Anti-BCMA antibodies

Abstract

Antibodies and antigen-binding fragments thereof that bind to B cell maturation antigen (BCMA), and methods of using the antibodies and antigen-binding fragments thereof, are provided.

Description

Technical Field

[0001] The present disclosure relates to novel antibodies and antigen-binding fragments thereof that bind to B-cell maturation antigen (BCMA), and methods of using these antibodies and antigen-binding fragments thereof. Background Art

[0002] B cell maturation antigen (BCMA) is a member of the tumor necrosis receptor (TNFR) family and is expressed on cells of the B-cell lineage. BCMA expression is highest on terminally differentiated B cells and is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. BCMA expression is associated with many cancers and plasma cell malignancies such as multiple myeloma (MM). RNA has been commonly detected in multiple myeloma cells, and several researchers have detected BCMA protein on the surface of plasma cells from multiple myeloma patients. Therefore, BCMA has been studied as a potential therapeutic target for multiple myeloma.

[0003] Therefore, there is a need in the art for compositions useful for use in methods of treating multiple myeloma. Summary of the invention

[0004] The present disclosure provides anti-BCMA antibodies and antigen-binding fragments thereof.

[0005] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to BCMA is provided, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising an amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising an amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprises a CDR-H4 sequence comprising a CDR-H5 sequence comprising a CDR-H6 sequence comprising a CDR-H7 sequence comprising a CDR-H8 sequence comprising a CDR-H9 sequence comprising a CDR-H1 sequence comprising a CDR-H2 sequence comprising a CDR-H3 ...1 sequence comprising a CDR-H1 sequence comprising a CDR-H1 sequence comprising a CDR-H1 sequence comprising a CDR-H1 sequence comprising a CDR-H1 sequence comprising a CDR-H1 sequence comprising a CDR-H1 sequence comprising a CDR-H1 sequence comprising a CDR- 1 SSTGX 2 VTPX 3 X 4 The CDR-L1 sequence of the amino acid sequence of YAN (SEQ ID NO: 25), wherein X 1 is R or A, X 2 It is T or A, X 3 is S or G, and X 4 N or Y, containing DNNX 5 X 6 The CDR-L2 sequence of the amino acid sequence of PP (SEQ ID NO: 26), wherein X 5 is S, I, or N, and X 6is R or K, and a CDR-L3 sequence comprising an amino acid sequence of ALX7X8GX9QWV (SEQ ID NO: 27), wherein X 7 W or Y, X 8 is F or Y, and X 9 It is N or G.

[0006] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, wherein the VH domain comprises a CDR-H1 sequence comprising an amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising an amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising an amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprises a CDR-L1 sequence comprising an amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4), CRSSTGTVTPSNYAN (SEQ ID NO: 5), CASSTGAVTPSNYAN (SEQ ID NO: 6), or CASSTGAVTPGYYAN (SEQ ID NO: 7), a CDR-L2 sequence comprising an amino acid sequence of DNNSRPP (SEQ ID NO: 9), DNNIKPP (SEQ ID NO: 10), or DNNNKPP (SEQ ID NO: 11), and a CDR-L3 sequence comprising an amino acid sequence of ALWFGNQWV (SEQ ID NO: 12). ID NO: 13), ALWYGGQWV (SEQ ID NO: 14) or ALYYGGQWV (SEQ ID NO: 15).

[0007] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, wherein the VH domain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:16, and the VL domain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:24.

[0008] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:16, and an antibody light chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:24.

[0009] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain comprising the amino acid sequence of SEQ ID NO:16, and the VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:24.

[0010] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:16 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:24.

[0011] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9), and a CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

[0012] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof has a heavy chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:16, and a light chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:18.

[0013] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:16 and a light chain comprising the amino acid sequence of SEQ ID NO:18.

[0014] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CRSSTGTVTPSNYAN (SEQ ID NO: 5), a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9), and a CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

[0015] In certain exemplary embodiments, the antibody or antigen-binding fragment comprises a heavy chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:16, and a light chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:19.

[0016] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:16 and a light chain comprising the amino acid sequence of SEQ ID NO:19.

[0017] In certain exemplary embodiments, the antibody or antigen-binding fragment comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9), and a CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

[0018] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:16, and an antibody light chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:20.

[0019] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:16 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:20.

[0020] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CASSTGAVTPSNYAN (SEQ ID NO: 6), a CDR-L2 sequence comprising the amino acid sequence of DNNIKPP (SEQ ID NO: 10), and a CDR-L3 sequence comprising the amino acid sequence of ALWYGGQWV (SEQ ID NO: 14).

[0021] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:16, and an antibody light chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:21.

[0022] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:16 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:21.

[0023] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CASSTGAVTPGYYAN (SEQ ID NO: 7), a CDR-L2 sequence comprising the amino acid sequence of DNNNKPP (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of ALYYGGQWV (SEQ ID NO: 15).

[0024] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:16, and an antibody light chain that is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:23.

[0025] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:16 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:23.

[0026] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9), and a CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

[0027] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

[0028] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

[0029] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0030] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof is a multispecific antibody.

[0031] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises one or more full-length antibody heavy chains comprising an Fc region.

[0032] In certain exemplary embodiments, the Fc region is a human IgG1 Fc region.

[0033] In certain exemplary embodiments, the Fc region is a human IgG4 Fc region.

[0034] In certain exemplary embodiments, provided herein is a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present disclosure, and a pharmaceutically acceptable carrier.

[0035] In certain exemplary embodiments, provided herein is an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof.

[0036] In certain exemplary embodiments, provided herein is an expression vector comprising a nucleic acid molecule.

[0037] In certain exemplary embodiments, a host cell comprising a nucleic acid molecule is provided herein. In certain exemplary embodiments, a host cell comprising an expression vector is provided herein. In certain exemplary embodiments, a host cell is provided herein, and the host cell is a mammalian cell.

[0038] In certain exemplary embodiments, provided herein is a method for preparing an antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell under suitable conditions and recovering the antibody or the antigen-binding fragment thereof.

[0039] In certain exemplary embodiments, provided herein is a method of treating or preventing a disease or disorder, the method comprising administering a pharmaceutical composition provided herein to a subject in need thereof.

[0040] In certain exemplary embodiments, provided herein is a method of treating or preventing cancer, the method comprising administering a pharmaceutical composition provided herein to a subject in need thereof.

[0041] In certain exemplary embodiments, provided herein is a method of treating or preventing a plasma cell disorder, the method comprising administering a pharmaceutical composition provided herein to a subject in need thereof.

[0042] In certain exemplary embodiments, provided herein is a method of treating multiple myeloma in a subject, the method comprising administering an antibody or antigen-binding fragment thereof to a subject in need thereof.

[0043] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof is used as a medicament.

[0044] In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof are for use in a method of treating or preventing a disease or disorder.

[0045] In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof are for use in a method of treating or preventing cancer.

[0046] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof is for use in a method of treating or preventing multiple myeloma.

[0047] In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof are for use in a method of treating or preventing plasma cell malignancies.

[0048] The summary of the disclosure described above is non-limiting, and other features and advantages of the disclosed antigen binding proteins and methods will be apparent from the following description of the figures, detailed description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments and in conjunction with the accompanying drawings. This patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fee.

[0050] Figure 1A Seven anti-BCMA antibody VL domain variants and their percent sequence identity to human germline VL are shown.

[0051] Figure 1B Shown is a sequence alignment of the parental VL domain and seven VL domain variants.

[0052] Figure 1C Shown is a sequence alignment of the CA10 parental VL domain and the human germline VL domain.

[0053] Figure 2 is a table showing the expression and purity of anti-BCMA antibody variants. Purity was determined using SDS-PAGE and SEC.

[0054] Figure 3 Cross-reactivity of clonal CA10 variants with human and cynomolgus BCMA proteins is shown.

[0055] Figure 4 The binding between anti-BCMA mAb antibody and BCMA protein on the surface of HEK cell membrane is shown.

[0056] Figure 5A-Figure 5B Anti-BCMA mAb antibody variants are shown ( Figure 5A ) and parental antibody ( Figure 5B ) and the BCMA protein on the surface of MM1R cell membrane.

[0057] Figure 6 The sensorgrams and affinity constants between anti-BCMA mAb antibodies and human BCMA Avi-His protein are shown.

[0058] Figure 7The sensorgrams and affinity constants between anti-BCMA mAb antibodies and human BCMA DTA-His protein are shown.

[0059] Figure 8 Sensorgrams and affinity constants between anti-BCMA mAb antibodies and cynomolgus monkey BCMA Avi-His protein are shown.

[0060] Fig. 9 The sensorgrams and affinity constants between anti-BCMA mAb antibodies and cynomolgus monkey BCMA DTA-His protein are shown. DETAILED DESCRIPTION

[0061] Before describing the present disclosure, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0063] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, exemplary methods and materials are now described.All publications mentioned herein are incorporated by reference in their entirety.

[0064] As used herein, the term "BCMA" refers to B cell maturation antigen. BCMA (also referred to as TNFRSF17, BCM or CD269) is a member of the tumor necrosis receptor (TNFR) family, and is mainly expressed on terminally differentiated B cells (such as memory B cells and plasma cells). Its ligand is called the B cell activator (BAFF) and proliferation-inducing ligand (APRIL) of the TNF family. BCMA participates in mediating the survival of plasma cells to maintain long-term humoral immunity. The BCMA gene is encoded on chromosome 16, producing a primary mRNA transcript (NCBI accession number NM_001192.2) of 994 nucleotides in length, which encodes a protein (NP_001183.2) of 184 amino acids. A second antisense transcript derived from the BCMA locus has been described, which may play a role in regulating BCMA expression. (Laabi Y. et al., Nucleic Acids Res., 1994, 22: 1147-1154). Other transcript variants have been described, the significance of which is unclear (Smirnova AS et al. Mol Immunol., 2008, 45(4): 1179-1183). A second isoform has been identified, also known as TV4 (Uniprot identifier Q02223-2). As used herein, "BCMA" includes proteins comprising mutations, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type BCMA.

[0065] As used herein, the term "antibody" or "antigen binding protein" refers to an immunoglobulin molecule that specifically binds to or immunoreacts with an antigen or epitope (e.g., a BCMA antigen or epitope), and includes polyclonal and monoclonal antibodies, as well as functional antibody fragments thereof, including but not limited to antigen binding fragment (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain variable fragments (scFv) and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term "antibody" includes genetically engineered or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, meditope-enabled antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, double antibodies, three antibodies, four antibodies, tandem double scFv, tandem three scFv), etc. Unless otherwise specified, the term "antibody" should be understood to cover its functional antibody fragments. As used herein, the term "functional antibody fragment" refers to an antibody fragment that has at least 80%, at least 85%, at least 90% or at least 95% of the affinity of the antibody of interest from which the fragment is derived.

[0066] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid sequence within the variable region of an antibody that confers antigen specificity and binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. It is known that "framework region" or "FR" refers to the non-CDR portion of the variable region of the heavy and light chains in the art. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each light chain variable region.

[0067] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB [Journal of Molecular Biology] 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. [Journal of Molecular Biology] 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography [Antibody-antigen interactions: contact analysis and binding site topography], J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); and Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70 (AHo numbering scheme).

[0068] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions indicated by inserting letters (e.g., "30a"), and deletions are also shown in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0069] The "CDR" or "complementarity determining region" or individually designated CDR (e.g., "CDR-H1", "CDR-H2", "CDR-H3") of a given antibody or region thereof (e.g., its variable region) should be understood to encompass the (or specific) complementary determining region defined by any known scheme. Similarly, the "FR" or "framework region" or individually designated FR (e.g., "FR-H1", "FR-H2") of a given antibody or region thereof (e.g., its variable region) should be understood to encompass the (or the designated) framework region defined by any known scheme. In some cases, schemes for identifying specific CDRs or FRs are specified, such as CDRs defined by IMGT, Kabat, Chothia, AbM or contact methods. In other cases, specific amino acid sequences of CDRs or FRs are given. Unless otherwise specified, all specific CDR amino acid sequences mentioned in this disclosure are IMGT CDRs. However, the present disclosure also encompasses alternative CDRs defined by other schemes, such as those determined by the abYsis Key Annotation (website: abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0070] As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. The human mAb disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo), such as those in CDR and particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs formed by transplanting CDR sequences derived from germline of another mammalian species (e.g., mice) onto human FR sequences. The term is included in antibodies recombinantly produced in non-human mammals or non-human mammalian cells. The term is not intended to include antibodies separated or produced from human subjects.

[0071] As used herein, the term "multispecific antigen binding molecule" refers to bispecific, trispecific or multispecific antigen binding molecules and antigen binding fragments thereof. Multispecific antigen binding molecules may have specificity for multiple different epitopes of a target polypeptide, or may contain antigen binding domains that are specific for epitopes of more than one target polypeptide. Multispecific antigen binding molecules can be single multifunctional polypeptides, or can be multimeric complexes of two or more polypeptides covalently or non-covalently bound to each other. The term "multispecific antigen binding molecule" includes antibodies of the present disclosure that can be connected or co-expressed with another functional molecule (e.g., another peptide or protein). For example, antibodies or fragments thereof can be functionally connected (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities (e.g., proteins or fragments thereof) to produce bispecific or multispecific antigen binding molecules with a second binding specificity. According to the present disclosure, the term "multispecific antigen binding molecule" also includes bispecific, trispecific or multispecific antibodies or antigen binding fragments thereof. In certain exemplary embodiments, the antibodies of the present disclosure are functionally connected to another antibody or antigen binding fragment thereof to produce bispecific antibodies with a second binding specificity.

[0072] As used herein, the terms "specifically binds", "specifically binding", "binding specificity" or "specific recognition" refer to an antigen binding protein or antigen binding fragment thereof that exhibits significant affinity for an antigen (e.g., BCMA antigen) and does not exhibit significant cross-reactivity to targets other than BCMA proteins. As used herein, the term "affinity" refers to the strength of the interaction between an antigen binding protein or antigen binding fragment thereof antigen binding site and the epitope to which it binds. In certain exemplary embodiments, affinity is measured by surface plasmon resonance (SPR), such as in a Biacore instrument. As will be readily appreciated by those skilled in the art, antigen binding protein affinity can be reported as a dissociation constant (KD) expressed in molar concentration (M). The KD value of the antigen binding protein or antigen binding fragment thereof disclosed herein is about 10 -6 M to about 10 -12 M range (i.e., low micromolar to picomolar range), about 10 -7 M to 10 -11 M, about 10 -8 M to about 10 -10 M, about 10 -9 M. In certain embodiments, the antigen binding protein or antigen binding fragment thereof has about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10-10 M, 10 -11 M or 10 -12 In certain embodiments, the antigen binding protein or antigen binding fragment thereof has a binding affinity of about 10 -7 M to about 10 -9 Binding affinities in the nanomolar range.

[0073] Specific binding can be determined according to any art-recognized means of determining such binding. In some embodiments, specific binding is determined by competitive binding assays (e.g., ELISA) or Biacore assays. In certain embodiments, the assay is performed at about 20°C, 25°C, 30°C, or 37°C.

[0074] As used herein, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., a separated binding polypeptide provided herein) present outside the body to a patient, for example, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, the substance is typically administered after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the substance is typically administered before the onset of the disease or its symptoms, and can be continued for a long time to delay or reduce the appearance or degree of disease-related symptoms.

[0075] As used herein, the term "composition" is intended to encompass a product containing the specified ingredients (e.g., an isolated binding polypeptide provided herein), optionally in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients, optionally in the specified amounts.

[0076] "Effective amount" means an amount of an active pharmaceutical agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to achieve a desired physiological result in an individual in need of the agent. The effective amount may vary between individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the assessment of the individual's medical symptoms, and other relevant factors.

[0077] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, as used herein, the term "subject" refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, untamed animals, farm animals, sports animals, and pets.

[0078] As used herein, the term "therapy" refers to any regimen, method and / or agent that can be used to prevent, manage, treat and / or improve a disease or symptoms associated therewith. In some embodiments, the term "therapy" refers to any regimen, method and / or agent that can be used to regulate the immune response of a subject to an infection or symptoms associated therewith. In some embodiments, the term "therapy" refers to biological therapies known to those skilled in the art (such as medical personnel), supportive therapies, and / or other therapies that can be used to prevent, manage, treat and / or improve a disease or symptoms associated therewith. In other embodiments, the term "therapy" refers to biological therapies known to those skilled in the art (such as medical personnel), supportive therapies, and / or other therapies that can be used to regulate the immune response to an infection or symptoms associated therewith in a subject.

[0079] As used herein, the terms "treat, treatment and treating" refer to reducing or improving the progression, severity and / or duration of a disease or symptoms associated therewith by administering one or more therapies (including but not limited to administering one or more prophylactic or therapeutic agents, such as the isolated binding polypeptides provided herein). As used herein, the term "treat" may also refer to changing the course of the subject being treated. The therapeutic effects of treatment include but are not limited to preventing the occurrence or recurrence of the disease, alleviation of one or more symptoms, weakening of the direct or indirect pathological consequences of the disease, reduction in the rate of disease progression, improvement or alleviation of the disease state, and alleviation or improvement of the prognosis.

[0080] The term "about" or "approximately" means within about 20% (eg, about 10%, about 5%, or about 1% or less) of a given value or range.

[0081] Anti-BCMA Antibodies

[0082] In one aspect, the disclosure provides antibodies or antigen-binding fragments thereof that have binding specificity for BCMA.

[0083] Exemplary anti-BCMA antibodies or antigen-binding fragments thereof CDRs are listed in Table 1 below. Exemplary anti-BCMA antibodies or antigen-binding fragments thereof variable heavy chain (VH) and variable light chain (VL) domains are listed in Table 2 below.

[0084] Table 1. Antibody heavy and light chain CDR regions

[0085]

[0086]

[0087]

[0088] Table 2. Antibody VH and VL sequences

[0089]

[0090]

[0091] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1 sequence comprising an amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising an amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising an amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and the VL domain comprising a CDR-H4 sequence comprising a CDR-H5 sequence comprising a CDR-H6 sequence comprising a CDR-H7 sequence comprising a CDR-H8 sequence comprising a CDR-H9 sequence comprising a CDR-H1 sequence comprising a CDR-H2 sequence comprising a CDR-H3 ... 1 SSTGX 2 VTPX 3 X 4 The CDR-L1 sequence of the amino acid sequence of YAN (SEQ ID NO: 25), wherein X 1 is R or A, X 2 It is T or A, X 3 is S or G, and X 4 N or Y, containing DNNX 5 X 6 The CDR-L2 sequence of the amino acid sequence of PP (SEQ ID NO: 26), wherein X 5 is S, I, or N, and X 6 is R or K, and a CDR-L3 sequence comprising an amino acid sequence of ALX7X8GX9QWV (SEQ ID NO: 27), wherein X 7 W or Y, X 8 is F or Y, and X 9 It is N or G.

[0092] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and a VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4), CRSSTGTVTPSNYAN (SEQ ID NO: 5), CASSTGAVTPSNYAN (SEQ ID NO: 6), or CASSTGAVTPGYYAN (SEQ ID NO: 7), a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9), DNNIKPP (SEQ ID NO: 10), or DNNNKPP (SEQ ID NO: 11), and a CDR-H3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 12). ID NO: 13), ALWYGGQWV (SEQ ID NO: 14) or ALYYGGQWV (SEQ ID NO: 15).

[0093] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2), and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3), and a VL domain comprising a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4), a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9), and a CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

[0094] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:16 and a VL domain that is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:16 and the VL domain is SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:24. The amino acid sequence of NO:24 is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical.

[0095] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain, the VH domain being at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:16, and the VL domain being at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:16 The amino acid sequence of NO:24 is at least about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical.

[0096] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 16 and a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 24.

[0097] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and an antibody light chain comprising the amino acid sequence of SEQ ID NO:2.

[0098] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

[0099] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0100] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is an anti-BCMA antibody-drug conjugate.

[0101] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is a monospecific antibody.

[0102] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is a bispecific antibody.

[0103] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is a trispecific antibody.

[0104] In certain embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is a multispecific antibody.

[0105] Immune cell conjugates

[0106] The anti-BCMA antigen binding proteins disclosed herein can exist in the form of immune cell engagement. An immune cell engager is an antigen binding protein comprising at least one binding domain capable of binding to a cell surface protein of an immune cell and at least one binding domain that binds to a separate cell surface protein (e.g., a surface protein on a tumor cell, such as BCMA). Binding domains specific to cell surface proteins of immune cells can specifically recruit immune cells to target tumor cells to be eliminated. Examples of immune cells that can be recruited include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, monocytes, and macrophages. Examples of surface proteins that can be used to recruit immune cells include, but are not limited to, CD3, TCRa, TCRp, CD16, NKG2D, CD89, CD64, and CD32a.

[0107] Expression of antigen binding proteins

[0108] In one aspect, polynucleotides encoding binding proteins disclosed herein (eg, antigen binding proteins and antigen binding fragments thereof) are provided. Methods of preparing binding proteins are also provided, comprising expressing these polynucleotides.

[0109] Typically, the polynucleotide encoding the binding protein disclosed herein is inserted into an expression vector to be introduced into a host cell, which can be used to produce the desired amount of these binding proteins. Therefore, in certain aspects, the disclosure provides expression vectors comprising the polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.

[0110] The term "vector" or "expression vector" as used herein is used to refer to a vector used to introduce a desired gene into a cell and express it in the cell according to the present disclosure. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. Typically, vectors compatible with the present disclosure will include a selection marker, appropriate restriction sites (to facilitate cloning of the desired gene), and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0111] For the purpose of the present disclosure, a variety of expression vector systems can be used. For example, a class of vectors utilizes DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus. Other categories relate to the use of polycistronic systems with internal ribosome binding sites. In addition, the cells that have been integrated into their chromosomes by DNA can be selected by introducing one or more markers that allow the selection of transfected host cells. The marker can provide the original nutrition to the auxotrophic host, provide biocide resistance (such as antibiotics) or resistance to heavy metals such as copper. The selective marker gene can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. The optimal synthesis of mRNA may also require other elements. These elements may include signal sequence, splicing signal and transcription promoter, enhancer and termination signal. In certain embodiments, the cloned variable region gene is inserted into the expression vector together with the heavy chain and light chain constant region genes (such as human constant region genes) synthesized as described above.

[0112] In other embodiments, polycistronic constructs can be used to express binding proteins. In this expression system, multiple target gene products, such as heavy and light chains of antibodies, can be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will recognize that this expression system can be used to effectively produce all polypeptides disclosed in this application.

[0113] More generally, once a vector or DNA sequence encoding a binding protein (e.g., an antibody or fragment thereof) is prepared, the expression vector can be introduced into an appropriate host cell. That is, the host cell can be transformed. The introduction of a plasmid into a host cell can be achieved by various techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and complete viral infection. See Ridgway, AAG "Mammalian Expression Vectors" Chapter 24.2, Pages 470-472 Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, Massachusetts. 1988). Plasmids can be introduced into a host by electroporation. The transformed cells are grown under conditions suitable for producing light and heavy chains, and their heavy and / or light chain protein synthesis is determined. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence activated cell sorter analysis (FACS), immunohistochemistry, etc.

[0114] As used herein, the term "transformation" should be used in a broad sense to refer to the introduction of DNA into a recipient host cell, thereby changing the genotype.

[0115] Likewise, "host cells" refer to cells transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. When describing the process of isolating polypeptides from recombinant hosts, unless otherwise specifically stated, the terms "cells" and "cell culture" can be used interchangeably to indicate the source of the antibody. In other words, recovering polypeptides from "cells" may mean recovering from centrifuged whole cells, from the supernatant of lysed cell cultures, or from cell cultures containing culture medium and suspended cells.

[0116] In one embodiment, the host cell line used for antibody expression is of mammalian origin. One skilled in the art can determine the specific host cell line that is most suitable for expressing the desired gene product therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HEK (human kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney). In one embodiment, the cell line may cause the expressed antibody to undergo glycosylation changes, such as afucosylation (e.g., (Crucell) or FUT8 knockout CHO cell line ( Cells) (Biowa, Princeton, NJ)). In one embodiment, NSO cells can be used. CHO cells are particularly useful. Host cell lines are generally available through commercial services, such as the American Tissue Culture Collection, or from authors of published literature.

[0117] In vitro production allows for scale-up to produce large quantities of desired polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogenous suspension cultures (e.g., in airlift reactors or continuous stirred reactors), or immobilized or embedded cell cultures (e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges). If necessary and / or desired, solutions of polypeptides may be purified by conventional chromatography (e.g., gel filtration, ion exchange chromatography, DEAE-cellulose chromatography and / or (immuno) affinity chromatography).

[0118] Genes encoding the binding proteins characterized in the present disclosure can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard, it should be recognized that various unicellular non-mammalian microorganisms (such as bacteria) can also be transformed, that is, those that can grow in culture or fermentation. Easily transformed bacteria include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella strains; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. It should be further recognized that when expressed in bacteria, the binding protein can become part of the inclusion body. In some embodiments, the binding protein is subsequently separated, purified and assembled into a functional molecule. In some embodiments, the binding protein of the present disclosure is expressed in a bacterial host cell. In some embodiments, the bacterial host cell is transformed with an expression vector comprising a nucleic acid molecule encoding the binding protein of the present disclosure.

[0119] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of eukaryotic microorganisms, although many other strains are commonly used. For expression in the genus Saccharomyces, plasmid YRp7 (Stinchcomb et al., Nature [Nature], 282: 39 (1979); Kingsman et al., Gene [Gene], 7: 141 (1979); Tschemper et al., Gene [Gene], 10: 157 (1980)) is generally used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants (such as ATCC No. 44076 or PEP4-1) that lack the ability to grow in tryptophan (Jones, Genetics [Genetics], 85: 12 (1977)). Then, the presence of trp1 damage (as a feature of the yeast host cell genome) provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0120] Methods of administering antigen binding proteins

[0121] Methods for preparing and administering antigen binding proteins (e.g., anti-BCMA antibodies or antigen binding fragments thereof disclosed herein) to subjects are well known or easily determined by those skilled in the art. The route of administration of the antigen binding proteins disclosed herein may be oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. All of these forms of administration are clearly considered to be within the scope of the present disclosure, and one form of administration will be a solution for injection, particularly a solution for intravenous or intraarterial injection or infusion. Typically, suitable pharmaceutical compositions for injection may include a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), and the like. However, in other methods compatible with the teachings herein, the modified antibody may be delivered directly to the site of the bad cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0122] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include but are not limited to 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements, such as those based on Ringer's dextrose, etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, etc., may also be present. More specifically, the pharmaceutical composition suitable for injection includes sterile aqueous solution (in the case of water-soluble) or dispersion and sterile powder for the temporary preparation of sterile injectable solution or dispersion. In such cases, the composition must be sterile and should be in a fluid state, to the extent that it is easy to extract with a syringe. It should be stable under manufacturing and storage conditions, and should also be properly preserved to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained by, for example, using a coating (such as lecithin), maintaining the required particle size in the case of a dispersion, and using a surfactant.

[0123] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). Isotonic agents, for example, sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride can also be included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption (e.g., aluminum monostearate and gelatin).

[0124] In any case, sterile injectable solutions can be prepared in the following manner: active compound (e.g., modified binding polypeptide itself or in combination with other active agents) is mixed in the required amount in an appropriate solvent, followed by filtration sterilization, the solvent having a composition or composition combination listed herein as required. Typically, dispersions are prepared by mixing active compound in a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for preparing sterile injectable solutions, preparation methods generally include vacuum drying and freeze drying, which methods produce powders of active ingredients plus any additional required ingredients from previously sterile filtered solutions. According to methods known in the art, injection preparations are processed, filled into containers (such as ampoules, bags, bottles, syringes or vials), and sealed under aseptic conditions. In addition, these preparations can be packaged and sold in the form of kits, such as those described in co-pending USSN09 / 259,337 and USSN09 / 259,338, each of which is incorporated herein by reference. Such articles of manufacture may include labels or package inserts indicating that the composition may be used to treat a subject suffering from or susceptible to an autoimmune or neoplastic disorder.

[0125] The effective dose of the disclosed compositions for treating the above-mentioned conditions varies according to many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other agents administered, and whether the treatment is preventive or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, may also be treated. The therapeutic dose may be adjusted stepwise using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0126] For passive immunization with antigen binding proteins, the dosage range can be, for example, about 0.0001 to 100 mg / kg, and more typically 0.01 to 5 mg / kg host body weight (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.). For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg, such as at least 1 mg / kg. Intermediate doses within the above range are also intended to be within the scope of the present disclosure. Subjects can apply such dosages every day, every other day, every week, or according to any other schedule determined by empirical analysis. Exemplary treatments require multiple doses to be applied over an extended period of time (e.g., at least six months). Additional exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. Exemplary dosage regimens include 1-10 mg / kg or 15 mg / kg for several consecutive days, 30 mg / kg every other day, or 60 mg / kg per week. In some methods, two or more antigen binding proteins with different binding specificities are administered simultaneously, in which case the dosage of each antigen binding protein administered falls within the ranges indicated.

[0127] Antigen binding proteins as described herein can be administered multiple times. The interval between single doses can be weekly, monthly or annual. The interval can also be irregular, as indicated by measuring the blood level of the modified binding polypeptide or antigen of the patient. In some methods, the dosage is adjusted to achieve a plasma-modified antigen binding protein concentration of 1-1000 μg / ml, and in other methods, 25-300 μg / ml is reached. Alternatively, the antigen binding protein can be administered as a sustained release formulation, in which case it is necessary to administer less frequently. For antigen binding proteins, dosage and frequency vary depending on the half-life of the antigen binding protein in the patient. Typically, humanized antibodies show the longest half-life, followed by chimeric antibodies and non-human antibodies.

[0128] The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, a composition containing the antigen-binding proteins of the present invention or its mixture is applied to a patient who is not yet in a disease state to enhance the patient's resistance. Such an amount is defined as a "preventive effective dose". In this use, the exact amount also depends on the patient's health and general immunity, but generally ranges from 0.1 to 25 mg / agent, especially 0.5 to 2.5 mg / agent. A relatively low dose is administered at relatively low frequency intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer a relatively high dose (e.g., about 1 to 400 mg / kg antibody / agent, 5 to 25 mg doses are more commonly used for radioimmunoconjugates, and higher doses are more commonly used for cytotoxin-drug modified antibodies) at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete improvement of disease symptoms. Thereafter, a preventive regimen can be administered to the patient.

[0129] The antigen binding proteins described herein can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic). 90 An effective single therapeutic dose (i.e., a therapeutically effective amount) of a Y-labeled modified antibody ranges from about 5 to about 75 mCi, such as from about 10 to about 40 mCi. 131 An effective single therapeutic non-myeloablative dose of the modified antibody of I ranges from about 5 to about 70 mCi, such as from about 5 to about 40 mCi. 131 An effective single therapeutic myeloablative dose of an antibody labeled with I (i.e., may require autologous bone marrow transplantation) ranges from about 30 to about 600 mCi, such as from about 50 to less than about 500 mCi. After incorporation of the chimeric antibody, due to the longer circulating half-life relative to the murine antibody, the chimeric antibody may be administered to the patient. 131 An effective single therapeutic non-myeloablative dose of an I-labeled chimeric antibody ranges from about 5 to about 40 mCi, such as less than about 30 mCi. 111 In labeling, imaging standard doses are typically less than about 5 mCi.

[0130] Although antigen binding proteins can be applied in the manner just described above, it must be emphasized that in other embodiments, antigen binding proteins can be applied to otherwise healthy patients as first-line therapy. In such embodiments, antigen binding proteins can be applied to patients with normal or average red bone marrow reserves and / or patients who have not been and currently are not receiving one or more other therapies. As used herein, modified antibodies or fragments thereof are used in combination with adjuvant therapy or in combination to mean sequential, simultaneous, coexistent, parallel, concomitant or concurrent administration or application of therapy and disclosed antibodies. It will be appreciated by those skilled in the art that the administration or application of the various components of the combined treatment regimen can be timed to enhance the overall effectiveness of the treatment. Skilled technicians (e.g., experienced oncologists) will be able to easily discern effective combined treatment regimens based on the selected adjuvant therapy and the teachings of this specification without excessive experiments.

[0131] As previously mentioned, the antigen binding proteins, immunoreactive fragments or recombinants disclosed herein can be administered in a pharmaceutically effective amount for in vivo treatment of mammalian disorders. In this regard, it should be recognized that the disclosed antigen binding proteins will be formulated to facilitate administration of the active ingredient and enhance its stability.

[0132] According to the pharmaceutical composition disclosed herein, a pharmaceutically acceptable non-toxic sterile carrier is generally included, such as physiological saline, non-toxic buffer, preservative, etc. For the purpose of the present application, the pharmaceutically effective amount of the modified antigen-binding protein, its immunoreactive fragment or recombinant conjugated or not conjugated to the therapeutic agent should be understood as being sufficient to achieve effective combination with the antigen and achieve the amount of benefit (such as improving the symptoms of a certain disease or disorder or detecting a certain substance or cell). In the case of tumor cells, the modified binding polypeptide is generally able to interact with the selected immunoreactive antigen on the neoplastic or immunoreactive cells, and increase the number of deaths of those cells. Of course, the pharmaceutical composition disclosed herein can be administered in a single dose or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0133] In keeping with the scope of this disclosure, the antigen-binding proteins of this disclosure can be applied to humans or other animals in an amount sufficient to produce a therapeutic or preventive effect according to the above-mentioned treatment methods. The antigen-binding proteins of this disclosure can be applied to this human or other animal in a conventional dosage form (prepared according to known techniques by combining the antibodies of this disclosure with conventional pharmaceutically acceptable carriers or diluents). Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient combined therewith, the route of administration, and other well-known variables. Those skilled in the art will further understand that a mixture comprising one or more of the binding polypeptides described in this disclosure can be proven to be particularly effective.

[0134] The biological activity of the pharmaceutical composition defined herein can be determined, for example, by cytotoxicity assays, as described in the following examples, WO 99 / 54440 or Schlereth et al. (Cancer Immunol. Immunother. [Cancer Immunology and Immunotherapy] 20 (2005), 1-12). As used herein, "efficacy" or "in vivo efficacy" refers to the response to the therapy of the pharmaceutical composition of the present invention, using, for example, standardized NCI response criteria. The success or in vivo efficacy of a therapy using the pharmaceutical composition of the present invention refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to cause the desired effect, i.e., the depletion of pathological cells, such as tumor cells. In vivo efficacy can be monitored by established standard methods for the corresponding disease entity, including but not limited to white blood cell count, differentiation, fluorescence activated cell sorting, bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used. In addition, computer-assisted tomography, x-ray, or magnetic resonance tomography (eg, for response assessment based on National Cancer Institute criteria) can be used [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos G P. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999 Apr;17(4):1244]), positron emission tomography, leukocyte count, differentiation, fluorescence-activated cell sorting, bone marrow aspirate, lymph node biopsy / histology, and various lymphoma-specific clinical chemistry parameters (e.g., lactate dehydrogenase), as well as other established standard methods.

[0135] Examples

[0136] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and use the methods and compositions described herein, and are not intended to limit the scope of their invention as determined by the inventors. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0137] Example 1. Anti-BCMA Antibodies

[0138] Generation of anti-human BCMA antibodies

[0139] A group of 15 8-week-old female Trianni human Ig transgenic mice were implanted subcutaneously with estradiol pellets and injected with 100 μg of anti-mouse CD25 antibody three weeks later. One week later, all mice were intraperitoneally administered approximately 5 x 10 6 (5 million) 300.19 cells overexpressing human BCMA were used for primary immunization. Subsequently, three immunizations were performed with the extracellular domain (ECD) of human BCMA conjugated to diphtheria toxin A (DTA) protein, and two immunizations were performed with the ECD of cynomolgus monkeys conjugated to diphtheria toxin A (DTA) protein in Sigma adjuvant. After the last immunization, the anti-BCMA specific IgG titer in the blood was measured by ELISA using human BCMA-ECD protein as an antigen to evaluate the immune response. Mice that produce high anti-BCMA antibody titers (1: 100000 or higher) are selected for hybridoma generation. Four days before these mice were sacrificed to harvest spleens to prepare fusion cells, these mice were boosted with a combination of the ECD domains of human and cynomolgus BCMA-DTA proteins without adjuvant.

[0140] The fusion and generation of hybridomas were performed according to existing protocols (Yale J Biol Med, 1981, 54(5) 387-402) and Gefter et al. (Somatic Cell Genet, 1977, 3(2) 231-236). A single cell suspension of splenocytes was mixed with F0 myeloma cells at a ratio of 5:1 in a 50 ml conical polypropylene tube and the cells were washed twice with serum-free IMDM. To this end, 1 mL of polyethylene glycol 1500 (PEG) (Roche Applied Science, Indianapolis, Indiana), preheated at 37° C., was slowly added to the cell pellet over a period of about 1 minute while gently shaking the tube. The cells were incubated in PEG for one minute, and then 1 mL of serum-free IMDM was added dropwise to the pellet over a period of 30 seconds, and then 9 mL of serum-free IMDM was added to the pellet for one minute. The test tube was centrifuged at room temperature at 350 x g for 10 minutes, and the supernatant was aspirated. The pellet was resuspended in 200 mL of filtered complete hybridoma production medium (supplemented with 10% FBS (Hyclone), 1 x non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 1 x penicillin-streptomycin (Gibco), hybridoma fusion and cloning factor (Roche) and 1 x HAT (Sigma) IMDM (Hyclone)). The cells were transferred to a T-150 suspension cell flask and stored in an incubator at 37 ° C, 7% CO2.

[0141] The next day, the cells in the T-150 flask were centrifuged and the supernatant removed. The cells were carefully transferred to a hybridoma semisolid selection and cloning medium (Molecular Devices) containing anti-mouse IgG conjugated to FITC (Molecular Devices). The cells were gently mixed and the semisolid medium was inoculated into a six-well plate at 2 mL / well. The plates were incubated at 37° C., 7% CO2 to promote the growth of hybridomas. After incubation for about 10 days, the hybridomas secreting antibodies reacted with anti-mouse IgG conjugated to FITC and produced a green halo around the hybridomas. These hybridomas were detected by Clonepix (Molecular Devices), and single colonies were picked into the wells of multiple 96-well plates. The 96-well plates containing cloned hybridomas were incubated for another 10 days at 37° C., 7% CO2.

[0142] Anti-BCMA antibody sequences

[0143] The heavy chain variable region is shown below:

[0144] QVQLVESGGGVVQPGRSLRLSCAAS GFTFSNFGMH WVRQAPGRGL EWVA VIWSDETNR YYADSVKGRFTVSRDNVKSTVYLQMNSLISEDTAV YYCAR DQQYCSSDSCFTWFDP WGQGTLVTVSS (SEQ ID NO: 16). The underlined residues are CDRs 1, 2 and 3.

[0145] CDR-H1 is shown as GFTFSNFGMH (SEQ ID NO: 1). CDR-H2 is shown as VIWSDETNR (SEQ ID NO: 2). CDR-H3 is shown as DQQYCSSDSCFTWFDP (SEQ ID NO: 3).

[0146] The parent light chain variable region is shown below:

[0147] QVVVTQESALTTSPGETVTLT CRSSTGTVTPSNYAN WVQEKPDHFFT GLIG DNNSRPP GVPARFSASLIGDKAALTITGAQTEDEAMYFC ALWFQ FGGGTKVTVL (SEQ ID NO: 17). The underlined residues are CDRs 1, 2, and 3.

[0148] CDR-L1 is shown as CRSSTGTVTPSNYAN (SEQ ID NO: 5). CDR-L2 is shown as DNNSRPP (SEQ ID NO: 9). CDR-L3 is shown as ALWFGNQWV (SEQ ID NO: 13).

[0149] The light chain variable region of variant 1 is shown below:

[0150] QTVVTQEPSLTVSPGGTVTLT CRSSTGTVTPSNYAN WVQQKPDHFFT GLIG DNNSRPP GVPARFSASLIGDKAALTLSGVQPEDEAEYYC ALWFQ FGGGTKLTVL (SEQ ID NO: 18). The underlined residues are CDRs 1, 2, and 3.

[0151] CDR-L1 is shown as CRSSTGTVTPSNYAN (SEQ ID NO: 5). CDR-L2 is shown as DNNSRPP (SEQ ID NO: 9). CDR-L3 is shown as ALWFGNQWV (SEQ ID NO: 13).

[0152] The light chain variable region of variant 2 is shown below:

[0153] QTVVTQEPSLTVSPGGTVTLT CASSTGTVTPSNYAN WVQQKPGQAP RGLIG DNNSRPP GTPARFSASLLGGKAALTLSGVQPEDEAEYYC ALWFG FGGGTKLTVL (SEQ ID NO: 19). The underlined residues are CDRs 1, 2, and 3.

[0154] CDR-L1 is shown as CASSTGTVTPSNYAN (SEQ ID NO: 4). CDR-L2 is shown as DNNSRPP (SEQ ID NO: 9). CDR-L3 is shown as ALWFGNQWV (SEQ ID NO: 13).

[0155] The light chain variable region of variant 3 is shown below:

[0156] QTVVTQEPSLTVSPGGTVTLT CASSTGAVTPSNYAN WVQQKPGQAP RGLIG DNNIKPP WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC ALWYG GQW FGGGTKLTVL (SEQ ID NO: 20). The underlined residues are CDRs 1, 2, and 3.

[0157] CDR-L1 is shown as CASSTGAVTPSNYAN (SEQ ID NO: 6). CDR-L2 is shown as DNNIKPP (SEQ ID NO: 10). CDR-L3 is shown as ALWYGGQWV (SEQ ID NO: 14).

[0158] The light chain variable region of variant 4 is shown below:

[0159] QTVVTQEPSLTVSPGGTVTLT CASSTGAVTPGYYAN WVQQKPGQAP RGLIG DNNNKPP WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC ALYYG FGGGTKLTVL (SEQ ID NO: 21). The underlined residues are CDRs 1, 2, and 3.

[0160] CDR-L1 is shown as CASSTGAVTPGYYAN (SEQ ID NO: 7). CDR-L2 is shown as DNNNKPP (SEQ ID NO: 11). CDR-L3 is shown as ALYYGGQWV (SEQ ID NO: 15).

[0161] The light chain variable region of variant 5 is shown below:

[0162] QTVVTQEPSLTVSPGGTVTLT CASSTGAVTSGYYAN WVQQKPGQAP RALIG DNNIKPS WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC ALYYG FGGGTKLTVL (SEQ ID NO: 22). The underlined residues are CDRs 1, 2, and 3.

[0163] CDR-L1 is shown as CASSTGAVTSGYYAN (SEQ ID NO: 8). CDR-L2 is shown as DNNIKPS (SEQ ID NO: 12). CDR-L3 is shown as ALYYGGQWV (SEQ ID NO: 15).

[0164] The light chain variable region of variant 6 is shown below:

[0165] QTVVTQEPSLTVSPGGTVTLT CASSTGTVTPSNYAN WVQQKPGQFP RGLIG DNNSRPP GTPARFSASLLGGKAALTLSGVQPEDEAEYYC ALWFG FGGGTKLTVL (SEQ ID NO: 23). The underlined residues are CDRs 1, 2, and 3.

[0166] CDR-L1 is shown as CASSTGTVTPSNYAN (SEQ ID NO: 4). CDR-L2 is shown as DNNSRPP (SEQ ID NO: 9). CDR-L3 is shown as ALWFGNQWV (SEQ ID NO: 13).

[0167] The light chain variable region of variant 7 is shown below:

[0168] QTVVTQEPSLTVSPGGTVTLT CASSTGTVTPSNYAN WVQQKPGQAF RGLIG DNNSRPP GTPARFSASLLGGKAALTLSGVQPEDEAEYYC ALWFGFGGGTKLTVL (SEQ ID NO: 24). The underlined residues are CDRs 1, 2, and 3.

[0169] CDR-L1 is shown as CASSTGTVTPSNYAN (SEQ ID NO: 4). CDR-L2 is shown as DNNSRPP (SEQ ID NO: 9). CDR-L3 is shown as ALWFGNQWV (SEQ ID NO: 13).

[0170] Example 2. Expression and purification of anti-BCMA antibodies

[0171] Expi293F TM The cells were subcultured until the cell density reached about 3-5 x 10 6 Cells were seeded to a final density of 2.5-3 x 10 6 viable cells / mL and then allowed to grow overnight. TM Dilute the cells to a final density of 3 x 10 6 viable cells / mL and mix the cells gently.

[0172] Plasmid DNA was transferred into TM Complexation buffer (or OPTI-MEM TM I medium) to a total plasmid DNA concentration of about 1.0 μg / mL. TM OPTI-PLEX for 293 reagents TM Complexation buffer (or OPTI-MEM TM The two solutions were incubated at room temperature for 5 minutes. TM 293 reagent to the diluted plasmid DNA, mix the solution, and add the EXPIFECTAMINE TM Incubate the solution of 293 / plasmid DNA complex at room temperature for 10-20 minutes. Gently swirl during the addition to slowly transfer the complex to Expi293F TM The cells were then incubated in a 37°C incubator with ≥80% relative humidity and 8% CO2 on an orbital shaker.

[0173] About 18-22 hours after transfection, add EXPIFECTAMINE TM 293 Transfection Enhancer 1 and EXPIFECTAMINE TM293 Transfection Enhancer 2 was added to the transfection flask, the flask was gently swirled during the addition, and the flask was immediately returned to the 37°C incubator with ≥80% relative humidity and 8% CO2 on an orbital shaker platform.

[0174] The protein-containing culture supernatant was harvested approximately 5-7 days after transfection.

[0175] purification

[0176] The antibodies were purified on mAbSelect SuRe columns (GE#11-0034-95, 16 mm x 25 mm). Prior to loading onto the column, the antibody solution was filtered through a 0.2 μm PES filter to remove any particles. The following buffers were used during purification: Buffer A: 0.2 M NaOH; Buffer B: 20 ​​mM sodium phosphate, 150 mM NaCl pH 7.2 (or PBS 1X); Buffer C: 50 mM sodium citrate (dihydrate), pH 3.5; Buffer D: 50 mM succinic acid; and Buffer E: 20% ethanol. The antibodies were purified using the following protocol: 1) sanitize the mAbSelect SuRe column with 6 column volumes (CV) of Buffer A, 15 minutes contact time (2 mL / min, 15 minutes); 2) rinse the column with 5 CV MilliQ water; 3) equilibrate the column with 15 CV of Buffer B. Prior to antibody loading, collect and store 0.5 mL of treated media; 4) load antibody onto the column; 5) wash the column with 6CV buffer B; 6) elute the antibody with 6CV buffer C, and neutralize the eluate with 1 / 10 volume 600 mM sodium phosphate dibasic or Tris pH 10; 7) strip the column with 6CV buffer D, and add 1 / 10 volume 600 mM sodium phosphate dibasic or Tris pH 10 to neutralize; 8) wash the column with 10CV buffer A; 9) rinse the column with 5CV MilliQ water; 10) re-equilibrate the column with 10CV buffer V; and 11) wash the column with 10CV buffer E and store it at 4°C.

[0177] Use a MWCO Millipore concentrator to reduce the eluate to a volume of about 0.5 mL to 2 mL: a) add about 2 mL of DPBS to the concentrator to wet the membrane, centrifuge the concentrator for about 1 minute, and discard the liquid; b) add the eluate to the concentrator and centrifuge to pass the liquid through the membrane. Repeat this operation to reduce the volume to about 0.5 mL to 2 mL.

[0178] Use GE PD-10 columns or THERMO SCIENTIFIC TM ZEBA TMColumn buffer exchange to DPBS: a) Cut column diagonally to allow for better dripping and remove cap to drain liquid; b) Add DPBS (5 mL) to column and pass through column, repeat 4 times; c) Load the entire concentrated sample into column by filter sterilization and pass through column. Rinse the bottom of MWCO filter to collect any extra antibody; d) Add DPBS to bring volume to 2 mL to completely load column; e) Place 15 mL conical tube under column and add 4 mL DPBS to column for elution. Concentrate eluate using MWCO Millipore concentrator and use NANODROP TM The antibody concentration was determined by UV-Vis spectrophotometer.

[0179] Example 3. Cross-reactivity of cloned CA10 variants with human and cynomolgus monkey BCMA proteins

[0180] Prepare BCMA antigen solution (2 μg / mL or 1 mg / mL biotinylated BCMA antigen when using streptavidin coated plates) in phosphate buffered saline (PBS). Add the antigen solution to a 96-well plate (50 μL per well) and incubate overnight at 4°C. Block the plate with 250 μL PBSA (PBS containing 1% BSA) per well for 2 hours at 37°C, then wash three times with 200 μL phosphate buffered saline (PBS) per well. Add hybridoma supernatant (50 μL) or 1 mg / mL purified test or control (positive and negative) antibodies (50 μL) per well in a 96-well plate and incubate for 1 hour at room temperature. Wash the plate three times with 200 μl PBS per well, then add horseradish peroxidase (HRP)-conjugated secondary antibody (50 μl per well) diluted 1:5,000 in PBSA and incubate for 1 hour at room temperature. Then, wash the plate three times with 200 μl PBS per well. Tetramethylbenzidine dihydrochloride (TMB) substrate was prepared by mixing with solution A and solution B (1:1) in a 15 mL conical tube, and then 50 μL of substrate was added to the wells. The color was allowed to develop for 1-5 minutes, and then 50 μL of stop solution was added to each well. The absorbance was read at 450 nm using SpectraMax (Molecular Devices).

[0181] Among the antibody clones that exhibited BCMA-specific binding, clone CA10_parent (CA10_V2_parent) showed strong binding ( Figure 3 Variants 1-4, 6, and 7 all had similar binding to the parent antibody. However, variant 5 (CA10_PE_V5) lost its binding ability compared to the other variants ( Figure 3 ).

[0182] Example 4. Binding of anti-BCMA mAb antibodies to BCMA protein on the membrane surface of HEK or MM1R cells

[0183] Cell staining for flow cytometry

[0184] HEK 293 cells or MM1R cells (0.5 x 10 6 ) were aliquoted into separate FACS tubes and labeled as unstained, test samples or positive controls. Hybridoma supernatant (20 μL) or purified CA10 or positive control (1 μg / mL, 5 μL) was added to the cells and incubated on ice for 30 minutes. The cells were washed by adding 3 mL 1XPBX / 5% FBS (FACS buffer) and centrifuged at 1500 rpm (450 x g) for 5-7 minutes. The supernatant was poured out and the cells were gently vortexed to mix. 50 μL 1:500 diluted FITC-conjugated goat anti-human (or mouse) H+L specific secondary antibodies were added to the cells and the cells were incubated on ice or at 4 ° C in the dark for 30 minutes. FACS buffer (3 mL) was added and the cells were washed at 1500 rpm for 5-7 minutes. The supernatant was discarded and the cells were resuspended in 150-200 μL FACS buffer. Cells were collected on BD FACS Canto and data were analyzed using Flojo V10 software.

[0185] Variants 1-4, 6, and 7 all had similar BCMA protein binding to the CA10_parent (CA10_V2_parent) antibody on both HEK293 cells and MM1R cells ( Figure 4 and 5A -5B). Variant 5 showed no binding to BCMA protein on HEK293 cells and MM1R cells.

[0186] Example 5. Antibody and Antigen Protein Interactions and Affinity Constants Determined by Surface Plasmon Resonance Measurements

[0187] Prepare 1X HBS-EP+ in MilliQ water, filter and degas. BIACORE TM T100 was pre-treated by perfusion with 1X HBS-EP+. Immobilized chip Protein A CM % BIACORE was used TM Control software.

[0188] Antibodies were diluted to 20 μg / mL in HBS-EP+. Seven 1:1 serial dilutions of antigen were prepared (final concentration 80 nM) and 1X HBS-EP+ was used as a 0 nm control. 1X HBS-EP+ was used for priming injections and HCl (12 mM) was used for regeneration. Samples were run and analyzed using BIACORE TMThe data were analyzed by T100 software.

[0189] Table 3. List of reagents and equipment

[0190]

[0191]

[0192] Table 4. Antigen list

[0193]

[0194] Calculations for preparing antigen dilutions:

[0195]

[0196] Table 5. Antibody list

[0197]

[0198]

[0199] Calculation for preparing antibody dilution:

[0200]

[0201] Variants 1, 2, 6 and 7 showed similar affinity to the CA10_parent (CA10WT) antibody for human and cynomolgus monkey BCMA protein ( Figure 6-Figure 9 Variant 3 showed similar affinity to the parent antibody for human BCMA protein, but lower affinity for cynomolgus monkey BCMA. Variants 4 and 5 lost affinity for both human and cynomolgus monkey BCMA proteins.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to B cell maturation antigen (BCMA), the antibody or antigen-binding fragment thereof comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises Contains CX 1 SSTGX 2 VTPX 3 X 4 The CDR-L1 sequence of the amino acid sequence of YAN (SEQ ID NO: 25), wherein X 1 is R or A, X 2 It is T or A, X 3 is S or G, and X 4 is N or Y; Contains DNNX 5 X 6 The CDR-L2 sequence of the amino acid sequence of PP (SEQ ID NO: 26), wherein X 5 is S, I, or N, and X 6 is R or K; as well as Contains ALX 7 X 8 GX 9 The CDR-L3 sequence of the amino acid sequence of QWV (SEQ ID NO: 27), wherein X 7 W or Y, X 8 is F or Y, and X 9 It is N or G.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises A CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4), CRSSTGTVTPSNYAN (SEQ ID NO: 5), CASSTGAVTPSNYAN (SEQ ID NO: 6), or CASSTGAVTPGYYAN (SEQ ID NO: 7); A CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9), DNNIKPP (SEQ ID NO: 10), or DNNNKPP (SEQ ID NO: 11); and A CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13), ALWYGGQWV (SEQ ID NO: 14), or ALYYGGQWV (SEQ ID NO: 15).

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The VH domain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 16; and The VL domain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 24, Optionally among them: The VH domain comprises the amino acid sequence of SEQ ID NO: 16; and The VL domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO:

24.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 16; and The antibody light chain comprises the amino acid sequence of SEQ ID NO:

24.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4); a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9); and A CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein: The antibody heavy chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 16; and The antibody light chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 18, Optionally among them: The antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 16; and The antibody light chain comprises the amino acid sequence of SEQ ID NO:

18.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of CRSSTGTVTPSNYAN (SEQ ID NO: 5); a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO: 9); and A CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein: The antibody heavy chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 16; and The antibody light chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:

19.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4); a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO:9); and A CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein: The antibody heavy chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 16; and The antibody light chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:

20.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of CASSTGAVTPSNYAN (SEQ ID NO: 6); a CDR-L2 sequence comprising the amino acid sequence of DNNIKPP (SEQ ID NO: 10); and A CDR-L3 sequence comprising the amino acid sequence of ALWYGGQWV (SEQ ID NO: 14).

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein: The antibody heavy chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 16; and The antibody light chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:

21.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of CASSTGAVTPGYYAN (SEQ ID NO: 7); a CDR-L2 sequence comprising the amino acid sequence of DNNNKPP (SEQ ID NO: 11); and A CDR-L3 sequence comprising the amino acid sequence of ALYYGGQWV (SEQ ID NO: 15).

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein: The antibody heavy chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO: 16; and The antibody light chain is at least about 90% identical or at least 95% identical to the amino acid sequence of SEQ ID NO:

23.

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSNFGMH (SEQ ID NO: 1); a CDR-H2 sequence comprising the amino acid sequence of VIWSDETNR (SEQ ID NO: 2); and a CDR-H3 sequence comprising the amino acid sequence of DQQYCSSDSCFTWFDP (SEQ ID NO: 3); and The VL domain comprises a CDR-L1 sequence comprising the amino acid sequence of CASSTGTVTPSNYAN (SEQ ID NO: 4); a CDR-L2 sequence comprising the amino acid sequence of DNNSRPP (SEQ ID NO:9); and A CDR-L3 sequence comprising the amino acid sequence of ALWFGNQWV (SEQ ID NO: 13).

16. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein: The antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof; or The antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

17. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is a multispecific antibody.

18. An antibody or antigen-binding fragment thereof according to any one of the preceding claims for use as a medicament.

19. An antibody or antigen-binding fragment thereof according to any one of the preceding claims for use in a method of treating or preventing a disease or disorder.

20. An antibody or antigen-binding fragment thereof according to any one of the preceding claims for use in a method of treating or preventing cancer.

21. The antibody or antigen-binding fragment thereof according to any one of the preceding claims for use in a method of treating or preventing multiple myeloma.

22. The antibody or antigen-binding fragment thereof according to any one of the preceding claims for use in a method of treating or preventing plasma cell malignancies.

23. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein according to any one of claims 1 to 17.

25. An expression vector comprising the nucleic acid molecule according to claim 24.

26. A host cell comprising the nucleic acid molecule according to claim 24.

27. A host cell comprising the expression vector according to claim 25.

28. The host cell of claim 27, wherein the cell is a mammalian cell.

29. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the method comprising culturing the host cell according to any one of claims 26 to 28 under suitable conditions, and recovering the antibody or antigen-binding fragment thereof.

30. A method of treating or preventing a disease or disorder, the method comprising administering the pharmaceutical composition of claim 23 to a subject in need thereof.

31. A method for treating or preventing cancer, comprising administering the pharmaceutical composition according to claim 23 to a subject in need thereof.

32. A method for treating or preventing multiple myeloma, comprising administering the pharmaceutical composition according to claim 23 to a subject in need thereof.

33. A method for treating or preventing a plasma cell disorder, the method comprising administering the pharmaceutical composition of claim 23 to a subject in need thereof.

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