CN114667294B

CN114667294B - Antibodies that specifically bind to B cell maturation antigens and uses thereof

Info

Publication number: CN114667294B
Application number: CN202080077674.5A
Authority: CN
Inventors: 刘江海; 曾昕; 刘彬; 孔洋; 曾顺泽; 林静
Original assignee: Chengdu Shengshi Ruike Biotechnology Co ltd; Chengdu Shengshijunlian Biotechnology Co ltd
Current assignee: Chengdu Shengshi Ruike Biotechnology Co ltd; Chengdu Shengshijunlian Biotechnology Co ltd
Priority date: 2019-09-02
Filing date: 2020-09-02
Publication date: 2024-04-16
Anticipated expiration: 2040-09-02
Also published as: CN114667294A; WO2021043169A1

Abstract

Antibodies that specifically bind to B cell maturation antigens and uses thereof are provided. To an antibody or active fragment thereof which specifically binds to a B cell maturation antigen, and fusion proteins comprising the antibody or active fragment thereof. The fusion protein may comprise said antibody or active fragment thereof, a transmembrane domain and a costimulatory signaling region, said antibody or active fragment thereof being capable of specifically binding to the tumor-specific antigen B cell maturation antigen and activating T cells via the transmembrane domain and the costimulatory signaling region. Also provided are CAR-T cells capable of expressing the fusion protein, which target B cell maturation antigens, utilizing CAR-T cells to specifically kill tumor cells, such as multiple myeloma or acute myelogenous leukemia. The CAR-T can be used as a therapeutic drug for tumor diseases, and provides a new method for preventing and treating tumors.

Description

Antibodies that specifically bind to B cell maturation antigens and uses thereof

Technical Field

The invention relates to the field of biological medicine, in particular to an antibody specifically binding to a B cell maturation antigen, and preparation and application thereof.

Background

T cells are one of the lymphocytes and play an important role in cell-mediated immunity. It differs from other lymphocytes, such as B cells and natural killer cells (NK cells), in the presence of T Cell Receptors (TCRs) on the cell surface. T helper cells, also known as CD4 ⁺ T or CD 4T cells express CD4 glycoprotein on their surface. Helper T cells are activated when exposed to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, such cells proliferate rapidly and secrete availableCytokines that regulate the immune response. Cytotoxic T cells, also known as CD8 ⁺ T cells or CD8T cells express CD8 glycoproteins on the cell surface. CD8 ⁺ T cells are activated when exposed to peptide antigens presented by MHC class I molecules. Memory T cells are a subpopulation of T cells that persist and respond to related antigens, thus providing the immune system with memory against past infection and/or tumor cells.

The T cell modified by chimeric antigen receptor (chimeric antigen receptor, abbreviated as CAR) is CAR-T, and is modified by genetic engineering means under in vitro culture conditions so as to express exogenous anti-tumor genes. The CAR gene is an artificially designed gene fragment, and the encoded protein mainly comprises an extracellular recognition domain and an intracellular signal transduction domain: the former is a specific antibody fragment for targeted recognition of tumor surface specific molecules; the latter is used to initiate a specifically recognized immune cell response, exerting cellular immunity. After genetic engineering, T cells can produce chimeric antigen receptors on their surface. CARs are proteins that allow T cells to recognize a specific protein (antigen) on tumor cells. Genetically engineered CAR T cells can be grown in the laboratory until their number reaches billions. The expanded CART cells may then be infused into a patient.

B cell maturation antigen (B Cell Maturation Antigen, BCMA) is a cell surface receptor encoded by the human TNFRSF17 gene, which binds to B cell activating factor (B cell activating factor, BAFF) and proliferation inducing ligand (APRIL), playing an important role in B lymphocyte differentiation and autoimmune response. Therefore, the polypeptide has high expression in part of B lymphocyte-related hematological tumors, is an excellent target with strong patent medicine potential, and the indications include but are not limited to Multiple Myeloma (MM), acute myelogenous leukemia (Acute myeloid leukemia, AML) and the like.

A number of anti-BCMA CAR constructs have been described. In 2013, carpenter et al disclosed a T cell approach to anti-BCMA CAR transduction, a preclinical study using a combination of in vitro and animal (mouse) assays (Carpenter et al 2013;Clin Cancer Res;19 (8); 2048-2060). 6 months 2015, bluebird and Celgene initiated research collaboration for BCMA CAR-T cell therapy. At the beginning of 2016, the university of pennsylvania cancer center (Abramson Cancer Center) began recruitment of patients with multiple myeloma using anti-BCMA CAR T. CARs for BCMA or corresponding CAR T have been described, for example, in WO2018/028647, WO2017/211900, WO2016/014789, WO2016/094304, WO2016/014565 and WO2013/154760, etc. CN109134665a also discloses a BCMA chimeric antigen receptor based on single domain antibody and application.

Although many potential replacement therapies are under development, there remains a need to provide effective methods to address conditions associated with the presence of pathogenic B cells, particularly multiple myeloma, acute myelogenous leukemia. This is mainly because off-target and recurrence is easily caused during the relevant immunotherapy.

Disclosure of Invention

In view of this, the present invention provides an antibody directed against BCMA and CAR-T cells based on the antibody, and their preparation and use. The CAR-T cells can specifically recognize and kill tumors, and have more efficient tumor killing activity, such as Multiple Myeloma (MM) and acute myelogenous leukemia (Acute myeloid leukemia, AML).

In particular, the present invention provides an anti-BCMA antibody or antigen binding fragment thereof which binds to a human BCMA polypeptide which competitively binds to the amino acid sequence of SEQ ID NO: 2. SEQ ID NO:15 or SEQ ID NO: 19. or SEQ ID NO:23-38, a single heavy chain variable region (VHH) binds to a human BCMA epitope. Illustratively, the anti-BCMA antibody or antigen binding fragment thereof comprises the amino acid sequence as set forth in SEQ ID NO: 2. SEQ ID NO:15 or SEQ ID NO: 19. or SEQ ID NO:23-38, or a humanized variant sequence thereof, of a single heavy chain complementarity determining region (VHH) of a H-CDR1, H-CDR2, and H-CDR3, e.g., the H-CDR1, H-CDR2, and H-CDR3 are each selected from the group consisting of SEQ ID NOs: 10-12 or an identity sequence thereof; SEQ ID NO:16-18 or an identity sequence thereof; or SEQ ID NO:20-22 or an identity sequence thereof.

Illustratively, an antibody or antigen-binding fragment thereof of the invention comprises the amino acid sequence of SEQ ID NO: 2. SEQ ID NO:15 or SEQ ID NO:19 or a humanized sequence thereof or a sequence identical to SEQ ID NO: 2. SEQ ID NO:15 or SEQ ID NO: 19. or SEQ ID NO:23-38, having at least 80% sequence identity.

Illustratively, SEQ ID NO:10 refers to the sequence identical to SEQ ID NO:10 (H-CDR 1, the coding sequence of which may be illustratively SEQ ID NO: 7);

SEQ ID NO:11 refers to the sequence identical to SEQ ID NO:11 (H-CDR 2, the coding sequence of which may illustratively be SEQ ID NO: 8); and

SEQ ID NO:12 refers to the sequence identical to SEQ ID NO:12 (H-CDR 3, the coding sequence of which may be, for example, SEQ ID NO: 9).

Further, the antibody or antigen binding fragment of the invention may be selected from the group consisting of: camel Ig, ig NAR, fab fragment, fab 'fragment, F (ab)' ₂ Fragments, F (ab)' ₃ Fragments, fv, scFv, bis-scFv, (scFv) ₂ Minibodies, diabodies, triplex antibodies, quadruplex antibodies, disulfide stabilized Fv proteins, single domain antibodies (sdabs, nanobodies), bispecific antibodies or trispecific antibodies, and the like.

The invention also provides a fusion protein comprising the antibody or antigen binding fragment described above.

Illustratively, the fusion proteins described in the present invention may further comprise a tag sequence (e.g., poly-His, hemagglutinin, c-Myc, GST, flag-tag, etc.) or an IgG1-Fc protein sequence, an epitope tag (e.g., other epitope directed against human BCMA) or an antibody-active fragment tag (e.g., an antibody or antibody-active fragment directed against other epitope or the same epitope of human BCMA, or a ligand capable of binding human BCMA), preferably the fusion protein has the amino acid sequence of SEQ ID NO: 4.

The invention also provides an antibody-drug conjugate comprising an antibody or antigen binding fragment as described in the invention.

Illustratively, in the antibody-drug conjugates of the invention, the drug is selected from the group consisting of: a radiolabel, ³² P、 ³⁵ S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, digitoxin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the foregoing; or an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, and an antiparasitic agent, or a combination of any of the foregoing.

The invention also provides a Chimeric Antigen Receptor (CAR) comprising: (1) An extracellular antigen-binding domain comprising an antibody or antigen-binding fragment, fusion protein or antibody-drug conjugate described herein; and optionally comprising (2) a transmembrane domain; and, (3) an intracellular signaling domain.

Illustratively, in the CARs of the invention, the transmembrane domain is derived from a transmembrane domain selected from one or more of the group consisting of the α, β or ζ chain, CD3 epsilon, CD4, CD5, CD8 a, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154, ICOS, and PD1 of a T cell receptor.

Illustratively, in a CAR of the invention, the intracellular signaling domain comprises a costimulatory signaling domain and is selected from the group consisting of: one or more of CD2, CD3 ζ, CD3 γ, CD3 δ, CD3 epsilon, CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, CD79B, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C and B7-H3.

In addition, in the CARs of the invention, a hinge domain between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain may also be illustratively included. Preferably, the hinge domain is derived from CD8 a.

Further, in the CAR of the invention, wherein the antibody or antigen fragment is conjugated to a drug as in the antibody-drug conjugate of the invention, or is fused to an additional polypeptide or protein as in the fusion protein of the invention, e.g., to an antibody against another epitope of human BCMA, such as a single domain antibody, or to a ligand capable of binding human BCMA.

Illustratively, the CARs of the invention have the amino acid sequence of SEQ ID NO:6 or a sequence as set forth in SEQ ID NO:6 having at least 80% sequence identity.

The invention also provides polynucleotides encoding the antibodies or antigen binding fragments thereof, fusion proteins or CARs of the invention,

illustratively, the polynucleotide encoding an antibody or antigen-binding fragment of the invention is set forth in SEQ ID NO:1 or a degenerate sequence thereof;

the polynucleotide for encoding the fusion protein disclosed by the invention is shown as SEQ ID NO:3 or a degenerate sequence thereof; or (b)

The polynucleotide for encoding the CAR disclosed by the invention is shown as SEQ ID NO:5 or a degenerate sequence thereof.

The invention also provides an isolated CAR-T cell or CAR-NK cell, characterized in that said CAR-T cell or CAR-NK cell is capable of expressing an antibody or antigen binding fragment thereof according to the invention; the CAR-T cell or CAR-NK cell is capable of expressing the fusion protein of the invention; the CAR-T cell or CAR-NK cell is capable of expressing the antibody-drug conjugate of the invention; the CAR-T cell or CAR-NK cell is capable of expressing a CAR of the invention; the CAR-T cell or CAR-NK cell comprises a polynucleotide of the invention.

Illustratively, CART cells of the invention are cd4+ T cells or a cell mixture comprising cd4+ T cells and cd8+ T cells.

The invention also provides a vector comprising a polynucleotide according to the invention.

Illustratively, the vector is an expression vector, such as a viral vector, preferably a retroviral vector, such as a lentiviral vector, preferably selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), wirnner-Medi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine Infectious Anemia Virus (EIAV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

The invention also provides an immune effector cell comprising a CAR of the invention, or comprising a polynucleotide of the invention, or comprising a vector of the invention.

Illustratively, the immune effector cells of the present invention are T lymphocytes or natural killer cells.

The invention also provides a pharmaceutical composition comprising a CAR-T cell or CAR-NK cell as described herein, or comprising an immune effector cell as described herein, and optionally a pharmaceutically acceptable carrier or adjuvant.

The invention also provides a method for preparing the CAR-T cell or the CAR-NK cell or the immune effector cell, which comprises introducing the vector into T lymphocyte or natural killer cell.

Illustratively, the method of preparing a CAR-T cell of the invention comprises the steps of:

(1) Synthesis and amplification (antibody or antibody fragment) -CD8 alpha range-CD 8 ^TM -4-1BB-CD3 zeta fusion protein gene, and (antibody or antibody fragment) -CD8 alpha range-CD 8 ^TM Cloning the-4-1 BB-CD3 zeta fusion protein gene onto slow virus expression vector;

(2) Infecting 293T cells by using the lentiviral packaging plasmid and the lentiviral expression vector plasmid obtained in the step (1), packaging and preparing lentivirus; and

(3) And (3) infecting the T cells by using the lentivirus obtained in the step (2) to obtain the CAR-T cells.

The invention also provides the application of the materials in the following items in preparing medicines for treating and/or preventing cancers:

(a) An antibody or antigen-binding fragment thereof of the invention;

(b) The fusion protein disclosed by the invention;

(c) The antibody-drug conjugate of the invention;

(d) The CAR of the invention;

(e) The CAR-T cells or the CAR-NK cells of the invention; or (b)

(f) The immune effector cell.

Illustratively, the cancer is a tumor that highly expresses B cell maturation antigens and related diseases, such as multiple myeloma and acute myelogenous leukemia, preferably relapsed multiple myeloma.

The invention also provides a method of treating and/or preventing cancer comprising administering an effective amount of (a) an antibody or antigen binding fragment thereof of the invention, (b) a fusion protein of the invention, (c) an antibody-drug conjugate of the invention, and (d) a CAR of the invention; (e) CAR-T cells or CAR-NK cells of the invention; or (f) the immune effector cell of the invention is administered to a subject.

For antibodies or antigen binding fragments thereof, fusion proteins, CARs, etc., of the invention, variants thereof, e.g., identical sequences or humanized sequences, etc., are also contemplated by the invention. Illustratively, the sequence of identity refers to about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99% or more, 99.6% or more, 99% or more, or 9.7% or more, or 99% or more with respect to the original sequence or the reference sequence.

For polynucleotides of the invention, degenerate sequences or complementary sequences are also contemplated by the invention. Illustratively, the degenerate sequence has about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.7% or more, 99.5% or more, or 99.7% or more.

The CAR provided by the invention can specifically bind to a tumor specific antigen B cell maturation antigen and activate the T cell through a transmembrane domain and a costimulatory signaling region. The CAR-T cell can express a fusion protein taking the B cell maturation antigen as a target antigen, so that the CAR-T cell can specifically kill tumor cells and is used for treating tumor diseases, such as tumor treatment with high expression of the B cell maturation antigen.

Detailed Description

Multiple myeloma, also known as plasmacytoma, is a currently refractory B-cell lymphoma that is derived from malignant transformed plasma cell clones, and the disease usually recurs and develops resistance after a multiple line treatment regimen.

CART or NK cells directed against BMCA of the invention can target B cell maturation antigens (BMCA) and thus can be used to treat cancers associated with BMCA, such as multiple myeloma or acute myelogenous leukemia, because BCMA is highly expressed in multiple myeloma tumor cells and acute myelogenous leukemia, but not in normal B cells or precursor B cells. In addition, in anti-CD 19 antibodies or anti-CD 19 CAR-T or NK cell therapies directed against B cells, non-hodgkin lymphoma (B-NHL) resistance occurs due to the loss of the targeted cancer cell surface antigen, and thus new alternative targets are needed. For B-NHL, BCMA can be a suitable target, therefore, the high affinity anti-BCMA CAR-T or NK cells of the invention should also be therapeutically useful for B-NHL.

Since the anti-BCMA CAR of the present invention confers extremely high avidity to T cells or NK cells, it can also be used to identify B cell lymphomas that are low in BCMA expression. In addition, the anti-BCMA CAR-T or-NK of the invention is not reactive to normal T cells, B cells, NK cells, endothelial cells, all myeloid lineages and their precursor cells. Thus, the anti-BCMA CAR-T or-NK of the invention does not have undesired reactivity towards bone marrow cell precursors.

The high affinity of the CAR of the invention enables the CAR-T cells to a) recognize tumor target cells with high, medium and low BCMA surface expression, to have low off-target reactivity, b) be activated against the tumor target cells, and c) kill the tumor target cells. Thus, the anti-BCMA CAR-T of the present invention can be used to treat a variety of lymphomas, multiple myeloma tumor cells and acute myelogenous leukemia and B-NHL, such as follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia, among others.

In an in vitro co-culture system, after specifically recognizing BCMA antigen on RPMI 8226 (human multiple myeloma cell line), the anti-BCMA CAR-T cells of the present invention activate the immune response of T cells through the activation domain of the CAR molecule, inducing the lysis of target cells RPMI 8226; the target cells released LDH (lactate dehydrogenase ) after lysis, and by detecting LDH levels in the reaction system, the actual cytotoxicity of CAR-T cells can be measured compared to the control group.

Animal experiments prove that the anti-BCMA CAR-T cell can obviously kill multiple myeloma cells.

Meanwhile, the in vitro killing effect of the fusion protein BCMA-PE24 containing the antibody of the invention on RPMI 8226 cells is also proved by experiments.

Chimeric antigen receptor:

the CAR comprises an extracellular domain derived from an antibody and an intracellular domain comprising a signaling module derived from a T cell signaling protein. In one embodiment, the extracellular domain may comprise a heavy chain variable region from an immunoglobulin, or variable regions comprising a heavy chain and a light chain, e.g., constructed as a single chain variable fragment (scFv), preferably a single domain antibody (sdAb) with only heavy chain variable regions. The sdAb is linked to a hinge region that provides flexibility and transduction of signals to intracellular signaling domains through a transmembrane domain. The transmembrane domain is preferably derived from CD8 a. In the first generation of CARs (the term "generation" is for the intracellular signaling domain) the intracellular signaling domain consists of the zeta chain of the TCR complex. The second generation CARs were designed to contain a single co-stimulatory domain derived from CD28 or 4-1 BB. Third generation CARs include two costimulatory domains, e.g., 4-1BB-CD3 ζ. The present invention preferably relates to a second generation or third generation CAR.

The present invention provides genetically engineered receptors that redirect cytotoxicity of immune effector cells to B cells. These genetically engineered receptors are referred to herein as Chimeric Antigen Receptors (CARs). CARs are chimeric protein molecules with specific anti-BCMA cellular immune activity based on the combination of antibody specificity targeting an antigen (e.g., BCMA) with the intracellular domain of an activated T cell receptor or NK cell receptor. In this context, the term "chimeric" refers to compositions of different proteins or DNA from different sources.

The CARs of the invention include an extracellular domain (also referred to as a binding domain or antigen binding domain) that binds BCMA, a transmembrane domain, and an intracellular domain or intracellular signaling domain. Binding of the anti-BCMA antigen binding domain of the CAR to BCMA on the surface of the target cell results in aggregation of the CAR and delivery of an activation stimulus to the CAR-containing cells. The CAR is capable of specifically redirecting immune effector cells, thereby eliciting proliferation, cytokine production, phagocytosis or cell killing of target antigen expressing cells.

In some embodiments of the invention, the CAR comprises the following domains: a humanized extracellular binding domain that specifically binds BCMA; a transmembrane domain; one or more intracellular signaling domains. In some embodiments, the CAR sequentially comprises an extracellular binding domain of a humanized BCMA antigen binding fragment; one or more spacer regions; a transmembrane domain; one or more intracellular signaling domains.

An "extracellular antigen-binding domain" or "extracellular binding domain" is used interchangeably and provides the CAR with the ability to specifically bind to the target antigen BCMA of interest. The binding domain may be derived from natural, synthetic, semisynthetic or recombinant sources. Preferred are sdabs of recombinant origin.

"specific binding" is understood by those skilled in the art to be well known to various experimental methods or means that can be used to test binding and binding specificity. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. In many protein-protein interactions, some cross-reaction or background binding may occur, but this does not compromise the "specificity" of binding between CAR and epitope. "specific binding" describes the binding of an anti-BCMA antibody or antigen binding fragment thereof (CAR also comprising them) to BCMA with a binding affinity that is higher than background binding.

An "antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or a T cell response in an animal. In some embodiments of the invention, the target antigen is an epitope of a BCMA polypeptide. An "epitope" refers to a region of an antigen that binds to a binding agent. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids that result in the tertiary structure of a protein.

A "single chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present as a single polypeptide chain and in either direction (e.g., VL-VH or VH-VL). Typically, the scFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding. In a preferred embodiment, the CAR of the invention comprises an antigen-specific binding domain that is an scFv and may be a murine, human or humanized scFv. Single chain antibodies can be cloned from the V region gene of a hybridoma specific for a desired target. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds a human BCMA polypeptide. Illustrative examples of variable heavy chains suitable for use in constructing the BCMA-resistant CARs of the present invention include, but are not limited to, the amino acid sequences of SEQ ID NOs: 2, and a polypeptide comprising the amino acid sequence shown in (a). Illustrative examples of variable light chains suitable for use in constructing an anti-BCMA CAR of the present invention include any variable light chain of an anti-BCMA antibody, including, but not limited to, the variable light chain in CN109641012 a.

Antibodies and antibody fragments:

the CAR comprises an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds a B Cell Maturation Antigen (BCMA) polypeptide. Thus, antibodies or antibody fragments of the invention include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain fragments (scFv), single variable fragments (ssFv), single domain antibodies (e.g., VHH fragments from nanobodies), fab fragments, F (ab') ₂ Fragments, fragments generated from Fab expression libraries, anti-idiotype antibodies and epitope-binding fragments, or a combination of any of the foregoing, provided that they have similar binding properties of the CARs of the invention, preferably comprising corresponding CDRs, or VH and VL regions, as described herein. Micro-and multivalent antibodies such as diabodies, trivalent antibodies, tetravalent antibodies and pentavalent antibodies may also be used in the methods of the invention. The immunoglobulin molecules of the invention may be of any class (i.e., igG, igE, igM, igD and IgA) or subclass of immunoglobulin molecules. Thus, as used herein, the term antibody also includes antibodies and antibody fragments encompassed by the CARs of the invention, which are produced by modification of intact antibodies or re-synthesized using recombinant DNA methods.

As used herein, "antibody" generally refers to a protein consisting essentially of one or more polypeptides encoded by immunoglobulin genes or immunoglobulin gene fragments. When the term "antibody" is used, it may also be considered to refer to "antibody fragments". Optionally, the antibody or antibody fragment may be chemically conjugated to or expressed as a fusion protein with other proteins or other proteins. In some embodiments, the antibodies or antigen binding fragments of the invention are comprised on a multispecific antibody, e.g., a bispecific antibody. Such multispecific antibodies may be produced by known methods, e.g., crosslinking two or more antibodies, antigen-binding fragments (e.g., scFv) of the same type or of different types. Exemplary methods of making multispecific antibodies include those described in PCT patent publication No. WO2013/163427, which is incorporated herein by reference in its entirety.

The affinity of the binding domain polypeptides and antibodies or antibody fragments or CAR proteins of the invention can be readily determined using conventional techniques, for example by competitive ELISA (enzyme linked immunosorbent assay), or using a surface plasmon resonance device (such as Biacore).

Humanized antibodies comprising one or more CDRs of an antibody or antibody fragment of the invention or comprising one or more CDRs derived from the antibody or antibody fragment can be prepared using methods known in the art. For example, four steps can generally be used to humanize monoclonal antibodies: (1) Determining the nucleotide and predicted amino acid sequences of the light and heavy chain variable domains of the starting antibody; (2) Designing a humanized antibody, i.e., determining which antibody framework regions to use in the humanization process; (3) developing humanization methods/techniques; and (4) transfection and expression of humanized antibodies. See, for example, U.S. Pat. No. 6,180,370.

The term humanized antibody means an immunoglobulin derived from or modified to human immunoglobulin sequences from at least a portion of the framework and optionally a portion of the CDR regions or other regions involved in binding. Humanized, chimeric or partially humanized forms of mouse monoclonal antibodies can be prepared, for example, by recombinant DNA techniques. Humanized versions of mouse antibodies can be generated by joining the CDR regions of a non-human antibody to human constant regions by recombinant DNA techniques (Queen et al, 1989; WO 90/07861). Alternatively, the monoclonal antibody used in the methods of the invention may be a human monoclonal antibody. Human antibodies can be obtained, for example, using phage display (WO 91/17271; WO 92/01047).

As used herein, humanized antibodies also refer to forms of non-human (e.g., murine, camel, llama, shark) antibodies, which are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., fv, fab, fab ', F (ab') ₂ Or other antigen binding subsequences of antibodies, e.g., vHH.

As used herein, a human or humanized antibody or antibody fragment refers to an antibody having an amino acid sequence corresponding to that of a human produced antibody, and can be prepared using any technique known in the art for preparing antibodies. The human antibody or fragment thereof may be selected by competitive binding experiments or other means to determine that it has the same epitope binding specificity as a particular mouse antibody.

Variable regions and CDRs

The variable region of an antibody refers to the variable region of an antibody light chain alone or the variable region of an antibody heavy chain or a combination of both. The heavy and light chain variable regions each consist of four Framework Regions (FR) connected by three Complementarity Determining Regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FR and, together with CDRs from the other chains, contribute to the formation of the antigen binding site of the antibody.

CDRs are mainly responsible for binding to epitopes. There are a number of methods available for determining the boundaries of CDR amino acid sequences, such as Kabat et al, sequences of Proteins of Immunological Interest, (5 th edition, 1991, national institutes of health, bethesda Md, "Kabat" numbering scheme); AI-Lazikani B, lesk AM, chothia C.Standard conformations for the canonical structures of immunoglobins.J.mol.biol.1997; 273:927-48 ("Chothia" numbering scheme); lefranc et al ("IMGT unique numbering for immunoglobulin and T cell receptor varia ble domain sand lg superfamily V-like domains," Dev. Comp. Immunol.,27:55-77, 2003; "IMGT" numbering scheme); or North, B, lehmann A, dunback R.A new clustering of antibody CDR loop conformations: j.mol.biol. (2011), 406 (2): 228-256. Alternative methods include, in addition to the above methods, new solutions developed with the development of bioinformatics. Although Kabat is the most commonly used method, CDRs may refer to CDRs defined by one or more methods, or by a combination of these methods.

Illustratively, the positions of the heavy chain variable region CDRs in the respective VHHs, as determined according to North, kabat, chothia and IMGT numbering schemes, are shown in table 1 below. The CDRs referred to herein may be CDRs or CDR combinations determined by the same method, or CDRs or CDR combinations determined by different methods. For example, an antibody of the invention may contain HCDR1, HCDR2 and HCDR3 of one of Lead1-19 as determined by any of North, kabat, chothia and IMGT. Or an antibody of the invention contains HCDR1, HCDR2 of Lead1 as determined by North and HCDR3 of one of Lead2-19 as determined by Kabat. One skilled in the art can freely select CDRs identified by different methods and can freely combine these CDRs.

TABLE 1

In some embodiments, the invention provides an antibody or fragment thereof comprising a CAR, wherein the antibody or fragment thereof comprises at least one CDR, at least two, or at least three CDRs substantially identical to at least one CDR, at least two, or at least three CDRs of an antibody of the invention. In some embodiments, the at least one, two, or three CDRs have at least about 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity with at least one, two, or three CDRs of an antibody of the invention. It will be appreciated that for the purposes of the present invention, binding specificity and/or overall activity is generally retained, although the degree of activity may vary (may be greater or less) than the antibody.

In certain embodiments, substitutions, insertions, or deletions may be present in one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the CDRs that do not substantially reduce binding affinity. In certain embodiments of the variant VH and VL sequences provided above, each CDR may be unchanged or comprise no more than one, two or three amino acid substitutions, insertions or deletions. For example, the CDR sequences SEQ ID NO: 10. 11 or 12, or one, two or three amino acids may be substituted, inserted or deleted with the same class of amino acids, and still retain the ability to bind human BCMA. Or the CDR sequences SEQ ID NO: 16. 17 or 18 may be substituted, inserted or deleted with one, two or three amino acids of the same class and still retain the ability to bind human BCMA. Alternatively, the CDR sequences SEQ ID NO: 20. 21 or 22 may be substituted, inserted or deleted with one, two or three amino acids of the same class and still retain the ability to bind human BCMA. Alternatively, the antibodies of the invention comprise the HCDR1, HCDR2 and HCDR3 identified by the same method as shown in table 1, read 2, read 3, read 4, read 5, read 6, read 7, read 8, read 9, read 10, read 11, read 12, read 13, read 14, read 15, read 16, read 17, read 18, or read 19, or a combination of HCDR1, HCDR2 and HCDR3 identified by different methods.

For mutable sites, reference may be made to, for example, cunningham and Wells (1989) Science,244: 1081-1085. This method that can be used to identify residues or regions of an antibody that can be targeted for mutation is referred to as "alanine scanning mutagenesis". In this method, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. It is contemplated that additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex is used to identify the point of contact between the antibody and the antigen. Such contact residues and adjacent residues may be targeted as candidates for replacement. Variants may be screened to determine whether they contain the desired property.

For example, as shown in this disclosure, CDR1 (AAS) in Lead 1 (SEQ ID NO: 2)GFTLDYYAIG) is replaced with AASDSTVELThe Lead 3 antibody remained active after YAIG. That is, after 5 amino acid residues are replaced, the mutated antibody remains active.

It should therefore be clearly understood that although the claims herein do not specifically define the specific sequence of the mutated CDR, the skilled person can make mutations (e.g. in the case of substitution, deletion or addition of 1, 2 or 3 amino acid residues in the CDR regions identified in the present invention, although the substitution of 5 amino acid residues is described above) according to the prior art, to find mutated CDRs which retain the activity of the antibody, and antibodies comprising mutated CDRs belong to the obvious variants of the antibodies of the invention and are thus covered by the scope of the invention.

Further elucidation of chimeric antigen receptors

In certain embodiments, a CAR of the invention may comprise a linker residue between the individual domains added for proper spacing and conformation of the molecule, e.g., a linker comprising an amino acid sequence that connects the VH domain and the VL domain and provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains a specific binding affinity for the target molecule. The CARs of the invention may comprise one, two, three, four, or five or more linkers. In particular embodiments, the linker is about 1 to about 25 amino acids in length, about 5 to about 20 amino acids, or about 10 to about 20 amino acids in length, or any suitable length of amino acids.

Illustrative examples of linkers include glycine polymers; glycine-serine polymer; glycine-alanine polymer; alanine-serine polymers; other flexible joints are known in the art, such as a Wheatstone joint. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as linkages between domains of fusion proteins or some of them (e.g., CARs as described herein).

In particular embodiments, the binding domain of the CAR is followed by one or more "spacers" or "spacer polypeptides," corresponding to linkers that distance the antigen binding domain from the effector cell surface to enable proper cell-to-cell contact, antigen binding, and activation. In certain embodiments, the spacer region is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer region may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer region comprises CH2 and CH3 domains of IgG1 or IgG 4.

In some embodiments, the binding domain of the CAR may be followed by one or more "hinge domains" that are located away from the effector cell surface to enable proper cell-to-cell contact, antigen binding, and activation. The CAR may comprise one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be of natural, synthetic, semisynthetic or recombinant origin. The hinge domain may comprise a naturally occurring immunoglobulin hinge region or an amino acid sequence of an altered immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (e.g., CD8 a, CD4, CD28, PD1, CD152, and CD 7), which may be wild-type hinge regions from these molecules, or may be altered. In another embodiment, the hinge domain comprises a PD1, CD152, or CD8 a hinge region.

The "transmembrane domain" is the portion of the CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain may be derived from natural, synthetic, semisynthetic, or recombinant sources. The TM domain may be derived from the α, β or ζ chain, CD3 epsilon, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1 of a T cell receptor. In one embodiment, the CAR of the invention comprises a TM domain derived from CD8 a or CD 28.

In a particular embodiment, a CAR of the invention comprises an intracellular signaling domain. An "intracellular signaling domain" refers to a domain that is involved in transduction of information that is effective against BCMACAR binding to human BCMA polypeptide into immune effector cells to elicit effector cell functions (e.g., activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to target cells to which the CAR binds) or other cellular responses elicited by antigen binding extracellular CAR domains. The term "effector function" refers to the specialized function of immune effector cells. For example, the effector function of a T cell may be cytolytic activity or assistance or activity including cytokine secretion. The term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs cells to perform a specialized function.

The CARs of the invention comprise one or more co-stimulatory signaling domains to enhance the efficacy, expansion and/or memory formation of T cells expressing the CAR receptor. As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a CAR molecule that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen.

Illustratively, a CAR of the invention comprises an antibody of the invention. Further exemplary, the CARs of the invention are in addition to SEQ ID NOs: 6, other antibodies of the invention, such as read 2, read 3, read 4, read 5, read 6, read 7, read 8, read 9, read 10, read 11, read 12, read 13, read 14, read 15, read 16, read 17, read 18, or read 19, may be substituted for SEQ ID NO: CAR obtained after Lead1 in 6.

Proteins

"protein", "polypeptide fragment" and "polypeptide" are used interchangeably, unless indicated to the contrary, and are used in accordance with conventional meanings, i.e. as amino acid sequences. Proteins are not limited to a particular length, e.g., they may comprise a full-length protein sequence or fragment of a full-length protein, and may include post-translational modifications of polypeptides (e.g., glycosylation, acetylation, phosphorylation, etc.) and include other modifications known in the art both naturally occurring and non-naturally occurring.

In various embodiments, a CAR polypeptide or protein of the invention comprises a signal (or leader) sequence at the N-terminus of the protein that is capable of directing protein transfer upon or after translation. The polypeptides may be prepared using a variety of well known recombinant and/or synthetic techniques. The polypeptides of the invention specifically include the CARs of the disclosure, or sequences having deletions, additions, and/or substitutions of one or more (e.g., 1-20, 1-10, or 1-5) amino acids of the CARs disclosed herein.

Nucleic acid

As used herein, the term "polynucleotide" refers to mRNA, RNA, genomic RNA (gRNA), positive strand RNA (+)), negative strand RNA (-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, a polynucleotide of the invention comprises a polynucleotide or variant having at least about 70%,71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,94%,95%,96%,97%,98%,99%,99.5%,99.9% or 100% sequence identity to any of the reference sequences described herein, typically wherein the variant retains at least one biological activity of the reference sequence.

Illustratively, the encoded BCMA binding domains of the present invention-CD 8 alpha range-CD 8 ^TM The sequence of the polynucleotide of the-4-1 BB-CD3 zeta fusion protein is any DNA sequence capable of encoding the fusion protein, preferably the sequence is SEQ ID NO:5 or a complement thereof. In another aspect, the encoded BCMA binding domain of the present invention-CD 8 alpha range-CD 8 ^TM The sequence of the polynucleotide of the-4-1 BB-CD3 ζ fusion protein may be identical under stringent conditions to the sequence represented by SEQ ID NO:5, and encoding a polynucleotide of the fusion protein or a complement thereof;

the "stringent conditions" described herein may be any of low stringency conditions, medium stringency conditions, high stringency conditions, preferably high stringency conditions. Illustratively, the "low stringency conditions" can be conditions of 30 ℃, 5 x SSC, 5 x Denhardt's solution, 0.5% sds, 52% formamide; "Medium stringent conditions" may be conditions of 40 ℃, 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 52% formamide; "high stringency conditions" can be conditions of 50℃in 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 52% formamide. It will be appreciated by those skilled in the art that higher temperatures result in polynucleotides with higher homology. In addition, one skilled in the art can select the combination of factors such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., that affect the stringency of hybridization to achieve the corresponding stringency.

In addition to this, the hybridizable polynucleotide may be, for example, a polynucleotide which, when calculated by the same search software as FASTA, BLAST, etc., has the same default parameters set by the system as the polynucleotide encoding SEQ ID NO: the polynucleotide of 5 has about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.8% or more.

Nucleotide sequence identity can be determined using the algorithm rules BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990;Proc.Natl.Acad.Sci.USA 90:5873, 1993). Programs BLASTN, BLASTX based on BLAST algorithm rules have been developed (AltschulSF, et al J Mol Biol 215:403, 1990). When a BLASTN is used to analyze a base sequence, parameters are score=100 and wordlength=12; furthermore, when BLASTX is used to analyze amino acid sequences, parameters are score=50, wordlength=3; when BLAST and Gapped BLAST programs are used, the system employing each program can set default parameter values.

Polynucleotides may be prepared, manipulated and/or expressed using any of a variety of maturation techniques known and available in the art. In order to express the desired polypeptide or protein, the nucleotide sequence encoding the polypeptide may be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs)), phages (e.g., lambda phage or M13 phage), and animal viruses. Examples of animal viral vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV 40). An example of an expression vector is the pClneo vector (Promega) for expression in mammalian cells; lenti4/V5-DESTTM, pLenti/V5-DESTTM and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequences of the chimeric proteins disclosed herein can be linked to such expression vectors for expression of the chimeric proteins in mammalian cells. "control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector (e.g., origins of replication, promoters, enhancers, translational initiation signals (SD sequences or Kozak sequences) introns, polyadenylation sequences, 5 'and 3' untranslated regions) that interact with host cell proteins for transcription and translation. The strength and specificity of such elements or sequences may vary. Any number of suitable transcription and translation elements or sequences may be used, including broadly expressed promoters and inducible promoters, depending on the vector system and host used.

Related ADC

Antibody-drug conjugate (ADC) technology is a target-directed technology that allows selective killing or inhibition of the growth or division of cancer cells. Typically, ADCs act by targeting cancer cells with antibodies and then releasing toxic substances (i.e., drugs) in the cells, thereby triggering cell death. ADC technology increases the efficacy of therapeutic or targeted antibodies and reduces the risk of adverse reactions, as it allows for accurate delivery of drugs to target cancer cells and release under specific conditions while minimizing collateral damage to healthy cells.

The basic structure of an antibody-drug conjugate may be an "antibody-linker-pharmaceutically active molecule" or an "antibody-pharmaceutically active molecule" (no linker). For conjugates with linkers, the linkers allow the drug to exhibit an effect on the target cancer cells, e.g., after separation from the antibody (e.g., by enzyme-mediated hydrolysis) and after the drug reaches the target cells. The linker also serves a functional role by linking the antibody and the drug. The efficacy and toxicity of the antibody-drug conjugate thus depends in part on the stability of the linker, which therefore plays an important role in drug safety.

The linker of the antibody-drug conjugate may be broadly classified as non-cleavable or cleavable. Many non-cleavable linkers are attached to the antibody using a thioether that comprises the cysteine of the antibody. Lateral-conjugated drugs are often not separated from antibodies in vivo and reduced efficacy may also occur. In the case of the widely used thiol-maleimide method, the antibody-drug conjugate is unstable, which may result in the separation of the drug from the conjugate before or after it reaches the target cell. The cleavable linker may be, for example, by lysosomal enzymatic hydrolysis. The cleavable linker may comprise a disulfide bond, e.g., comprising a cysteine of an antibody. Disulfide linkers that allow dissociation via thiol exchange reactions rely to some extent on uptake of the antibody-drug conjugate into target cells and exposure of the disulfide to the cytosol as a reducing environment. However, because various types of thiols (e.g., albumin and glutathione) are present in the blood, the drug may separate from the antibody before reaching its target.

In order to replace chemically unstable linkers that are poorly stable in physiological extracellular conditions, such as hydrazone and disulfide-based linkers, there is a need for linkers that are stable under physiological extracellular conditions. Furthermore, there is a need for linkers with high plasma stability to improve therapeutic applicability, as the drug should only be released into the cell targeted by the protein to which the drug is attached, not outside the cell.

The prior literature has reported new methods for preparing antibody-drug conjugates, see for example U.S. patent publication No.2012/0308584. Further improvements are possible.

The CAR of the invention or the antibody of the invention may still be conjugated to a pharmaceutically active molecule, such as erlotinib, lymphokines, botulinum toxins, affinity ligands, radiolabels, immunomodulatory compounds, anti-cancer agents, ribozymes, and the like, on the antigen binding domain side.

Carrier body

In particular embodiments, cells (e.g., immune effector cells, such as T cells) are transduced with a retroviral vector (e.g., a lentiviral vector) encoding a CAR. For example, immune effector cells are transduced with a CAR-encoding vector comprising a humanized anti-BCMA antibody or antigen binding fragment that binds a BCMA polypeptide having a transmembrane domain and an intracellular signaling domain such that these transduced cells can elicit a CAR-mediated cytotoxic response.

Retroviruses are a common tool for gene delivery. In particular embodiments, the retrovirus is used to deliver a polynucleotide encoding a Chimeric Antigen Receptor (CAR) to a cell. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into linear double-stranded DNA copies, and subsequently covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus". Proviruses serve as templates for RNA polymerase II and direct the expression of RNA molecules that encode structural proteins and enzymes required for the production of new viral particles.

Exemplary retroviruses suitable for use in particular embodiments include, but are not limited to: moloney murine leukemia virus (M-MuLV), moloney murine sarcoma virus (MoMSV), harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline Leukemia Virus (FLV), murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)), and lentiviruses.

As used herein, the term "lentivirus" refers to a group (or genus) comprising a plurality of retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedivirus (VMV) virus; goat arthritis-encephalitis virus (CAEV); equine Infectious Anemia Virus (EIAV); feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In particular embodiments, the lentivirus is used to deliver a polynucleotide comprising a CAR to a cell.

The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, e.g., inserted into, a vector nucleic acid molecule. The vector may include sequences that direct autonomous replication in the cell, or may include sequences sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication defective retroviruses and lentiviruses.

It will be apparent to those skilled in the art that the term "viral vector" is used broadly to refer to a nucleic acid molecule (e.g., a transfer plasmid) or viral particle that mediates nucleic acid transfer, including virus-derived nucleic acid elements that generally promote transfer or integration of a nucleic acid molecule into the genome of a cell. Viral particles typically include various viral components, and sometimes host cell components in addition to nucleic acids.

The term viral vector may refer to a virus or viral particle capable of transferring a nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements derived primarily from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof derived primarily from a retrovirus. The term "lentivirus" refers to a genus of the retrovirus family that is capable of effectively infecting non-periodic and postmitotic cells; they can deliver significant amounts of genetic information into the DNA of host cells, so that they are one of the most efficient methods of gene delivery vectors.

Thus, in a preferred embodiment, the invention relates to a method of transfecting a cell with an expression vector encoding a CAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences that promote expression of the CAR, e.g., a promoter, enhancer, poly-a signal, and/or one or more introns. In a preferred embodiment, the CAR coding sequence is flanked by transposon sequences, such that a transposase is present to allow integration of the coding sequence into the genome of the transfected cell.

In some embodiments, the genetically transformed cell is further transfected with a transposase that facilitates integration of the CAR coding sequence into the genome of the transfected cell. In some embodiments, the transposase is provided as a DNA expression vector. However, in a preferred embodiment, the transposase is provided as an expressible RNA or protein such that long term expression of the transposase does not occur in the transgenic cell. For example, in some embodiments, the transposase is provided as an mRNA (e.g., an mRNA comprising a cap and a poly-a tail). Any transposase system may be used in accordance with an embodiment of the invention. However, in some embodiments, the transposase is salmon-type Tel-like transposase (SB). In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, but are not limited to, SB10, SB11, or SB 100X transposases (see, e.g., mates et al, 2009,Nat Genet.41 (6): 753-61 or US9228180, which are incorporated herein by reference). For example, the method may comprise electroporating cells having mRNA encoding SB10, SB11, or SB 100X transposase.

Sequence variants:

sequence variants of the claimed nucleic acids, proteins, antibodies, antibody fragments and/or CARs (e.g., those defined by percent sequence identity) are also included within the scope of the invention, which maintain similar binding properties of the invention. These variants show alternative sequences but retain substantially the same binding properties such as target specificity, as the particular sequence provided is known to be a functional analogue or a functional analogue. Sequence identity relates to the percentage of identical nucleotides or amino acids when sequence alignments are performed.

The expression "sequence identity" as used herein refers to the degree of sequence identity on a nucleotide-nucleotide basis or on an amino acid-amino acid basis over a comparison window. Thus, the "percentage of sequence identity" can be calculated by: comparing the two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, he, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) is present on the two sequences to produce a number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain a percentage of sequence identity. Included are nucleotides or polypeptides having at least about 50%,55%,60%,65%,70%,75%,80%,85%,90%,95%,96%,97%,98%,99% or 100% sequence identity to any of the reference sequences described herein, typically wherein the polypeptide variant retains at least one biological activity of the reference polypeptide.

One of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding a polypeptide or protein as described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions and other changes which fall within the sequence identity are also encompassed by the invention.

Protein sequence modifications that may occur by substitution are also included within the scope of the invention. Substitutions as defined herein are modifications to the amino acid sequence of a protein whereby one or more amino acids are replaced by the same number of (different) amino acids, thereby producing a protein containing an amino acid sequence different from the primary protein. Substitutions may be made which preferably do not significantly alter the function of the protein. As with the addition, the substitution may be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of the protein. This is especially true when the modification involves the replacement of a "conservative" amino acid with another amino acid having similar properties. Such "conservative" amino acids may be natural or synthetic amino acids that can be substituted due to size, charge, polarity, and conformation without significantly affecting the structure and function of the protein. In general, many amino acids can be substituted with conservative amino acids without adversely affecting the function of the protein.

Generally, non-polar amino acids Gly, ala, val, ile and Leu; non-polar aromatic amino acids Phe, trp and Tyr; neutral polar amino acids Ser, thr, cys, gln, asn and Met; positively charged amino acids Lys, arg and His; the negatively charged amino acids Asp and Glu represent a conserved amino acid group. This list is not exhaustive. For example, it is well known that Ala, gly, ser and sometimes Cys can be substituted for each other even though they belong to different groups.

Substitution variants remove at least one amino acid residue in the antibody molecule and insert a different residue at its position. For the occurrence of substitution mutagenesis, the most interesting positions include hypervariable regions, but FR alterations are also contemplated. If such substitutions result in a change in biological activity, a greater number of changes can be introduced and the product screened.

Gene-modified gene cells and immune cells

In particular embodiments, the invention contemplates cells genetically modified to express a CAR of the invention for use in treating a B cell-related disorder. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell", "modified cell" and "redirected cell" are used interchangeably. As used herein, the term "gene therapy" refers to the introduction of additional genetic material in the form of DNA or RNA into the total genetic material in a cell, which restores, modifies or modifies the expression of a gene, or is used to express a therapeutic polypeptide (e.g., CAR or ADC). In particular embodiments, the CARs of the invention are introduced into and expressed in immune effector cells in order to redirect their specificity for a target antigen of interest, such as a BCMA polypeptide.

An "immune cell" or "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and/or CDC).

The immune effector cells of the invention may be autologous or non-autologous ("non-autologous", e.g., allogeneic, syngeneic or xenogeneic). As used herein, "autologous" refers to cells from the same subject, which is a preferred embodiment of the invention. As used herein, "allogeneic" refers to cells of the same species as the subject or patient but that are genetically different. As used herein, "syngeneic" refers to cells that are genetically identical but from different subjects. As used herein, "heterologous" refers to cells from a different species. In a preferred embodiment, the cells of the invention are autologous or allogeneic.

Exemplary immune effector cells for use with the CARs of the invention include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells) or activated T lymphocytes. Cytokine Induced Killer (CIK) cells are typically CD 3-and CD 56-positive non-Major Histocompatibility Complex (MHC), which is a limiting Natural Killer (NK) like T lymphocyte. The T cells may be T helper cells (Th), such as T helper cell 1 (Th 1) or T helper cell 2 (Th 2). The T cells may be helper T cells or cytotoxic T cells or any other T cell subpopulation. Other exemplary T cell populations suitable for use in particular embodiments include naive T cells and memory T cells.

For example, T cells modified with the CAR of the invention described herein can recognize and kill tumor cells when reintroduced back into a patient after autologous cell transplantation. CIK cells may have enhanced cytotoxic activity compared to other T cells and thus represent a preferred embodiment of the immune cells of the invention.

As will be appreciated by the skilled artisan, other cells may also be used as immune effector cells having the CARs described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include progenitor cells of effector cells, wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro.

The invention provides methods of making immune effector cells expressing the CARs of the invention. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs as described herein. In certain embodiments, the immune effector cells are isolated from the individual and genetically modified without further manipulation in vitro. These cells can then be reapplied directly to the individual. In a further embodiment, the immune effector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express the CAR. In this regard, immune effector cells may be cultured prior to and/or after genetic modification (i.e., transduction or transfection to express a CAR of the present invention).

In particular embodiments, the cell source is obtained from the subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein. In particular embodiments, the CAR-modified immune effector cell comprises a T cell. T cells may be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells may be obtained from a blood unit collected from a subject using any technique or combination of techniques known to those skilled in the art, for example, by sedimentation and antibody-conjugated bead-based methods. In one embodiment, cells from the circulating blood of the individual are obtained by apheresis. The component blood products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and placed in a suitable buffer or medium for subsequent processing. The cells may be washed with PBS or another suitable solution that is free of calcium, magnesium and most divalent cations. As will be appreciated by those of ordinary skill in the art, the washing step may be accomplished by methods known to those of ordinary skill in the art, such as by using a semi-automated flow-through centrifuge (e.g., cobe2991 cell processor, baxter CytoMate, etc.). After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, the undesired components of the apheresis sample may be removed in cells that are directly resuspended in culture medium.

In certain embodiments, T cells are isolated from Peripheral Blood Mononuclear Cells (PBMCs) by lysing the erythrocytes and depleting the monocytes (e.g., by PERCOLLTM gradient centrifugation). Specific T cell subsets can be further isolated by positive or negative selection techniques. One method that may be used is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a monoclonal antibody mixture directed against cell surface markers present on negatively selected cells.

PBMCs can be directly genetically modified to express CARs using the methods of the invention. In certain embodiments, T lymphocytes are further isolated after isolation of PBMCs, and in certain embodiments, cytotoxic and helper T lymphocytes may be sorted into subpopulations of naive, memory and effector T cells prior to or after genetic modification and/or expansion. Cd8+ cells can be obtained by using standard methods. In some embodiments, the cd8+ cells are further sorted into naive, central memory and effector cells by identifying cell surface antigens associated with each of these types of cd8+ cells.

Immune effector cells (e.g., T cells) may be genetically modified after isolation using known methods, or immune effector cells may be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In particular embodiments, immune effector cells (e.g., T cells) are genetically modified (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) with a chimeric antigen receptor of the invention, and then activated and expanded in vitro. In various embodiments, for example, U.S. patent No. 5,858,358 may be used; 6,905,681; 7,067,318; 7,232,566; 5,883,223; the methods described in 6,797,514 and 6,867,041 activate and expand T cells before or after genetic modification to express a CAR.

In another embodiment, for example, a mixture of one, two, three, four, five or more different expression vectors, each encoding a different chimeric antigen receptor protein (e.g., CAR variant sequence) as in the present invention, can be used to genetically modify a donor population of immune effector cells. The resulting modified immune effector cells form a mixed population of modified cells, wherein a portion of the modified cells express more than one different CAR protein.

In one embodiment, the invention provides a method of storing immune effector cells targeted to a BCMA protein expressing genetically modified murine, human, or humanized CAR protein comprising cryopreserving the immune effector cells such that the cells remain viable when thawed. A portion of the immune effector cells expressing the CAR protein may be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients suffering from B cell related disorders. The cryopreserved transformed immune effector cells can be thawed, grown, and expanded, if desired, to obtain more such cells.

Composition and formulation

The compositions of the invention may comprise one or more polypeptides, polynucleotides, vectors comprising such polynucleotides, genetically modified immune effector cells, and the like, as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. "pharmaceutical composition" refers to a composition formulated in a pharmaceutically or physiologically acceptable solution that is administered to a cell or animal alone or in combination with one or more other therapeutic modalities. It will also be appreciated that the compositions of the present invention may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies or other various pharmaceutically active agents, if desired. There is virtually no limit to the other components that may also be included in the composition, provided that the additional components do not adversely affect the ability of the composition to deliver the intended therapy.

The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifying agent that has been approved by the U.S. food and drug administration or by the chinese food and drug administration for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, wax, animal and vegetable fat, paraffin, organosilicon, bentonite, silicic acid and zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-thermal raw water; isotonic saline; ringer's solution; ethanol; phosphate buffer; as well as any other compatible substances used in pharmaceutical formulations.

In a particular embodiment, the composition of the invention comprises an amount of CAR-expressing immune effector cells of the invention. As used herein, the term "amount" refers to an "effective amount" of a genetically modified therapeutic cell (e.g., T cell) that achieves a beneficial or desired prophylactic or therapeutic result, including a clinical result.

"prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is used in the subject prior to or at an early stage of the disease. The term prevention does not necessarily mean to completely prohibit or prevent a particular medical condition. The term prophylaxis also refers to reducing the risk of occurrence or worsening of symptoms of a certain medical condition.

The "therapeutically effective amount" of the genetically modified therapeutic cells can vary depending on various factors, such as the disease state, age, sex, and weight of the individual, as well as the ability of the stem and progenitor cells to elicit the desired response in the individual. A therapeutically effective amount is also an amount that has a therapeutic benefit that outweighs any toxic or detrimental effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When the therapeutic amount is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician taking into account the age, weight, tumor size, the extent of infection or metastasis and individual differences in the condition of the patient (subject). It may be generally stated that a pharmaceutical composition comprising the T cells described herein may be at least 10 ² To 10 ¹⁰ Individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Individual cells/kg body weight (including all whole values within these ranges). The number of cells will depend on the end use of the composition and the type of cells contained therein. For the uses provided herein, the cells are typically 1L or less in volume, and may be 500mL or less, even 250mL or 100mL or less. Thus, the density of the desired cells is typically greater than 10 ⁶ Individual cells/ml, typically greater than 10 ⁷ Individual cells/ml, typically 10 ⁸ Individual cells/ml or higher. The clinically relevant number of immune cells may be distributed as multiple infusions that accumulate at or above 10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 、10 ¹¹ Or 10 ¹² Individual cells. In some embodiments of the invention, a lower number of cells may be administered, particularly because all infused cells will be redirected to a particular target antigen. The CAR-expressing cell composition can be administered multiple times at doses within these ranges. For a patient to be treated, the cells may be allogeneic, syngeneic, allogeneic or autologous.

In general, compositions comprising cells activated and expanded as described herein are useful for treating and preventing diseases that occur in immunocompromised individuals. In particular, compositions comprising the CAR modified T cells of the invention are useful for treating B cell malignancies. The CAR modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with a carrier, diluent, excipient, and/or with other components (e.g., IL-2) or other cytokines or cell populations. In particular embodiments, the pharmaceutical compositions of the invention comprise an amount of genetically modified T cells, together with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

The pharmaceutical composition of the invention comprising a population of immune effector cells (e.g., T cells) expressing a CAR can comprise: buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids (e.g., glycine); an antioxidant; chelating agents (e.g., EDTA) or glutathione; adjuvants, such as aluminum hydroxide; and a preservative. The compositions of the invention are preferably formulated for parenteral administration, for example intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration.

The liquid pharmaceutical composition, whether in solution, suspension or other similar form, may include one or more of the following: sterile diluents (e.g., water for injection), saline solutions (preferably physiological saline, ringer's solution, isotonic sodium chloride), fixed oils (e.g., synthetic mono-or diglycerides which may be used as a solvent or suspending medium), polyethylene glycol, glycerol, propylene glycol or other solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; and a buffer, such as acetate, citrate or phosphate, and an agent that regulates osmotic pressure, such as sodium chloride or glucose. Parenteral formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

In particular embodiments, the compositions of the invention comprise an effective amount of CAR-expressing immune effector cells alone or in combination with one or more therapeutic agents. Thus, the CAR-expressing immune effector cell composition can be administered alone or in combination with other known cancer therapies, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, and the like. The composition may also be administered in combination with an antibiotic. Such therapeutic agents are accepted in the art as standard treatments for specific disease states (e.g., specific cancers) as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapeutic agents, therapeutic antibodies, or other active and auxiliary agents.

Therapeutic method

The genetically modified immune effector cells of the present invention provide improved methods for adoptive immunotherapy for treating B cell related disorders including, but not limited to, immunomodulatory disorders and hematologic malignancies.

In a particular embodiment, a composition comprising immune effector cells comprising a CAR of the invention is used to treat a disorder associated with aberrant B-cell activity, also referred to as a "medical disorder associated with the presence of pathogenic B-cells".

As used herein, "a medical condition associated with the presence of pathogenic B cells" or "B cell malignancy" refers to a medical condition formed in B cells, such as cancer. In particular embodiments, the compositions of the invention comprising CAR-modified T cells are used to treat hematological malignancies, including but not limited to B-cell malignancies, such as Multiple Myeloma (MM), acute myelogenous leukemia, and non-hodgkin's lymphoma (NHL).

In another aspect of the invention, there is provided a CAR and CAR-T according to the invention as described herein for use in the treatment of a B-cell mediated or plasma cell mediated disease or antibody mediated disease or condition selected from the group consisting of: multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), non-secretory multiple myeloma, stasis multiple myeloma, unidentified Monoclonal Gammaglobosis (MGUS), isolated plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), plasmacytic leukemia, primary Amyloidosis (AL), heavy chain disease, systemic Lupus Erythematosus (SLE), ms syndrome/osteosclerotic myeloma, cold globulinemia type I and II, light chain deposition disease, idiopathic Thrombocytopenic Purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders, epidermolysis bullosa; or any non-hodgkin lymphoma B-cell leukemia or Hodgkin Lymphoma (HL) with BCMA expression or any disease in which a patient produces neutralizing antibodies to recombinant protein replacement therapy, wherein the method comprises administering to the patient a therapeutically effective amount of a CAR or CAR-T as described herein.

Multiple myeloma is a B-cell malignancy in the form of mature plasma cells characterized by neoplastic transformation of individual clones of these types of cells. These plasma cells proliferate in the BM and may invade adjacent bones, sometimes blood. Variant forms of multiple myeloma include dominant multiple myeloma (overt multiple myeloma), stasis multiple myeloma, plasma cell leukemia, non-secretory myeloma, igD myeloma, osteosclerotic myeloma, isolated plasmacytoma, and extramedullary plasmacytoma.

Non-hodgkin lymphomas include a large group of lymphocytic carcinomas (white blood cells). Non-hodgkin lymphomas can occur at any age and are typically marked by larger lymph nodes, fever and weight loss than normal. Non-hodgkin lymphomas may also be present in extranodal sites, such as the central nervous system, mucosal tissues, including the lungs, intestines, colon, and viscera. There are many different types of non-hodgkin lymphomas. For example, non-hodgkin lymphomas can be classified into invasive (fast growing) and indolent (chronic growing) types. Although non-hodgkin lymphomas may be derived from B cells and T cells, as used herein, the terms "non-hodgkin lymphoma" and "B cell non-hodgkin lymphoma" are used interchangeably. B-cell non-hodgkin's lymphoma (NHL) includes burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are typically B-cell non-hodgkin lymphomas.

As used herein, the terms "individual" and "subject" are generally used interchangeably and refer to any animal exhibiting symptoms of a disease, disorder or condition that can be treated with the gene therapy vectors, cell-based therapeutics and methods disclosed elsewhere herein. In preferred embodiments, the subject includes any animal that exhibits symptoms of a disease, disorder or condition of the hematopoietic system (e.g., B cell malignancy) that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein. Typical subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (e.g., cats or dogs). Including non-human primates, preferably including human patients. Typical subjects include human patients having or at risk of having a B-cell malignancy, which have been diagnosed with a B-cell malignancy.

As used herein, "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reduction of one or more measurable markers of the disease or disorder being treated. Treatment may optionally involve alleviation or amelioration of symptoms of a disease or disorder, or delay of progression of a disease or disorder. "treating" does not necessarily mean completely eradicating or curing the disease or disorder or associated symptoms thereof.

As used herein, "preventing" means a method of preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or disorder. It also refers to delaying the onset or recurrence of a disease or disorder, or delaying the onset or recurrence of symptoms of a disease or disorder. As used herein, "preventing" and like terms also include reducing the intensity, impact, symptoms and/or burden of a disease or disorder prior to the onset or recurrence of the disease or disorder.

In one embodiment, a method of treating a B cell-related disorder in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising a genetically modified immune effector cell of the invention. The number and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

Administration of the compositions of the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, the composition is administered parenterally. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the composition of the invention is administered to a subject by direct injection into a tumor, lymph node or site of infection.

English names appearing herein are case-insensitive; BCMA CAR-T represents a CAR-T cell capable of expressing a BCMA specific binding domain; CD8 ^TM Represents a transmembrane domain.

The present invention describes T cells (including CAR-T cells) more fully with respect to NK cells (including CAR-NK), but these descriptions for T cells are generally applicable to NK cells as well. The description of the presence of "T cells" or synonyms thereof in the context of T cell descriptions is herein incorporated by reference as if replaced with "NK cells" or synonyms thereof. This is necessary from the standpoint of brevity of description. If such a replacement is judged to be unsuitable in some cases according to the prior art, the replacement is not performed.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Drawings

FIG. 1 is a test result of determining the affinity of a Lead 1 VHH-FC fusion protein (anti-BCMA antibody, VHH-IgG1 Fc) to a recombinant protein of human BCMA antigen using ELISA.

FIG. 2 is a measurement of the affinity of the Lead 1 VHH-FC fusion protein to the BCMA overexpressing cell line RPMI8226 using a flow cytometer.

FIG. 3 is a schematic diagram of the structure of a CAR in the BCMA-CART structure according to an embodiment of the present invention, wherein the CAR is fused to the CD3 zeta signaling region through the CD8a hinge-Transmembrane (TM) domain and the 4-1BB co-stimulatory domain.

Figure 4 results of CAR expression in CAR-T cells detected with a flow cytometer.

Figure 5 shows that CAR-T of the invention has a significant inhibitory effect on multiple myeloma cell lines.

Figure 6 shows that CAR-T of the invention has a specific inhibitory effect on multiple myeloma.

FIG. 7 shows the in vitro killing effect of BCMA-PE24 Recombinant Immunotoxins (RITs) on multiple myeloma cell lines.

For a clearer description of the present invention, reference will now be made in detail to the following examples, which are illustrative of the present invention and are not to be construed as limiting the present application.

Example 1: alpaca (Alpaca) immune and antiserum potency detection

1. Immunization method

The recombinant protein of human BCMA antigen is injected subcutaneously and intramuscularly on the back of the neck into a plurality of bags, and the absorption condition of the subcutaneous injection bags is tracked and observed to confirm the correct immunization.

2. Immune cycle

(1) Primary immunization: mixing 0.5mg antigen and Freund's complete adjuvant at a ratio of 1:1, emulsifying, and injecting into 1 ml/alpaca; (2) second immunization: 3 weeks after the first immunization, 0.25mg antigen was mixed with Freund's incomplete adjuvant 1:1, emulsified and injected at a volume of 1 ml/single camel; (3) third immunization: 3 weeks after the second immunization, 0.25mg antigen was mixed with Freund's incomplete adjuvant 1:1, emulsified and injected at a volume of 1ml per alpaca; (4) fourth immunization: after 3 weeks of three immunizations, 0.25mg antigen was mixed 1:1 with Freund's incomplete adjuvant, emulsified and injected at a volume of 1ml per single camel.

3. Serum treatment and antiserum potency detection: one week after the fourth immunization, 50ml of peripheral blood was collected and serum and lymphocytes were isolated. Antigen was coated in ELISA 96-well plates and antibody titers in serum were determined by ELISA. The antisera titers are shown in the following table.

Example 2: construction and screening of phage display immune antibody libraries.

As the fourth serum titer > 1:32000, the high affinity antibody against human BCMA was shown to be present in the serum. Further constructing phage display immune antibody library, and obtaining positive monoclonal of anti-human BCMA nano antibody through biological screening.

1. Collecting blood after the fourth immunization of alpaca, and separating lymphocyte PBMC; taking 2X 10 ⁷ Extracting total RNA using an RNA extraction kit; a suitable amount of RNA (e.g., 3-5 ug) was taken and cDNA was obtained by RT-PCR reverse transcription kit.

2. The IgG2 and IgG3 heavy chain variable region sequences (nanobody heavy chain variable region VHH) were obtained stepwise by nested PCR or the heavy chain variable region sequences of IgG1, igG2 and IgG3 (conventional antibody IgG1 heavy chain variable region VH and nanobody IgG2, igG3 heavy chain variable region VHH) were obtained by one-step PCR.

3. Inserting a heavy chain variable region sequence into a linearized phagemid vector pShort subjected to enzyme digestion treatment by homologous recombination or enzyme digestion connection to obtain a recombinant vector; after purification and recovery, super competent SS320 cells (containing helper phage M13K 07) were transformed; resuspension and activation are carried out on the converted bacterial liquid for 1 hour by using an SOC culture medium; taking a small amount of bacterial liquid to perform 10-time ratio gradient dilution, selecting proper dilution titer, and adding the bacterial liquid into LB/tet ₁₀ LB/Carb ₅₀ Coating a plate on a culture plate, placing the culture plate in a biochemical incubator at 37 ℃ overnight, and calculating the storage capacity the next day; transferring the residual bacterial liquid into a large volume of 2YT/Carb ₅₀ /Kan ₂₅ Placing in liquid culture medium, shaking table at 37deg.C, overnight culturing, collecting supernatant the next day, adding 1/4 times volume of PEG/NaCl solution, precipitating phage, taking appropriate amount of PBT solution, and re-suspending and diluting to desired concentration to obtain phage display immune antibody library (-80 deg.C) for storage.

4. Statistical LB/Carb ₅₀ The number of clones on the plate was calculated as: library 1 (nested PCR): 2.25X10 ⁸ The method comprises the steps of carrying out a first treatment on the surface of the Library 2 (one-step method): 1.63×10 ⁸ . From the plate, 20 individual clones were randomly picked and sequenced, all with the correct insertion of VHH.

Example 3: screening of antibody libraries

1. Adding 10 mug/mL of human BCMA antigen recombinant protein into a 96-well plate, and coating at 4 ℃ overnight; NEB 5. Alpha. F' E.coli was grown in 2YT/Tet ₁₀ Streaking and growing the plate, and culturing overnight in a incubator at 37 ℃;

2. the following day, from overnight 2YT/Tet ₁₀ NEB5 alpha F' monoclonal was picked on plates and added to 3ml 2YT/Tet ₁₀ In the liquid culture medium, shaking bacteria at 37 ℃ to grow to OD ₆₀₀ ＝0.8；

3. Simultaneously, the antigen supernatant of the 96-well plate was removed, 200 μl of 1% bsa was added to each well for blocking, and 200 μl of 1% bsa was added to the blank wells as a negative control well, and placed in a 3D rotary shaker at room temperature for 2 hours; afterwards, the supernatants of the protein wells and control wells were removed, washed with 200uL PT, each added with 100uL phage antibody library, and placed in a 3D rotary shaker for 2 hours at room temperature; supernatant from the protein wells and control wells was removed and washed with 200uL PT; 100. Mu.L of 100mM HCl was added to the wells and left at room temperature for 5 minutes; the supernatant was aspirated, added to a 1.5ml centrifuge tube, and neutralized with 1M Tris-HCl.

4. Adding the mixed solution obtained in the step 3 into a centrifuge tube containing 1mL NEB5αF' bacteria, and culturing for 1 hour at the temperature of a shaking table of 37 ℃; diluting 20 μL of culture solution in centrifuge tube with appropriate ratio, and concentrating in LB/Carb ₅₀ Plating the culture plate, placing the culture plate in a biochemical incubator at 37 ℃ overnight, and calculating the titer and enrichment degree the next day; 1. Mu.L of helper phage M13K07 (final concentration 10) was added to the remaining culture broth ¹⁰ 25/mL), shaking table 37 ℃, culturing for 1 hour; transferring the culture solution into 35mL 2YT/Carb ₅₀ /Kan ₂₅ In the culture solution, placing in a shaking table, culturing overnight at 37 ℃, and collecting phage to form antibody libraries of each round.

5. The above procedure was repeated 3-5 rounds until phage enrichment occurred. If the antigen binding well is in LB/Carb ₅₀ The number of colonies on the plate was 10 times or more than that of the negative control wells, and enrichment was considered successful. In this experiment, after the third round of screening, the number of colonies in the antigen binding wells was 100 times that in the negative control wells, indicating successful enrichment, and then high affinity positive clones were selected by Phage-ELISA.

Example 4: identification of Positive clones and sequencing by Phage-ELISA

1. In 96 deep well plates, 400. Mu.L of 2YT/Carb was added per well ₅₀ /Kan ₂₅ M13K07 medium; from obtaining enriched LB/Carb ₅₀ The monoclonal is selected on the culture plate, transferred into a 96 deep well plate, placed on a shaking table, centrifuged at 200rpm and 37 ℃ overnight for the next day, and the supernatant is the phage produced by each monoclonal.

2. Simultaneously, diluting the recombinant protein of the human BCMA antigen to 1 mug/mL, adding the recombinant protein into an ELISA 96-well plate according to 50 mug/hole, and placing the mixture in a refrigerator at 4 ℃ for overnight;

3. the following day, ELISA plates were back-off to remove supernatant, and 100. Mu.L of 1% BSA was added to each well for blocking; 100. Mu.L of 1% BSA was added to the blank wells as negative control wells; incubate for 1 hour at room temperature. After the completion of the blocking, the antigen wells and the negative control wells were washed with PT solution, 50. Mu.L of supernatant of 96-deep well plate was added, and incubated at room temperature for 2 hours; one antigen well and one negative control well were added to each supernatant obtained from the monoclonal.

4. After the binding was completed, ELISA plates were washed with PT solution, 50. Mu.L of HRP-M13 antibody was added, and incubated at room temperature for 1 hour; after washing with PT solution and PBS solution, 50. Mu.L of TM8 was added, incubated at room temperature for 5 minutes, and then 50. Mu.L of 1M phosphoric acid was added to terminate the reaction; absorbance values at 450nm were measured with a microplate reader. Phage-ELISA results are shown in the following Table.

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Note that: when the OD value is more than 3, the reading range of the microplate reader is exceeded, and the microplate reader automatically sets the reading as overflow.

Monoclonal DNA sequencing is carried out by identifying the monoclonal antibodies with higher affinity as positive clones with the OD value of antigen holes being more than 0.5 (including overflow holes) and the OD value of negative control holes being less than 0.2; the corresponding sequences are shown in the following table.

Monoclonal DNA sequencing is carried out by identifying the monoclonal antibodies with higher affinity as positive clones with the OD value of antigen holes being more than 0.5 (including overflow holes) and the OD value of negative control holes being less than 0.2; the corresponding sequences are shown in the following Table (definition of CDR regions using North method, italicized and/or underlined for Lead1-Lead 3; CDR for Lead4-Lead19 is shown in Table 1).

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Example 5: eukaryotic expression of BCMA (VHH-Fc fusion protein) and affinity identification.

Eukaryotic expression is carried out on the screened Lead1 sequence, and the affinity of the eukaryotic expression with antigen recombinant proteins and antigen over-expression cell strains is studied.

1. Amplifying the VHH fragment of Lead1 by PCR, inserting the fragment into a eukaryotic expression vector pPCLG containing a human IgG1 partial fragment (finger+CH2+CH3, the amino acid sequence of the fusion protein after VHH fusion is as described in SEQ ID NO:4, and the nucleotide sequence is as described in SEQ ID NO: 3) by using a homologous recombination or cleavage ligation method; electrotransferring into colibacillus trans5 alpha host bacteria, screening with ampicillin, and sequencing to obtain correct recombinant plasmid; then amplifying and culturing host bacteria containing the recombinant plasmid, and obtaining the sterile endotoxin-free plasmid by using a endotoxin removal kit;

2. culturing 293F cells in serum-free medium; the recombinant expression plasmid was transferred into 293F cells for expression using polyplus suspension cell transfection reagents. Feed was added 24 and 72 hours after transfection and the supernatant was collected on day 5 for purification. The calculation is as follows: the yield of VHH-FC fusion protein of Lead1 was 95mg/L; SDS-PAGE electrophoresis identifies that the band size of the Lead1 VHH-FC is normal, with a purity of > 95%.

3. The affinity of the Lead 1 VHH-FC fusion protein to human BCMA antigen recombinant protein was determined using ELISA: 1) Adding the human BCMA antigen recombinant protein into an ELISA 96-well plate, and coating at 4 ℃ overnight; 2) Diluting the VHH-Fc fusion protein to different concentrations, performing ELISA reaction with the antigen, and measuring the absorbance value at 450nm by using an enzyme-labeled instrument; the results are shown in FIG. 1. The experimental results show that: BCMA Lead 1 VHH-FC fusion proteins capable of binding human BCMA antigen recombinant protein, EC thereof ₅₀ ＝4.2nM。

4. Using a flow cytometer, the affinity of the Lead 1 VHH-FC fusion protein to BCMA overexpressing cell strain RPMI8226 was detected: 1) Taking 0.3X10 ⁶ RPMI8226 cells were washed 2 times with PBS and then resuspended with 100uL of PBS; 2) Incubation with 1ug/ml Lead 1 VHH-FC fusion protein for 1 hour; 3) After washing the cells 3 times with PBS, the cells were resuspended in 100uL PBS, and 0.2ug/ml FITC-labeled Anti-human FC antibody was added and incubated for 1 hour; 4) After washing the cells 3 times with PBS, the cells were resuspended with 300uL of PBS and fluorescence was detected using a flow cytometer, the results of which are shown in FIG. 2. The experimental results show that: the BCMA Lead 1 VHH-FC fusion protein can bind to the RPMI8226 cell strain, and the binding curve of the BCMA Lead 1 VHH-FC appears to be obviously right-shifted relative to that of a blank cell strain.

Example 6: investigation of the therapeutic Effect of BCMA CAR-T on Multiple Myeloma (MM)

BCMA is an important target for the treatment of Multiple Myeloma (MM), CAR-T cells were constructed using BCMA Lead 1VHH sequences to investigate the killing effect of BCMA Lead 1VHH CAR-T on Multiple Myeloma (MM) cells.

1. Preparation of lentiviral vector: 1) Gene synthesis BCMA lead 1VHH-CD8TM-4-1BB-CD3 zeta fusion gene sequence (its amino acid sequence is shown in SEQ ID NO:6, the DNA sequence is shown as SEQ ID NO:5, the structural schematic diagram of which is shown in fig. 3); 2) Inserting the fragment into the PWPXLD-kana vector by using homologous recombination or enzyme digestion ligation; transforming the recombinant vector into an escherichia coli strain StbI3, screening by kanamycin, and sequencing the monoclonal to obtain a correct recombinant plasmid; then amplifying and culturing host bacteria containing the recombinant plasmid, and obtaining a sterile endotoxin-free plasmid, namely a PWPXLD plasmid vector containing a CAR gene fragment by using a endotoxin removal kit; 3) Meanwhile, lentivirus packaging helper plasmids psPax2 and PMD2.0G are respectively transformed into DH5 alpha and ampicillin for screening, and plasmids are extracted.

2. Preparation of CAR-expressing lentiviruses (Lenti-CAR): 1) Inoculation of 3X10 ⁶ 293T cells in culture dish; 2) After 24 hours, viral plasmids (CAR-PWPXLD: 9 μg, psPax2:9 μg, and PMD2.0G: 4.5. Mu.g) was mixed, 0.45mL of sterile water and 50. Mu.L of 2.5M Ca was added Cl ₂ The solution was then added dropwise to 500. Mu.L of 2 XBBS (50mM BES,280mM NaCl,1.5mM Na) ₂ HPO 4), maintaining the solution vortex mixed; standing at room temperature for 30 minutes; then adding the mixed solution into a 293T culture medium, and gently and uniformly mixing; 3) After 18 hours, the medium was replaced with DMEM medium containing 2% fbs; 4) After 48 hours, collecting the culture supernatant, centrifuging to remove cell debris, and filtering the supernatant with a 0.45 μm filter; then one third of the volume of TAKARA lentiviral concentrate reagent (Lenti-X) was added ^TM A Concentrator, commodity number 631231), and standing overnight at 4 ℃. Centrifuging at 4deg.C and 1500g for 45 min, re-suspending the precipitate with PBS to obtain virus solution, packaging, and storing at-80deg.C.

3. Preparation of BCMA VHH CAR-T cells: 1) Peripheral blood was collected at Day 0, and lymphocyte PBMCs and plasma were isolated; sorting cd3+ T cells from PBMCs; adjusting the cell suspension to a concentration of 1X 10 ⁶ Culturing in 12-well plate; adding dynabeads magnetic beads (Thermo Fisher) for stimulation; 2) In Day 0, a solution of fibronectin (5. Mu.g/cm was added to a 12-well plate ² ) Coating at 4 ℃ overnight; 3) In Day 1, the solution of fibronectin in 12-well plates was discarded and blocked with 2% bsa for 30 min; removing the blocking solution at 750. Mu.L/4.5 cm ² Adding virus liquid, placing in a 37 ℃ incubator, and standing for 4-6 hours; collecting stimulated T cells, taking 10 ⁶ Each well was filled with 5% CO in an incubator at 37 ℃ ₂ Culturing; 4) At Day 3, T cell concentration was adjusted to 5×10 ⁵ /ml, total replacement of fresh medium; 5) At Day5, T cell concentration was adjusted to 5×10 ⁵ /ml, CAR-T cell CAR expression was detected with a flow cytometer (fig. 4).

4. LDH assay detects CAR-T killing RPMI 8226 cells in vitro: 1) BCMA-highly expressed RPMI 8226 cells or BCMA-lowly expressed K562, raji cells were configured as 2X 10 cells ⁵ Car-T cell configuration of/mL, day5 was 4X 10 ⁵ /mL; the following groups were set: blank (200 μl media), spontaneous group a (100 μl CAR T cells+100 μl media), spontaneous group B (100 μl target cells+100 μl media), experimental group (100 μl CAR T cells+100 μl target cells); 200. Mu.L of culture medium was additionally set as volume correction group, target cell 100 μL+100 μL medium as the maximum release group; respectively placing in 96-well U-shaped plates, placing in a 37 ℃ incubator, and 5% CO ₂ Incubating for 24 hours; 2) The next day, 20. Mu.l of LDH maximum release reagent was added to each well of the maximum release group and the volume correction group, and the mixture was placed in a 37℃incubator with 5% CO ₂ Incubating for 45 minutes, and detecting maximum release; 3) Meanwhile, ELISA detection 96-well plates are taken, and LDH detection reagent 50 positive groups/holes are added in a dark place; taking out the 96-well U-shaped plate, uniformly mixing the wells, centrifuging 500g for 3 minutes, taking 50 mu L and adding the 50 mu L into a corresponding ELISA detection 96-well plate; placing the ELISA detection 96-well plate into an enzyme-labeled instrument, shaking and uniformly mixing for 30 minutes; after adding 50. Mu.L of the terminating reagent, the detection wavelength was 492nm; calculating a killing rate = (experimental group-spontaneous group a-spontaneous group B + blank group)/(maximum release value-volume correction value-spontaneous group B + blank group); the experimental results are shown in fig. 5, and it is obvious from fig. 5 that the CAR-T provided by the invention can have a remarkable curative effect or inhibition effect on the myeloid leukemia, and has no remarkable curative effect or inhibition effect on tumor cells (K562 and Raji) with low expression of BCMA.

5. Animal experiments detect in vivo killing of RPMI 8226 cells by CAR-T: B-NDG severe immunodeficiency mice were purchased for 4-6 weeks of age and adapted to 2X 10 subcutaneous inoculation of the forelimb flank after one week of feeding ⁶ RPMI 8226 multiple myeloma tumor cells/RPMI 8226 multiple myeloma tumor cells alone. Observing and measuring tumor size, and after 3-4 weeks, the tumor grows to 80-100mm ³ At this time, mice were randomly divided into 6 groups, and CAR-T cells were cultured in vitro for 7-8 days by continuous two-day tail intravenous injection, each dose being 2X 10 ⁶ /only. The tumor size was measured continuously, and the result is shown in fig. 6, in which the tumor size calculation method= (1/2) square of major diameter and minor diameter. As can be seen from fig. 6, the CAR-T prepared in the examples of the present application has a significant therapeutic effect or inhibitory effect on human multiple myeloma and acute myelogenous leukemia.

Example 7: in vitro killing of RPMI8226 cells by BCMA-PE24 Recombinant Immunotoxins (RITs)

Recombinant Immunotoxins (RITs) are targeted biopharmaceuticals made by linking highly specific monoclonal antibodies to biotoxin molecules with strong killing effects. Pseudosingle cellsBacillus exotoxins (pseudomonas extoxin, PE) are one of the commonly used biotoxins, where PE24 retains only the third Domain of the PE toxin (Domain III), thus reducing the immunogenicity of the drug while largely retaining the toxicity of PE. Previous studies have shown that: scFv-PE24 RITs prepared by using BCMA single domain antibodies linked to PE24 have a strong killing ability against multiple myeloma cells (see:https：// academic.oup.com/abt/article/1/1/19/5076366). And (3) fusion expression of the Lead 1 VHH sequence obtained by screening and PE24, and researching the in vitro killing effect of the recombinant immunotoxin on RPMI8226 cells.

1. The DNA sequence of BCMA Iead1 VHH-PE24 (SEQ ID NO:13, corresponding amino acid sequence: SEQ ID NO: 14) was synthesized artificially, and then the fragment was inserted into a pet25b (+) prokaryotic expression vector using homologous recombination or enzyme ligation methods, and a his tag was introduced at the C-terminus.

2. Transferring the recombinant plasmid into rosetta2 host bacteria, and transferring the recombinant plasmid into LB/Amp ₁₀₀ /Chl ₁₅ Culturing overnight at 37deg.C in culture medium; the following day, the overnight cultured bacteria were transferred into LB/Amp at 1:100 ₁₀₀ /Chl ₁₅ Culture medium, culture at 37deg.C for 2-3 hr, OD ₆₀₀ At=0.6-1, 1mm iptg was added and expression was induced overnight at 25 ℃.

3. Centrifuging, collecting thalli and washing with PBS; the thalli are resuspended by 10ml PBS, broken by ultrasound, and the supernatant is collected; the protein is obtained through Ni column adsorption and elution, and SDS-PAGE electrophoresis shows that the obtained protein has single band and correct position.

4. According to 1X 10 ⁴ Well, RPMI 8226 cells were seeded into 96-well plates while BCMA VHH-PE24 biotoxin protein was added at concentrations of 100ng/ml, 30ng/ml, 10ng/ml, 3ng/ml, 1ng/ml, 0.3ng/ml, 0.1ng/ml, and 0ng/ml (blank); after 96 hours, the killing effect was measured using CCK8 assay kit, the results are shown in fig. 7. The result shows that the BCMA Iead1 VHH-PE24 prepared in the embodiment of the application has specific obvious curative effect or inhibition effect on human multiple myeloma.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. An anti-BCMA antibody capable of binding a human BCMA polypeptide comprising the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 of a single heavy chain variable region (VHH) as shown in SEQ ID No. 2 wherein the antibody is a single domain antibody wherein the antibody comprises the heavy chain variable region CDRs defined according to North, kabat, chothia and IMGT numbering scheme in the table below wherein the numbers represent the heavy chain variable region CDRs in SEQ ID NO: position in VHH shown in 2:

2. the antibody of claim 1, wherein the HCDR1, HCDR2, and HCDR3 are SEQ ID NO: 10-12.

3. The antibody of claim 1, which is a heavy chain variable region sequence shown in SEQ ID NO. 2 or a humanized sequence thereof.

4. A bispecific or trispecific antibody comprising the antibody of claim 1, 2 or 3.

5. A fusion protein comprising the antibody of any one of claims 1-4.

6. The fusion protein of claim 5, further comprising a tag sequence.

7. The fusion protein of claim 6, wherein the tag sequence is Poly-His.

8. The fusion protein of claim 6, having a sequence set forth in SEQ ID NO: 4.

9. An antibody-drug conjugate comprising the antibody of any one of claims 1-4.

10. A Chimeric Antigen Receptor (CAR) comprising the antibody of any one of claims 1-4.

11. The CAR of claim 10, having a sequence set forth in SEQ ID NO: shown at 6.

12. A polynucleotide encoding the antibody of any one of claims 1-4, the fusion protein of claim 5, or the CAR of claim 10.

13. The polynucleotide of claim 12, wherein:

the polynucleotide encoding the antibody of claim 1 is set forth in SEQ ID NO:1 or a degenerate or complementary sequence thereof;

the polynucleotide for encoding the fusion protein of claim 5 is shown in SEQ ID NO:3 or a degenerate or complementary sequence thereof; or (b)

The polynucleotide encoding the CAR of claim 10 is set forth in SEQ ID NO:5 or a degenerate or complementary sequence thereof.

14. An isolated CAR-T cell or CAR-NK cell, wherein said CAR-T cell or CAR-NK cell is capable of expressing the antibody of any one of claims 1-4; the CAR-T cell or CAR-NK cell is capable of expressing the fusion protein of claim 5; the CAR-T cell or CAR-NK cell is capable of expressing the CAR of claim 10; or, the CAR-T cell or CAR-NK cell comprises the polynucleotide of claim 12.

15. A vector comprising the polynucleotide of claim 12.

16. The vector of claim 15, wherein the vector is an expression vector.

17. An immune effector cell comprising the CAR of claim 10, or comprising the polynucleotide of claim 12, or comprising the vector of claim 15.

18. The immune effector cell of claim 17, wherein the immune effector cell is a T lymphocyte or a natural killer cell.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising the fusion protein of claim 5, comprising the CAR-T cell or CAR-NK cell of claim 14, or comprising the immune effector cell of claim 17 or 18.

20. A method of making the CAR-T cell or CAR-NK cell of claim 14, or the immune effector cell of claim 17 or 18, comprising introducing the vector of claim 15 to a T lymphocyte or natural killer cell.

21. Use of the antibody of any one of claims 1-4, the fusion protein of claim 5, the antibody-drug conjugate of claim 9, the CAR of any one of claims 10-11, or the CAR-T cell or CAR-NK cell of claim 14, or the immune effector cell of claim 17 or 18 in the manufacture of a medicament for the treatment and/or prevention of cancer, wherein the cancer is multiple myeloma.