Patent Description:
Multiple myeloma (MM) is a common hematological malignancy, accounting for <NUM>% of all deaths resulted from cancer. MM is a heterogeneous disease and mainly caused by chromosomal translocation of t(<NUM>;<NUM>), t(<NUM>;<NUM>), t(<NUM>;<NUM>), del(<NUM>), del(<NUM>) (among others) (<NPL>; <NPL>; <NPL>). The main condition of multiple myeloma (MM) is the infinite expansion and enrichment of plasma cells in bone marrow, thereby leading to osteonecrosis. MM-affected patients may experience a variety of disease-related symptoms due to bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychological burden of cancer diagnosis. At present, the main treatments are chemotherapy and stem cell transplantation. The mainly used chemotherapy drugs are steroid, thalidomide, lenalidomide, bortezomib or a combination of various cytotoxic agents. For younger patients, high-dose chemotherapy can be used in combination with autologous stem cell transplantation.

BCMA (B-cell maturation antigen) is B-cell maturation antigen, a type III transmembrane protein consisting of <NUM> amino acid residues, and belongs to TNF receptor superfamily. The ligand of BCMA belongs to TNF superfamily, such as proliferation-inducing ligand (APRIL), B lymphocyte stimulating factor (BAFF). After binding to its ligand, BCMA activates B cell proliferation and survival. BCMA is specifically and highly expressed on the surface of plasma cells and multiple myeloma cells, but not expressed in hematopoietic stem cells and other normal tissue cells, therefore BCMA can be an ideal target for targeted therapy of MM.

Documents <CIT> and <CIT> disclose anti-BCMA antibodies and their use in chimeric antigen receptor (CAR) to BCMA.

Summing up, there is an urgent need in the art for antibodies specific to BCMA and immune effector cells targeting BCMA with improved efficacy.

It is an object of the present invention to provide antibodies specific to BCMA and immune effector cells that target BCMA. The invention is as defined in the attached claims.

In a first aspect, an antibody that targets BCMA is provided in the invention, and the antibody is selected from the group consisting of:.

In a specific embodiment, the antibody is selected from the group consisting of:.

In a further embodiment, an antibody of the present invention comprises an amino acid sequence selected from SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

In a second aspect, a nucleic acid is provided in the present invention, encoding the antibody of the invention.

In a third aspect, an expression vector is provided in the present invention, comprising the nucleic acid of the second aspect of the invention.

In a fourth aspect, a host cell is provided in the present invention, comprising the expression vector of the third aspect of the invention or has the nucleic acid of the second aspect of the invention integrated in the genome.

In a fifth aspect, the use of the antibody of the present invention is provided in the present invention, for the preparation of a targeting drug, an antibody drug conjugate or a multifunctional antibody which specifically targets tumor cells expressing BCMA; or.

In a sixth aspect, a multifunctional immunoconjugate is provided in the present invention, comprising:.

In a specific embodiment, the molecule that inhibits tumors is an antitumor cytokine or an antitumor toxin. Preferably, the cytokine comprises: IL-<NUM>, IL-<NUM>, type I interferon, TNF-alpha.

In a specific embodiment, the molecule that targets a surface marker of an immune cell is an antibody or a ligand that binds to a surface marker of an immune cell; and preferably, the surface marker of an immune cell comprises: CD3, CD16, CD28, and more preferably, the antibody that binds to a surface marker of an immune cell is an anti-CD3 antibody.

In a specific embodiment, the molecule that targets a surface marker of an immune cell is an antibody that binds to a surface marker of a T cell.

In a seventh aspect, a nucleic acid is provided in the present invention, encoding the multifunctional immunoconjugate of the invention.

In a eighth aspect, the use of the multifunctional immunoconjugate of the seventh aspect of the present invention is provided in the present invention, for the preparation of an antitumor drug, or.

In a ninth aspect, a chimeric antigen receptor is provided in the present invention, comprising an extracellular domain, a transmembrane domain and an intracellular signal domain, the extracellular domain comprises the antibody of the invention, and the antibody preferably is a single-chain antibody or domain antibody.

In a specific embodiment, the intracellular signal domain comprises one or more co-stimulatory signal domains and/or primary signal domains.

In a specific embodiment, the chimeric antigen receptor further comprises a hinge domain.

In a specific embodiment, the transmembrane domain is selected from the group consisting of alpha, beta, zeta chain of TCR, transmembrane regions of CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD1; and/or.

In a specific embodiment, the chimeric antigen receptor comprises the following sequentially linked an antibody, a transmembrane region and an intracellular signal region:.

In atenth aspect, a nucleic acid is provided in the present invention, encoding the chimeric antigen receptorof the invention.

In an eleventh aspect, an expression vector is provided in the present invention, comprising the nucleic acid of the tenth aspect of the invention.

In a twelfth aspect, a virus is provided in the present invention, comprising the vector of the eleventh aspect of the invention.

In a preferred embodiment, the virus is a lentivirus.

In a thirteenth aspect, the use of the chimeric antigen receptor of the tenth aspect of the present invention, or the nucleic acid of the eleventh aspect of the present invention, or the expression vector of the twelfth aspect of the present invention, or the virus of the thirteenth aspect of the present invention is provided in the present invention, for the preparation of genetically modified immune cells targeting a tumor cell expressing BCMA.

In a preferred embodiment, the tumor expressing BCMA is multiple myeloma.

In a fourteenth aspect, a genetically modified immune cell is provided in the present invention, which is transduced with the nucleic acid of the tenth aspect of the invention, or the expression vector of the eleventh aspect of the invention or the virus of the twelfth aspect of the present invention; or expresses the chimeric antigen receptor of the ninth aspect of the invention.

The immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.

In a specific embodiment, the genetically modified immune cell further expresses a sequence other than the chimeric antigen receptor of the tenth aspect of the invention, and the other sequence comprises a cytokine, or another chimeric antigen receptor, or a chemokine receptor, or an siRNA that reduces PD-<NUM> expression, or a protein that blocks PD-L1, or a TCR, or a safety switch;.

In a fifteenth aspect, the genetically modified immune cell of the present invention is provided in the present invention, for use as a tumor-suppressing drug, and the tumor is a tumor expressing BCMA.

In a sixteenth aspect, a pharmaceutical composition is provided in the present invention, comprising:.

It is to be understood that the various technical features of the present invention mentioned above and the various technical features specifically described hereinafter (as in the Examples) may be combined with each other within the scope of the present invention to constitute a new or preferred technical solution, which will not be repeated one by one herein.

Through extensive and intensive research, the inventors have unexpectedly discovered antibodies that specifically bind to BCMA, and these antibodies can be applied to prepare various targeted antitumor drugs and drugs for diagnosing tumors. The present invention has been completed based on the above findings.

The technical terms used herein have the same or similar meanings as conventionally understood by a skilled person. Some terms are defined as follows for understanding the invention,.

The term "BCMA" as used herein refers to a B cell maturation antigen, which is a type III transmembrane protein consisting of <NUM> amino acid residues (NCBI Reference Sequence: NP_001183. <NUM>), and the amino acid sequence is shown in SEQ ID No: <NUM>. In a specific embodiment, BCMA refers to human BCMA.

The term "APRIL" as used herein refers to A proliferation-inducing ligand, which is a proliferation-inducing ligand consisting of <NUM> amino acid residues (NCBI Reference Sequence: NP_003799. <NUM>), and belongs to TNF superfamily.

The term "antibody" as used herein refers to an antigen-binding protein of the immune system. The term "antibody" as used herein includes an intact full length antibody having an antigen binding region and any fragments thereof retaining an "antigen-binding portion" or "antigen-binding region", or a single strand thereof, such as a single chain variable fragment (scFv). A native antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains or antigen-binding fragments thereof interconnected by a disulfide bond. The term "antibody" also includes all recombinant forms of antibodies, particularly the antibodies described herein, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and antibody fragments that bind to antigens and derivatives hereinafter. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. VH and VL can be further subdivided into hypervariable regions named complementarity determining regions (CDRs), which are interspersed in more conserved regions named framework regions (FR). Each VH and VL consists of three CDRs and four FRs, from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

Antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragment consisting of VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region (<NPL>); (v) F(ab')<NUM> fragment, a bivalent fragment comprising two linked Fab fragments; (vi) a single-chain Fv molecule antigen binding site (<NPL>; <NPL>); (vii) bispecific single-chain Fv dimer (<CIT>); (viii) "dibody" or "tribody", multi-valent or multi-specific fragments constructed by gene fusion (<NPL>; <CIT>; <NPL>); and (ix) scFv genetically fused to identical or different antibodies (<NPL>).

The term "Fc" or "Fc region" as used herein includes a polypeptide comprising an antibody constant region other than the first constant region immunoglobulin domain. Therefore, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and flexible hinges at N-terminus of these domains. For IgA and IgM, Fc can include J chain. For IgG, Fc includes hinges between immunoglobulin domains Cγ2 and Cγ3 as well as Cγ1 and Cy2. Boundaries of Fc region may vary, however, the human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxy terminus, where numbering is based on EU index of Kabat. For human IgGl, Fc is defined herein to include residue P232 to its carboxy terminus, where numbering is based on EU index of Kabat. Fc may refer to the isolated region, or the region in the environment of Fc polypeptide, such as an antibody. The "hinge" as said above includes a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, IgG CH1 domain ends at position EU220 and IgG CH2 domain begins at residue EU237. Therefore, for IgG, the antibody hinge herein is defined to include <NUM> (D221 of IgG1) to <NUM> (A231 of IgG1), where the numbering is based on EU index of Kabat.

The term "parent antibody" or "parent immunoglobulin" as used herein includes an unmodified antibody which is to be modified to produce variants. The parent antibody can be a naturally occurring antibody, or a variant or modified version of a naturally occurring antibody. A parent antibody can refer to the antibody itself, a composition comprising the parent antibody, or a nucleic acid sequence encoding the same. The term "parent antibody" or "parent immunoglobulin" as used herein includes a murine or chimeric antibody that is to be modified to produce a humanized antibody.

The term "variant antibody" or "antibody variant" as used herein includes an antibody sequence that differs from the parent antibody sequence by at least one amino acid modification compared with the parent antibody. A variant antibody sequence herein has at least about <NUM>%, preferably at least about <NUM>%, more preferably at least about <NUM>% amino acid sequence identity to the parent antibody sequence. An antibody variant can refer to the antibody itself, a composition comprising the parent antibody, or a nucleotide sequence encoding the same.

The term "variant" as used herein includes an antibody sequence that differs from the parent antibody sequence by at least one amino acid modification compared with the parent antibody. In a specific embodiment, a variant antibody sequence herein has at least about <NUM>%, preferably at least about <NUM>%, more preferably at least about <NUM>%, more preferably at least about <NUM>%, more preferably at least about <NUM>%, most preferably at least about <NUM>% amino acid sequence identity to the parent antibody sequence. An antibody variant can refer to the antibody itself, a composition comprising the parent antibody, or a nucleotide sequence encoding the same. The term "amino acid modification" includes amino acid substitution, addition and/or deletion, and "amino acid substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, substitution R94K means that the arginine at position <NUM> is replaced by lysine, and "amino acid insertion" as used herein refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" refers to removal of an amino acid at a particular position in a parent polypeptide sequence.

The term "conservative modification" or "conservative sequence modification" as used herein refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. A family of amino acid residues having similar side chains has been defined in the art. These families include amino acids containing basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged acute side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR regions or the framework regions of the antibody of the present invention can be replaced with amino acid residues of other families with identical side chain, and the function retained by the altered antibody (variant antibody) can be tested.

All positions of immunoglobulin heavy chain constant region discussed in the present invention are numbered based on EU index of Kabat (<NPL>).

The term "antigenic determinant" as used herein, also named as antigenic epitope, may consist of a contiguous sequence of BCMA protein sequence or a discontinuous three-dimensional structure of BCMA protein sequence.

The term "chimeric antigen receptor" or "CAR" as used herein, refers to a polypeptide comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and a cytoplasmic signaling domain (i.e., an intracellular signal domain), and the intracellular signal domain refers to a protein that transmits signals into a cell by producing a second messenger through a defined signaling pathway, thereby regulating cellular activities, or a protein that correspondes to such a messenger and acts as an effector, including a primary signal domain and a functional signaling domain (i.e., a co-stimulatory signal domain) derived from a stimulatory molecule as defined below. The intracellular signal domain produces a signal that promotes the immune effector function of cells of the CAR (e.g., CAR T cells), and examples of immune effector functions, such as in CART cells, includes cell lytic activity and helper activity, including secretion of cytokine.

The term "primary signal domain" refers to modulating the initial activation of a TCR complex in an irritating manner. In one aspect, the primary signal domain is elicited by, for example, binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating a T cell response (including, but not limited to, proliferation, activation, differentiation, etc.). The primary signal domain that functions in a stimulatory manner may comprise an immunoreceptor tyrosine activation motif or a signaling motif of ITAM. Examples of primary signal domains comprising ITAM that are particularly useful in the present invention include, but are not limited to, the sequence derived from TCR ξ, FcRy, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d. In an exemplary CAR of the invention, in any one or more of the CARs of the invention, the intracellular signaling domain comprises an intracellular signaling sequence, such as the primary signal domain of CD3ξ.

The term "co-stimulatory signal domain" refers to a "co-stimulatory molecule" which is a related binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of a T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules or ligands thereof which are required for an effective immune response and non-antigen receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-<NUM>, LFA-<NUM> (CD11a/CD18) and <NUM>-1BB (CD137).

In the present invention, in one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, and the intracellular signaling domain comprises a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, and the intracellular signaling domain comprises a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, and the intracellular signaling domain comprises at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino acid (ND end) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during processing and localization of the CAR to the cell membrane.

The term "CD3ξ" as used herein is defined as a protein provided by GenBan Accession No. BAG36664. <NUM>, or equivalent residues from a non-human species such as a mouse, rodent, monkey, ape, and the like. "CD3ξ domain" as used herein is defined as amino acid residues from the cytoplasmic domain of ξ chain sufficient to functionally deliver the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of ξ comprises residues <NUM> to <NUM> of GenBan Accession No. BAG36664. <NUM>, a functional ortholog thereof - equivalent residues from non-human species such as a mouse, rodents, monkey, ape, etc..

The term "<NUM>-1BB" as used herein refers to a member of TNFR superfamily having the amino acid sequence of GenBank Acc. No. AAA62478. <NUM>, or equivalent residues from a non-human species such as a mouse, rodent, monkey, ape and the like. "<NUM>-1BB co-stimulatory domain" is defined as amino acid sequence <NUM>-<NUM> of GenBank ACC. No. AAA62478. <NUM>, or equivalent residues from non-classified species such as mouse, rodent, monkey, ape, etc. In one aspect, the "<NUM>-1BB co-stimulatory domain" is the sequence provided in SEQ ID NO: <NUM>, or equivalent residues from a non-human species such as a mouse, rodent, monkey, ape, and the like.

The term "interferon" as used herein refers to a full-length interferon, or an interferon fragment (truncated interferon) or interferon mutant substantially retaining the biological activities of a full-length wild-type interferon (e.g., retaining at least <NUM>%, preferably at least <NUM>%, more preferably at least <NUM>%, <NUM>% or <NUM>% of those of a full length interferon). Interferons include type I interferons (e.g., interferon α and interferon β) and type II interferons (e.g., interferon y).

The antibody of the present invention or a variant thereof can be applied to prepare various targeted antitumor drugs as well as drugs for diagnosing tumors, in particular, for preparing immune effector cells targeting BCMA.

In the present disclosure, antigen binding proteins having an antigen-binding region based on scFv, including antibodies, are described. A recombinant BCMA was used to select scFv from a human scFv phage display library. These molecules display fine specificity. For example, the antibody only recognizes K562 cells stably expressing BCMA and does not recognize K562 cells.

In some embodiments, the invention encompasses an antibody having scFv sequence, which is fused to one or more heavy chain constant regions to form an antibody having a human immunoglobulin Fc region to produce a bivalent protein, thereby increasing overall affinity and stability of an antibody. In addition, the Fc portion allows for direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to, for example, antibodies used in antigen quantification studies in order to immobilize antibodies for affinity measurement, targeted delivery of therapeutic drugs, use of immune effector cells to test Fc-mediated cytotoxicity and many other applications.

The results presented herein highlight the specificity, sensitivity and utility of the antibodies of the invention in targeting BCMA.

The molecules of the invention are based on single-chain variable fragments (scFv) identified and selected by phage display, the amino acid sequence of which confers specificity to BCMA and forms the basis of all antigen binding proteins of the present disclosure. Therefore, the scFv can be used to design various different "antibody" molecules, including, for example, full length antibodies, fragments thereof such as Fab and F(ab')<NUM>, fusion proteins (including scFv_Fc), multivalent antibodies, i.e., an antibody having more than one specificity to the same or different antigens, for example, bispecific T cell-binding antibody (BiTE), tri-antibody, etc. (<NPL>).

In one embodiment where the antigen binding protein is a full length antibody, the heavy and light chains of the antibodies of the invention may be of full length (for example, the antibody may comprise at least one, preferably two intact heavy chains, and at least one, preferably two intact light chains), and alternatively may comprise an antigen binding moiety (Fab, F(ab')<NUM>, Fv or scFv). In other embodiments, the antibody heavy chain constant region is selected, for example, from IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The selection of antibody type will depend on the immune effector function that the designed antibody is intended to elicit. Suitable amino acid sequences for the constant regions of various immunoglobulin isotypes and methods for producing a wide variety of antibodies are known to a skilled person in the construction of recombinant immunoglobulins.

In a first aspect, an antibody or fragment thereof binding to BCMA as defined in the claims is provided in the present invention.

Each of the heavy and light chain variable region sequences can bind to BCMA, therefore, the heavy and light chain variable region sequences can be "mixed and matched" to produce anti-BCMA binding molecules of the invention.

In another aspect, variants of an antibody or fragment thereof binding to BCMA is provided in the present invention. Accordingly, an antibody or fragment thereof is provided in the present invention, having a heavy chain and/or light chain variable region that is at least <NUM>% identical to the variable region sequence of the heavy or light chain. Preferably, the amino acid sequence identity of the heavy and/or light chain variable regions is at least <NUM>%, preferably at least <NUM>%, more preferably at least <NUM>%, more preferably <NUM>%, more preferably <NUM>%, even more preferably <NUM>%, the most preferably <NUM>%, including, for example, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%. The variant can be obtained from the antibody described in the present application as a parent antibody by yeast library screening, phage library screening, point mutation or the like. As in the method used in Example <NUM> of the present application, the antibody 23F10 was used as the parent antibody, and the phage library screening method was used for mutation modification.

Standard assays for assessing the binding ability of an antibody, such as an anti-BCMA antibody, are known in the art and include, for example, ELISA, Western blot and flow cytometry analysis. Suitable assays are described in detail in the examples.

An isolated nucleic acid encoding an antibody binding to BCMA and fragment thereof, a vector and a host cell comprising the nucleic acid or vector, are also provided in the present invention. The nucleic acid can be present in an intact cell, cell lysate, or can be in a partially purified or substantially purified form.

The nucleic acid of the invention can be obtained using standard molecular biology techniques, for example, standard PCR amplification or cDNA cloning techniques, thereby obtaining cDNA encoding the light and heavy chains of an antibody or encoding VH and VL segments. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), one or more nucleic acids encoding the antibodies can be recovered from the library. Methods for introducing foreign nucleic acids into host cells are generally known in the art and can vary with the used host cell.

Preferred nucleic acid molecules of the invention are those selected from the group consisting of SEQ ID NOs: <NUM>, <NUM> and <NUM> which encode a light chain variable region, and/or those selected from the group consisting of SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM> and <NUM> which encode a heavy chain variable region. A more preferred nucleic acid molecule comprises a sequence of SEQ ID NO: <NUM> encoding a heavy chain and a sequence of SEQ ID NO: <NUM> encoding a light chain, or comprises a sequence of SEQ ID NO: <NUM> encoding a heavy chain and a sequence of SEQ ID NO: <NUM> encoding a light chain, or comprises a sequence of SEQ ID NO: <NUM> encoding a heavy chain and a sequence of SEQ ID NO: <NUM> encoding the light chain, or comprises a sequence of SEQ ID NO: <NUM> encoding a heavy chain and a sequence of SEQ ID NO: <NUM> encoding the light chain, or comprises a sequence of SEQ ID NO: <NUM> encoding a heavy chain and a sequence of SEQ ID NO: <NUM> encoding the light chain.

For expressing a protein, a nucleic acid encoding an antibody of the invention can be integrated into an expression vector. A variety of expression vectors are available for protein expression. Expression vectors can include self-replicating extra-chromosomal vectors, or vectors integrated into the host genome. Expression vectors used in the present invention include, but are not limited to, those which enable expression of proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of expression vectors which are commercially available or otherwise available, can be used in the present invention to express antibodies.

In the present invention, a multifunctional immunoconjugate is also provided, comprising the antibodies described herein and further comprising at least one functional molecule of other type. The functional molecule is selected from, but not limited to, a molecule that targets a tumor surface marker, a tumor-suppressing molecule, a molecule that targets a surface marker of an immune cell, or a detectable label. The antibody and the functional molecule may form a conjugate by covalent attachment, coupling, attachment, cross-linking, or the like.

As a preferred mode, the immunoconjugate may comprise an antibody of the invention and at least one molecule that targets a tumor surface marker or a tumor-suppressing molecule. The tumor-suppressing molecule may be anti-tumor cytokines or anti-tumor toxins. Preferably, the cytokines include but are not limited to IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, type I IFN, TNF-alpha. In a specific embodiment, the molecule that targets a tumor surface marker is a molecule that targets the same tumor surface marker as the antibody of the invention. For example, the molecule that targets a tumor surface marker can be an antibody or ligand that binds to a tumor surface marker, for example, can act synergistically with the antibodies of the invention to more precisely target tumor cells.

As a preferred mode, the immunoconjugate may comprise an antibody of the present invention and a detectable label. Such detectable labels include, but are not limited to, fluorescent labels, chromogenic labels such as enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals and non-radioactive paramagnetic metal ion. More than one marker can also be included. The label used to label the antibody for the purpose of detection and / or analysis and / or diagnosis depends on the used particular detection / analysis / diagnosis technique and / or method, eg, immunohistochemical staining (tissue) samples, flow cytometry, and the like. Suitable labels for detection / analysis / diagnosis techniques and / or methods known in the art are well known to those skilled in the art.

As a preferred mode, the immunoconjugate may comprise: an antibody of the invention and a molecule that targets a surface marker of an immune cell. The molecule targeting a surface marker of a immune cell may be an antibody or a ligand binding to a surface marker of a immune cell, capable of recognizing the immune cell, and carry the antibody of the present invention to the immune cell. The antibody of the present invention can target the immune cell to tumor cells, thereby inducing the immune cell to specifically kill tumors. The immune cell surface marker may be selected from the group consisting of CD3, CD16, CD28, and preferably, the antibody binding to the immune cell surface marker is an anti-CD3 antibody. The immune cells can be selected from the group consisting of T cells, NK cells, and NKT cells.

As a means of chemically generating an immunoconjugate by conjugation, either directly or indirectly (eg, by a linker), the immunoconjugate can be produced as a fusion protein comprising an antibody of the invention and other suitable proteins. The fusion protein can be produced by a method known in the art, for example recombinantly produced by constructing and subsequently expressing the nucleic acid molecule which comprises the nucleotide sequence encoding the antibody in frame with a nucleotide sequence encoding a suitable label.

In another aspect of the invention, a nucleic acid molecule encoding at least one antibody of the invention, a functional variant, or an immunoconjugate thereof is provided. Once obtaining the relevant sequence, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferating host cells by conventional methods.

The present invention also relates to vectors comprising the appropriate DNA sequences described above as well as appropriate promoters or control sequences. These vectors can be used to transform an appropriate host cell to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

A plurality of chimeric antigen receptors (CAR) are provided in the present invention, comprising an antibody or antibody fragment of the present invention. The CAR T cell exhibits anti-tumor properties. In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding CAR. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express CAR.

In a preferred embodiment, the BCMA binding portion of a CAR is a scFv antibody fragment that retains an equivalent binding affinity, for example it binds to the same antigen with comparable efficacy, as compared with the IgG antibody from which it is derived. The antibody fragment is functional, thereby providing a biochemical reaction, which can include, but is not limited to, activating an immune response, inhibiting the initiation of signaling from its target antigen, inhibiting kinase activity, and the like. Accordingly, a BCMA-CAR which comprises a WT1 binding domain and engineered into a T cell, and a method for using it in adoptive immunotherapy are provided in the present invention.

In one aspect, the anti-BCMA antigen binding domain of CAR is a scFv antibody fragment that is humanized relative to the murine sequence scFv from which it is derived.

In one aspect, the CAR of the invention combines the antigen binding domain of a particular antibody with an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include, but are not limited to, CD3 ξ chain, <NUM>-1BB and CD28 signaling modules, and combinations thereof.

In one aspect, the BCMA-CAR comprises at least one intracellular signaling domain that is selected from a CD137 (<NUM>-1BB) signaling domain, a CD28 signaling domain, a CD3ξ signaling domain, or any combination thereof. In one aspect, the BCMA-CAR comprises at least one intracellular signaling domain derived from one or more co-stimulatory molecules that are not CD137 (<NUM>-1BB) or CD28.

Exemplarily, the sequence of BCMA-CAR can be 7A12-BBZ (SEQ ID NO: <NUM>), 25C2-BBZ (SEQ ID NO: <NUM>), 25D2-BBZ (SEQ ID NO: <NUM>), 7G2-BBZ (SEQ ID NO: <NUM>), 7A12-28Z (SEQ ID NO: <NUM>), 7A12-28BBZ (SEQ ID NO: <NUM>), 7G2-28Z (SEQ ID NO: <NUM>), 7G2-28BBZ (SEQ ID NO: <NUM>), 25C2-28Z (SEQ ID NO: <NUM>), 25C2-28BBZ (SEQ ID NO: <NUM>), 25D2-28Z (SEQ ID NO: <NUM>), 25D2-28BBZ (SEQ ID NO: <NUM>). Conventional transmembrane domain and intracellular domain can be selected by a skilled person to replace the transmembrane domain and intracellular domain of the above SEQ ID NO: <NUM>-<NUM>, which will fall within the scope of this application.

An immune cell comprising a chimeric antigen receptor of the invention is also provided in the present invention.

In another aspect, the chimeric antigen receptor-modified T cell provided in the present invention further carries an encoding sequence for a foreign cytokine; preferably, the cytokine comprises: IL-<NUM>, IL-<NUM> or IL-<NUM>. The immune cells are preferably selected from T lymphocytes, NK cells or NKT cells.

In another aspect, the chimeric antigen receptor-modified T cell provided in the present invention further comprise a PD-L1 blocker or a protein that blocks PD-L1, such as native PD-<NUM>, or a mutant PD-<NUM> capable of binding to PD-L1, or a fragment of native or mutant PD-<NUM> capable of binding to PD-L1, or an antibody against PD-L1. Exemplarily, the PD-L1 blocker may comprise an amino acid sequence encoded by SEQ ID NO:<NUM>.

The antibodies, immunoconjugates comprising the antibodies, and genetically modified immune cells of the present invention can be used in the preparation of a pharmaceutical composition or diagnostic reagent. In addition to an effective amount of the antibody, immunological conjugate, or immune cell, the composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that when the molecular entities and compositions are properly administered to animals or humans, they do not cause adverse, allergic or other untoward reactions.

Specific examples of some of the substances which may be used as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, dextrose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethylcellulose sodium, ethylcellulose and methylcellulose; gum tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween®; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers and the like.

The composition of the present invention can be prepared into various dosage forms as needed, and the dosage to be administered to a patient can be determined by a physician according to factors, such as type, age, body weight, and general disease condition of a patient, mode of administration, and the like. For example, injection or other treatment may be used.

The invention will be further illustrated hereinafter in conjunction with specific examples. It is to be understood that the examples are not intended to limit the scope of the claims. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as <NPL>, or according to the conditions recommended by the manufacturer.

The gene (SEQ ID NO: <NUM>) of extracellular segment of human BCMA, Met1-Ala54 (SEQ ID NO: <NUM>), was in vitro synthesized, inserted into the eukaryotic expression plasmid containing the Fc fragment Asp104-Lys330 of human IgG1 heavy chain constant region, and linked with "GS" to form a fusion expression protein BCMA_huFc (SEQ ID NO: <NUM>), and the corresponding gene sequence is shown in SEQ ID NO: <NUM>.

The gene (SEQ ID NO: <NUM>) of extracellular segment of human BCMA was inserted into the eukaryotic expression plasmid containing Fc fragment Arg100-Lys324 of murine IgG1 heavy chain constant region, and linked with "GS" to form a fusion expression protein BCMA_muFc (SEQ ID NO: <NUM>), and the corresponding gene sequence is shown in SEQ ID NO: <NUM>.

<NUM> ug was taken for SDS-PAGE and the results are shown in <FIG>.

The full length gene (SEQ ID NO: <NUM>) of human BCMA was synthesized in vitro, and cleavage sites MluI, SalI (SEQ ID NO: <NUM>) were introduced, which were inserted into the lentiviral packaging plasmid pWPT by double digestion.

The detection method was the same as identification of the mixed clone, and the experimental results are shown in <FIG>. <FIG> of the monoclonal clones were BCMA positive clones.

The phage display library used in the present invention is a whole human natural scFv phage library constructed by the present company, and has a storage capacity of 1E+<NUM>. The scFv fragment highly specific for BCMA was obtained using screening methods known to a skilled person. Briefly, <NUM> ug/ml antigen BCMA_huFc and human Fc fragment were coated in immunotubes, respectively. To reduce the effect from Fc fragment, the phage library was added to the immunotube coated with human Fc fragment for <NUM> hr. The supernatant was taken and added to the immunotube coated with BCMA_huFc for <NUM> hours, then the non-specific phage was washed away, and the bound phage was eluted and used to infect E. coli TG1 in logarithmic growth phase. The phage eluted was expanded and the expanded phage library was purified using PEG/NaCl precipitation for the next round of screening. Panning was performed for <NUM>-<NUM> cycles to enrich scFv phage clones that specifically bind to BCMA. Positive clones were determined by standard ELISA methods for BCMA_huFc. Human Fc fragment was used as an unrelated antigen in ELISA to verify the specificity of the antibody. A total of <NUM> clones were screened, in which <NUM> clones specifically bound to BCMA_huFc, while did not bind to human Fc fragment in ELISA assays. <NUM> clones with high signal values were picked for sequencing, and <NUM> single sequences were obtained. These <NUM> clones were purified and expressed to obtain three clones specifically binding to K562-BCMA cells (<FIG>), and the clone were named as 7G2, 7A12 and 23F10. By sequencing analysis, the heavy chain variable region of 7A12 is the amino acid sequence shown in SEQ ID NO: <NUM>, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: <NUM>; the heavy chain variable region of 7G2 is the amino acid sequence shown in SEQ ID NO: <NUM>, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: <NUM>; and the heavy chain variable region of 23F10 is the amino acid sequence shown in SEQ ID NO: <NUM>, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: <NUM>.

Amino acid sequence of the heavy chain variable region of 7A12 (SEQ ID NO: <NUM>):
<IMG>.

Nucleotide sequence of the heavy chain variable region of 7A12 (SEQ ID NO: <NUM>):
<IMG>.

Amino acid sequence of the light chain variable region of 7A12 (SEQ ID NO: <NUM>)
<IMG>.

Nucleotide sequence of the light chain variable region of 7A12 (SEQ ID NO: <NUM>):
<IMG>.

Amino acid sequence of the heavy chain variable region of 7G2 (SEQ ID NO: <NUM>):
<IMG>.

Nucleotide sequence of the heavy chain variable region of 7G2 (SEQ ID NO: <NUM>):
<IMG>.

Amino acid sequence of the light chain variable region of 7G2 (SEQ ID NO: <NUM>):
<IMG>.

Nucleotide sequence of the light chain variable region of 7G2 (SEQ ID NO: <NUM>):
<IMG>.

Amino acid sequence of the heavy chain variable region of 23F10 (SEQ ID NO: <NUM>):
<IMG>.

Nucleotide sequence of the heavy chain variable region of 23F10 (SEQ ID NO: <NUM>):
<IMG>
<IMG>.

Amino acid sequence of the light chain variable region of 23F10 (SEQ ID NO: <NUM>):
<IMG>.

Nucleotide sequence of the light chain variable region of 23F10 (SEQ ID NO: <NUM>):
<IMG>.

Primers were designed for VH and VL fragments of 7G2, 7A12, 23F10, respectively, and a linker consisting of <NUM> flexible amino acids (GGGGSGGGGSGGGGS) was introduced to form a scFv; a NheI cleavage site and protective bases were introduced upstream to VH, and a BamHI cleavage site and protective bases were introduced downstream to VL. The PCR product was analyzed by <NUM>% agarose gel electrophoresis, purified and recovered. After digestion, it was ligated into V152 eukaryotic expression vector (purchased from Shanghai Ruijin Biotechnology Co. 293F cells in logarithmic growth phase were transiently transfected with 293fectin™ Transfection reagent (Invitrogen, <NUM>-<NUM>) or polyethyleneimine (PEI) (Sigma-Aldrich, <NUM>). At <NUM>-<NUM> days after transfection, the supernatant was collected and subjected to affinity purification of Protein A. The obtained antibodies were quantitatively and qualitatively analyzed by SDS PAGE (<FIG>).

The binding of the antibody to K562 stably expressing BCMA was tested by flow cytometry. The method for FACs detection is as follows: cells were harvested, washed once with growth medium, and resuspended in PBS. The cell concentration was adjusted to 4E+<NUM> cells/ml. The gradient-diluted scFv_Fc fusion antibody was incubated with the cells for <NUM> minutes on ice, the initial concentration of the antibody was <NUM>, which was <NUM>-fold diluted for <NUM> gradients in total. Thereafter, the antibody was incubated with FITC-labeled anti-mouse IgG secondary antibody, and, after washed twice, detected using Guava easyCyte™ HT System. <FIG> shows the binding of scFv_Fc fusion forms of antibody 7A12, 7G2 and 23F10 to K562-BCMA. All the three antibodies exhibited a concentration-dependent binding with an EC50 of <NUM>, <NUM> and <NUM>, respectively.

The affinities of different antibodies to BCMA were determined using biacore T200. The used method was as follows:
BCMA_huFc was coated on a CM5 chip by amino coupling to about <NUM> RU, and the gradient-diluted antibody as a mobile phase was passed through the antigen-coated channel at a flow rate of <NUM> ul/min. The running buffer was HBS-N and the temperature was <NUM>. The experimental data was analyzed by BIAevaluation <NUM> and the kinetic curves were fitted using <NUM>:<NUM> langmuir model. KD of 7A12 (scFv_Fc) was <NUM> pM, KD of 7G2 (scFv_Fc) was <NUM> pM, and KD of 23F10 (scFv_Fc) was <NUM> pM (see <FIG>). The parameters are shown in the following table:.

RPMI8226 is a peripheral blood B lymphocyte of human multiple myeloma. The method for FACs detection is as follows: cells were harvested, washed once with growth medium, and resuspended in PBS. The cell concentration was adjusted to 4E+<NUM> cells/ml. The gradient-diluted scFv_Fc fusion antibody was incubated with the cells for <NUM> minutes on ice, and the initial concentration of the antibody was <NUM>, and <NUM>-fold diluted for <NUM> gradients in total. Thereafter, the antibody was incubated with a FITC-labeled anti-mouse IgG secondary antibody, and, after washed twice, detected by Guava easyCyte™ HT System. <FIG> shows the concentration-dependent binding of scFv_Fc fusion forms of antibody 7A12, 7G2 and 23F10 on cell line RPMI8226.

The fusion protein of human APRIL His115-Leu250 and Fc fragment Asp104-Lys330 of human IgG1 heavy chain constant region linked by "GS" was recombinantly expressed. The fusion protein APRIL_huFc (SEQ ID NO: <NUM>), the corresponding gene sequence was SEQ ID NO: <NUM>. Transient transfection, expression and purification were performed as described in Example <NUM>.

A ELISA plate was coated with <NUM> ng/ml <NUM> ul/empty BCMA_muFc at <NUM> overnight. On the next day, the plate was washed with PBS for <NUM> times, and PBS containing <NUM>% skim milk powder was added and blocked at room temperature for <NUM> hour. <NUM> ng/ml APRIL_huFc and gradient-diluted antibody 7A12, 7G2 or 23F10 (starting concentration <NUM>, <NUM>-fold dilution, <NUM> gradients) were simultaneously added. The resulted mixture was incubated for <NUM> hour at room temperature, washed for three times with PBST, and three times with PBS. A <NUM>: <NUM> dilution of HRP-labeled mouse anti-human Fc antibody was added, incubated for <NUM> hour at room temperature, and washed three times with PBST, and three times with PBS. TMB was added for development and read with a microplate reader.

The experimental results are shown in <FIG>. All of 7A12, 7G2 and 23F10 can significantly inhibit the binding of APRIL to BCMA, which demonstrates that the antibodies of the invention can inhibit the binding of BCMA to its natural ligand.

Lentiviral plasmids expressing the second and third generation chimeric antigen receptors of antibody 7A12 were constructed using PRRLSIN-cPPT. EF-1α as a vector, including PRRLSIN-cPPT. EF-1α-7A12-28Z, PRRLSIN-cPPT. EF- 1α-7A12-BBZ and PRRLSIN-cPPT. EF-1α-7A12-28BBZ. 7A12-28Z sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 7A12 scFv (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), CD28 transmembrane region (SEQ ID NO: <NUM>), intracellular signaling domain (SEQ ID NO: <NUM>) and intracellular segment CD3ξ (SEQ ID NO: <NUM>) of CD3; 7A12-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 7A12 scFv (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), transmembrane region (SEQ ID NO: <NUM>), CD137 intracellular signaling domain (SEQ ID NO: <NUM>) and CD3ξ (SEQ ID NO: <NUM>); 7A12-28BBZ sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 7A12-scFv (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), CD28 transmembrane region (SEQ ID NO: <NUM>), intracellular segment (SEQ ID NO: <NUM>), CD137 intracellular signaling domain (SEQ ID NO: <NUM>) and CD3ξ (SEQ ID NO: <NUM>).

Lentiviral plasmids expressing the second and third generation chimeric antigen receptors of antibody 7G2 were constructed using PRRLSIN-cPPT. EF-1α as a vector, including PRRLSIN-cPPT. EF-1α-7G2-28Z, PRRLSIN-cPPT. EF- 1α-7G2-BBZ and PRRLSIN-cPPT. EF-1α-7G2-28BBZ. 7G2-28Z sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 7G2 scFv (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), CD28 transmembrane region (SEQ ID NO: <NUM>), intracellular signaling domain (SEQ ID NO: <NUM>) and intracellular segment CD3ξ(CD ID NO: <NUM>) of CD3; 7G2-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 7G2 scFV (SEQ ID NO: <NUM>) ), CD8 hinge (SEQ ID NO: <NUM>), transmembrane region (SEQ ID NO: <NUM>), CD137 intracellular signaling domain (SEQ ID NO: <NUM>) and CD3ξ (SEQ ID NO: <NUM>); 7G2-28BBZ sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 7G2-scFv (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), CD28 transmembrane region (SEQ ID NO: <NUM>), intracellular segment ( SEQ ID NO: <NUM>), CD137 intracellular signaling domain (SEQ ID NO: <NUM>) and CD3ξ (SEQ ID NO: <NUM>).

The positive infection rates of Mock, 7A12-28Z, 7A12-BBZ, 7A12-28BBZ, 7G2-28Z, 7G2-BBZ and 7G2-28BBZ T cell in the in vitro toxicity killing experiment are shown in <FIG>, which are <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, respectively.

CytoTox <NUM> non-radioactive cytotoxicity assay kit (Promega) was used with reference to the instructions of CytoTox <NUM> non-radioactive cytotoxicity assay kit.

Target cells: <NUM>µl of <NUM>×<NUM><NUM>/mL K562, K562-BCMA and RPMI-<NUM> cells were inoculated into <NUM> well plates, respectively. Effector cells: T-Mock and CAR T cells expressing different chimeric antigen receptors were added at an effector target ratio of <NUM>:<NUM>, <NUM>:<NUM> or <NUM>:<NUM>. Qudraplicate wells were set for each group, and the average of <NUM> replicate wells was taken. The detection time was hour <NUM> of incubation of the cells. Each experimental group and each control group are as follows:.

The results showed that each of the CAR T cells expressing different chimeric antigen receptors had significant in vitro killing activities against BCMA-positive K562-BCMA and RPMI-<NUM> cells, especially for RPMI-<NUM> cells endogenously expressing BCMA, while almost no killing effect on BCMA-negative K562 cells (<FIG>).

RPMI-<NUM> cells were inoculated into <NUM> NOD/SCID mice at <NUM>×<NUM><NUM>/mice, respectively. On Day <NUM> after subcutaneous inoculation of tumor cells, the average tumor volume was <NUM><NUM>, the mice were randomly divided into <NUM> groups, and <NUM> × <NUM><NUM> CAR T were injected into the tail vein. And cyclophosphamide was intraperitoneally injected before the injection at a doseage of <NUM>/kg for clearing residual T cells in mice in advance. On Day <NUM> of CAR T injection, the mice were sacrificed by cervical dislocation.

The tumor size of the mice was analyzed. The results are shown in <FIG>. Compared with UTD group, the antitumor effects in 7A12-28Z, 7A12-BBZ and 7A12-28BBZ treatment groups were significant, and on Day <NUM> of CAR T injection, there was <NUM> case of tumor regression in <NUM> mice of 7A12-28Z treatment group, <NUM> cases of tumor regression in <NUM> mice of 7A12-BBZ treatment group, and <NUM> cases of tumor regression in <NUM> mice of 7A12-28BBZ treatment group. The tumor inhibition rates were 7A12-28Z (<NUM>%), 7A12-BBZ (<NUM>%) and 7A12-28BBZ (<NUM>%), respectively.

In this example, 23F10 was used as a parent antibody, and 23F10 was modified by phage display method. A phage library was constructed based on 23F10 with CDR3 regions of the light and heavy chain being retained, and two phage libraries were constructed by randomizing CDR1 and CDR2 of the light chain or CDR1 and CDR2 of the heavy chain with degenerate primers, respectively. Primer information is as follows:.

A template plasmid was firstly constructed based on antibody 23F10 (scFv) (SEQ ID NO: <NUM>). For phage libraries of randomized light chain CDR1 and CDR2, primers LMF and BL1R were used to PCR-amplify fragment <NUM>; primers BL2F and FdR were used to PCR-amplify fragment <NUM>; then fragment <NUM> and fragment <NUM> were ligated by bridge-PCR to obtain a full length scFv containing the randomized sequence, and afterwards the full-length fragment was digested with NcoI and NotI and ligated into an identically digested template plasmid by T4 ligase. The plasmid was transduced into TG1 competent cells by electroporation, the storage capacity of which was <NUM>. For phage libraries of randomized heavy chain CDR1 and CDR2, primers LMF and BH1R were used to PCR-amplify fragment <NUM>; primers BH2F and FdR were used to PCR-amplify fragment <NUM>; then fragment <NUM> and fragment <NUM> were ligated by bridge-PCR to obtain a full length scFv containing the randomized sequence, and afterwards the full-length fragment was digested with NcoI and NotI and ligated into an identically digested template plasmid by T4 ligase. The plasmid was transduced into TG1 competent cells by electroporation, the storage capacity of which was <NUM>.

Screening of phage libraries. Referring to the method in Example <NUM>, the initial concentration of antigen BCMA_huFc was <NUM>, and a <NUM>-fold gradient dilution was performed for the next round of screening. Panning was performed for <NUM>-<NUM> cycles to enrich scFv phage clones specifically binding to BCMA_huFc. Positive clones were determined by standard ELISA methods for BCMA_huFc. In ELISA, human Fc fragment was used as an unrelated antigen to verify the specificity of the antibody. A total of <NUM> ELISA-positive clones were picked and the dissociation constant Kd of the supernatant was determined by biacore after reinduction. Among them, there are two clones, 25C2 and 25D2, the Kd of which is <NUM> times lower than the parental clone 23F10, as shown in the following table:.

The light chains of clones 25C2 and 25D2 were sequenced as being identical to 23F10. In <FIG>, the heavy chain amino acid sequences of clones 25C2, 25D2 and 23F10 were compared, wherein, compared with the parent antibody 23F10, there are <NUM> point mutations on the heavy chain in clone 25C2 (SEQ ID NOs: <NUM>, <NUM> are the amino acid sequence and the nucleotide sequence of 25C2 heavy chain variable region, respectively), and there are <NUM> point mutations on CDR1, serine to glycine at <NUM>st position and tyrosine to asparagine at <NUM>nd position; there are <NUM> point mutations on CDR2, serine to asparaginyl at the <NUM>th position and tyrosine to phenylalanine at the <NUM>th position, and there is <NUM> point mutation in the framework region, serine to glycine at the <NUM>th position. Compared with the parent antibody 23F10, there are <NUM> point mutations on the heavy chain of Clone 25D2 (SEQ ID NO: <NUM>, <NUM> are the amino acid sequence and nucleotide sequence of the heavy chain variable region of 25D2, respectively), wherein there are <NUM> point mutations in CDR2 region, serine to glycine at the <NUM>th position, serine to asparagine at the <NUM>th position and tyrosine to phenylalanine at the <NUM>th position, and there is <NUM> point mutation in the framework region, serine to arginine at the <NUM>th position.

The sequence of HCDR1 of 25C2 is set forth in SEQ ID NO: <NUM>, and the sequence of HCDR2 of 25C2 is set forth in SEQ ID NO: <NUM>. The sequence of HCDR1 of 25D2 is set forth in SEQ ID NO: <NUM>, and the sequence of HCDR2 of 25D2 is set forth in SEQ ID NO: <NUM>. The nucleotide sequence and amino acid sequence of 25C2 scFv are shown in SEQ ID NO: <NUM>, <NUM>, respectively, and the nucleotide sequence and amino acid sequence of the 25D2 scFv are shown in SEQ ID NO: <NUM>, <NUM>, respectively.

According to Example <NUM>, appropriate cleavage sites and protecting bases were introduced upstream to VH, and appropriate cleavage sites and protecting bases were introduced downstream to VL. The PCR product was analyzed by <NUM>% agarose gel electrophoresis, purified and recovered. After digestion, it was ligated into eukaryotic expression vector V152 containing human Fc fragment (purchased from Shanghai Ruijin Biotechnology Co. ), and transiently transfected into 293F cells by 293Fectin and expressed.

The aggregation of 25C2 and 25D2 was analyzed by SEC. As shown in <FIG> and <FIG>, the antibody in a monomer form accounted for <NUM>% and <NUM>%, respectively. Compared with the parent antibody 23F10 (<NUM>% monomer rate), the monomer rate was increased by <NUM>% and <NUM>%, respectively, and the aggregation was significantly reduced. After concentration by ultrafiltration, the obtained antibodies were quantitatively and qualitatively analyzed by SDS PAGE. The yields were <NUM> ug/ml and <NUM> ug/ml, respectively (yield = weight of final product/transfection volume).

K562 and K562 cells (K562-BCMA) stably expressing human BCMA were used and harvested, washed with complete growth medium, and plated into U-bottom microtiter plates at about <NUM> to <NUM> x <NUM><NUM> cells/well. The gradient-diluted scFv_Fc fusion antibody was incubated with K562-BCMA/K562 for <NUM> minutes on ice, and then incubated with FITC-labeled anti-human Fc as a secondary antibody. After two washing steps, the analysis was performed using a Guava easyCyteTM HT System, and the experimental data was processed using GraphPad Prism to obtain an EC50. <FIG> shows the binding of 25C2, 25D2 to K562-BCMA and K562 cells. The results showed that EC50s of two clones, 25C2, 25D2 with improved stability and reduced aggregation binding to K562-BCMA were <NUM> and <NUM>, respectively, which, compared with 23F10, were increased by <NUM> to <NUM> times.

The specificity of the antibodies 23F10, 25C2, 25D2 was determined by ELISA.

<NUM> ug/ml recombinant human BCMA_Fc, mouse BCMA_Fc, TACI_huFc (R&D, #174TC), BAFF R (R&D, #<NUM>-BR) were coated on immunoplates at <NUM> overnight. The next day, <NUM>µl/well of <NUM>% MPBS was added for <NUM> hours, then <NUM> purified antibody (scFv format) was added and incubated at <NUM> for <NUM> hour, washed three times with PBST (PBS containing <NUM>% Tween-<NUM>), and washed three times with PBS. And then <NUM>: <NUM> diluted HRP-labeled anti-Flag tag antibody (sigma, #A8592-<NUM>) was added, incubated for <NUM> hour at <NUM>, washed three times with PBST (PBS containing <NUM>% Tween-<NUM>) and washed three times with PBS. <NUM> ul/well of TMBS substrate was added and developed for <NUM>-<NUM> minutes. The reaction was quenched by adding <NUM> ul of <NUM> sulfuric acid.

Results are shown in <FIG>, wherein antibodies 7A12, 23F10, 25C2, 25D2 specifically bind to human BCMA, and do not bind human TACI and human BAFF R. Among them, the binding of antibodies 25C2, 25D2 to mouse BCMA is weaker.

According to the procedure of Example <NUM>, plasmids of chimeric antigen receptor of 25C2, 25D2 were constructed, respectively.

Lentiviral plasmid PRRLSIN-cPPT. EF-1α-25C2-BBZ expressing the second-generation chimeric antigen receptor of antibody 25C2 was constructed using PRRLSIN-cPPT. EF-1α as a vector. Lentiviral plasmid PRRLSIN-cPPT. EF-1α-25D2-BBZ expressing the second-generation chimeric antigen receptor of antibody 25D2 was constructed using PRRLSIN-cPPT. EF-1α as a vector.

25C2-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 25C2 scFv (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), transmembrane region (SEQ ID NO: <NUM>), CD137 intracellular signaling domain (SEQ ID NO: <NUM>) and CD3ξ (SEQ ID NO: <NUM>).

25D2-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: <NUM>), 25D2 scFV (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), transmembrane region (SEQ ID NO: <NUM>), CD137 intracellular signaling domain (SEQ ID NO: <NUM>) and CD3ξ (SEQ ID NO: <NUM>).

According to the procedure of Example <NUM>, the plasmids PRRLSIN-cPPT. EF-1α-25C2-BBZ, PRRLSIN-cPPT. EF-1α-25D2-BBZ were subjected to lentiviral packaging, T cell infection and amplification, respectively, to obtain chimeric antigen receptor-modified T cells 25C2-BBZ and 25D2-BBZ.

In this example, CAR-T cells expressing soluble PD1 were prepared using scFv of antibody 7A12. The preparation method is listed as follows:.

Using the T-sPD1-Fc plasmid as a template, the upstream primer <NUM>'-acgcgtcctagcgctaccggtcgccaccatgcagatcccacaggcgccc-<NUM>' (SEQ ID NO: <NUM>) and the downstream primer <NUM>'-ctctcggggctgcccaccatacaccagggtttggaactggc-<NUM>' (SEQ ID NO: <NUM>) were used in PCR amplification to obtain sPD1 sequence; and the upstream primer <NUM>'-tatggtgggcagccccgagagccacag-<NUM>' (SEQ ID NO: <NUM>), downstream primer <NUM>'-aaaattcaaagtctgtttcactttacccggagacagggag-<NUM>' (SEQ ID NO: <NUM>) were used in amplification to obtain sPD1-CH3 fragment.

The sPD1-CH3 fragment and the fragment of 7A12-BBZ (SEQ ID NO: <NUM>) were PCR-spliced and amplified to obtain sPD1-CH3-7A12-BBZ, and the sequence of 7A12-BBZ consists of CD8α signal peptide (SEQ ID NO: <NUM>), 7A12 scFv (SEQ ID NO: <NUM>), CD8 hinge (SEQ ID NO: <NUM>), transmembrane region (SEQ ID NO: <NUM>), CD137 intracellular signaling domain (SEQ ID NO: <NUM>) and CD3ξ (SEQ ID) NO: <NUM>).

The above constructed fragment sPD1-CH3-7A12-BBZ has a MluI cleavage site at <NUM>' end and a SalI cleavage site at <NUM>' end, which was double-digested with MluI and SalI and ligated into indentically double-digested PRRLSIN-cPPT. EF-1α vector to obtain a plasmid expressing sPD-<NUM>-CH3 protein and a chimeric antigen receptor targeting BCMA.

According to the procedure of Example <NUM>, T cells sPD-<NUM>-7A12-BBZ expressing sPD1 and 7A12-BBZ were obtained.

In vitro killing experiments were performed using 25C2-BBZ T cells, 25D2-BBZ T cells, 7A12-BBZ T cells, C11D5. <NUM>-BBZ T cells, and sPD-<NUM>-7A12-BBZ T cells as effector cells, among which C11D5. <NUM>-BBZ (SEQ ID NO: <NUM>) is a second generation CAR prepared by using anti-BCMA mouse anti-C11D5. <NUM> (see <CIT>). Target cells were human myeloma cells NCI-H929 and multiple myeloma peripheral blood B lymphocytes RPMI-<NUM>.

CytoTox <NUM> non-radioactive cytotoxicity assay kit (Promega) was used according to the instruction of CytoTox <NUM> non-radioactive cytotoxicity test kit.

Effector cells were inoculated in <NUM>-well plates at a effector target ratio of <NUM>:<NUM>, <NUM>:<NUM> or <NUM>:<NUM>, and <NUM>µL of <NUM>×<NUM><NUM>/mL NCI-H929 and RPMI-<NUM> cells were inoculated into the corresponding <NUM>-well plates.

Pentaplicate wells were set for each group, and the plates were incubated in an incubator for <NUM>.

The experimental groups and the control groups were set as follows: experimental group: each target cell + T lymphocytes expressing different chimeric antigen receptors; control group <NUM>: maximal release of LDH from target cells; control group <NUM>: spontaneous release of LDH from target cells; Control group <NUM>: spontaneous release of LDH from Effector cells. The calculation formula is: % cytotoxicity = [(experimental group - effector cell spontaneous group - target cell spontaneous group) / (target cell maximum - target cell spontaneous)] * <NUM>.

The experimental results of cell killing are shown in <FIG>.

<NUM>×<NUM><NUM> RPMI-<NUM> cells were subcutaneously inoculated into the right iliac crest of B-NDG mice, and on Day <NUM>, the average tumor volume was about <NUM><NUM>, thereby obtaining a subcutaneous xenograft model of B-NDG mice loaded with peripheral blood B lymphocytes RPMI-<NUM> of multiple myeloma.

The mouse subcutaneous xenograft model was divided into <NUM> groups (<NUM> in each group), and injected with 25C2-BBZ, 25D2-BBZ and untransfected T cells (UTD) at a dose of <NUM>×<NUM><NUM>, respectively. The results are shown in the following table. On Day <NUM> and Day <NUM> of inoculation of tumor cells, in all <NUM> mice of the 25C2-BBZ and 25D2-BBZ treatment groups, tumors regressed.

The mouse subcutaneous xenograft model was divided into <NUM> groups (<NUM> in each group), and injected with 25C2-BBZ, 25D2-BBZ, C11D5. <NUM>-BBZ, 7A12-BBZ and untransfected T cells (UTD) at an injection dose of <NUM> x <NUM><NUM> CAR T. The tumor regression was shown in the following table and <FIG>.

Claim 1:
An antibody that targets BCMA, wherein the antibody is selected from the group consisting of:
(<NUM>) an antibody, comprising HCDR1 as shown in SEQ ID NO: <NUM>, HCDR <NUM> as shown in SEQ ID NO: <NUM>, HCDR3 as shown in SEQ ID NO: <NUM>, and LCDR1 as shown in SEQ ID NO: <NUM>, LCDR2 as shown in SEQ ID NO: <NUM> and LCDR3 as shown in SEQ ID NO: <NUM>;
(<NUM>) an antibody, comprising HCDR1 as shown in SEQ ID NO: <NUM>, HCDR <NUM> as shown in SEQ ID NO: <NUM>, HCDR3 as shown in SEQ ID NO: <NUM>, LCDR1 as shown in SEQ ID NO: <NUM>, LCDR2 as shown in SEQ ID NO: <NUM> and LCDR3 as shown in SEQ ID NO: <NUM>;
(<NUM>) an antibody, comprising HCDR1 as shown in SEQ ID NO: <NUM>, HCDR2 as shown in SEQ ID NO: <NUM>, HCDR3 as shown in SEQ ID NO: <NUM>, LCDR1 as shown in SEQ ID NO: <NUM>, LCDR2 as shown in SEQ ID NO: <NUM> and LCDR3 as shown in SEQ ID NO: <NUM>;
(<NUM>) an antibody, comprising HCDR1 as shown in SEQ ID NO: <NUM>, HCDR2 as shown in SEQ ID NO: <NUM>, HCDR3 as shown in SEQ ID NO: <NUM>, LCDR1 as shown in SEQ ID NO: <NUM>, LCDR2 as shown in SEQ ID NO: <NUM> and LCDR3 as shown in SEQ ID NO: <NUM>;
(<NUM>) an antibody, comprising HCDR1 as shown in SEQ ID NO: <NUM>, HCDR2 as shown in SEQ ID NO: <NUM>, HCDR3 as shown in SEQ ID NO: <NUM>, LCDR1 as shown in SEQ ID NO: <NUM> , LCDR2 as shown in SEQ ID NO: <NUM> and LCDR3 as shown in SEQ ID NO: <NUM>.