CN112778417A

CN112778417A - Isolated protein combined with antigen BCMA and application thereof

Info

Publication number: CN112778417A
Application number: CN202011232311.2A
Authority: CN
Inventors: 杨月; 周金财; 李霄培; 史中军; 王华菁; 陈思晔; 杨焕风; 何晓文
Original assignee: Shanghai OriginCell Medical Technology Co Ltd
Current assignee: Shanghai OriginCell Medical Technology Co Ltd
Priority date: 2019-11-07
Filing date: 2020-11-06
Publication date: 2021-05-11
Anticipated expiration: 2040-11-06
Also published as: CN112778417B

Abstract

The present application relates to an isolated antigen binding protein comprising at least one CDR in a VH having the amino acid sequence shown as SEQ ID No. 30 or 59; and it comprises at least one CDR in the VL whose amino acid sequence is shown in SEQ ID NO. 26. The application also relates to nucleic acids encoding the isolated antigen binding proteins, vectors comprising the isolated antigen binding proteins, cells comprising the nucleic acids or the vectors, methods of making the isolated antigen binding proteins, and uses of the isolated antigen binding proteins.

Description

Isolated protein combined with antigen BCMA and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to an isolated protein combined with antigen BCMA and application thereof.

Background

Tumors are a serious health-threatening disease in humans, of which myeloma (also known as plasmacytoma) is a malignant tumor with a wide range of harms. B cell maturation antigens, also known as BCMA, are members of the tumor necrosis receptor (TNFR) family and are expressed predominantly on terminally differentiated B cells, such as memory B cells and plasma cells, and are particularly common in myelomas.

In recent years, with the progress of molecular biology, genomics and proteomics, a series of molecular targeted drugs for the treatment of myeloma have been developed. The molecular targeted therapy takes some marker molecules expressed by tumor cells as targets, and selects a targeted blocker to effectively intervene, thereby achieving the effect of inhibiting the growth, the development and the metastasis of tumors. Compared with three traditional treatment methods of operation, radiotherapy and chemotherapy, the molecular targeted therapy can efficiently and selectively kill tumor cells and reduce the damage to normal tissues. However, the effect of the current molecular targeting technology for tumor therapy is still not satisfactory, and there are many points to be improved.

Disclosure of Invention

In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in the VH set forth in amino acid sequence SEQ ID NO 30 or 59; and which comprises at least one CDR in the VL shown in amino acid sequence SEQ ID NO. 26.

In certain embodiments, the isolated antigen binding protein has one or more of the following properties:

1) can be 7 × 10^-11M or less binds to a BCMA protein, wherein the KD value is determined by Octet;

2) a BCMA protein capable of specifically binding to the surface of MM1S myeloma cells in a FACS assay;

3) can inhibit tumor growth and/or tumor cell proliferation.

In certain embodiments, the BCMA protein comprises a human BCMA protein.

In certain embodiments, the tumor comprises a BCMA-positive tumor.

In certain embodiments, the BCMA-positive tumor comprises myeloma.

In certain embodiments, the isolated antigen binding protein comprises HCDR1 in the VH as shown in amino acid sequence SEQ ID No. 30 or 59.

In certain embodiments, the isolated antigen binding protein comprises HCDR2 in the VH as shown in amino acid sequence SEQ ID No. 30 or 59.

In certain embodiments, the isolated antigen binding protein comprises HCDR3 in the VH as shown in amino acid sequence SEQ ID No. 30 or 59.

In certain embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 4.

In certain embodiments, the HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 12 or 53.

In certain embodiments, the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 5, 8, and 52.

In certain embodiments, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO 6.

In certain embodiments, the isolated antigen binding protein comprises LCDR1 in the VL set forth in amino acid sequence SEQ ID No. 26.

In certain embodiments, the isolated antigen binding protein comprises LCDR2 in the VL set forth in amino acid sequence SEQ ID No. 26.

In certain embodiments, the isolated antigen binding protein comprises LCDR3 in the VL set forth in amino acid sequence SEQ ID No. 26.

In certain embodiments, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 10.

In certain embodiments, the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs 1 and 7.

In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 11.

In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 2 and 9.

In certain embodiments, the LCDR3 comprises the amino acid sequence set forth in SEQ ID No. 3.

In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen-binding fragment comprises a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv and/or dAb.

In certain embodiments, the VL comprises the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

In certain embodiments, the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1 and the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO 13.

In certain embodiments, the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO 13.

In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2 and the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 14.

In certain embodiments, the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 14.

In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO. 15.

In certain embodiments, the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO. 15.

In certain embodiments, the N-terminus of L-FR4 is linked to the C-terminus of LCDR3 and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO 16.

In certain embodiments, the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO 16.

In certain embodiments, the VL comprises the amino acid sequence set forth in any one of SEQ ID NOS 23-25.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Ig kappa constant region.

In certain embodiments, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 31.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain LC, and the LC comprises an amino acid sequence set forth in any one of SEQ ID NOS 33-35.

In certain embodiments, the VH comprises the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In certain embodiments, the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1 and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO 17.

In certain embodiments, the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO 17 or 54.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 18, 21, and 55.

In certain embodiments, the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 18, 21, and 55.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID No. 19 or 56.

In certain embodiments, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO 19 or 56.

In certain embodiments, the N-terminus of H-FR4 is linked to the C-terminus of HCDR3 and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO 20.

In certain embodiments, the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO 20.

In certain embodiments, the VH comprises the amino acid sequence set forth in any one of

SEQ ID NOs

27, 28, 29, 57, and 58.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 32.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain HC, and the HC comprises an amino acid sequence set forth in any one of SEQ ID NOs 36, 37, 38, 60, and 61.

In another aspect, the present application provides an isolated one or more nucleic acid molecules encoding an isolated antigen binding protein described herein.

In another aspect, the present application provides a vector comprising a nucleic acid molecule as described herein.

In another aspect, the present application provides a cell comprising a nucleic acid molecule described herein or a vector described herein.

In another aspect, the present application provides a method of making an isolated antigen binding protein described herein, the method comprising culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In another aspect, the present application provides a pharmaceutical composition comprising an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.

In another aspect, the present application provides the use of an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein and/or a pharmaceutical composition as described herein in the manufacture of a medicament for the prevention, alleviation and/or treatment of a tumor.

In certain embodiments, the tumor comprises a myeloma.

In another aspect, the present application provides a method of preventing, ameliorating, or treating a tumor, the method comprising administering to a subject in need thereof an isolated antigen binding protein described herein.

In certain embodiments, the tumor comprises a myeloma.

In another aspect, the present application provides a method of detecting BCMA in a sample comprising administering an isolated antigen binding protein as described herein.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows the results of the detection of the binding activity of the intact antibodies 8A7, 8A10, 8A11, hBCMA19, hBCMA22 and hBCMA23 to cell surface BCMA;

FIG. 2 shows the ADCC activity assay results of 8A7, 8A10, 8A11, hBCMA19, hBCMA22, hBCMA23 whole antibody;

FIG. 3 shows the results of CDC activity assay of 8A10, hBCMA22, hBCMA23 intact antibody;

FIG. 4 shows the results of the hBCMA22 whole antibody tumor burden experiment;

figure 5 shows the hBCMA22 whole antibody tumor growth inhibition results.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

The present application is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not affect the activity of the substance, mixed with artificial or synthetic substances.

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability obtained from a natural state by artificial means. The "isolated antigen binding protein" may comprise a portion that binds an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding portion to an antigen. The antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, scaffolds comprising antibody sources introduced, for example, with mutations to stabilize the three-dimensional structure of the antigen binding protein and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., Korndorfer et al, 2003, Proteins: Structure, Function, andBioinformatics,53(1): 121-; roque et al, Biotechnol.prog.20:639-654 (2004). In addition, peptide antibody mimetics ("PAMs") as well as scaffolds based on antibody mimetics utilizing fibronectin components can be used as scaffolds.

In the present application, the term "KD" (likewise, "KD" or "KD") generally refers to an "affinity constant" or an "equilibrium dissociation constant", and refers to a value obtained at equilibrium in a titration measurement, or by dividing the dissociation rate constant (KD) by the association rate constant (ka). The association rate constant (ka), the dissociation rate constant (KD), and the equilibrium dissociation constant (KD) are used to indicate the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) for an antigen (e.g., a BCMA protein). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine the sample at equilibrium in physiological buffer. For example, the KD values can be determined by Octet, as well as using other experimental pathways and instruments such as BIAcore (biomolecular interaction analysis) (e.g., instruments available from BIAcore international ab, algehathcarecompany, Uppsala, sweden). In addition, the KD value can also be determined using KinExA (dynamic exclusion assay) available from sapidyne instruments (Boise, Idaho).

In the present application, the term "BCMA" generally refers to a B cell maturation antigen, also known as TNFRSF17, BCM or CD269, which is a member of the tumor necrosis receptor (TNFR) family and is expressed primarily on terminally differentiated B cells, such as memory B cells and plasma cells. BCMA is present in a variety of tumors, with myeloma being particularly common. The ligands are called B-cell activators of the TNF family (BAFF) and proliferation-inducing ligands (APRIL). BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. The gene for BCMA is encoded on chromosome 16, producing a primary mRNA transcript of 994 nucleotides in length (NCBI accession NM — 001192.2) which encodes a 184 amino acid protein (NP — 001183.2). In the present application, "BCMA" may comprise mutated proteins, such as point mutations, fragments, insertions, deletions and splice variants of full-length wild-type BCMA. Further, in the present application, the BCMA protein may comprise a human BCMA protein.

In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can be determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds a target (which may be an epitope) is an antibody that binds that target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). For example, in the present application, the isolated antigen binding protein is capable of binding to BCMA protein with a dissociation constant (KD) of <7x10-11M or lower. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

In the present application, the term "inhibition" generally refers to a reduction in the growth rate of cells or the number of cells. For example, an isolated antigen binding protein described herein, which is capable of inhibiting tumor growth and/or tumor cell proliferation.

In the present application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor may be a solid tumor or a hematological tumor. For example, in the present application, a tumor can be a BCMA-positive tumor, wherein the BCMA-positive tumor can comprise a myeloma.

In the present application, the term "variable domain" generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain an antigen binding site.

In the present application, the term "variable" generally refers to the fact that certain segments of the variable domains differ greatly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection, and in some cases form part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F (ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function (e.g., specifically bind BCMA). Typically, such fragments should include an antigen binding domain. The basic 4 chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 elementary heterotetramer units with another polypeptide called the J chain and contain 10 antigen binding sites, while IgA antibodies comprise 2-5 elementary 4 chain units that can aggregate in association with the J chain to form multivalent combinations. For IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for the alpha and gamma chains, respectively, and four CH domains for the mu and epsilon isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH and CL to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The VH and VL pair together to form a single antigen-binding site. For the structure and properties of antibodies of different classes see, for example, Basic and Clinical Immunology,8th Edition, Daniel P.Sties, Abba I.Terr and Tristram G.Parsolw (eds), Appleton & Lange, Norwalk, conn.,1994, page 71 and chapter 6. L chains from any vertebrate species can be classified into one of two distinctly different classes, termed κ and λ, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of its heavy Chain (CH) constant domain, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK 1.

In the present application, the term "CDR" generally refers to a region of an antibody variable domain whose sequence is highly variable and/or forms a structurally defined loop. Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR 3). In natural antibodies, HCDR3 and LCDR3 show the majority of diversity of the six CDRs, and in particular HCDR3 is thought to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al, Immunity 13:37-45 (2000); johnson and Wu, in Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camel antibodies consisting of only heavy chains function normally and are stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996).

In this application, the term "FR" generally refers to a more highly conserved portion of an antibody variable domain, which is referred to as the framework region. Typically, the variable domains of native heavy and light chains each comprise four FR regions, namely four in the VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in the VL (L-FR1, L-FR2, L-FR3, and L-FR 4). For example, the VL of an isolated antigen binding protein described herein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4. The VH of an isolated antigen binding protein described herein may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In the present application, the term "antigen-binding fragment" generally refers to a fragment having antigen-binding activity. In the present application, the antigen binding fragment may comprise a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv and/or dAb.

In this application, the term "directly connected" is used in contrast to the term "indirectly connected," which generally refers to a direct connection. For example, the direct linkage may be a direct linkage without a spacer between the substances. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirectly linked" generally refers to a condition in which the substances are not directly linked to each other. For example, the indirect connection may be a connection via a spacer. For example, in the isolated antigen binding proteins described herein, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be linked directly or indirectly.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been the recipient of a subject plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may comprise cells transfected in vitro with a vector of the invention. The cell may be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell.

In the present application, the term "pharmaceutical composition" generally refers to a composition that is suitable for administration to a patient, preferably a human patient. For example, a pharmaceutical composition described herein, which can comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable adjuvant" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

Detailed Description

Isolated antigen binding proteins

In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in the VH having an amino acid sequence shown in SEQ ID NO 30 or 59; and it comprises at least one CDR in the VL whose amino acid sequence is shown in SEQ ID NO. 26.

For example, the VH of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in any one of

SEQ ID NOs

27, 28, 29, 57, and 58.

For example, the VL of an isolated antigen binding protein described herein can comprise an amino acid sequence set forth in any one of SEQ ID NOs 23-25.

For example, in the present application, the isolated antigen binding protein may comprise HCDR1 in the VH shown in SEQ ID NO:30 or 59. For example, in the present application, the isolated antigen binding protein may comprise HCDR2 in the VH shown in SEQ ID NO:30 or 59. For example, in the present application, the isolated antigen binding protein may comprise HCDR3 in the VH shown in SEQ ID NO:30 or 59. As another example, in the present application, the isolated antigen binding protein may comprise LCDR1 in VL having the amino acid sequence shown in SEQ ID NO. 26. For example, in the present application, the isolated antigen binding protein may comprise LCDR2 in VL having the amino acid sequence shown in SEQ ID NO. 26. For example, in the present application, the isolated antigen binding protein may comprise LCDR3 in VL having the amino acid sequence shown in SEQ ID NO. 26.

Properties of the isolated antigen binding protein

In the present application, the isolated antigen binding protein may have one or more of the following properties:

3) can inhibit tumor growth and/or tumor cell proliferation.

In the present application, the isolated antigen binding protein can be present at 7 × 10^-11K of M or less_DBinding to BCMA protein, wherein said K_DThe value can be determined by Octet. For example, the isolated antigen binding proteins described herein bind K derived from human BCMA protein_DThe value may be ≦ 7 × 10^-11M、≤6.5×10^-11M、≤6×10^-11M、≤5.5×10^-11M、≤5×10^-11M、≤4.5×10^-11M、≤4×10^-11M、≤3.5×10^-11M、≤3×10^-11M、≤2.5×10^-11M、≤2×10^-11M、≤1.5×10^-11M、≤1×10^-11And M. As another example, the isolated antigen binding proteins described herein bind K derived from murine BCMA protein_DThe value may be ≦ 7 × 10^-11M、≤6.5×10^-11M、≤6×10^-11M、≤5.5×10^-11M、≤5×10^- ¹¹M、≤4.5×10^-11M、≤4×10^-11M、≤3.5×10^-11M、≤3×10^-11M、≤2.5×10^-11M、≤2×10^-11M、≤1.5×10^-11M、≤1×10^-11And M. For another example, an isolated antigen binding protein described herein binds K of a monkey-derived BCMA protein_DThe value may be ≦ 7 × 10^-11M、≤6.5×10^-11M、≤6×10^-11M、≤5.5×10^-11M、≤5×10^-11M、≤4.5×10^-11M、≤4×10^-11M、≤3.5×10^-11M、≤3×10^-11M、≤2.5×10^-11M、≤2×10^-11M、≤1.5×10^-11M、≤1×10^-11M。

In the present application, the KD values can also be determined by ELISA, competition ELISA or BIACORE or KINEXA.

In the present application, the isolated antigen binding protein is capable of specifically binding to BCMA protein on the surface of MM1S myeloma cells, which specific binding can be determined by FACS. For example, the specific binding of an isolated antigen binding protein described herein to BCMA protein on the surface of MM1S myeloma cells can be reflected by the half maximal effector concentration (EC50) in FACS assays, e.g., a lower half maximal effector concentration (EC50) indicates better specific binding. For example, the isolated antigen binding protein may have an EC50 value in FACS assay for binding to BCMA protein on the surface of MM1S myeloma cells of 0.01. mu.g/ml to 0.10. mu.g/ml, 0.01. mu.g/ml to 0.15. mu.g/ml, 0.01. mu.g/ml to 0.20. mu.g/ml, 0.01. mu.g/ml to 0.25. mu.g/ml, 0.01. mu.g/ml to 0.30. mu.g/ml, 0.01. mu.g/ml to 0.35. mu.g/ml, 0.01. mu.g/ml to 0.40. mu.g/ml, 0.01. mu.g/ml to 0.45. mu.g/ml, or 0.01. mu.g/ml to 0.50. mu.g/ml.

In the present application, an isolated antigen binding protein described herein is capable of inhibiting tumor growth and/or tumor cell proliferation, e.g., is capable of reducing tumor volume by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%. As another example, the number of tumor cells can be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.

In the present application, the BCMA protein may comprise a human BCMA protein, which may comprise the GeneBank accession number: BAB 60895.1.

In the present application, the neoplasm may comprise a BCMA-positive neoplasm, which may comprise a bone marrow cancer.

In the present application, the isolated antigen binding protein is capable of inhibiting the growth of tumor cells by inducing the production of antibody-dependent cell-mediated cytotoxicity (ADCC). For example, the isolated antigen binding proteins described herein are capable of bringing cytotoxic infiltrating T lymphocytes into tumor tissue, thereby inhibiting the growth of tumor cells. For another example, an isolated antigen binding protein described herein can also indirectly inhibit proliferation of BCMA positive cells via a macrophage-associated mechanism.

In the present application, the isolated antigen binding protein has some endocytic activity. The endocytic activity can be detected by the Confocal method.

The class of isolated antigen binding proteins

In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof. For example, an isolated antigen binding protein described herein can include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, triabodies, tetrabodies, Fv fragments, scFv fragments, Fab 'fragments, F (ab')2 fragments, and camelized single domain antibodies.

In the present application, the antibody may be a humanized antibody. In other words, an isolated antigen binding protein described herein, which can be an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to a relevant antigen (e.g., human BCMA) and comprises a Framework (FR) region having substantially the amino acid sequence of a human antibody and a Complementarity Determining Region (CDR) having substantially the amino acid sequence of a non-human antibody. By "substantially" in the context of a CDR is meant that the amino acid sequence of the CDR is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a CDR of a non-human antibody. The humanized antibody can comprise substantially all of at least one and typically two variable domains (Fab, Fab ', F (ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., antibody) and all or substantially all of the framework regions are framework regions having human immunoglobulin consensus sequences. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least a variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only humanized light chains. In some embodiments, the humanized antibody contains only humanized heavy chains. In particular embodiments, the humanized antibody contains only the humanized variable domains of the light chain and/or the humanized heavy chain.

In the present application, the antigen binding fragment may comprise a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv and/or dAb.

CDR

In the present application, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO. 10.

In the present application, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs 1 and 7.

In the present application, the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 11.

In the present application, the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs 2 and 9.

In the present application, the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

For example, the LCDR1 of the isolated antigen binding proteins described herein can comprise the amino acid sequence shown in SEQ ID NO. 1, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 2, and LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

For example, the LCDR1 of the isolated antigen binding proteins described herein can comprise the amino acid sequence shown in SEQ ID NO. 7, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 2, and LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

For example, the LCDR1 of the isolated antigen binding proteins described herein can comprise the amino acid sequence shown in SEQ ID NO. 7, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 9, and LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

In the present application, the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO. 4.

In the present application, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO 12 or 53.

In the present application, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs 5, 8 and 52.

In the present application, the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 6.

For example, the HCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 4, the HCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 5, and the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 6.

For example, the HCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 4, the HCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 8, and the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 6.

For example, the HCDR1 of the isolated antigen binding proteins described herein can comprise the amino acid sequence shown as SEQ ID NO. 4, the HCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 52, and the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 6.

As another example, the LCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 1, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 2, LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 5, and HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6.

As another example, the LCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 2, LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 8, and HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6. As another example, the LCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 9, LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 5, and HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6.

As another example, the LCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 2, LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 52, and HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6.

As another example, the LCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 9, LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 52, and HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6.

As another example, the LCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 7, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 9, LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 8, and HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6.

FR

In the present application, the VL of the isolated antigen binding protein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

In the present application, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 13.

For example, the C-terminus of the L-FR1 may be linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 13.

In the present application, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 14.

For example, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 14.

In the present application, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 15.

For example, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 15.

In the present application, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 16.

For example, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO 16.

As another example, L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR2 can comprise the amino acid sequence shown as SEQ ID NO. 14, L-FR3 can comprise the amino acid sequence shown as SEQ ID NO. 15, and L-FR4 can comprise the amino acid sequence shown as SEQ ID NO. 16.

In the present application, the VH of the isolated antigen binding protein may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In the present application, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO 17 or 54.

In the present application, the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO 17 or 54.

In the present application, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 22 or 55.

In the present application, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise an amino acid sequence shown in any one of SEQ ID NOs 18, 21 and 55.

In the present application, the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs 18, 21 and 55.

In the present application, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO 19 or 56.

In the present application, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO 19 or 56.

In the present application, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20.

In the present application, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 20.

For example, H-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown in SEQ ID NO. 17, H-FR2 can comprise the amino acid sequence shown in SEQ ID NO. 18, H-FR3 can comprise the amino acid sequence shown in SEQ ID NO. 19, and H-FR4 can comprise the amino acid sequence shown in SEQ ID NO. 20.

For example, H-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown in SEQ ID NO. 17, H-FR2 can comprise the amino acid sequence shown in SEQ ID NO. 21, H-FR3 can comprise the amino acid sequence shown in SEQ ID NO. 19, and H-FR4 can comprise the amino acid sequence shown in SEQ ID NO. 20.

For example, H-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 54, H-FR2 can comprise the amino acid sequence shown as SEQ ID NO. 55, H-FR3 can comprise the amino acid sequence shown as SEQ ID NO. 56, and H-FR4 can comprise the amino acid sequence shown as SEQ ID NO. 20.

As another example, L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 13, L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 14, L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 15, L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 16, and H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO. 17, H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 18, H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 19, and H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 20.

As another example, L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 13, L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 14, L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 15, L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 16, and H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO. 17, H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 21, H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 19, and H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 20.

As another example, L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 13, L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 14, L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 15, L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 16, and H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO. 54, H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 55, H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 56, and H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 20.

VL and VH

The isolated antigen binding proteins described herein can comprise an antibody light chain variable region VL and an antibody heavy chain variable region VH. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 26 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 30 or 59. As another example, the VL of the isolated antigen binding protein may comprise an amino acid sequence set forth in any one of SEQ ID NOs 23-25. The VH of the isolated antigen binding protein may comprise the amino acid sequence shown in any one of

SEQ ID NOs

27, 28, 29, 57, and 58.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 23 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 27.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 24 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 28.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 25 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 29.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 24 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 57.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 25 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 57.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 25 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 58.

Light and heavy chains

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human Ig kappa constant region. For example, the antibody light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO. 31.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. For example, the antibody heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO. 32.

In the present application, the isolated antigen binding protein may comprise an antibody light chain LC, and the LC may comprise an amino acid sequence set forth in any one of SEQ ID NOS 33-35.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain HC, and the HC may comprise an amino acid sequence set forth in any one of SEQ ID NOs 36, 37, 38, 60, and 61.

An isolated antigen binding protein described herein can comprise an antibody light chain and an antibody heavy chain.

For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 33 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 36.

For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 34 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 37.

For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 35 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 38.

For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 34 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 64.

For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 35 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 64.

For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 35 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 65.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 33 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 36. Wherein the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 1, LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 2, LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO. 5, and HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 6. Wherein L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 14, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 15, L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 16, and H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 17, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 18, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 19, and H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 20. The VL may comprise the amino acid sequence shown in SEQ ID NO. 23, and the VH may comprise the amino acid sequence shown in SEQ ID NO. 27. For example, the isolated antigen binding protein can be an 8a7 whole antibody.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 34, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 37. Wherein the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 2, LCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence shown as SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 8, and HCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 6. Wherein L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 14, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 15, L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 16, and H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 17, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 18, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 19, and H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 20. The VL may comprise the amino acid sequence shown in SEQ ID NO. 24 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 28. For example, the isolated antigen binding protein can be an 8a10 whole antibody.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 35 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 38. Wherein the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 9, LCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence shown as SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 5, and HCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 6. Wherein L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 14, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 15, L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 16, and H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 17, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 21, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 19, and H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 20. The VL may comprise the amino acid sequence shown in SEQ ID NO. 25 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 29. For example, the isolated antigen binding protein can be an 8a11 whole antibody.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 34, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 60. Wherein the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 2, LCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence shown as SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 52, and HCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 6. Wherein L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 14, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 15, L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 16, and H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 54, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 55, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 56, and H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 20. The VL may comprise the amino acid sequence shown in SEQ ID NO. 24, and the VH may comprise the amino acid sequence shown in SEQ ID NO. 57. For example, the isolated antigen binding protein may be an intact antibody to hBCMA 19.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 35 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 60. Wherein the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 9, LCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence shown as SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 52, and HCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 6. Wherein L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 14, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 15, L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 16, and H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 54, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 55, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 56, and H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 20. The VL may comprise the amino acid sequence shown in SEQ ID NO. 25, and the VH may comprise the amino acid sequence shown in SEQ ID NO. 57. For example, the isolated antigen binding protein may be an intact antibody to hBCMA 22.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 35, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 61. Wherein the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 7, LCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 9, LCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 3, and HCDR1 may comprise the amino acid sequence shown as SEQ ID NO. 4, HCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 8, and HCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 6. Wherein L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 14, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 15, L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 16, and H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 54, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 55, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 56, and H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 20. The VL may comprise the amino acid sequence shown in SEQ ID NO. 25 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 58. For example, the isolated antigen binding protein may be an intact antibody to hBCMA 23.

Nucleic acid molecules, vectors, cells, methods of preparation and pharmaceutical compositions

In another aspect, the present application also provides an isolated nucleic acid molecule or molecules that can encode an isolated antigen binding protein described herein. The isolated nucleic acid molecule or molecules described herein can be an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized, but can encode an isolated antigen binding protein described herein.

In another aspect, the present application also provides a vector, which may comprise a nucleic acid molecule as described herein. The vector may be used to express the genetic material element carried by the vector in a host cell by transformation, transduction or transfection of the host cell. For example, the carrier may include: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). As another example, the vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. In addition, the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In another aspect, the present application also provides a cell, which may comprise a nucleic acid molecule described herein or a vector described herein. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. In certain embodiments, the cells may further comprise cells transfected in vitro with a vector of the invention. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell may be a mammalian cell. In certain embodiments, the mammalian cell can be a HEK293 cell.

In another aspect, the present application also provides methods of making an isolated antigen binding protein described herein, which methods can include culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In another aspect, the present application also provides a pharmaceutical composition that can comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.

In certain embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flow into the patient, as described in WO 2015/036583.

Use and application

In another aspect, the present application also provides the use of an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein, and/or a pharmaceutical composition as described herein, in the manufacture of a medicament for the prevention, amelioration and/or treatment of a tumor.

In another aspect, the present application also provides a method of preventing, ameliorating, or treating a tumor, which method can comprise administering to a subject in need thereof an isolated antigen binding protein described herein. In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and/or the pharmaceutical composition described herein can be used for preventing, ameliorating, or treating a tumor.

In the present application, the tumor may be a solid tumor or a hematological tumor. For example, the tumor can comprise a BCMA-positive tumor, which can comprise a myeloma.

In the present application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In another aspect, the present application also provides a method of detecting BCMA in a sample comprising administering an isolated antigen binding protein as described herein. In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

Without wishing to be bound by any theory, the following examples are only intended to illustrate the protein molecules, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

Examples

Example 1 screening of anti-BCMA antibodies Using phage antibody library

Phage natural human antibody libraries (Shanghai Protocyte medicine Co., Ltd.) were alternately sorted by recombinant human BCMA-hFc and BCMA-His proteins (Shanghai Protocyte medicine Co., Ltd., wherein human BCMA-hFc represents a protein formed by the linkage of human BCMA and human Fc, the amino acid sequence of human BCMA protein is shown by GeneBank accession No.: BAB60895.1, and the amino acid sequence of human Fc is shown by SEQ ID NO: 51), and four rounds of sorting were performed. Antigen BCMA-hFc protein 1ml is coated in an enzyme-linked immune tube by using CBS buffer solution, the concentration of BCMA-hFc and BCMA-His is 40 mu g/ml (first round, second round) or 20ug/ml (third round, fourth round), and the temperature is kept overnight at 4 ℃; the next day, 2ml of PBS buffer solution containing 5% of skimmed milk powder is used for sealing the immune tube; adding 1ml of the blocked phase to an immune tube, and incubating for 2h (first, second round) or 1h (third, fourth round) at room temperature; PBST (tween 0.05%) wash 10 times (first, second round) or 15 times (third, fourth round); adding 800 μ l of Gly-HCl buffer solution with pH2.2 for elution, and immediately adding 400 μ l of Tris-HCl buffer solution with pH8.0 for neutralization after 8 minutes; adding into 20ml E.coli SS320 with logarithmic growth phase OD about 0.8, mixing, standing at 37 deg.C for 1 hr; taking out 1ml of bacterial liquid for determining the titer of the phage and preserving the glycerol; coating the residual bacterial liquid on a flat plate, and culturing overnight in an incubator at 37 ℃; the next day, the scraped bacteria were inoculated into 80ml of 2YT-Amp medium in a certain ratio to make OD equal to 0.3, and 20: 1 proportion of the helper phase, then uniformly mixing and standing for 1h at 37 ℃; adding IPTG and Kan antibiotics, performing shaking culture at 250rpm and 30 ℃ overnight; collecting supernatant, precipitating phage with PEG/NaCl solution, and suspending in 1.5ml PBS buffer solution; resuspended phage were used for the next round of enrichment screening, and after 4 rounds of panning, significant enrichment was observed. And (3) identifying the phage antibody clone obtained by panning by ELISA: human BCMA protein (Shanghai Protocyte medicine technology Co., Ltd.) was coated on a 96-well ELISA plate at a concentration of 1. mu.g/ml overnight at 4 ℃. Then, non-specific binding sites were blocked with 5% skim milk powder, washed thoroughly, and the monoclonal phage supernatant was added to a 96-well plate and incubated at 37 ℃ for 2 hours. After fully washing, adding Anti-M13-HRP (GE health care, 27-9421-01), acting at 37 ℃ for 45min, fully washing, adding TMB for color development, acting at room temperature for 5-10min, finally stopping reaction with sulfuric acid, and measuring the OD value of each hole at 450 nm.

6 phage antibody clones that specifically bind to human BCMA (i.e., the isolated BCMA antibodies described herein) were identified by ELISA, and the VH and VL gene sequences were obtained after sequencing, and the 6 phage antibody clones were designated 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23, and the VH and VL gene sequences of the 6 phage antibody clones are shown in table 1.

TABLE 1.6 cloned VH and VL sequences

Example 2 expression and purification of anti-BCMA intact antibodies

Phage antibody clones 8A7, 8A10, 8A11, hBCMA19, hBCMA22, hBCMA23 and positive control antibody chC11D5.3 (patent CN102421801B) were reconstituted onto eukaryotic expression vectors. Briefly, antibody clones 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23, and chc11d5.3 were redesigned as whole IgG1, kappa antibodies (i.e., isolated antigen binding proteins described herein) by: designing a primer to perform PCR amplification on the VH cloned by the phage antibody, and cloning the PCR product to a pCMV-IgG1NDL vector subjected to AgeI and SalI double enzyme digestion; primers were designed to perform PCR amplification on VL of phage antibody clones, and PCR products were cloned by recombination into the AgeI and BsiWI double digested pCMV-kappa vector. After the sequencing is correct, the heavy and light chain expression vectors are co-transfected into 293F cells for transient expression and purified by a ProteinA column to obtain complete IgG1 and kappa antibodies of 8A7, 8A10, 8A11, hBCMA19, hBCMA22, hBCMA23 and chC11D5.3. The gene sequences of the light and heavy chains of 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23, and chc11d5.3 whole antibodies are shown in table 2.

TABLE 2 light and heavy chain sequences of intact antibodies

Example 3 binding affinity assay for anti-BCMA intact antibodies

The binding affinities of 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23 and chc11d5.3 intact antibodies obtained in example 2 to recombinant human BCMA protein were measured using an Octet RED384 instrument (Pall ForteBio). First, human BCMA protein was labeled with Biotin (EZ-Link Sulfo-NHS-LC-Biotin, Pierce, 21327). Analysis of binding kinetics between antigen and antibody was performed by the biofilm interference (BLI) technique using a molecular interaction analyzer, fortebioocettred 384(PALL) (0.1% BSA and 0.02% tween 20 in PBS buffer was used for antigen and antibody dilutions). Fixing the antigen coupled by biotin with the concentration of 20nM to the SA sensor at 1500rpm/min for 2 min; then, the antibodies were combined with the antibody solutions (i.e., 8A7, 8A10, 8A11, hBCMA19, hBCMA22, hBCMA23 and chC11D5.3 intact antibodies obtained in example 2) in double dilution for 5min at 1500 rpm/min. Finally, the dissociation was carried out for 20 minutes at 1500 rpm/min. The residual antibody was regenerated by glycine pulsing. The obtained results were subjected to Data Analysis using Octet Data Analysis 9.0 software (fortebio) to calculate the binding strength between the antigen and the antibody, and K was obtained_DValues, Ka (1/Ms) values and Kd (1/s) values. The anti-BCMA whole antibodies 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23 and chc11d5.3 all bind with high affinity to recombinant human BCMA protein (as shown in table 3).

TABLE 3 binding affinities of anti-BCMA intact antibodies to human BCMA protein

In table 3, Ka: an association rate constant; kd: an off rate constant; k_D: affinity constant, equal to Kd/Ka.

As can be seen from table 3, 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23 intact antibody has a smaller off-rate than the control antibody chc11d5.3, so 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23 intact antibody has a higher affinity than the control antibody chc11d5.3.

Example 4 detection of binding Activity of anti-BCMA intact antibodies to cell surface BCMA

Detection of cell surface target antigen (BCMA) binding activity to antibodies (i.e., 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23, and chc11d5.3 whole antibodies obtained in example 2) was performed by flow cytometric fluorescence sorting (FACS) using an iquee Screener flow cytometer (purchased from IntelliCyt) using PBS containing 0.1% BSA as a buffer, as follows:

1. buffer was used to make concentrations of 1 × 10⁶cells/ml of target cells (i.e., MM1S myeloma cells) were added to a 96-well tip-and-bottom plate (corning 3894), 30. mu.l per well;

2. using a buffer solution to prepare a detection antibody with the concentration of 20 mu g/ml, and diluting the antibody according to a 3-fold ratio to form 8 concentration gradients;

3. adding the prepared antibodies with different concentrations into the paved target cells according to 30 mul/hole and mixing evenly;

incubating for 1 hour at 4.4 ℃ in a refrigerator;

5. adding 150 mul of buffer solution into each hole, centrifuging for 5 minutes at 300g, removing supernatant, and shaking loose cells;

6. repeating the step 5;

7. preparing a fluorescent secondary antibody (ab98593) by using a buffer solution according to a ratio of 1:200, adding 30 mu l of the fluorescent secondary antibody into each hole, uniformly mixing, and incubating for 30 minutes in a refrigerator at 4 ℃;

8. adding 150 mul of buffer solution into each hole, centrifuging for 5 minutes at 300g, removing supernatant, and shaking loose cells;

9. repeating the step 8;

10. add 35. mu.l buffer to each well and mix well before detecting with flow meter.

The results of the flow affinity binding assay are shown in table 4 and figure 1.

TABLE 4 binding results of anti-BCMA intact antibodies to cell surface BCMA proteins

Intact antibodies	8A7	8A10	8A11	hBCMA19	hBCMA22	hBCMA23	chC11D5.3
								EC50(μg/ml)	1.31	0.50	1.25	0.33	0.68	0.71	1.55

As can be seen from table 4, 8a7, 8a10, 8a11, hBCMA19, hBCMA22, hBCMA23 whole antibody has stronger binding activity with myeloma cell MM1S which expresses low level of BCMA antigen; and all had lower EC50 values than the positive control antibody chc11d5.3. As can be seen from FIG. 1, the mean fluorescence intensity of the binding of the intact antibodies 8A7, 8A10, 8A11, hBCMA19, hBCMA22, hBCMA23 to MMIS of myeloma cells was high.

Example 5 ADCC Activity assay of anti-BCMA intact antibodies

ADCC Activity of anti-BCMA intact antibodies (i.e., 8A7, 8A10, 8A11, hBCMA19, hBCMA22, hBCMA23, and chC11D5.3 intact antibodies) against BCMA antigen-expressing myeloma cells MM1S by Cytotox

Detection was performed using a non-radioactive cytotoxicity detection kit (Promega). The specific processing procedures of the cell and antibody samples are as follows:

resuspend MM1S cells were harvested in 5% FBS (GIBICO) -containing X-VIVO (LONZA) medium, resulting in a total of 2.0X10⁵cells/ml of cell suspension are ready for use. Take 8x10⁷PBMC cells (healthy human) were resuspended to 16ml in 5% FBS (GIBICO) -containing X-VIVO (LONZA) medium to form 5.0X10⁶cells/ml of cell suspension are ready for use. 8A7, 8A10, 8A11, hBCMA19, hBCMA22, hBCMA23 and positive control chC11D5.3 whole antibody were diluted with medium to different antibody concentrations. Then, after plating with 50. mu.l MM1S cell suspension, 100. mu.l PBMC cell suspension and 50. mu.l each antibody solution per well (96-well V-plate (Thermo)), CO was added at 37 ℃ C₂The incubator was incubated for 24 hours at rest. As control wells, 50. mu.l of MM1S cell suspension plus 100. mu.l of PBMC cell suspension were used. According to CytoTox

The ADCC activity of the antibody is detected by a non-radioactive cytotoxicity detection kit (Promega) according to the following formula: cytotoxicity ═ 100X (experimental wells-control wells)/(target cells maximal killing-target cells spontaneous release). Experiment ofThe results are shown in FIG. 2, where isotype control refers to intact IgG1, kappa antibody not associated with this target. As can be seen from fig. 2, the 8a7, 8a10, 8a11, hBCMA19, hBCMA22 and hBCMA23 whole antibodies can effectively mediate effective killing of BCMA positive myeloma cells MM1S, and have better killing effect than the positive control chc11d5.3, i.e. have more significant ADCC activity than the positive control.

Example 6 CDC Activity assay of anti-BCMA intact antibodies

The ability of anti-BCMA whole antibody to elicit CDC effects on CHO-BCMA cells was tested using a cytotoxicity assay kit (Promega, Cat # G1780) as follows:

1. preparing a culture medium (A: DMEM + 2% FBS, B: DMEM + 2% FBS + 10% rabbit complement);

2. target cells CHO-BCMA were centrifuged at 400g for 5 minutes, and then the cells were resuspended in the above medium A to a target cell density of 4X10⁵Adding 100 ul/ml of each cell into a 96-well cell culture plate;

3. antibodies were diluted with medium B above, at 10ug/ml starting concentration, in 3-fold gradient dilutions, for a total of 8 gradients. Adding 100 ul/well into detection wells, setting two multiple wells at each concentration point, and taking chC11D5.3 as a positive control antibody;

4. complement is Rabbit complement Rabbit Complent 3-4week (Cat #31061-3) purchased from PelFreez Bio, and the final concentration of complement used is 5%;

5. setting a control hole according to the requirements of the kit, after the obtained mixture is incubated in an incubator at 37 ℃ for 4 hours, recording the 490nM light absorption value by a microplate reader, calculating the lysis percentage of the target cells by using a formula given by the kit, and analyzing and processing data by Graphpad prism 8.

The results are shown in FIG. 3, where isotype control refers to intact IgG1, kappa antibody not associated with this target. As can be seen from fig. 3, the 8a10, hBCMA22, hBCMA23 intact antibodies all induced strong CDC effect in a dose-dependent manner, i.e. all had significant CDC activity, with the 8a10 intact antibody killing better than the positive control chc11d5.3.

Example 7 anti-BCMA intact antibodies reduce tumor burden in xenograft mice

We used NOD scid common gamma chain knockout (NSG) mice lacking functional B, T and NK cell populations injected 6X 10 in tail vein⁶After each MM1S-luc cell, mice were randomly assigned to 3 groups of 8 mice each. The first group received an equivalent dose of PBS until the end of the experiment, while the second and third groups received a 200ug weekly injection of the experimental group antibody and isotype control antibody, respectively. Antibody was administered intraperitoneally (i.p.) for a period of 4 weeks starting on the date of tumor cell injection. Mouse tumor growth was monitored once a week using a mouse in vivo imaging system and bioluminescence was measured 3 minutes after intraperitoneal injection of fluorescein.

Experimental results as shown in figure 4, the group treated with hBCMA22 antibody showed significantly lower tumor burden starting from the first measurement point on day 7. In addition, the panel showed a smaller overall tumor burden throughout the monitoring period. At day 28 post target cell injection, a higher tumor burden was seen in mice receiving isotype control antibody, as shown in figure 5. Mice receiving the BCMA antibody were able to significantly inhibit tumor growth.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Sequence listing

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<223> 8A7 VL

<400> 23

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ser

20 25 30

Gly Ile His Leu Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Gly Arg Asn Arg Ala Ala Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Arg Phe Pro Arg His Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 24

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A10/hBCMA19 VL

<400> 24

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Pro

20 25 30

Gly Ile Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Gly Arg Asn Arg Ala Ala Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Arg Phe Pro Arg His Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 25

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A11/hBCMA22/hBCMA23 VL

<400> 25

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Pro

20 25 30

Gly Ile Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Gly Ser Asn Arg Pro Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Arg Phe Pro Arg His Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 26

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL formula

<220>

<221> misc_feature

<222> (32)..(32)

<223> Xaa = Pro or Ser

<220>

<221> misc_feature

<222> (35)..(35)

<223> Xaa = His or Ser

<220>

<221> misc_feature

<222> (36)..(36)

<223> Xaa = Phe or Leu

<220>

<221> misc_feature

<222> (37)..(37)

<223> Xaa = Ile or Leu

<220>

<221> misc_feature

<222> (56)..(56)

<223> Xaa = Arg or Ser

<220>

<221> misc_feature

<222> (59)..(59)

<223> Xaa = Ala or Pro

<220>

<221> misc_feature

<222> (60)..(60)

<223> Xaa = Ala or Ser

<400> 26

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Xaa

20 25 30

Gly Ile Xaa Xaa Xaa His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Gly Xaa Asn Arg Xaa Xaa Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Arg Phe Pro Arg His Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 27

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A7 VH

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 28

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A10 VH

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Ser Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 29

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A11 VH

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 30

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A7/8A10/8A11 VH

<220>

<221> misc_feature

<222> (48)..(48)

<223> Xaa = Ile or Met

<220>

<221> misc_feature

<222> (66)..(66)

<223> Xaa = Gly or Ser

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Xaa

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Xaa Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 31

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A7/8A10/8A11/hBCMA19/hBCMA22/hBCMA23 light chain constant region

<400> 31

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 32

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A7/8A10/8A11/hBCMA19/hBCMA22/hBCMA23 heavy chain constant region

<400> 32

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 33

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A7 light chain

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ser

20 25 30

Gly Ile His Leu Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Gly Arg Asn Arg Ala Ala Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Arg Phe Pro Arg His Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 34

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A10/hBCMA19 light chain

<400> 34

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Pro

20 25 30

Gly Ile Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Gly Arg Asn Arg Ala Ala Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Arg Phe Pro Arg His Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 35

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A11/hBCMA22/hBCMA23 light chain

<400> 35

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Pro

20 25 30

Gly Ile Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Gly Ser Asn Arg Pro Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Arg Phe Pro Arg His Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 36

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A7 heavy chain

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 37

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A10 heavy chain

<400> 37

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Ser Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 38

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 8A11 heavy chain

<400> 38

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 39

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> code 8A7 VL

<400> 39

gaaatcgttc tgacccagag tccagcgacg ctgagtctga gtccgggtga acgcgcgacg 60

ctgagttgcc gtgccagcga aagcgttagc gtgagtggca ttcacttact ccactggtac 120

cagcagaaac cgggccaagc cccacgtctg ctgatctatc tgggacgcaa tcgggcggcc 180

ggcattccag cccgctttag cggcagcggt agcggtaccg attttacgct gaccatcagc 240

agtctggagc cagaagatgc cgccatctac tactgtttac agagcagaag gtttcctcga 300

cacttcggcc aaggtaccaa actggaaatc aag 333

<210> 40

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> code 8A10/hBCMA19 VL

<400> 40

gaaatcgttc tgacccagag tccagcgacg ctgagtctga gtccgggtga acgcgcgacg 60

ctgagttgcc gtgccagcga aagcgttagc gtgcctggca tatctttcat ccactggtac 120

cagcagaaac cgggccaagc cccacgtctg ctgatctatc tgggacgcaa tcgggcggcc 180

ggcattccag cccgctttag cggcagcggt agcggtaccg attttacgct gaccatcagc 240

agtctggagc cagaagatgc cgccatctac tactgtttac agagcagaag gtttcctcga 300

cacttcggcc aaggtaccaa actggaaatc aag 333

<210> 41

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding 8A11/hBCMA22/hBCMA23 VL

<400> 41

gaaatcgttc tgacccagag tccagcgacg ctgagtctga gtccgggtga acgcgcgacg 60

ctgagttgcc gtgccagcga aagcgttagc gtgcctggca tatctttcat ccactggtac 120

cagcagaaac cgggccaagc cccacgtctg ctgatctatc tgggcagcaa tcgaccatcc 180

ggcattccag cccgctttag cggcagcggt agcggtaccg attttacgct gaccatcagc 240

agtctggagc cagaagatgc cgccatctac tactgtttac agagcagaag gtttcctcga 300

cacttcggcc aaggcaccaa actggaaatc aag 333

<210> 42

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding 8A7 VH

<400> 42

caagttcagc tcgtgcagag cggctccgag gtgaagcagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata cagcttcgcc gattacacca tcaactgggt tcgtcaagct 120

cccggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaggtcg tttcgtgttc tctttagaca caagcgcgag caccgcctat 240

ttacagatca gctctttaaa ggccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag c 351

<210> 43

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding 8A10 VH

<400> 43

caagttcagc tcgtgcagag cggctccgag gtgaagcagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata cagcttcgcc gattacacca tcaactgggt tcgtcaagct 120

cccggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaagtcg tttcgtgttc tctttagaca caagcgcgag caccgcctat 240

ttacagatca gctctttaaa ggccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag c 351

<210> 44

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding 8A11 VH

<400> 44

caagttcagc tcgtgcagag cggctccgag gtgaagcagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata cagcttcgcc gattacacca tcaactgggt tcgtcaagct 120

cccggtcaag gtctggagtg gataggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaggtcg tttcgtgttc tctttagaca caagcgcgag caccgcctat 240

ttacagatca gctctttaaa ggccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag c 351

<210> 45

<211> 654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding 8A7 light chain

<400> 45

gaaatcgttc tgacccagag tccagcgacg ctgagtctga gtccgggtga acgcgcgacg 60

ctgagttgcc gtgccagcga aagcgttagc gtgagtggca ttcacttact ccactggtac 120

cagcagaaac cgggccaagc cccacgtctg ctgatctatc tgggacgcaa tcgggcggcc 180

ggcattccag cccgctttag cggcagcggt agcggtaccg attttacgct gaccatcagc 240

agtctggagc cagaagatgc cgccatctac tactgtttac agagcagaag gtttcctcga 300

cacttcggcc aaggtaccaa actggaaatc aagcgtacgg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 480

ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 540

agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 600

acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgc 654

<210> 46

<211> 654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding 8A10/hBCMA19 light chain

<400> 46

gaaatcgttc tgacccagag tccagcgacg ctgagtctga gtccgggtga acgcgcgacg 60

ctgagttgcc gtgccagcga aagcgttagc gtgcctggca tatctttcat ccactggtac 120

cagcagaaac cgggccaagc cccacgtctg ctgatctatc tgggacgcaa tcgggcggcc 180

ggcattccag cccgctttag cggcagcggt agcggtaccg attttacgct gaccatcagc 240

agtctggagc cagaagatgc cgccatctac tactgtttac agagcagaag gtttcctcga 300

cacttcggcc aaggtaccaa actggaaatc aagcgtacgg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 480

ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 540

agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 600

acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgc 654

<210> 47

<211> 654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding 8A11/hBCMA22/hBCMA23 light chain

<400> 47

gaaatcgttc tgacccagag tccagcgacg ctgagtctga gtccgggtga acgcgcgacg 60

ctgagttgcc gtgccagcga aagcgttagc gtgcctggca tatctttcat ccactggtac 120

cagcagaaac cgggccaagc cccacgtctg ctgatctatc tgggcagcaa tcgaccatcc 180

ggcattccag cccgctttag cggcagcggt agcggtaccg attttacgct gaccatcagc 240

agtctggagc cagaagatgc cgccatctac tactgtttac agagcagaag gtttcctcga 300

cacttcggcc aaggcaccaa actggaaatc aagcgtacgg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 480

ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 540

agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 600

acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgc 654

<210> 48

<211> 1341

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding the 8A7 heavy chain

<400> 48

caagttcagc tcgtgcagag cggctccgag gtgaagcagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata cagcttcgcc gattacacca tcaactgggt tcgtcaagct 120

cccggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaggtcg tttcgtgttc tctttagaca caagcgcgag caccgcctat 240

ttacagatca gctctttaaa ggccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag cgcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaacctg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccgggtaa a 1341

<210> 49

<211> 1341

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding the 8A10 heavy chain

<400> 49

caagttcagc tcgtgcagag cggctccgag gtgaagcagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata cagcttcgcc gattacacca tcaactgggt tcgtcaagct 120

cccggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaagtcg tttcgtgttc tctttagaca caagcgcgag caccgcctat 240

ttacagatca gctctttaaa ggccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag cgcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaacctg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccgggtaa a 1341

<210> 50

<211> 1341

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding the 8A11 heavy chain

<400> 50

caagttcagc tcgtgcagag cggctccgag gtgaagcagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata cagcttcgcc gattacacca tcaactgggt tcgtcaagct 120

cccggtcaag gtctggagtg gataggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaggtcg tttcgtgttc tctttagaca caagcgcgag caccgcctat 240

ttacagatca gctctttaaa ggccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag cgcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaacctg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccgggtaa a 1341

<210> 51

<211> 231

<212> PRT

<213> human (Homo sapiens)

<400> 51

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

1 5 10 15

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 52

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22 HCDR2

<400> 52

Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe Arg

1 5 10 15

Gly

<210> 53

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22/hBCMA23 HCDR2 general formula

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Ala or Thr

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa = Gly or Ser

<400> 53

Trp Ile Asn Thr Glu Thr Arg Glu Pro Xaa Tyr Ala Tyr Asp Phe Arg

1 5 10 15

Xaa

<210> 54

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22/hBCMA23 H-FR1

<400> 54

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala

20 25 30

<210> 55

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22/hBCMA23 H-FR2

<400> 55

Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 56

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22/hBCMA23 H-FR3

<400> 56

Arg Phe Thr Phe Thr Leu Asn Thr Ser Ile Ser Thr Ala Tyr Leu Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 57

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22 VH

<400> 57

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe

50 55 60

Arg Gly Arg Phe Thr Phe Thr Leu Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 58

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA23 VH

<400> 58

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Ser Arg Phe Thr Phe Thr Leu Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 59

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22/hBCMA23VH general formula

<220>

<221> misc_feature

<222> (59)..(59)

<223> Xaa = Ala or Thr

<220>

<221> misc_feature

<222> (66)..(66)

<223> Xaa = Gly or Ser

<400> 59

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Xaa Tyr Ala Tyr Asp Phe

50 55 60

Arg Xaa Arg Phe Thr Phe Thr Leu Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 60

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA19/hBCMA22 heavy chain

<400> 60

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe

50 55 60

Arg Gly Arg Phe Thr Phe Thr Leu Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 61

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hBCMA23 heavy chain

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr

20 25 30

Thr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Thr Tyr Ala Tyr Asp Phe

50 55 60

Arg Ser Arg Phe Thr Phe Thr Leu Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Arg Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 62

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding hBCMA19/hBCMA22 VH

<400> 62

caagttcagc tcgtgcagag cggcgccgag gtgaagaagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata caccttcgcc gattacacca tcaactgggt tcgtcaagct 120

accggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccgcctat 180

gcctacgact tcagaggtcg tttcacgttc actttaaaca caagcatcag caccgcctat 240

ttagagctca gctctttaag gtccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag c 351

<210> 63

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding hBCMA23VH

<400> 63

caagttcagc tcgtgcagag cggcgccgag gtgaagaagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata caccttcgcc gattacacca tcaactgggt tcgtcaagct 120

accggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaagtcg tttcacgttc actttaaaca caagcatcag caccgcctat 240

ttagagctca gctctttaag gtccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag c 351

<210> 64

<211> 1341

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding hBCMA19/hBCMA22 heavy chain

<400> 64

caagttcagc tcgtgcagag cggcgccgag gtgaagaagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata caccttcgcc gattacacca tcaactgggt tcgtcaagct 120

accggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccgcctat 180

gcctacgact tcagaggtcg tttcacgttc actttaaaca caagcatcag caccgcctat 240

ttagagctca gctctttaag gtccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag cgcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaacctg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccgggtaa a 1341

<210> 65

<211> 1341

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> encoding hBCMA23 heavy chain

<400> 65

caagttcagc tcgtgcagag cggcgccgag gtgaagaagc ccggcgcttc cgtgaaggtg 60

agctgcaagg ccagcggata caccttcgcc gattacacca tcaactgggt tcgtcaagct 120

accggtcaag gtctggagtg gatgggctgg atcaacaccg aaaccagaga gcccacctat 180

gcctacgact tcagaagtcg tttcacgttc actttaaaca caagcatcag caccgcctat 240

ttagagctca gctctttaag gtccgaggat accgccgtgt actactgcgc tcgtgacatc 300

agatacgtga tggactactg gggccaaggt acactggtga ccgtgtccag cgcgtcgacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaacctg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1080

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccgggtaa a 1341

Claims

1. An isolated antigen binding protein comprising at least one CDR in a VH set forth in amino acid sequence SEQ ID NO 30 or 59; and which comprises at least one CDR in the VL shown in amino acid sequence SEQ ID NO. 26.

2. The isolated antigen binding protein of claim 1, having one or more of the following properties:

3) can inhibit tumor growth and/or tumor cell proliferation.

3. The isolated antigen binding protein of any of claims 1-2, wherein the HCDR1 of the VH comprises the amino acid sequence set forth in SEQ ID No. 4; and/or the HCDR2 of said VH comprises the amino acid sequence shown as SEQ ID NO 12 or 53; and/or the HCDR3 of the VH comprises an amino acid sequence shown as SEQ ID NO. 6.

4. The isolated antigen binding protein of any of claims 1-3, wherein the LCDR1 of the VL comprises the amino acid sequence set forth in SEQ ID NO 10; and/or the LCDR2 of the VL comprises the amino acid sequence shown in SEQ ID NO. 11; and/or the LCDR3 of the VL comprises the amino acid sequence shown in SEQ ID NO. 3.

5. An isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-4.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A method of making the isolated antigen binding protein of any one of claims 1-4, the method comprising culturing the cell of claim 7 under conditions such that the isolated antigen binding protein of any one of claims 1-4 is expressed.

9. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-4, the nucleic acid molecule of claim 5, the vector of claim 6, and/or the cell of claim 7, and optionally a pharmaceutically acceptable adjuvant.

10. Use of the isolated antigen binding protein of any one of claims 1-4, the nucleic acid molecule of claim 5, the vector of claim 6, the cell of claim 7, and/or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the prevention, amelioration, and/or treatment of a tumor.