includes.

[0133] In one embodiment, the anti-BCMA antibody is belantamab, which comprises a HC having the amino acid sequence set forth in SEQ ID NO:9, and a LC having the amino acid sequence set forth in SEQ ID NO:10.

[0134] The sequence of an antibody can be determined by the Kabat numbering system (Kabat et al., Sequences of proteins of Immunological Interest NIH, 1987). Alternatively, it can be determined using the Chohtia numbering system (Al-Lazikani et al., 1997, JMB, vol. 273, pp. 927-948), the contact definition method (MacCallum RM, Martin ACR and Thornton JM, 1996, Journal of Molecular Biology, vol. 262, no. 5, pp. 732-745) or any other established method for numbering the residues of an antibody to determine CDRs known to those skilled in the art. Other numbering conventions for antibody sequences available to those skilled in the art include the "AbM" method (University of Bath) and the "contact" method (University of London). Finally, the antibody sequence can be numbered consecutively.

[0135] When referring to amino acids described herein numerically, they may be numbered according to the Kabat or sequential numbering system. Numerical references to specific amino acid numbers are described herein in the sequential numbering system unless otherwise specified. Throughout this specification, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" are numbered according to Kabat numbering. Amino acid residues of variable region sequences and full-length antibody sequences are numbered consecutively to represent any antibody sequence variant positions or post-translational modification variant positions, such as isomerized variants (e.g., D103), deamidated variants (e.g., N388) or oxidized variants (e.g., M34).

[0136] Reference to the position of a CDR (e.g., M34 or D103) provides the position number (consecutive numbering) relative to the entire antibody sequence. Thus, M34 of CDRH1 corresponds to the fourth residue of SEQ ID NO:1, i.e., NYW MH (SEQ ID NO:1). Similarly, D103 of CDRH3 refers to the fifth residue of SEQ ID NO:3, i.e., GAIY. D Refers to the underline in GYDVLDN (SEQ ID NO:3).

[0137] In one embodiment, the composition comprises an antibody variant comprising one or more amino acid changes in the primary sequence. In one embodiment, the composition comprises an antibody that is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 and / or the light chain sequence of SEQ ID NO:10, and has an aspartic acid (D) to asparagine (N) amino acid change, e.g., D103N in CDRH3 (e.g., D99N in the Kabat numbering).

[0138] In another embodiment, the composition comprises an antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO:1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO:2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO:3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO:4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:6, wherein the antibody comprises an amino acid change from aspartic acid (D) to asparagine (N), e.g., D103N in CDRH3.

[0139] In another embodiment, the anti-BCMA antibody comprises belantamab and includes an aspartic acid (D) to asparagine (N) amino acid change, e.g., D103N in CDRH3.

[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 amino acid 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.

[0141] In one embodiment, the composition comprises a mixture of antibodies comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO:1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO:2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO:3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO:4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:6, wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, or at least about 90% of the antibodies in the mixture contain D103N in CDRH3.

[0142] In one embodiment, a composition comprises belantamab, wherein about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 50% or more, about 75% or more, or about 90% or more of the belantamab comprises D103N in CDRH3.

[0143] In one embodiment, the composition comprises belantamab, wherein the belantamab comprises at least one antibody variant selected from the group consisting of G27Y, S30T, A93T, A24G, K73T, M48I, V67A, F71Y, D99N, M4L and K45E using the Kabat numbering system.

[0144] Antibody-drug conjugates Antibody-drug conjugates (ADCs) are a new class of potent anticancer agents that have shown remarkable clinical utility in recent years. ADCs consist of a cytotoxic agent chemically attached to an antibody via a linker. ADCs can destroy cancer cells with overexpression of cell surface proteins, presumably through a series of events including antigen binding at the cell surface, endocytosis, trafficking to lysosomes, degradation of the ADC, release of the payload, inhibition of cellular processes (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's lymphoma and systemic anaplastic lymphoma.

[0145] ADCs have been used in the treatment of cancer for localized delivery of cytotoxic agents, e.g., drugs that kill cells or inhibit cell growth or proliferation (Lambert, J., 2005, Curr. Opinion in Pharmacology 5, 543-549; Wu et al., 2005, Nature Biotechnology, vol. 23, no. 9, pp. 1137-1146; Payne, G., 2003, i3, pp. 207-212; Syrigos and Epenetos, 1999, Anticancer Research 19, pp. 605-614; Niculescu-Duvaz and Springer, 1997, Adv. Drug Deliv. Rev. 26, pp. 151-172; U.S. Patent Publication No. 4,975,278). ADCs allow targeted delivery of drug moieties to tumors and their intracellular accumulation there, whereas systemic administration of unconjugated drugs may result in unacceptable levels of toxicity not only to the tumor cells to be eliminated but also to normal cells (Baldwin et al., Lancet, March 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 (eds. A. Pinchera et al.), pp. 475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother., vol. 21, pp. 183-87).Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule 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 (European Patent Publication No. 1391213; Liu et al., 1996, Proc. Natl. Acad. Sci. USA 93:8618-8623) and calicheamicin (Lode et al., 1998, Cancer Res., vol. 58, p. 2928; Hinman et al., 1993, Cancer Res., vol. 53, p. 3336-3342).

[0146] In one embodiment, an anti-BCMA ADC comprises an antibody or antibody fragment conjugated to one or more cytotoxic agents, e.g., a chemotherapeutic drug, a drug growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).

[0147] In one embodiment, an anti-BCMA ADC has the following general structure: ABP-((Linker) n -Ctx) m [In the formula, ABP is an antigen-binding protein, an antibody or an antibody fragment, The linker is absent or any cleavable or non-cleavable linker; Ctx is any cytotoxic agent described herein; n is 0, 1, 2 or 3, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. has.

[0148] In exemplary embodiments, enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitgellin, restrictocin, phenomycin, enomycin or trichothecenes. See, e.g., International Publication WO 93 / 21232, published Oct. 28, 1993. Various radionuclides, e.g., 211 At, 212 Bi, 131 I, 131 In, 90 Y, or 186 Re is available for the production of radioconjugated antibodies.

[0149] The anti-BCMA antibodies or fragments thereof of the present disclosure may be conjugated to one or more cytotoxic agents, such as, but not limited to, calicheamicins, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, or derivatives of these toxins that have toxin activity. Suitable cytotoxic agents include, for example, austatins (including monomethylauristatin F (MMAF) and monomethylauristatin E (MMAE) and ester forms of MMAE), DNA minor groove binders, DNA minor groove alkylating agents, enediynes, lexitropsins, duocarmycins, taxanes (including paclitaxel and docetaxel), puromycins, dolastatins, maytansinoids, and vinca alkaloids. Particular cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, DM-1, DM-4, ​​netropsin. Other suitable cytotoxic agents include antitubulin agents, such as auristatins, vinca alkaloids, podophyllotoxins, taxanes, baccatin derivatives, cryptophycins, maytansinoids, combretastatins or dolastatins. Antitubulin agents include dimethylvaline-valinedolaisoloine-dolaproine-phenylalanine-p-phenylene-diamine (AFP), vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-1, DM-4, ​​or eleutherobin.

[0150] In one embodiment, an anti-BCMA ADC comprises an anti-BCMA antibody conjugated to MMAE or MMAF.

[0151] [ka]

[0152] Exemplary linkers include cleavable linkers and non-cleavable linkers. Cleavable linkers can be susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include peptide linkers that can be cleaved by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) linker or a phenylalanine-lysine (phe-lys) linker. Other suitable linkers include linkers that are hydrolyzable at a pH lower than 5.5, such as hydrazone linkers. Other suitable cleavable linkers include, for example, disulfide linkers. Exemplary linkers include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

[0153] In one embodiment, the linker may comprise a thiol-reactive maleimide, a caproyl spacer, the dipeptide valine-5 citrulline, p-aminobenzyloxycarbonyl, a self-immolative fragmenting group, or a protease-resistant maleimidocaproyl.

[0154] In another embodiment, an anti-BCMA ADC comprises an anti-BCMA antibody linked to MMAE or MMAF by an MC linker as shown in the structure below:

[0155] [ka]

[0156] The anti-BCMA ADCs described herein may comprise any of the anti-BCMA antibodies described herein together with any of the cytotoxic agents described herein.

[0157] In one embodiment, the anti-BCMA ADC comprises a CDRH1 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; CDRH2 comprising an amino acid sequence having at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; CDRH3 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; CDRL1 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; and / or a CDRL3 comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6, wherein the anti-BCMA antibody is conjugated to MMAE or MMAF.

[0158] In yet another embodiment, an anti-BCMA ADC comprises an anti-BCMA antibody comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO:1; a CDRH2 having the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 having the amino acid sequence set forth in SEQ ID NO:3; a CDRL1 having the amino acid sequence set forth in SEQ ID NO:4; a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5; and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:6, and is conjugated to MMAF or MMAE.

[0159] In one embodiment, the anti-BCMA ADC comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7. H and / or a V that comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. L and conjugated to MMAE or MMAF.

[0160] In yet another embodiment, the anti-BCMA ADC has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence set forth in SEQ ID NO:8. L and is conjugated to MMAF or MMAE.

[0161] In one embodiment, an anti-BCMA ADC comprises an anti-BCMA antibody comprising an HC comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; and / or a LC comprising an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:10, and is conjugated to MMAF or MMAE.

[0162] In yet another embodiment, the anti-BCMA ADC is belantamab mafodotin comprising an anti-BCMA antibody comprising a HC having the amino acid sequence set forth in SEQ ID NO: 9, and a LC having the amino acid sequence set forth in SEQ ID NO: 10, and is conjugated to MMAF.

[0163] Preparation and characterization of ADCs A particular native IgG1 molecule contains 16 disulfide bonds (32 cysteine ​​or sulfhydryl groups). In certain embodiments, the antibody is reduced in such a way that only four interchain disulfide bonds are reduced and can be conjugated with a cytotoxic agent, resulting in up to eight binding sites for the cytotoxic agent. In other words, the drug loading ("DL") number, i.e., the number of cytotoxic agents per antibody molecule, can range from 0 to 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.

[0164] The conjugation process may result in heterogeneity of drug-antibody binding for a given ADC composition, with both 1) the number of drugs attached to each antibody molecule and 2) the location of the cytotoxic agent being different. This results in an ADC composition with a variety of DL species. Herein, the average drug-antibody ratio across a heterogeneous ADC composition is referred to as the "average DAR" or "DAR". For example, an ADC composition may contain a mixture of antibody species, each with its own DL (some species in the mixture are DL2, some species in the mixture are DL4, some species in the mixture are DL6, and some species in the mixture are DL8), and the average DAR across the composition may be about 4.

[0165] In another embodiment, the term "proportion of DL" may be used to describe the proportion of a particular DL species within a heterogeneous ADC composition (e.g., the proportion of DL2 is about 10% to about 30% of the total heterogeneous ADC composition).

[0166] Drugs can be conjugated to antibodies via sulfhydryl groups on the antibody. The sulfhydryl groups can be sulfhydryl groups on cysteine ​​side chains. The cysteine ​​residues can be naturally occurring in antibodies (e.g., interchain disulfides) or introduced by other means, such as mutagenesis. Methods for conjugating drugs to sulfhydryl groups on antibodies are well known in the art (see, for example, U.S. Pat. Nos. 7,659,241, 7,498,298, and WO 2011 / 130613, 2014 / 152199, 2015 / 077605, and Bioconjugate Chem., 2005, vol. 16, pp. 1282-1290). Antibodies are generally reduced prior to conjugation to make sulfhydryl groups available for conjugation. Antibodies can be reduced using conditions known in the art. Reducing conditions are conditions that generally do not result in subsequent denaturation of the antibody and generally do not affect the antigen-binding affinity of the antibody.

[0167] The reducing agent used in the reduction step can be TCEP, which can be added in excess, for example, at room temperature for 30 minutes. For example, 250 μL of a 10 mM TCEP solution at pH 7.4 will readily reduce the interchain disulfides of 1-100 μg of antibody in 30 minutes at room temperature. However, other reducing agents and conditions can also be used. Examples of reaction conditions include conditions of pH 5-8 and temperatures of 5°C to 37°C.

[0168] There are various methods for calculating the proportion of DL species and / or average DAR of ADC compositions, and are known to those skilled in the art. For example, the heterogeneity of ADCs bound to cysteines is generally measured by hydrophobic interaction chromatography (HIC), which separates DL species based on the number of drugs loaded. LC-MS assays have also been developed to evaluate DL distribution. Exemplary methods for calculating drug loading distribution in ADC compositions can be found, for example, in Journal of Chromatography B 1060, 2017, pp. 182-189.

[0169] For example, DL0 has no drug loading on the antibody. For example, DL2 has a drug loading of 2. In one embodiment, the conjugation sites of DL2 are LC C214 and HC C224. For example, DL4 has a drug loading of 4. In one embodiment, the conjugation sites of DL4a are LC C214, HC C224, LC C214 and HC C224. In one embodiment, the conjugation sites of DL4b are HC C230, HC C233, HC C230 and HC C233. For example, DL6 has a drug loading of 6. In one embodiment, the conjugation sites of DL6 are LC C214, HC C224, HC C230, HC C233, HC C230 and HC C233. For example, DL8 has a drug loading of 8. In one embodiment, the conjugation sites of DL8 are LC C214, HC C224, LC C214, HC C224, HC C230, HC C233, HC C230 and HC C233.

[0170] In one embodiment, the percentage of a particular DL species (e.g., percentage DL0, percentage DL2, percentage DL4a, percentage DL4b, percentage DL6, percentage 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 the sum of all DL species. In one embodiment, the average DAR can be calculated from the area under the curve for each DL species using the following formula:

number

[0171] In one embodiment, the proportion of DAR of a particular subspecies (e.g., the proportion of DL2a in total DL2) is determined by collecting the particular DL species using a combination of analytical techniques, which may include HIC, non-reducing separation methods and mass spectrometry techniques.

[0172] In one embodiment, the average DAR of the anti-BCMA ADC composition is from about 2 to about 7, from about 2 to about 6, from about 2.1 to about 5.7, from about 2.1 to about 5.0, from about 2.1 to about 4.6, from about 2.1 to about 4.1, from about 2.1 to about 3.5, from about 2.1 to about 3.0, from about 3.0 to about 5.7, from about 3.0 to about 5.0, from about 3.0 to about 4.6, from about 3.0 to about 4.1, from about 3.0 to about 3.5, from about 3.5 to about 5 .7, about 3.5 to about 5.0, about 3.5 to about 4.6, about 3.5 to about 4.1, about 3.8 to about 4.5, about 4.1 to about 5.7, about 4.1 to about 5.0, about 4.1 to about 4.6, about 4.6 to about 5.7, about 4.6 to about 5.0, about 5.0 to about 5.7, about 2.1, about 3.0, about 3.5, about 4.1, about 4.6, about 5.0 or about 5.7.

[0173] In another embodiment, the composition comprises an anti-BCMA ADC with a mean 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.

[0174] In one embodiment, the composition comprises an anti-BCMA ADC, the antibodies include a CDRH1 having the amino acid sequence set forth in SEQ ID NO:1, a CDRH2 having the amino acid sequence set forth in SEQ ID NO:2, a CDRH3 having the amino acid sequence set forth in SEQ ID NO:3, a CDRL1 having the amino acid sequence set forth in SEQ ID NO:4, a CDRL2 having the amino acid sequence set forth in SEQ ID NO:5, and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:6, wherein the cytotoxic agent is MMAE or MMAF, and the average DAR is about 2 to about 6, about 2.1 to about 5.7, about 3.4 to about 4.6, or about 3.8 to about 4.5.

[0175] In one embodiment, the composition comprises an anti-BCMA ADC, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:7. H and V having the amino acid sequence shown in SEQ ID NO:8 L wherein the cytotoxic agent is MMAE or MMAF, and the average DAR is from about 2 to about 6, from about 2.1 to about 5.7, from about 3.4 to about 4.6, or from about 3.8 to about 4.5.

[0176] In one embodiment, the composition comprises belantamab mafodotin and the mean DAR is from about 2 to about 6, from about 2.1 to about 5.7, from about 3.4 to about 4.6, or from about 3.8 to about 4.5.

[0177] In one embodiment, the percentage 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%.

[0178] 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%.

[0179] In one embodiment, the proportion of DL4a species in the anti-BCMA ADC composition is at least about 30%, at least about 35%, about 35.5% to about 37.9%, about 35% to about 38%, about 30% to about 40%, or about 20% to about 50%. In another embodiment, the proportion of DL4a species is the majority species in the anti-BCMA ADC composition, accounting for about 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of all species combined.

[0180] In one embodiment, the proportion of DL4b species in the anti-BCMA ADC composition is at least about 5%, at least about 7%, about 7.1% to about 8.5%, about 7% to about 9%, about 5% to about 10%, or about 1% to about 15%.

[0181] In one embodiment, the proportion of DL6 species in the anti-BCMA ADC composition is at least about 10%, at least about 14%, about 14.0% to about 19.1%, about 14% to about 20%, about 10% to about 20%, or about 5% to about 30%.

[0182] In one embodiment, the proportion of DL8 species in the anti-BCMA ADC composition is at least about 1%, at least about 6%, about 6.0% to about 12.0%, about 4% to about 15%, or about 1% to about 20%.

[0183] In one embodiment, a composition comprises an anti-BCMA ADC and the percentage of DL2 is about 15% to about 27% or about 15% to about 32%, the percentage of DL4a is about 35% to about 38% or about 30% to about 40%, the percentage of DL4b is about 7% to about 9% or about 5% to about 10%, the percentage of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the percentage of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0184] In one embodiment, the composition comprises belantamab mafodotin, and the percentage of DL2 is about 15% to about 27% or about 15% to about 32%, the percentage of DL4a is about 35% to about 38% or about 30% to about 40%, the percentage of DL4b is about 7% to about 9% or about 5% to about 10%, the percentage of DL6 is about 14% to about 20% or about 10% to about 20%, and / or the percentage of DL8 is about 6.0% to about 12.0% or about 4% to about 15%.

[0185] The term "undesirable DAR species" as used herein refers to any DAR species that is undesirable in the final composition and may adversely affect certain properties of the final therapeutic product (e.g., target binding, efficacy, safety, etc.). In one embodiment, the undesirable DAR species is DL0, i.e., antibody that is not conjugated with a cytotoxic agent after the conjugation process. In one embodiment, the percentage of DL0 in the ADC composition is about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less. In another embodiment, the percentage of DL0 in the ADC composition is about 1% to about 10%, about 2% to about 5%, or about 2.0% to about 4.8%.

[0186] Post-translational modifications A "post-translational modification product" of an antibody described herein is an antibody composition in which all or a portion of the composition comprises a "post-translational modification." A post-translational modification is a change to an antibody that may be the result of production, upstream and downstream manufacturing, and / or storage of the antibody in a host cell (e.g., effects of exposure to light, temperature, pH, water, or reaction with excipients and / or immediate container closure systems). Thus, a composition of the disclosure may be formed from manufacturing or storage of an antibody. Exemplary post-translational modifications include alterations in the antibody sequence ("antibody variants" as described above), specific leader sequence cleavage, addition of various sugar moieties in various glycosylation patterns, nonenzymatic glycosylation, deamidation, oxidation, disulfide bond scrambling and other cysteine ​​variants (e.g., free sulfhydryls, racemic disulfides, thioether and trisulfide bonds), isomerization, C-terminal lysine truncation, and / or N-terminal glutamine cyclization.

[0187] In one example, post-translational modification products include "product-related impurities" that include chemical changes that result in reduced function and / or activity. In another example, post-translational modification products include "product-related substances" that include chemical changes that do not result in reduced function and / or activity. Product-related impurities of the antibodies described herein include isomerized and oxidized variants. Product-related substances of the antibodies described herein include deamidated variants, glycosylated variants, C-terminal truncated variants, and N-terminal pyroglutamic acid variants.

[0188] In one embodiment, the composition comprises a heavy chain sequence of SEQ ID NO:9 and a light chain sequence of SEQ ID NO:10, including one or more functional post-translational modifications thereof. In another embodiment, the composition comprises a heavy chain sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, and a light chain sequence of SEQ ID NO:10, including one or more functional post-translational modifications thereof.

[0189] The proportion of variants provided herein is expressed as a percentage of the total amount of antibody in the composition (e.g., the "population" of antibody).For example, 40% or less oxidized variants refers to 40% or less of the total 100% antibody in the composition being oxidized.For example, 25% or less isomerized variants refers to 25% or less of the total 100% antibody in the composition being isomerized.

[0190] Glycation is a post-translational modification that involves a nonenzymatic chemical reaction between reducing sugars, such as glucose, and free amine groups in proteins, commonly found at the epsilon amine of lysine side chains or at the N-terminus of proteins. Glycation can occur during manufacture and / or storage in the presence of reducing sugars.

[0191] Deamidation, which may occur during manufacture and / or storage, can be an enzymatic or chemical reaction. Deamidation can occur by a simple chemical reaction via intramolecular cyclization, where the amide nitrogen of the next amino acid in the chain nucleophilically attacks the amide (N+1 attacks N) to form a succinimide intermediate. Deamidation primarily converts asparagine (N) to isoaspartic acid (isoaspartate) and aspartic acid (aspartate) (D) in a ratio of about 3:1. This deamidation reaction is therefore thought to be related to the isomerization of aspartate (D) to isoaspartate. Both deamidation of asparagine and isomerization of aspartate may involve the intermediate succinimide. Deamidation can occur at glutamine residues as well, although to a much lesser extent. Deamidation can occur in the CDRs, Fab (non-CDR regions) or Fc regions. The isomerization is the conversion of aspartate (D) to isoaspartate and involves the intermediate succinimide.

[0192] Oxidation can occur during production and / or storage (e.g., in the presence of oxidizing conditions) and results in covalent modification of proteins induced directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation can occur primarily at methionine residues, but also at tryptophan and free cysteine ​​residues. Oxidation can occur in the CDRs, Fab (non-CDR) regions, or Fc regions.

[0193] Disulfide bond scrambling can occur during manufacturing and / or storage conditions. Under certain circumstances, disulfide bonds can break or form improperly, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryls (-SH) can facilitate shuffling.

[0194] Thioether formation and racemization of disulfide bonds can occur under basic conditions during preparation or storage, leading to β-elimination of the disulfide bridge back to a cysteine ​​residue via dehydroalanine and a persulfide intermediate. Subsequent cross-linking of dehydroalanine with cysteine ​​can form a thioether bond, or the free cysteine ​​residue can reform the disulfide bond with a mixture of D- and L-cysteine.

[0195] Trisulfides can result from the insertion of a sulfur atom into a disulfide bond (Cys-SSS-Cys) and can be formed by the presence of hydrogen sulfide in the production cell culture.

[0196] N-terminal glutamine (Q) and glutamate (E) in the heavy and / or light chains can undergo cyclization to form pyroglutamate (pGlu). pGlu formation can occur in the production bioreactor, but can also occur non-enzymatically, for example, depending on the pH and temperature of processing and storage conditions. Cyclization of the N-terminal Q or E is commonly observed in natural human antibodies.

[0197] Cleavage of the C-terminal lysine is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in recombinant and natural human antibodies. Variations of this process include the removal of lysine from one or both heavy chains by cellular enzymes from recombinant host cells. Upon administration to a human subject / patient, removal of residual C-terminal lysine may occur.

[0198] The present disclosure encompasses antibodies that may have been subjected to or may have undergone one or more of the post-translational modifications described herein. Exemplary compositions include mixtures or blends of antibodies 1) with and without the post-translational modification(s) described herein, or 2) with two or more post-translational modifications.

[0199] The composition may comprise a mixture of antibody variants and post-translationally modified variants. For example, the antibody composition may comprise one or more of, for example, two or more of, oxidation variants, deamidation variants, isomerization variants, N-terminal pyroglutamate variants, and C-terminal lysine truncation variants.

[0200] For example, in one embodiment, a composition may comprise a mixture of antibodies, wherein 10% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10, and 90% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10 with C-terminal lysine truncations.

[0201] In another exemplary embodiment, a composition may comprise a mixture of antibodies, wherein 10% of the antibodies in the mixture comprise the amino acid sequence of SEQ ID NOs: 9 and 10, 90% of the antibodies in the mixture comprise the amino acid sequence of SEQ ID NOs: 9 and 10 with C-terminal lysine truncations, and of the 100% total antibody mixture, up to 100% of the N-terminal glutamines are cyclized to pyroglutamate.

[0202] In another exemplary embodiment, a composition may comprise a mixture of antibodies, wherein 10% of the antibodies in the mixture comprise the amino acid sequence of SEQ ID NOs: 9 and 10, 90% of the antibodies in the mixture comprise the amino acid sequence of SEQ ID NOs: 9 and 10 with C-terminal lysine truncations, and of the 100% total antibody mixture, up to 100% are N-terminal pyroglutamate and up to 23% are isomerized at D103 of CDRH3.

[0203] In yet another exemplary embodiment, a composition may comprise a mixture of antibodies, wherein 20% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10, 80% of the antibodies in the mixture comprise the amino acid sequences of SEQ ID NOs: 9 and 10 with the N103 variant in CDRH3, and of the 100% total antibody mixture, up to 37% of the antibodies are oxidized at amino acid M34 in CDRH1.

[0204] In one embodiment, the post-translational modifications described herein do not result in significant changes in antigen binding affinity, biological activity, pharmacokinetics (PK) / pharmacodynamics (PD), aggregation, immunogenicity, and / or binding to Fc receptors, except as identified and described as product-related impurities.

[0205] "Function" or "activity" as described herein is defined as one or more of: 1) binding to BCMA, 2) binding to FcγRIIIa, and / or 3) binding to FcRn. In one embodiment, "reduced function" or "reduced activity" means that BCMA binding, FcγRIIIa binding, or FcRn binding is reduced as a percentage compared to a reference standard and is significant relative to assay variability. For example, a reduction in function or activity can be described as a reduction of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more.

[0206] In one embodiment, the anti-BCMA antibody comprises an antibody that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and SEQ ID NO:10, including any post-translational modifications of the antibody, if present.

[0207] In another embodiment, the anti-BCMA antibody comprises belantamab and any post-translational modifications, if present.

[0208] Antibody variants are commonly observed when the composition of an antibody is analyzed by charge-based separation techniques such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX).

[0209] Pharmaceutical Compositions The compositions described herein may be in the form of a pharmaceutical composition. A "pharmaceutical composition" may include the compositions described herein (e.g., active ingredient) and one or more pharma- ceutically acceptable excipients. An excipient must be acceptable in the sense that it is compatible with the other ingredients of the formulation, can be formulated into a pharmaceutical preparation, is not harmful to its recipient, and / or does not interfere with the effectiveness of the active ingredient.

[0210] As used herein, a "pharmaceutically acceptable excipient" may include any and all solvents, diluents, carriers, dispersion media, coating agents, antibacterial and antifungal agents, isotonicity agents and / or absorption retardants. Examples of pharmaceutically acceptable excipients include one or more of the following: buffers, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, the composition also contains isotonicity agents, such as polyols, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride; preservatives; cosolvents; antioxidants (including ascorbic acid and methionine); chelating agents (such as EDTA); metal complexes (e.g., Zn 2+ -protein conjugate); a biodegradable polymer; and / or a salt-forming counterion (such as sodium or potassium).

[0211] The precise nature of the excipient or other material will depend on the route of administration, which may be, for example, oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and intratumoral. It will be appreciated that preferred excipients may vary depending, for example, on the condition of the recipient and the disease being treated.

[0212] The mixture of excipients and each of the consortia together form a "pharmaceutical formulation" (or "formulation"). The formulation may be in liquid or lyophilized form. The composition in a liquid formulation may be filled into a container and frozen. In certain embodiments, an aliquot of the frozen formulation comprising the composition may be lyophilized. The lyophilisate may be reconstituted by the addition of water or other aqueous solution to produce a reconstituted formulation comprising the composition.

[0213] 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 the anti-BCMA antigen binding protein 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 a 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 a 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 a reconstituted formulation at a concentration of about 50 mg / mL.

[0214] In certain embodiments, the buffer is a citrate buffer. The citrate buffer can be achieved, 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 a preferred embodiment, the concentration of the citrate buffer is 25 mM. In some embodiments, the buffer is a histidine buffer at a concentration of about 5 mM to about 35 mM.

[0215] Buffering agents can be used to help maintain a preferred pH range, hi certain embodiments, the pH of the formulation is from about 5.5 to about 7 or from about 5.9 to about 6.5, preferably pH 6.2.

[0216] In some embodiments, the formulation comprises a polyol. In some embodiments, the polyol is a sugar, preferably a non-reducing sugar. In some embodiments, the non-reducing sugar is trehalose. In some embodiments, the formulation comprises about 120 mM to about 240 mM trehalose. In yet another embodiment, the formulation comprises about 200 mM trehalose.

[0217] In one embodiment, the formulation comprises a chelating agent. In another embodiment, the chelating agent is EDTA. In a particular embodiment, the formulation comprises EDTA at a concentration of 0.01 mM to about 0.1 mM. In yet another embodiment, the formulation comprises EDTA at a concentration of 0.05 mM.

[0218] In some embodiments, the formulation includes a surfactant. A "surfactant" is a surface active agent that can exert their effect at solid-solid, solid-liquid, liquid-liquid and liquid-air interfaces due to their chemical composition containing both hydrophilic and hydrophobic groups. Surfactants can reduce protein concentration in dilute solutions at air-water and / or water-solid interfaces where proteins may adsorb and aggregate. Surfactants can bind to hydrophobic interfaces of protein formulations. Some parenterally acceptable non-ionic surfactants include the polysorbate or polyether groups. Polysorbates 20 and 80 are suitable surfactant stabilizers in the formulations of the present disclosure. In some embodiments, the formulation includes polysorbate 20 or polysorbate 80 at about 0.01% to about 0.05%. In yet another embodiment, the formulation includes polysorbate 20 or polysorbate 80 at about 0.02%. In a preferred embodiment, the formulation includes polysorbate 80 at about 0.02%.

[0219] One aspect of the disclosure relates to a formulation comprising about 20 mg / mL to about 100 mg / mL of an anti-BCMA ADC, about 10 mM to about 25 mM of a buffer, and about 120 mM to about 240 mM of a polyol, wherein the formulation has a pH of 5.5 to 6.5.

[0220] In one embodiment, the formulation comprises about 20 mg / mL to about 60 mg / mL of an anti-BCMA ADC, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0221] In one embodiment, the composition comprises an ADC in a formulation, 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 cytotoxin is MMAE or MMAF, the formulation comprises about 20 mg / mL to about 60 mg / mL of the ADC, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0222] In one embodiment, the composition comprises an ADC in a formulation, and the antibody is a V H and V having the amino acid sequence shown in SEQ ID NO:8. L the cytotoxin is MMAF or MMAE, the formulation comprises about 20 mg / mL to about 60 mg / mL of ADC, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0223] In one embodiment, the composition comprises an ADC in a formulation, the ADC is belantamab mafodotin, the formulation comprises about 20 mg / mL to about 60 mg / mL belantamab mafodotin, about 10 mM to about 30 mM citrate buffer, about 120 mM to about 240 mM trehalose, about 0.01 mM to about 0.1 mM EDTA, and about 0.01% to about 0.05% polysorbate 20 or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0224] In one embodiment, the composition comprises belantamab mafodotin in a formulation comprising about 20 mg / mL, about 25 mg / mL, about 50 mg / mL or 60 mg / mL of belantamab mafodotin, 25 mM citrate buffer, 200 mM trehalose, 0.05 mM disodium EDTA, 0.02% polysorbate or polysorbate 80, and the pH of the formulation is about 5.9 to about 6.5.

[0225] In some embodiments, each mL of a composition disclosed herein contains belantamab mafodotin (50 mg), citric acid (0.42 mg), edetate disodium dihydrate (0.019 mg), polysorbate 80 (0.2 mg), trehalose dihydrate (75.6 mg), and trisodium citrate dihydrate (6.7 mg), and has a pH of about 6.2.

[0226] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability during manufacture, transportation, storage and administration. Stability can be measured at a selected temperature for a selected period of time. For example, for a product stored at a recommended temperature of 2°C to 8°C, the formulation is stable at room temperature, about 30°C or 40°C for at least one month, and / or at about 2-8°C for at least one year, preferably at least two years. For example, the degree of aggregation during storage 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 is present as aggregates in the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, pp. 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs., 1991 and Jones, A. Adv. Drug Delivery Rev., Vol. 10, pp. 29-90, 1993.

[0227] In certain embodiments of the present disclosure, the formulation allows the composition to remain stable upon freezing, thawing, and / or mixing.

[0228] In yet another aspect, the present disclosure is directed to an article of manufacture, e.g., a kit, comprising a container holding the composition in the formulation described herein. In one aspect, an injection device is provided that comprises the formulation. The injection device may comprise a pen injector device or an autoinjector device. In one embodiment, the formulation is contained in a pre-filled syringe.

[0229] Methods of Treatment and Compositions for Use The compositions of the disclosure may provide a therapeutic approach for the treatment of B cell related disorders or diseases (e.g., antibody mediated diseases or plasma cell mediated diseases, or plasma cell malignancies (e.g., cancers such as multiple myeloma)), or other diseases treatable by anti-BCMA ADCs. In particular, one object of the disclosure is to provide compositions comprising anti-BCMA ADCs that specifically bind BCMA (e.g., human BCMA) and modulate (e.g., inhibit or block) the interaction of BCMA with its ligands (e.g., BAFF and / or APRIL) in the treatment of diseases and disorders responsive to modulation of that interaction.

[0230] In another aspect of the disclosure, there is provided a method of treating a subject (e.g., a human patient) suffering from a B cell related disorder or disease (e.g., an antibody-mediated disease or a plasma cell mediated disease, or a plasma cell malignancy (e.g., cancer such as multiple myeloma), comprising administering to the subject a therapeutically effective amount of an anti-BCMA ADC composition described herein.

[0231] In yet another embodiment, the disclosure provides a method of treating a cancer patient, the method comprising administering to the patient a therapeutically effective amount of an anti-BCMA ADC composition described herein.

[0232] As used herein, the terms "cancer" and "tumor" are used interchangeably and refer to cells that have undergone transformation, e.g., malignant transformation, in either the singular or plural form, that renders the host organism pathological. Primary cancer cells can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from a cancer cell origin. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. A "clinically detectable" tumor, when referring to a type of cancer that usually manifests as a solid tumor, is a tumor that is detectable based on the tumor mass, e.g., a tumor that is detectable by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during a physical exam, and / or a tumor that is detectable by the expression of one or more cancer-specific antigens in a sample obtained from a patient. The tumor may be a hematopoietic (or hematologic or hematological or blood-related) cancer, such as a cancer derived from blood cells or immune cells, which may be referred to as a "liquid tumor." Specific examples of clinical conditions based on hematological tumors include leukemias, such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies, such as multiple myeloma, MGUS and Waldenstrom's macroglobulinemia; lymphomas, such as non-Hodgkin's lymphoma and Hodgkin's lymphoma, etc.

[0233] The cancer is either one in which there is an abnormal number of blast cells or unwanted cell proliferation, or one that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (anaplastic or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryocytic (or megakaryoblastic) leukemia. These leukemias are sometimes collectively referred to as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPDs), including, but not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndromes or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transition (RAEBT); and myelofibrosis (MFS), with or without myeloid metaplasia of unknown etiology.

[0234] Hematopoietic cancers also include lymphoid malignancies, which may affect lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal sites. Lymphoid cancers include, but are not limited to, B-cell malignancies, including B-cell non-Hodgkin's lymphoma (B-NHL). B-NHL can be indolent (or low-grade), intermediate (or aggressive) or high-grade (highly aggressive). Indolent B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL), such as nodal MZL, extranodal MZL, splenic MZL, and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHL includes mantle cell lymphoma (MCL) with or without leukocyte infiltration, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHL includes Burkitt lymphoma (BL), Burkitt-like lymphoma, small noncleaved cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immune cell type), primary effusion lymphoma, HIV-associated (or AIDS-associated) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or post-transplant lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocytic (LGL) leukemia, acute lymphocytic (or lymphocytic or lymphoblastic) leukemia, and Castleman's disease. NHL can also include T-cell non-Hodgkin's lymphoma (T-NHL), including, but not limited to, unspecified T-cell non-Hodgkin's lymphoma (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphadenopathy (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome, among others.

[0235] Hematopoietic cancers also include Hodgkin's lymphoma (or Hodgkin's disease), including classical Hodgkin's lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma, lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocytopenic Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell dyscrasias or plasma cell carcinomas, including multiple myeloma (MM) (including smoldering MM), monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include cancers of additional hematopoietic cells, such as polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes, and natural killer cells, etc. Tissues containing hematopoietic cells, referred to herein as "hematopoietic cell tissues," include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as the spleen, lymph nodes, lymphoid tissues associated with mucous membranes (such as gut-associated lymphoid tissue), tonsils, Peyer's patches, and appendix, and lymphoid tissues associated with other mucous membranes (e.g., the lining of the bronchi).

[0236] 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, non-small cell lung cancer, mesothelioma, pancreatic cancer and prostate cancer.

[0237] The term "treat" and its derivatives, as used herein, is meant to include therapeutic therapy. Treating with respect to a particular condition means (1) improving the condition or one or more of the biological manifestations of the condition; (2) (a) preventing one or more points in the biological cascade that leads to or causes the condition, or (b) preventing one or more of the biological manifestations of the condition; (3) alleviating one or more of the symptoms, effects, or side effects associated with the condition, or one or more of the symptoms, effects, or side effects associated with the condition or its treatment; (4) slowing the progression of the condition or one or more of the biological manifestations of the condition; and / or (5) curing one or more of the biological manifestations of the condition or condition by eliminating or reducing to undetectable levels one or more of the biological manifestations of the condition without additional treatment for a period of remission that is considered to be in remission of the manifestations. Those skilled in the art will understand the period of time that is considered to be in remission for a particular disease or condition.

[0238] B cell disorders can be divided into defects in B cell development / immunoglobulin production (e.g., immunodeficiencies) and excessive / uncontrolled proliferation (e.g., lymphomas, leukemias). As used herein, B cell disorders refer to both types of disease, and methods of treating B cell disorders using the compositions described herein are provided.

[0239] In certain embodiments, the disease or disorder is multiple myeloma (MM), chronic lymphocytic leukemia (CLL), solitary plasmacytoma (bone, extramedullary), amyloidosis (AL), smoldering multiple myeloma (SMM), solitary plasmacytoma (bone, extramedullary), or Waldenstrom's macroglobulinemia.

[0240] Preventive therapy is also contemplated.Those skilled in the art will recognize that "prevention" is not an absolute term.In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to substantially reduce the likelihood or severity of a condition or its biological expression, or to delay the onset of such a condition or its biological expression.Preventive therapy is appropriate, for example, when a subject is considered to be at high risk of developing cancer, for example, when the subject has a strong family history of cancer or when the subject has been exposed to a carcinogen.

[0241] "Subject" or "patient" are used interchangeably herein and are broadly defined to include any person in need of treatment, such as a person in need of cancer treatment. A subject may include a mammal. In one embodiment, a subject is a human patient. A subject in need of cancer treatment may include patients in various stages, such as newly diagnosed, relapsed, refractory, progressive disease, remission, and others. A subject in need of cancer treatment may also include patients who have undergone stem cell transplantation or who are considered transplant ineligible.

[0242] Subjects can be pre-screened to be selected for treatment with the compositions described herein, in one embodiment, a sample from a subject is tested for expression of BCMA prior to treatment with the compositions described herein.

[0243] The subject may have undergone at least one cancer therapy prior to receiving treatment with the composition of the present disclosure, hi one embodiment, the subject has undergone at least one, at least two, at least three, at least four, at least five, at least six, or at least seven cancer therapies prior to receiving treatment with the composition of the present disclosure.

[0244] In another embodiment, the subject has been newly diagnosed with cancer and has not undergone any therapy prior to being treated with the compositions of the present disclosure.

[0245] The composition of the present disclosure can be administered by any suitable route.In some compositions, suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and intratumoral.It is recognized that preferred routes may vary, for example, depending on the recipient's condition and the cancer to be treated.

[0246] In certain embodiments, the compositions of the present disclosure are administered as pharmaceutical compositions.

[0247] The term "administering" as used herein is meant to refer to the delivery of the compositions described herein to achieve a therapeutic objective. The compositions may be administered at a sufficient interval and duration to achieve clinical efficacy. The compositions may be administered to a subject to target therapy to a specific site.

[0248] In some embodiments, the composition is administered by injection. Thus, in one aspect, an injection device is provided that comprises the composition, pharmaceutical composition or formulation of the present disclosure. The injection device may include a pen injector device or an autoinjector device.

[0249] The term "therapeutically effective amount" or "therapeutically effective dose" of a composition, as used herein, refers to an amount that is effective for preventing or treating or alleviating the symptoms of B cell-mediated disorder or disorder.Therapeutically effective amounts and treatment regimens are generally determined empirically and may depend on factors such as the age, weight and health of the patient, and the disease or disorder being treated.Such factors are within the control of the attending physician.

[0250] An appropriate therapeutically effective amount of a composition comprising an anti-BCMA ADC can be readily determined by one of skill in the art. A suitable dose of the compositions described herein can be calculated for each patient according to body weight, for example, a suitable dose can 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, or for example, about 1 mg / kg to about 15 mg / kg, for example, about 10 mg / kg to about 15 mg / kg.

[0251] In one embodiment, the therapeutically effective dose of a composition comprising an anti-BCMA ADC is about 0.03 mg / kg to about 4.6 mg / kg. In yet another embodiment, the therapeutically effective dose of a composition comprising an anti-BCMA ADC is at least about 0.03 mg / kg, at least about 0.06 mg / kg, at least about 0.12 mg / kg, at least about 0.24 mg / kg, at least about 0.48 mg / kg, at least about 0.96 mg / kg, 1 mg / kg, at least about 1.92 mg / kg, at least about 3.4 mg / kg, or at least about 4.6 mg / kg. In yet another embodiment, the therapeutically effective dose of a composition comprising an anti-BCMA ADC is 1.9 mg / kg, 2.5 mg / kg, or 3.4 mg / kg.

[0252] In certain embodiments, the composition can be co-administered to the subject with one or more additional therapeutic agents. In another embodiment, the composition can be co-administered to the subject with one or more additional cancer therapeutic agents. Additional cancer therapeutic agents include, but are not limited to, other immunomodulatory agents, therapeutic antibodies (e.g., anti-CD38 antibodies such as daratumumab), CAR-T therapeutic agents, BiTEs, HDAC inhibitors, proteasome inhibitors (e.g., bortezomib), anti-inflammatory compounds, and immunomodulatory imid drugs (IMiDs) (e.g., thalidomide and its analogs).

[0253] "Concomitant administration" refers to the administration of two or more different pharmaceutical compositions or treatments (e.g., radiation therapy) administered to a subject in combination in the same or separate pharmaceutical compositions. Thus, concomitant 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 time or at different times.

[0254] In one aspect of the disclosure, the disclosure provides a method of treating a B cell disease or disorder in a subject in need thereof, comprising administering a therapeutically effective dose of a composition comprising an anti-BCMA ADC described herein.

[0255] In one aspect of the disclosure, the present disclosure provides a composition as described herein for use in treating a B cell disease or disorder. In another aspect of the disclosure, the present disclosure provides a composition as described herein for use in treating cancer.

[0256] In one embodiment of the disclosure, there is provided a use of the composition in the manufacture of a medicament for use in treating a B-cell disease or disorder. In another embodiment of the disclosure, there is provided a use of the composition in the manufacture of a medicament for use in treating cancer.

[0257] All patent and literature references disclosed herein are expressly and entirely incorporated by reference. EXAMPLES

[0258] Reagents and equipment Belantamab was produced and purified using standard mAb manufacturing procedures. Belantamab was then conjugated with MMAF to produce the belantamab mafodotin ADC drug substance. All samples were stored in formulation buffer at -80°C until analysis.

[0259] Maleimidocaproyl monomethyl auristatin F (mcMMAF) was purchased from MedChemExpress (Monmouth Junction, NJ). Isotopically labeled water (H 18 O, 97%), dithiothreitol (DTT), sodium iodoacetate (IAA), tris(2-carboxyethyl)phosphine (TCEP), calcium chloride and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Guanidine HCl was purchased from MP Biomedicals (Irvine, CA). Size-exclusion chromatography (SEC) spin columns were purchased from Bio-Rad (Hercules, CA) and trypsin was purchased from Worthington Biochemical (Lakewood, NJ). Tris base, ethylenediaminetetraacetic acid (EDTA) disodium salt, hydrochloric acid and LC-MS grade trifluoroacetic acid (TFA), water (HO) and acetonitrile (ACN) were purchased from ThermoFisher Scientific (Waltham, MA).

[0260] Liquid chromatography tandem mass spectrometry (LC-MS / MS) was performed on a Waters Acquity UPLC system equipped with a Waters BEH 300 C18 column (2.1 × 150 mm, 1.7 μm particle size) connected to a Thermo Scientific Orbitrap XL mass spectrometer.

[0261] Reduced LC-MS analysis was performed on a Waters Acquity UPLC system equipped with a Waters BEH200 SEC (4.6 × 150 mm, 1.7 μm particle size) coupled to a Xevo G2-XS mass spectrometer.

[0262] Example 1: Stable isotope labeling of MMAF Sample preparation Dissolve MMAF (20 mg) in 120 μL of acetonitrile and add H2 18 Mix with 80 μL of 2.5% (v / v) TFA in O to obtain 1% TFA 60:40 / ACN:H2 18Final reaction conditions were 100 mg / mL MMAF in O. The solution was allowed to react for 14 days at room temperature, protected from light. Aliquots (10 μL) were frozen at -80° C. until analysis.

[0263] The isotopic purity of the labeled MMAF was determined by diluting an aliquot 1000-fold (0.1 mg / mL) in 50:50 / ACN:HO and analyzing using reversed-phase liquid chromatography separation using a linear gradient from 0% to 100% B in 5 min at a flow rate of 0.2 mL / min. Mobile phase A was 0.1% TFA in water and mobile phase B was 0.09% TFA in ACN. The column was flushed with 100% B for 3 min after each injection and re-equilibrated with 0% B for 12 min. The column temperature was 30° C., the autosampler temperature was 5° C., and the injection volume was 5 μL.

[0264] MS analysis was performed with post-UV detection (215 nm) operating in data-dependent acquisition mode (1 MS scan followed by 2 MS / MS scans). The electrospray ionization (ESI) spray voltage was 4.5 kV, sheath gas flow rate was 40 L / min, solvent desorption auxiliary gas flow rate was 5 L / min, capillary voltage was 60 V, capillary temperature was 250 °C, tube lens was 120 V, and scan range was m / z 250-2000. Data were examined using Thermo Xcalibur software.

[0265] Results and Discussion Stable isotope labeling of MMAF was performed using the acid-mediated solvent exchange mechanism described in (Liu et al., Advances and applications of stable isotope labeling-based methods for proteome relative quantitation., Trends in Anal. Chem., 2020, vol. 124, pp. 115815). Briefly, when reacted under acidic conditions, two 18-oxygen atoms can be exchanged from isotopically labeled water to a carboxylic acid functional group via an orthoacid intermediate. Several labeling optimization conditions were explored, including acid concentration [0.1%-10% (v / v)], acid type (TFA or FA), reaction temperature (room temperature, 37°C, 70°C) and organic phase (ACN or DMSO). Reaction times ranged from 1 h to 6 weeks, and as expected, higher acid concentrations and temperatures increased the labeling rate. The optimization experiments were performed using a 50:50 / ACN:H2 18 The assay was performed using 0.1 mg / mL MMAF in O 50:50 / ACN:H2 18 Since a thin organic solvent layer is formed in O, the concentrated batch (100 mg / mL) is diluted in 60:40 / ACN:H2 18 Labeled in O.

[0266] Minimization of MMAF hydrolysis (Figure 1) was a major factor in optimizing the labeling reaction conditions due to potential competition between maleimide-containing fragments and labeled MMAF for unoccupied ADC conjugation sites. This limitation did not allow the labeling reaction to proceed to equilibrium and was stopped when minimal unlabeled (+0 Da) MMAF remained. This resulted in a mixture of singly labeled (+2 Da) and doubly labeled (+4 Da) MMAF. This discrepancy was accounted for during MS data processing. The final labeling reaction conditions (1% TFA, room temperature, 14 days) were ultimately selected based on sufficient labeling (minimization of +0 Da MMAF) and minimal hydrolysis (<5%).

[0267] Example 2: Reduction of ADC and conjugation with isotopically labeled MMAF Sample preparation Belantamab mafodotin (250 μg, 10 mg / mL) was partially reduced with 1.25 μL of 1 M DTT for 15 min at 37° C. in formulation buffer (pH 6.2). Excess DTT was removed by eluting the sample through a SEC spin column equilibrated with formulation buffer. The sample was then conjugated with 1 μL of 100 mg / mL isotope-labeled MMAF for 30 min at room temperature. Excess MMAF was removed by eluting the sample through a SEC spin column equilibrated with formulation buffer.

[0268] Conjugation efficiency was determined by diluting samples to 1 mg / mL in water and analyzing them using isocratic size-exclusion liquid chromatography separation using 0.1% TFA in a 65:35 / ACN:HO mobile phase at a flow rate of 0.2 mL / min. The column temperature was 25° C., the autosampler temperature was 5° C., and the injection volume was 1 μL.

[0269] MS analysis was performed with post-UV detection (215 nm) in high sensitivity mode. The electrospray ionization (ESI) spray voltage was 2.2 kV, sampling cone was 120, ion source temperature was 150 °C, offset voltage was 80 V, desolvation temperature was 500 °C, cone gas flow rate was 60 L / h and desolvation gas flow rate was 800 L / h. The scan range was m / z 700-5000, and the scan time was 1 s. Data were examined using Waters MassLynx software.

[0270] Results and Discussion Reduction / conjugation scheme: Briefly, belantamab was partially reduced with TCEP and then conjugated with MMAF. TCEP is generally used in preference to DTT to avoid competing side reactions between the maleimide and thiol groups of MMAF and DTT, respectively. Initial reduction / conjugation experiments confirmed that TCEP allows for higher conjugation efficiency compared to DTT, but subsequent peptide mapping analysis using legacy methods required the use of DTT to fully reduce all remaining disulfide bonds. The choice of DTT for both reduction steps and subsequent removal of excess DTT by SEC spin columns prior to MMAF conjugation resulted in minimal conjugation of hydrolytic fragments and yielded fully conjugated light and heavy chains (Figure 2).

[0271] Example 3: Stable isotope labeled peptide mapping LC-MS / MS analysis of belantamab mafodotin Sample preparation Belantamab mafodotin (250 μg, 10 mg / mL) was reduced and conjugated with isotopically labeled MMAF as described in Example 2. Samples were then prepared using standard peptide mapping procedures by evaporating to near dryness (approximately 5 μL) and denaturing by adding 60 μL of denaturation buffer (6M guanidine HCl, 1.2M Tris HCl, 2.5 mM Na2EDTA, pH 7.5) and vortexing for 2 minutes at room temperature. Samples were reduced by adding 3 μL of 1M DTT and incubating for 20 minutes at room temperature, then alkylated by adding 7.2 μL of 1M IAA and incubating for 30 minutes at room temperature. Alkylation was quenched by adding 4.2 μL of 1M DTT. The samples were then buffer exchanged into digestion buffer (50 mM Tris, 1 mM CaCl2, pH 7.5) using a SEC spin column and digested by adding 2.5 μL of 5 mg / mL trypsin (1:20 / enzyme:protein) and incubating for 30 minutes at 37° C. The digestion was quenched by adding 3 μL of 1 N HCl and 10 μL was injected for LC-MS / MS analysis.

[0272] Samples were analyzed using the same LC-MS / MS conditions as described in Example 2, except that a two-step gradient was used: 0% to 40% B in 90 min, followed by 40% to 60% B in 10 min. The column was flushed with 100% B for 12 min and re-equilibrated at 0% B for 18 min after each injection. Sequence coverage and PTM data were analyzed using Protein Metrics Byos software (Cupertino, CA). Isotope conjugation data were analyzed using Skyline software (University of Washington).

[0273] Results and Discussion Cysteine-conjugated ADCs are generally conjugated at cysteine ​​residues involved in interchain disulfide bonds. Reduction of these disulfide bonds generates eight potential conjugation sites (one in each light chain, one in each heavy chain Fab region, and two in each heavy chain hinge region). As each disulfide bond is reduced, two free sulfhydryl groups are generated, so even numbers of drug-loaded species are commonly found. This process generates drug-loaded species ranging from DL0 to DL8, along with the potential for positional isomers (Figure 3).

[0274] SIL peptide mapping yielded liquid chromatography peaks containing both native and labeled conjugated peptides, whereas standard peptide mapping yielded separate peaks for native and conjugated peptides (Figure 4). The conjugated peptide peak data for the SIL samples were processed by integrating extracted ion chromatograms (XICs) using Skyline software to obtain peak areas for individual isotopes of interest.

[0275] SIL did not induce a large enough mass change to completely separate the isotopic envelopes of native and SIL peptides. This resulted in a mixture of isotopic isomers due to overlap of isobaric isotopes corresponding to native or SIL-conjugated peptides. Therefore, the natural isotopic contributions of the M+2 to M+4 isotopomers of the light and heavy Fab peptides were calculated using the theoretical isotope ratios calculated from the unlabeled ADC samples (Figures 5 and 6). This calculation also corrected for sites conjugated with singly labeled (+2 Da) MMAF. The combined native isotopomer peak areas were then divided by the total isotopomer peak areas to determine the peptide conjugation level.

[0276] The theoretical isotope ratios of the doubly conjugated (DL2, C230 and C233) and native (DL0) forms of the hinge peptide were calculated from the unlabeled ADC sample and the SIL mAb intermediate sample, respectively (Figure 7). The DL0 isotope ratio of the mAb sample considered the isotope contribution from the DL0 peptide labeled with all combinations of the SIL MMAF of the two available hinge conjugation sites. These DL2 and DL0 theoretical isotope ratios were used to calculate the isotope contribution of each peptide form. First, the M and M+1 isotopomer peak areas were assumed to be fully associated with the DL2 peptide. The M+1 peak area was then multiplied by the DL2 relative theoretical isotope ratio of each isotopomer to determine the DL2 contribution to the M+2 to M+14 isotopomers. The M+15 and M+16 isotopomers were then assumed to be fully associated with the DL0 peptide. The M+15 peak area was then multiplied by the DL0 relative theoretical isotope ratio of each isotopomer to determine the DL0 contribution to the M+2 to M+14 isotopomers. Finally, the peak areas of both the DL2 and DL0 isotopomers were subtracted from the sum of each isotopomer peak area to determine the contribution of the single conjugated (DL1, C230 or C233) hinge peptide. The sum of the isotopomer peak areas of each form was then divided by the total isotopomer peak area to determine the level of conjugated peptide form of each hinge. Interestingly, this data processing method was compared to a more rigorous algorithm described by Jennings and Matthews (Determination of Complex Isotopomer Patterns in Isotopically Labeled Compounds by Mass Spectrometry., Anal.Chem., 2005, 77, 6435-6444), and both methods gave similar results.

[0277] Comparison of SIL peptide mapping with standard peptide mapping The linearity of the method was examined by testing samples consisting of a linear combination of belantamab (mAb) and belantamab mafodotin (ADC) to generate samples with DARs ranging from 0.0 to 5.7 (Tables 1 and 2). Standard peptide mapping showed reduced sensitivity and linearity when quantifying light and heavy chain Fab conjugation levels, making it impractical to use relative quantification between native and conjugated peptides. In contrast, SIL peptide mapping showed excellent linearity (R 2 ≥ 0.996) (Figure 8). Both methods gave linear results for singly and doubly conjugated hinge sites, but SIL peptide mapping showed greater differences in the amounts of singly and doubly conjugated hinges. The singly conjugated hinge was not detected as a major isoform in any drug-loaded species of belantamab mafodotin by the orthogonal method. This result correlated well with the SIL peptide mapping analysis of drug-loaded fractions described in experiment 4. Furthermore, the DAR values ​​calculated from SIL peptide mapping showed better linearity (R 2 = 0.998), which correlated within 5% of the theoretical DAR determined by HIC (Figure 9).

[0278] Conjugation levels of the belantamab mafodotin reference standard (DAR=4.0) were quantified and compared using both methods (Table 3). SIL peptide mapping preparations (n=4) were analyzed in parallel with standard peptide mapping preparations (n=4) to assess reproducibility. Lower levels of light and heavy chain Fab conjugation (~70%) were detected with SIL peptide mapping compared to standard peptide mapping (~90%-95%). Higher levels of doubly conjugated hinges (~25%) and lower levels of single-conjugated hinges (~5%-10%) were detected compared to standard peptide mapping (~15% and ~12% for doubly and single-conjugated, respectively). DAR values ​​calculated from SIL peptide mapping (3.9-4.0) were also more accurate than those calculated from standard peptide mapping (4.6) when compared to a theoretical DAR of 4.0. Typical PTMs of belantamab mafodotin were also quantified, and no significant differences were detected between the SIL and reference samples (Table 4). Complete sequence coverage was also detected for all samples.

[0279] [Table 1]

[0280] [Table 2]

[0281] [Table 3]

[0282] [Table 4]

[0283] Example 4: Analysis of Hydrophobic Interaction Chromatography Drug-Loaded Fractions Sample preparation Samples with different DARs (DAR = 2.1-5.7) and individual drug-loaded samples (DL2, DL4a, DL4b and DL6) were analyzed by SIL peptide mapping as described in experiment 3.

[0284] Results and Discussion Belantamab mafodotin samples with DARs ranging from 2.1 to 5.7 were produced as part of preclinical studies to evaluate the impact of DAR on drug efficacy. The DARs of these samples were calculated by integrating the peak areas of the various drug-loaded species in the HIC chromatograms. SIL peptide mapping results (Table 5 and Figure 10) showed 41.8% to 85.8% light and heavy chain Fab conjugation in samples with increasing DAR. This result was expected as the light and heavy chain Fab sites are paired with disulfide bonds. Doubly conjugated hinges showed 7.2% to 57.4%, while single conjugated hinges were much lower at 5.1% to 15.8%, highlighting the predominance of doubly conjugated hinges at higher DARs. Interestingly, single conjugated hinges were highest in the 4.0 and 4.6 DAR samples, with an inflection point where single conjugated hinges decreased in the higher DAR samples. The DAR values ​​calculated from HIC and SIL peptide mapping correlated within 10%, demonstrating the utility of SIL peptide mapping for "bottom-up" DAR characterization while also providing site-specific conjugation levels (see Figure 11).

[0285] Individual drug-loaded belantamab mafodotin samples (DL2, DL4a, DL4b, and DL6) were collected by fractionation in a scaled-up hydrophobic interaction chromatography (HIC) method (Figure 12). The major positional isomers of DL2, DL4, and DL6 were confirmed by orthogonal analysis and used to calculate theoretical conjugation site occupancy values. Theoretical values ​​were not calculated for the DL4b sample because this sample is a mixture of DL4a and DL4b isoforms and the HIC peaks overlap. SIL peptide mapping yielded values ​​that correlated well with the theoretical values ​​(Table 6). SIL peptide mapping also confirmed that the major hinge conjugation form for isoforms DL4b and DL6 was double conjugated, with minor amounts of single-conjugated hinges detected.

[0286] [Table 5]

[0287] [Table 6]

[0288] Example 5: Non-reduced capillary gel electrophoresis (NR-CGE) analysis of drug-loaded variants Sample preparation A HIC purification method was optimized and performed on an AKTA system using a Tosoh Toyopearl Butyl-650S column (35 μm, 8 mm × 10 cm) (Part No. 45126) with the following conditions: 25 °C, flow rate of 3.5 mL / min, injection volume of 60 mg, and UV absorbance detection at 280 nm. The method uses a gradient flow of an initial mobile phase composed of 1.5 M ammonium sulfate and 50 mM potassium phosphate, pH 7.0, and an elution mobile phase composed of 20% 2-propanol and 50 mM potassium phosphate, pH 7.0. Fractions of 4.5 mL were collected at the peak apex. Fractions of each DL variant (DL0, DL2, DL4a, DL4b, DL6, and DL8) were collected from multiple injections, pooled, and buffer exchanged with formulation buffer.

[0289] result The purified DL variants DL0, DL2, DL4a, DL4b, DL6 and DL8 were tested using the release and stability HIC method for purity analysis. The results obtained from HIC chromatography of these DL variants are summarized in Table 7. The purity of DL0, DL2, DL4a and DL6 was greater than 90%. DL4b and DL8 were 69.3% and 81.9%, respectively.

[0290] [Table 7]

[0291] NR-CGE analysis of purified DL variants was performed using the release and stability capillary gel electrophoresis (CGE) method. The denaturing sample preparation procedure results in dissociation of the heavy and light chains, which are no longer linked by disulfide bonds, and the resulting separation provides a characteristic fingerprint of drug conjugation. The possible isoforms of belantamab mafodotin are shown diagrammatically in Figure 3, and the theoretical NR-CGE fingerprints of each DL variant are shown in Table 8.

[0292] [Table 8]

[0293] The results of NR-CGE analysis of purified DL variants are summarized in Table 9. Purified DL0 was 94.6% pure by HIC (Table 7) and 92.9% IgG by NR-CGE, indicating that the DL0 peak in HIC is likely unconjugated IgG. Additional peaks in the NR-CGE profile of purified DL0 included light chain (LC) and HC-HC-LC (HHLC) species, likely derived from the 4.5% DL2 present in the fraction.

[0294] Purified DL2 contained mainly LC and HHLC species by NR-CGE analysis, consistent with DL2a, indicating that the predominant DL2 variant is DL2a. Some DL2b coeluted under the DL2 peak, as suggested by the detection of 4.4% IgG in purified DL2.

[0295] Purified DL4a contains mainly LC and HC-HC (HHC) species in NR-CGE analysis, indicating that DL4a is the major DL4 variant in belantamab mafodotin. The DL4a peak in HIC is a four cysteine ​​residue-conjugated DL variant containing an interchain disulfide bond between LC and HC. HHLC fragments are found at approximately 6%, likely originating from either DL2a or DL4c. However, as shown, the purity of DL4a is 97.1% by HIC, so there is probably some species co-eluting under the DL4a peak, which is the main cause of the high HHLC content.

[0296] Purified DL4b contains primarily LC, HHC and HC-LC (HLC) species in NR-CGE analysis. HIC results suggest that 28.6% of the fraction is DL4a, which is the LC and HHC fragments observed. The HLC fragments are a result of DL4b, indicating that the species eluting under the DL4b peak in HIC analysis of belantamab mafodotin are mostly conjugated to four cysteine ​​groups, including two interchain disulfide bonds, within the hinge region. Purified DL4b also contains small amounts of HHLC and HC fragments, likely due to co-purification of DL4c or DL6a.

[0297] Purified DL6 contains a mixture of LC, HC and HLC species by NR-CGE analysis. This is consistent with DL6a, indicating that the major DL6 variant in belantamab mafodotin is conjugated at four cysteine ​​residues, including two cysteine ​​residues derived from two interchain disulfide bonds in the hinge region and two cysteine ​​residues derived from disulfide bonds between the LC and HC chains. The HHC species was present at 3.2%, likely derived from copurification of DL4a.

[0298] Purified DL8 contained primarily LC and HC species, consistent with conjugation at the eight cysteine ​​residues that comprise the four interchain disulfide bonds of belantamab mafodotin. HLC fragments, likely derived from copurification of DL4b or DL6a, were present at approximately 11%, consistent with peaks reported in the HIC analysis (Table 7).

[0299] Representative NR-CGE electropherograms of purified DL0, DL2, DL4a, DL4b, DL6 and DL8 are shown in FIG.

[0300] [Table 9]

[0301] Example 6: Intact and reduced LC-MS results The purified DL variants prepared as described in Example 5 were analyzed using intact and reduced LC-MS. The total drug loading of the purified DL variants was determined using intact LC-MS analysis. The total drug loading of the heavy and light chains was determined using reduced LC-MS. The results are shown in Table 10 and the spectra are shown in Figures 14 and 15.

[0302] The results of the intact LC-MS analysis of the purified DL variants were consistent with the purity results of the analytical HIC analysis (Table 7), with similar species identified in both analyses at nearly identical abundances. The exception to this is that in the purified DL4b fraction, the DL3 variant is detected by HIC, but the intact LC-MS analysis did not detect a species in this fraction with a mass corresponding to a drug load of three drug molecules. This suggests that the DL3 peak in the purified DL4b fraction is in fact another DL4 variant, rather than a species with a drug load of three drug molecules.

[0303] Reduced LC-MS analysis of the purified DL variants was consistent with the conjugation pattern predicted by the NR-CGE results: purified DL2 had a reduced LC-MS profile consistent with DL2a, with 50% DL0 and 50% DL1 for both LC and HC, indicating conjugation at the two cysteine ​​residues that comprise the interchain disulfide bond between the LC and HC.

[0304] Reduced LC-MS analysis of purified DL4a showed 93.6% and 96.9% DL1 in the LC and HC, respectively, consistent with conjugation at the four cysteine ​​residues that comprise the interchain disulfide bond between the LC and HC, indicating that almost all of the purified DL4a is DL4a variants. DL2a and DL4b were also observed at low levels in the HIC results, contributing to the low levels of LC DL0 and HC DL2 observed in reduced LC-MS analysis.

[0305] Reduced LC-MS of the pure DL4b variant was expected to consist of 100% LC DL0 and 100% HC DL2. LC DL0 was present in 50.8% and HC DL2 in 54.7%, indicating that approximately 50% of the sample contained DL4b. 50% LC was DL1 and 50% HC was DL1, indicating the possible presence of DL4a in the population, consistent with the detection of 30% DL4a by HIC. Other DL4 variants may also contribute to the LC DL1 and HC DL1 peaks detected by reduced LC-MS analysis.

[0306] Reduced LC-MS analysis of purified DL6 indicated that the purified DL6 fraction was the DL6a variant. Pure DL6a variants were expected to consist of 50:50 / DL0:DL1 in the LC and 50:50 / DL2:DL3 in the HC. There was slightly more LC DL1 and HC DL2 than expected from the pure DL6a fraction, indicating that some DL6b variants may be present. These results indicated that the major DL6 variant of belantamab mafodotin is conjugated at four cysteine ​​residues that contain interchain disulfide bonds in the hinge and two cysteine ​​residues that contain interchain disulfide bonds between the LC and HC.

[0307] Reduced LC-MS analysis of purified DL8 was expected to consist of 100% LC DL1 and 100% HC DL3. These results indicated that over 80% of the LC was DL1 and over 80% of the HC was DL3. These results were consistent with the expected abundance based on HIC purity of approximately 80% (Table 7). LC DL1 and HC DL2 were detected at 17% and 14%, respectively, which may possibly be a result of DL6 and DL4b co-purifying with the DL8 variant from the HIC results.

[0308] [Table 10]

[0309] Example 7: Peptide mapping by LC-MS / MS Conjugation sites and post-translational modifications of purified DL variants, prepared as described in Example 5, were assessed using peptide mapping LC-MS / MS.

[0310] Binding of antigen, FcγRIIIa and FcRN by SPR The antigen, FcγRIIIa and FcRn specific binding activity of the purified DL variants was measured using SPR. Antigen, FcγRIIIa and FcRN analysis was performed using surface plasmon resonance (SPR) methods of release and stability. The measured specific binding activity was between 80 and 110% for the purified DL variants. The three SPR activity assays showed that the specific activity decreased with increasing number of conjugated drug molecules (Table 11). Despite this trend being observed, the decrease in binding activity as a function of drug loading was minimal and remained within the acceptance criteria of the specification. It was confirmed that there was no significant change in antigen FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity by SPR with increasing DAR.

[0311] [Table 11]

[0312] Cell proliferation inhibition potency and ADCC reporter bioassays The biological activity of purified DL variants was monitored using cell growth inhibition bioassays of release and stability and ADCC reporter bioassays.The results are shown in Table 12.The relative potency of purified DL variants in cell growth inhibition bioassays correlated with increasing drug loading.The relative potency of purified DL0 variants in cell growth inhibition bioassays was 0.0, as expected for unconjugated molecules.

[0313] The relative potencies of the purified DL variants in the ADCC reporter bioassay were similar. Similar to what was observed for FcγRIIIa and FcRn binding, there was likely a correlation between increased relative potency and decreased drug loading (Table 11). This was not unexpected, as conjugation of drugs near the hinge region may undergo slight conformational changes that affect protein-protein interactions in the Fc.

[0314] [Table 12]

[0315] Capillary Differential Scanning Calorimetry (DSC) Capillary DSC was used to measure the changes in the tertiary structure and thermal stability of belantamab mafodotin after drug conjugation (Figure 16). In a typical thermogram of belantamab, the first peak corresponds to the unfolding of the CH2 domain, and the second peak corresponds to the unfolding of the CH3 domain and Fab. The melting transitions of the CH3 and Fab of mAbs often overlap and generally occur at the same or higher temperatures compared to the CH2 domain. The melting temperature generally correlates with the stability of the protein, and increased stability is usually manifested as an increase in the melting temperature due to the increased amount of energy required to unfold the protein.

[0316] Table 13 and Figure 16 show the transition temperatures and DSC thermograms, respectively, of the purified DL variants. The DSC thermogram and transition temperatures of purified DL0 are similar to belantamab. A slight increase in Tm2 was observed compared to belantamab, possibly due to the difference in the formulation buffer.

[0317] DSC analysis of purified DL2 showed a decrease in the apparent transition temperature of the Fab from 84.2 °C to 78.2 °C, likely a result of drug conjugation at LC C214 and HC C224, cysteine ​​residues that form interchain disulfide bonds between the LC and HC. This data was consistent with the NR-CGE and reduced LC-MS results.

[0318] DSC analysis of purified DL4a showed a decrease in the apparent transition temperature of the Fab, similar to that observed for purified DL2. However, compared to purified DL2, the purified DL4a variant has a much larger peak area at 78.0 °C. This correlated with the data showing that both cysteines, including the interchain disulfide bond between the LC and HC, are conjugated. A slight decrease in the apparent transition temperature of the CH2 domain was observed, which may be due to another DL4 variant co-purified with DL4a.

[0319] DSC analysis of purified DL4b was partially similar to that of purified DL2, with a reduced apparent transition temperature of the Fab domain, but unlike DL2, the apparent transition temperature of the CH2 domain was also reduced, suggesting that purified DL4b contains species with conjugation to cysteines at both the hinge and the Fab interchain disulfide.

[0320] DSC analysis of purified DL6 showed a decrease in the apparent transition temperature of the CH2 domain and some of the Fab. The percentage of shifted Fab was similar to that observed for purified DL2. This data was consistent with drug conjugation of the two cysteine ​​residues that form the interchain disulfide bond between the LC and HC, and the four cysteine ​​residues in the hinge interchain disulfide bond.

[0321] DSC analysis of purified DL8 showed a decrease in the melting temperature of the CH2 domain similar to that observed with purified DL6. In addition, there was a decrease in the apparent transition temperature of the Fab similar to that observed with purified DL4a. These results were consistent with the conjugation of eight cysteine ​​residues in four interchain disulfide bonds.

[0322] Disruption of the interchain disulfide bonds by partial reduction and conjugation of cysteine ​​residues is predicted to lower the thermal unfolding transition temperature, and capillary DSC data obtained for the purified DL variants were consistent with this hypothesis.

[0323] [Table 13]

[0324] Conclusions from Examples 5 to 7 Purified DL variants were characterized to determine their purity, potency and identity, including the conjugation sites on belantamab mafodotin. Results showed that the DL distribution was a heterogeneous mixture of DL variants conjugated with an even number of drugs (mcMMAF) after partial reduction of the interchain disulfide bonds. The predominant drug-loaded variant of belantamab mafodotin was the DL4a species, which contained four MMAF molecules conjugated to each LC C214 and each HC C224, including the interchain disulfide bond between the LC and HC. The DL0 variant was confirmed to be unconjugated belantamab. Although the relative potency of DL variants increased in association with increasing drug loading in cell growth inhibition assays, belantamab mafodotin consisted of a distribution of these DL variants that contributed to the overall relative potency. The distribution of DL was controlled by the drug-to-antibody ratio (DAR). The HIC method is currently being performed on both the drug substance and the drug product to monitor the release and steady-state drug loading variants of belantamab mafodotin.

[0325] Sequence Listing SEQ ID NO:1: CDRH1 NYWMH

[0326] SEQ ID NO:2: CDRH2 ATYRGHSDTYYNQKFKG

[0327] SEQ ID NO:3: CDRH3 GAIYDGYDVLDN

[0328] SEQ ID NO:4: CDRL1 SASQDISNYLN

[0329] SEQ ID NO:5: CDRL2 YTSNLHS

[0330] SEQ ID NO:6: CDRL3 QQYRKLPWT

[0331] SEQ ID NO: 7: Heavy chain variable region (CDRs underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDN WGQGTLVTVSS

[0332] SEQ ID NO:8: Light chain variable region (CDRs underlined) DIQMTQSPSSLSASVGDRVTITC SASQDISNYLN WYQQKPGKAPKLLIY YTSNLHS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYRKLPWT FGQGTKLEIKR

[0333] SEQ ID NO: 9: Heavy chain region (CDRs underlined; HC C224, HC C230 and HC C233 in bold / underlined) [ka]

[0334] SEQ ID NO: 10: Light chain region (CDRs underlined; LC C214 in bold / underlined) [ka]

[0335] SEQ ID NO: 11: Heavy chain region including D103N (CDRs underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYNGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0336] SEQ ID NO: 12: Heavy chain region containing N388D (CDRs underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0337] SEQ ID NO: 13: Heavy chain region containing N393D (CDRs underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0338] SEQ ID NO: 14: Heavy chain region including N388D and N393D (CDRs underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYCAR GAIYDGYDVLDN WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESDGQPEDNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. (i) preparing an isotopically labeled antibody drug conjugate (ADC) sample by conjugating an unoccupied cysteine site of a cysteine-conjugated ADC using an isotopically labeled cytotoxin containing a carbonyl group and a reducing agent; and (ii) performing peptide mapping of the sample; Analytical methods including:

2. 2. The method of claim 1, wherein the cytotoxin is MMAF or MMAE.

3. 3. The method of claim 1 or 2, wherein the ADC is first reduced by the reducing agent and then conjugated with the isotope-labeled cytotoxin.

4. 3. The method of claim 1 or 2, wherein excess isotope-labeled cytotoxin is removed by eluting the sample from a size-exclusion chromatography column prior to the peptide mapping.

5. The method of claim 1 or 2, wherein the peptide mapping comprises the use of liquid chromatography tandem mass spectrometry (LC-MS / MS).

6. 3. The method of claim 1, wherein the peptide mapping comprises denaturing the sample, reducing any remaining disulfide bonds, and alkylating the resulting free sulfhydryls.

7. 3. The method of claim 1 or 2, wherein the peptide mapping comprises enzymatic digestion of the sample to generate isotopically labeled conjugated peptides, and optionally quenching the enzymatic digestion by the addition of a strong acid.

8. 8. The method of claim 7, wherein the isotopically labeled conjugated peptide is ionized, a mass-to-charge ratio is detected, and the detected mass-to-charge ratio for the isotopically labeled conjugated peptide is compared to a non-isotopically labeled conjugated peptide.

9. 3. The method of claim 1 or 2, comprising reacting a cytotoxin with isotopically labeled water to produce said isotopically labeled cytotoxin.

10. 3. The method of claim 1 or 2, wherein the ADC is belantamab mafodotin.

11. A composition comprising an anti-BCMA antibody conjugated to a cytotoxic agent, forming an antibody-drug conjugate (ADC), comprising: The antibody CDRH1 comprising the amino acid sequence of SEQ ID NO: 1; CDRH2 comprising the amino acid sequence of SEQ ID NO: 2; a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3; CDRL1 comprising the amino acid sequence of SEQ ID NO: 4, a CDRL2 comprising the amino acid sequence of SEQ ID NO:5, and CDRL3 comprising the amino acid sequence of SEQ ID NO:6 and the cytotoxic agent is MMAF or MMAE, and and / or the percent drug loading at LC C214 is about 56% to about 80%, and / or the percent drug loading at HC C224 is about 58% to about 81%, and / or the percent drug loading at HC hinge DL2 at HC C230 and HC C233 is about 15% to about 46%, and / or the percent drug loading at HC hinge DL1 at HC C230 or HC C233 is about 11% to about 15%.

12. The composition of claim 11 , wherein the anti-BCMA antibody is belantamab.

13. 12. The composition of claim 11, wherein the ADC is belantamab mafodotin.

14. A pharmaceutical composition comprising the composition of claim 11 and at least one pharmaceutically acceptable excipient.

15. 15. The composition of claim 11 or the pharmaceutical composition of claim 14 for use in the treatment of cancer.

16. 12. Use of the composition of claim 11 in the manufacture of a medicament for use in the treatment of cancer.

17. 1. A method for determining the conjugation level of a cysteine-conjugated antibody-drug conjugate, comprising: a) reducing the antibody-drug conjugate to form a reduced antibody-drug conjugate; b) conjugating the reduced antibody-drug conjugate with an isotopically labeled cytotoxin to form an isotopically labeled antibody-drug conjugate; c) generating an isotopically labeled conjugate peptide from the isotopically labeled antibody-drug conjugate and performing peptide mapping on the isotopically labeled conjugate peptide; d) detecting the mass-to-charge ratio of the isotopically labeled conjugate peptide; and e) comparing the mass-to-charge ratio of the isotopically labeled conjugate peptide with the mass-to-charge ratio of a non-isotopically labeled conjugate peptide to determine the conjugation level of the cysteine-conjugated antibody-drug conjugate.

18. 18. The method of claim 17, wherein the cytotoxin is MMAF or MMAE.

19. 19. The method of claim 17 or 18, wherein the cysteine-conjugated antibody-drug conjugate is first reduced by a reducing agent and then conjugated with the isotope-labeled cytotoxin.

20. 20. The method of claim 19, wherein excess reducing agent is removed by eluting the sample from a size exclusion chromatography column prior to said peptide mapping.

21. 19. The method of claim 17 or 18, wherein the peptide mapping comprises the use of liquid chromatography tandem mass spectrometry (LC-MS / MS).

22. 19. The method of claim 17 or 18, wherein the peptide mapping comprises denaturing the sample, reducing residual disulfide bonds, and alkylating the resulting free sulfhydryls.

23. 19. The method of claim 17 or 18, wherein the peptide mapping comprises enzymatic digestion of the sample to generate isotopically labeled conjugated peptides, and optionally quenching the enzymatic digestion by the addition of strong acid.

24. 19. The method of claim 17 or 18, comprising reacting a cytotoxin with isotopically labeled water to produce said isotopically labeled cytotoxin.

25. 19. The method of claim 17 or 18, wherein the cysteine-conjugated antibody-drug conjugate is belantamab mafodotin.

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