WO2021043169A1

WO2021043169A1 - Antibody that binds specifically to b-cell maturation antigen and use thereof

Info

Publication number: WO2021043169A1
Application number: PCT/CN2020/113046
Authority: WO
Inventors: 刘江海; 曾昕; 刘彬; 孔洋; 曾顺泽; 林静
Original assignee: 成都盛世君联生物技术有限公司; 成都盛世锐科生物技术有限公司
Priority date: 2019-09-02
Filing date: 2020-09-02
Publication date: 2021-03-11
Also published as: CN114667294A; CN114667294B

Abstract

Provided is an antibody that specifically binds to the B-cell maturation antigen (BCMA) and a use thereof. The present invention relates to an antibody or an active fragment thereof which can specifically bind to the BCMA, and a fusion protein which comprises the antibody or the active fragment thereof. The fusion protein may comprise the antibody or the active fragment thereof, a transmembrane domain and a costimulatory signal transduction region, the antibody or the active fragment thereof can specifically bind to a tumor-specific antigen BCMA, and T cells are activated by means of the transmembrane domain and the costimulatory signal transduction region. Also provided is a CAR-T cell capable of expressing the described fusion protein, the CAR-T cell uses the BCMA as a target antigen and is used to specifically kill tumor cells, such as multiple myeloma or acute myeloid leukemia. CAR-T cells can be used as a therapeutic drug for oncological diseases, thus providing a new method for tumor prevention and treatment.

Description

Antibody that specifically binds B cell mature antigen and its use

Technical field

The invention relates to the field of biomedicine, in particular to an antibody that specifically binds to a mature antigen of B cells, and its preparation and application.

Background technique

T cells are a type of 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 (TCR) on the cell surface. T helper cells, also known as CD4 ⁺ T or CD4 T 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 cytokines that can regulate immune responses. Cytotoxic T cells, also known as CD8 ⁺ T cells or CD8 T cells, express CD8 glycoprotein 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 subset of T cells that exist for a long time and respond to related antigens, thus providing the immune system with memories of past infections and/or tumor cells.

T cells modified by chimeric antigen receptor (CAR) are called CAR-T, which are modified by genetic engineering methods under in vitro culture conditions to express exogenous anti-tumor genes. The CAR gene is an artificially designed gene fragment. Its encoded protein mainly includes an extracellular recognition domain and an intracellular signal transduction domain: the former is a specific antibody fragment used to target and recognize specific molecules on the tumor surface; the latter is used to start Immune cell response after specific recognition plays a role in cellular immunity. After genetic modification, T cells can produce chimeric antigen receptors on their surface. CAR is a protein that allows T cells to recognize specific proteins (antigens) on tumor cells. Genetically engineered CAR T cells can grow in the laboratory until their number reaches billions. The expanded CART cells can then be infused into the patient.

B Cell Maturation Antigen (BCMA) is a cell surface receptor encoded by the human TNFRSF17 gene, which can bind to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). It plays an important role in cell differentiation and autoimmune response. Therefore, it is highly expressed in some blood tumors related to B lymphocytes and is an excellent target with strong potential for drug development. Indications include, but are not limited to, multiple myeloma (MM) and acute myeloid leukemia (Acute myeloid leukemia). , AML) and so on.

Many anti-BCMA CAR constructs have been described. In 2013, Carpenter et al. disclosed an anti-BCMA CAR-transduced T cell method, which is a pre-clinical study that uses a combination of in vitro tests and animal tests (mouse tests) (Carpenter et al., 2013; Clin Cancer) Res; 19(8); 2048-2060). In June 2015, Bluebird and Celgene launched a research collaboration for BCMA CAR-T cell therapy. At the beginning of 2016, the Abramson Cancer Center of the University of Pennsylvania (Abramson Cancer Center) began the recruitment of anti-BCMA CAR T to treat patients with multiple myeloma. The CAR or corresponding CAR T for BCMA has been described in WO2018/028647, WO2017/211900, WO2016/014789, WO2016/094304, WO2016/014565 and WO2013/154760, for example. CN109134665A also discloses a single domain antibody-based BCMA chimeric antigen receptor and its application.

Although many potential alternative therapies are under development, there is still a need to provide effective methods to solve the conditions associated with the presence of pathogenic B cells, especially multiple myeloma and acute myeloid leukemia. This is mainly because the relevant immunotherapy process is prone to miss the target and easily cause recurrence.

Summary of the invention

In view of this, the present invention provides an antibody against BCMA and CAR-T cells based on the antibody, as well as preparation and application thereof. The CAR-T cells can specifically recognize and kill tumors, and have more efficient tumor killing activity, such as multiple myeloma (MM) and acute myeloid leukemia (AML).

Specifically, the present invention provides an anti-BCMA antibody or antigen-binding fragment thereof that can bind to human BCMA polypeptide, which can competitively bind to SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 19, or SEQ ID NO: one of the human BCMA epitopes bound by a single variable heavy chain (VHH) shown in one of 23-38. Exemplarily, the anti-BCMA antibody or antigen-binding fragment thereof comprises a single weight as shown in one of SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 19, or SEQ ID NO: 23-38 The heavy chain complementarity determining regions of the variable chain region (VHH) H-CDR1, H-CDR2 and H-CDR3 or their humanized variant sequences, for example, the H-CDR1, H-CDR2 and H-CDR3 are selected respectively One or more CDRs shown in any one of SEQ ID NO: 10-12 or their identity sequences; one or more CDRs shown in any one of SEQ ID NO: 16-18 or Its identity sequence; or one or more CDRs or its identity sequence shown in any one of SEQ ID NO: 20-22.

Exemplarily, the antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region sequence shown in SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 19 or its humanized sequence Or an amino acid sequence that has at least 80% sequence identity with one of SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 19, or SEQ ID NO: 23-38.

Exemplarily, the identity sequence of SEQ ID NO: 10 refers to the heavy chain complementarity determining region 1 (H-CDR1) that has at least 80% sequence identity with SEQ ID NO: 10, the coding sequence of which may be exemplarily SEQ ID NO: 10 ID NO: 7);

The identity sequence of SEQ ID NO: 11 refers to the heavy chain complementarity determining region 2 (H-CDR2) that has at least 80% sequence identity with SEQ ID NO: 11, and its coding sequence may be, for example, SEQ ID NO: 8 );with

The identity sequence of SEQ ID NO: 12 refers to the heavy chain complementarity determining region 3 (H-CDR3) that has at least 80% sequence identity with SEQ ID NO: 12, and its coding sequence may be, for example, SEQ ID NO: 9 ).

Further, the antibody or antigen-binding fragment of the present invention can be selected from the following group: camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' ₂ fragment, F(ab)' ₃ fragment, Fv , ScFv, double-scFv, (scFv) ₂ , mini-antibodies, diabodies, tri-chain antibodies, four-chain antibodies, disulfide-stabilized Fv proteins and single domain antibodies (sdAbs, Nanobodies), bispecific antibodies Or trispecific antibodies and so on.

The present invention also provides a fusion protein, which comprises the above-mentioned antibody or antigen-binding fragment.

Exemplarily, the fusion protein described in the present invention may also include a tag sequence (such as Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, with additional epitopes (such as for human Other epitopes of BCMA) or additional antibody active fragments (such as antibodies or antibody active fragments directed against other epitopes of human BCMA or the same epitope, or ligands capable of binding to human BCMA), preferably, the fusion protein has SEQ ID NO: The sequence shown in 4.

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

Exemplarily, in the antibody-drug conjugate of the present invention, the drug is selected from the following: radiolabels, ³² P, ³⁵ S, fluorescent dyes, electron compacting reagents, enzymes, biotin, streptomyces Avidin, digitoxin, hapten, immunogenic protein, nucleic acid molecule having a sequence complementary to the target, or any combination of the foregoing; or immunomodulatory compound, anticancer agent, antiviral agent, antibacterial agent, antibacterial Fungal agents and antiparasitic agents, or any combination of the foregoing.

The present invention also provides a chimeric antigen receptor (CAR), which comprises: (1) an extracellular antigen-binding domain comprising the antibody or antigen-binding fragment, fusion protein or antibody-drug conjugate described in the present invention ; And optionally include (2) a transmembrane domain; and, (3) an intracellular signaling domain.

Exemplarily, in the CAR of the present invention, the transmembrane domain is derived from α, β, or ζ chains selected from the group consisting of T cell receptors, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, One or more transmembrane domains in the group consisting of CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154, ICOS and PD1.

Exemplarily, in the CAR of the present invention, the intracellular signal transduction domain includes a costimulatory signal transduction domain and is selected from: CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD7, CD22, CD27 One or more of, CD28, CD30, CD40, CD66d, CD79a, CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C and B7-H3.

In addition, the CAR of the present invention may also exemplarily include a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. Preferably, the hinge domain is derived from CD8α.

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

Exemplarily, the CAR of the present invention has the sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 6.

The present invention also provides a polynucleotide, which is a polynucleotide encoding the antibody or antigen-binding fragment of the present invention, the fusion protein of the present invention, or the CAR of the present invention,

Exemplarily, the polynucleotide encoding the antibody or antigen-binding fragment of the present invention is shown in SEQ ID NO: 1 or its degenerate sequence;

The polynucleotide encoding the fusion protein of the present invention is shown in SEQ ID NO: 3 or its degenerate sequence; or

The polynucleotide encoding the CAR of the present invention is shown in SEQ ID NO: 5 or its degenerate sequence.

The present invention also provides an isolated CAR-T cell or CAR-NK cell, characterized in that the CAR-T cell or CAR-NK cell can express the antibody or antigen-binding fragment of the present invention; CAR-T cells or CAR-NK cells can express the fusion protein of the present invention; the CAR-T cells or CAR-NK cells can express the antibody-drug conjugate of the present invention; the CAR-T cells Or CAR-NK cells can express the CAR of the present invention; the CAR-T cells or CAR-NK cells contain the polynucleotide of the present invention.

Exemplarily, the CART cells of the present invention are CD4+ T cells or a cell mixture containing CD4+ T cells and CD8+ T cells.

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

Exemplarily, 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) Wisner-Medi virus (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).

The present invention also provides an immune effector cell, which comprises the CAR according to the present invention, or comprises the polynucleotide according to the present invention, or comprises the vector according to the present invention.

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

The present invention also provides a pharmaceutical composition comprising the CAR-T cell or CAR-NK cell according to the present invention, or the immune effector cell according to the present invention, and optionally, a pharmaceutically acceptable Carrier or auxiliary material.

The present invention also provides a method for preparing the CAR-T cell or CAR-NK cell of the present invention, or preparing the immune effector cell of the present invention, which comprises introducing the carrier of the present invention into T lymphocytes Or natural killer cells.

Exemplarily, the preparation method of CAR-T cells of the present invention includes the following steps:

(1) Synthesis and amplification (antibody or antibody fragment) -CD8αhinge-CD8 ^TM -4-1BB-CD3ζ fusion protein gene, and the (antibody or antibody fragment) -CD8αhinge-CD8 ^TM -4-1BB-CD3ζ fusion protein Gene cloning into lentiviral expression vector;

(2) Use the lentiviral packaging plasmid and the lentiviral expression vector plasmid obtained in step (1) to infect 293T cells, package and prepare the lentivirus; and

(3) Infect T cells with the lentivirus obtained in step (2) to obtain CAR-T cells.

The present invention also provides the use of materials of the following items in the preparation of medicines for the treatment and/or prevention of cancer:

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

(b) The fusion protein of the present invention;

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

(d) CAR according to the present invention;

(e) The CAR-T cell or CAR-NK cell of the present invention; or

(f) The immune effector cells of the present invention.

Exemplarily, the cancer is a tumor that highly expresses B-cell mature antigens and related diseases, such as multiple myeloma and acute myeloid leukemia, preferably recurrent multiple myeloma.

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

For the antibodies or antigen-binding fragments, fusion proteins, CARs, etc. of the present invention, the present invention also considers their variants, such as their identity sequences or humanized sequences. Exemplarily, 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 with the original sequence or the reference sequence. Above, 76% or above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% or Above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or Above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or more, 99.8% or more, or 99.9% or more.

For the polynucleotide of the present invention, the present invention also considers its degenerate sequence or complementary sequence. Exemplarily, the homology between the degenerate sequence and the original sequence or the reference sequence is 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 above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above , 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% % Or more, 99.7% or more, 99.8% or more, or 99.9% or more.

The CAR provided by the present invention can specifically bind to the tumor-specific antigen B cell mature antigen, and activate the T cell through the transmembrane domain and the costimulatory signal conduction region. The CAR-T cell can express a fusion protein with a B cell mature antigen as a target antigen, and therefore can specifically kill tumor cells for the treatment of tumor diseases, for example, for the treatment of tumors with high expression of B cell mature antigen.

detailed description

Multiple myeloma, also known as plasmacytoma, is a B-cell lymphoma that is currently incurable. It is derived from a malignantly transformed plasma cell clone. The disease usually recurs and drug resistance develops after a multi-line treatment regimen.

The CART or NK cells against BMCA of the present invention can target B cell maturation antigen (BMCA), and then can be used to treat BMCA-related cancers, such as multiple myeloma or acute myeloid leukemia, because BCMA is in multiple bone marrow It is highly expressed in tumor cells and acute myeloid leukemia, but not in normal B cells or precursor B cells. In addition, in the anti-CD19 antibody or anti-CD19CAR-T or NK cell therapy directed against B cells, non-Hodgkin’s lymphoma (B-NHL) drug resistance occurs due to the loss of the targeted cancer cell surface antigen, so it is necessary New alternative targets. For B-NHL, BCMA can be used as a suitable target. Therefore, the high-affinity anti-BCMA CAR-T or NK cells of the present invention should also be used therapeutically for B-NHL.

Since the anti-BCMA CAR of the present invention confers extremely high affinity to T cells or NK cells, it can also be used to identify B-cell lymphomas with low BCMA expression. In addition, the anti-BCMA CAR-T or -NK of the present invention is not reactive to normal T cells, B cells, NK cells, endothelial cells, all bone marrow cell lineages and their precursor cells. Therefore, the anti-BCMA CAR-T or -NK of the present invention has no undesirable reactivity to bone marrow cell precursors.

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

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

Animal experiments have confirmed that the anti-BCMA CAR-T cells of the present invention can significantly kill multiple myeloma cells.

At the same time, through experiments, it is precisely the in vitro killing effect of the fusion protein BCMA-PE24 containing the antibody of the present invention on RPMI 8226 cells.

Chimeric antigen receptor:

The CAR contains an extracellular extracellular domain derived from an antibody and an intracellular domain containing a signaling module derived from a T cell signaling protein. In one embodiment, the extracellular domain may comprise a heavy chain variable region derived from an immunoglobulin, or a variable region comprising a heavy chain and a light chain, for example, is constructed as a single chain variable fragment (scFv), preferably only Single domain antibody (sdAb) with heavy chain variable region. The sdAb is connected to the hinge region, which provides flexibility and transmits signals to the intracellular signaling domain through the transmembrane domain. The transmembrane domain is preferably derived from CD8α. In the first-generation CAR (the term "generation" refers to the intracellular signaling domain), the intracellular signaling domain is composed of the zeta chain of the TCR complex. The second generation CAR is designed to contain a single costimulatory domain derived from CD28 or 4-1BB. The third generation CAR includes two costimulatory domains, such as 4-1BB-CD3ζ. The invention preferably relates to a second or third generation CAR.

The present invention provides genetically engineered receptors that redirect the cytotoxicity of immune effector cells to B cells. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CAR). CAR is a chimeric protein molecule with specific anti-BCMA cell immune activity based on the combination of an antibody targeting an antigen (e.g. BCMA) specifically with the intracellular domain of activated T cell receptor or NK cell receptor. As used herein, the term "chimeric" refers to being composed of different proteins or DNA from different sources.

The CAR of the present invention includes an extracellular domain (also referred to as a binding domain or an antigen binding domain) that binds to BCMA, a transmembrane domain, and an intracellular domain or an intracellular signaling domain. The binding of the anti-BCMA antigen binding domain of the CAR to the BCMA on the surface of the target cell results in the aggregation of the CAR and delivers an activation stimulus to the CAR-containing cells. CAR can specifically redirect immune effector cells, thereby triggering proliferation, cytokine production, phagocytosis, or cell killing of target antigen-expressing cells.

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

"Extracellular antigen binding domain" or "extracellular binding domain" can be used interchangeably and provide CAR with the ability to specifically bind to the target antigen of interest BCMA. The binding domain can be derived from natural, synthetic, semi-synthetic or recombinant sources. The sdAb of recombinant origin is preferred.

"Specific binding" should be as understood by those skilled in the art, and those skilled in the art are clearly aware of various experimental methods or means that can be used to test binding and binding specificity. Methods of determining equilibrium association or equilibrium dissociation constants are known in the art. In many protein-protein interactions, some cross-reactions or background binding may occur, but this does not damage the "specificity" of the binding between CAR and epitope. "Specific binding" describes the binding of an anti-BCMA antibody or its antigen-binding fragment (also including their CAR) to BCMA, and its binding affinity is higher than background binding.

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

A "single chain Fv" or "scFv" antibody fragment comprises the VH domain and the VL domain of an antibody, where these domains exist as a single polypeptide chain and in either direction (for example, VL-VH or VH-VL). Generally, the scFv polypeptide also contains a polypeptide linker between the VH domain and the VL domain, which allows the scFv to form the desired structure for antigen binding. In a preferred embodiment, the CAR of the present invention comprises an antigen-specific binding domain, which is a scFv and can be a murine, human or humanized scFv. The single-chain antibody can be cloned from the V region gene of the hybridoma specific for the desired target. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds to a human BCMA polypeptide. Illustrative examples of variable heavy chains suitable for constructing the anti-BCMA CAR of the present invention include but are not limited to the amino acid sequence shown in SEQ ID NO: 2. Illustrative examples of the variable light chain suitable for constructing the 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 CN109641012A.

Antibodies and antibody fragments:

The CAR contains an extracellular antigen binding domain, which contains an antibody or antibody fragment that binds to a B cell maturation antigen (BCMA) polypeptide. Therefore, the antibodies or antibody fragments of the present 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 (such as VHH fragments from Nanobodies), Fab fragments, F(ab′) ₂ fragments, fragments generated from Fab expression libraries, anti-idiotypic antibodies and epitope binding fragments or any combination of the above, provided that they It has the similar binding characteristics of the CAR of the present invention, and preferably comprises the corresponding CDR, or VH and VL regions as described herein. Mini-antibodies and multivalent antibodies such as diabodies, trivalent antibodies, tetravalent antibodies, and pentavalent antibodies can also be used in the methods of the present invention. The immunoglobulin molecules of the present invention can be of any class (ie, IgG, IgE, IgM, IgD, and IgA) or subclasses of immunoglobulin molecules. Therefore, as used herein, the term antibody also includes antibodies and antibody fragments comprised by the CAR of the present invention, which are produced by modifying intact antibodies or re-synthesized using recombinant DNA methods.

As used herein, "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. When the term "antibody" is used, it can also be considered to mean "antibody fragment". Optionally, antibodies or antibody fragments can be chemically conjugated with other proteins or fusion proteins of other proteins, or expressed as fusion proteins with other proteins. In some embodiments, the antibody or antigen-binding fragment of the invention is contained on a multispecific antibody, such as a bispecific antibody. Such multispecific antibodies can be produced by known methods, such as cross-linking two or more of the same type or different types of antibodies, antigen-binding fragments (e.g., scFv). Exemplary methods for preparing 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 polypeptide of the present invention and the antibody or antibody fragment or CAR protein can be easily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or using a surface plasmon resonance device (such as Biacore).

A humanized antibody can be prepared using methods known in the art, the humanized antibody comprising one or more CDRs of the antibody or antibody fragment of the present invention or one or more CDRs derived from the antibody or antibody fragment. For example, four steps can generally be used to humanize a monoclonal antibody: (1) Determine the nucleotide and predicted amino acid sequences of the light chain and heavy chain variable domains of the starting antibody; (2) Design the humanized antibody , That is, decide which antibody framework region to use in the humanization process; (3) carry out humanization methods/techniques; and (4) transfection and expression of humanized antibodies. For example, see, U.S. Patent No. 6,180,370.

The term humanized antibody means that at least a part of the framework region and optionally a part of the CDR region or other regions involved in binding of the immunoglobulin are derived from or adjusted to human immunoglobulin sequences. Humanized, chimeric or partially humanized forms of mouse monoclonal antibodies can be prepared, for example, by recombinant DNA technology. Linking the CDR regions of non-human antibodies with human constant regions by recombinant DNA technology can produce humanized mouse antibodies (Queen et al., 1989; WO 90/07861). Alternatively, the monoclonal antibody used in the method of the present invention may be a human monoclonal antibody. Human antibodies can be obtained, for example, using the phage display method (WO 91/17271; WO 92/01047).

As used herein, a humanized antibody also refers to the form of a non-human (e.g., murine, camel, llama, shark) antibody, which is a specific chimeric immunoglobulin, immunoglobulin containing minimal sequence derived from non-human immunoglobulin. Globulin chains or fragments thereof (e.g. Fv, Fab, Fab', F(ab') ₂ or other antigen binding subsequences of antibodies, such as vHH.

As used herein, a human or humanized antibody or antibody fragment refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of a human-produced antibody, and can be prepared using any technique known in the art for preparing antibodies. Human antibodies or fragments thereof can be selected through competitive binding experiments or other methods to determine that they have the same epitope binding specificity as a specific mouse antibody.

Variable region and CDR

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 chain variable region and the light chain variable region each consist of four framework regions (FR) connected by three complementarity determining regions (CDR), which are also referred to as hypervariable regions. The CDRs in each chain are held in close proximity by FRs, and together with CDRs from other chains, contribute to the formation of the antigen binding site of the antibody.

CDR is mainly responsible for binding antigen epitopes. There are many methods that can be used to determine the boundaries of CDR amino acid sequences, such as Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda Md, "Kabat" numbering scheme); Al-Lazikani B , Lesk AM, Chothia C. Standard conformations for the canonical structures of immunoglobulins. J. Mol. Biol. 1997; 273: 927-48 (“Chothia” numbering plan); Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor varia ble domain sand Ig superfamily V-like domains,"Dev.Comp.Immunol.,27:55-77,2003; "IMGT" numbering scheme); or North,B,LehmannA,Dunbrack RA new clustering of antibodyCDR loop conformations: J.Mol.Biol.(2011),406(2):228-256. In addition to the above methods, alternative methods include new solutions developed with the development of bioinformatics. Although Kabat is the most commonly used method, CDR can refer to a CDR defined by one or more methods, or by a combination of these methods.

Illustratively, Table 1 below shows the positions of the heavy chain variable region CDRs in the respective VHHs according to the North, Kabat, Chothia, and IMGT numbering schemes. 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, the antibody of the present invention may contain HCDR1, HCDR2, and HCDR3 of one of Lead1-19 determined by any of North, Kabat, Chothia, and IMGT. Or the antibody of the present invention contains HCDR1, HCDR2 of Lead1 determined by North and HCDR3 of one of Lead2-19 determined by Kabat. Those skilled in the art can freely choose the CDRs identified by different methods, and can freely combine these CDRs.

Table 1

In some embodiments, the present invention provides an antibody or fragment thereof contained in a CAR, wherein the antibody or fragment thereof comprises at least one CDR, at least two, or at least three CDRs that are substantially the same as the antibody of the present invention. One CDR, at least two or at least three CDRs. In some embodiments, the at least one, two, or three CDRs have at least about 70%, 75%, 85%, 86%, 87%, and at least one, two, or three CDRs of the antibody of the present invention. 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity. It should be understood that for the purposes of the present invention, the binding specificity and/or overall activity is generally retained, although the degree of activity may vary (which may be larger or smaller) compared to the antibody.

In certain embodiments, there may be substitutions, insertions, or deletions in one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity can be made in the CDRs (e.g., conservative substitutions as provided herein). In certain embodiments of the variant VH and VL sequences provided above, each CDR may be unaltered or contain no more than one, two or three amino acid substitutions, insertions or deletions. For example, one, two or three amino acids of the CDR sequence SEQ ID NO: 10, 11 or 12 contained in the antibody of the present invention can be replaced, inserted or deleted by the same amino acid, and still retain the ability to bind to human BCMA . Alternatively, one, two or three amino acids of the CDR sequence SEQ ID NO: 16, 17, or 18 contained in the antibody of the present invention can be replaced, inserted or deleted by the same type of amino acid, and still retain the ability to bind to human BCMA. Alternatively, one, two or three amino acids of the CDR sequence SEQ ID NO: 20, 21 or 22 contained in the antibody of the present invention can be replaced, inserted or deleted by the same amino acid, and still retain the ability to bind to human BCMA . Alternatively, the antibody of the present invention includes Lead1, Lead2, Lead3, Lead4, Lead5, Lead6, Lead7, Lead8, Lead9, Lead10, Lead11, Lead12, Lead13, Lead14, Lead15, Lead16, Lead17, Lead18, Or Lead19's HCDR1, HCDR2, and HCDR3 identified by the same method, or a combination of HCDR1, HCDR2, and HCDR3 identified by different methods.

For mutable sites, reference can be made to the description of Cunningham and Wells (1989) Science, 244:1081-1085. This method that can be used to identify residues or regions of antibodies that can be targeted for mutation is called "alanine scanning mutagenesis." In this method, residues or a set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified, and neutral or negatively charged amino acids (e.g., alanine or polyalanine) are used. Acid) to determine whether the interaction between the antibody and the antigen is affected. It may be considered to introduce additional substitutions at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as replacement candidates. Variants can be screened to determine whether they contain the desired properties.

For example, as shown in this disclosure, after replacing CDR1 (AAS GF T LDY YAIG) in Lead 1 (SEQ ID NO: 2) with AAS DS T VEL YAIG, the Lead 3 antibody still remains active. In other words, after replacing 5 amino acid residues, the mutant antibody still retains activity.

Therefore, it should be clearly understood that although the claims of this application file do not specifically define the specific sequence of the mutated CDR, those skilled in the art can perform mutations according to the prior art (with the preservation of the activity of the antibody, for example, in The CDR regions identified in the present invention, such as substitution, deletion, or addition of 1, 2, or 3 amino acid residues, even though 5 amino acid residues are replaced above), to find mutant CDRs that retain antibody activity and include mutant CDRs. The antibody is an obvious variant of the antibody of the present invention, and therefore is covered by the scope of protection of the present invention.

Further explanation of chimeric antigen receptor

In some embodiments, the CAR of the present invention may include linker residues between each domain added for proper spacing and conformation of the molecule, such as a linker containing an amino acid sequence, which connects the VH domain and the VL domain and provides The compatible spacer function of the interaction of the two sub-binding domains allows the resulting polypeptide to maintain specific binding affinity for the target molecule. The CAR of the present invention may contain one, two, three, four, or five or more linkers. In particular embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any suitable length of amino acids.

Illustrative examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; other flexible linkers known in the art, such as Whitlow linkers. Glycine and glycine-serine polymers are relatively unstructured, and therefore can be used as links between domains or some of the domains (such as the CAR described herein) of the fusion protein.

In a particular embodiment, the CAR binding domain is followed by one or more "spacers" or "spacer polypeptides", which are equivalent to linkers, which move the antigen-binding domain away from the surface of the effector cell so that the cell-to-cell interaction Appropriate contact, antigen binding and activation are possible. 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 include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer region includes the CH2 and CH3 domains of IgG1 or IgG4.

In some embodiments, the binding domain of the CAR may be followed by one or more "hinge domains", which move the antigen binding domain away from the surface of the effector cell to enable proper cell-to-cell contact, antigen binding, and activation. The CAR may contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be of natural, synthetic, semi-synthetic or recombinant origin. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular region of type 1 membrane proteins (e.g., CD8α, CD4, CD28, PD1, CD152, and CD7), which can be wild-type hinges from these molecules Zone, or can be changed. In another embodiment, the hinge domain comprises a PD1, CD152, or CD8α hinge region.

The "transmembrane domain" is a part of the CAR, which fuses the extracellular binding part and the intracellular signal transduction domain and anchors the CAR on the plasma membrane of immune effector cells. The TM domain can be derived from natural, synthetic, semi-synthetic or recombinant sources. The TM domain can be derived from the α, β or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 , CD137, CD152, CD154 and PD1. In one embodiment, the CAR of the present invention comprises a TM domain derived from CD8α or CD28.

In a particular embodiment, the CAR of the present invention includes an intracellular signaling domain. "Intracellular signaling domain" refers to the information involved in the effective anti-BCMACAR and human BCMA polypeptide binding to the inside of immune effector cells to trigger effector cell functions (such as activation, cytokine production, proliferation and cytotoxic activity, the Cytotoxic activity includes the release of cytotoxic factors to the target cells bound by the CAR or other cellular responses triggered by antigen binding to the extracellular CAR domain. The term "effector function" refers to the specialized function of immune effector cells. For example, the effector function of T cells may be cytolytic activity or assistance or activity including cytokine secretion. The term "intracellular signal transduction domain" refers to the part of a protein that transduces effector function signals and directs cells to perform specialized functions.

The CAR of the present invention contains one or more costimulatory signal transduction domains to enhance the efficacy, expansion and/or memory formation of T cells expressing CAR receptors. As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of CAR molecules, which provides a second signal required for the effective activation and function of T lymphocytes after binding to an antigen.

Exemplarily, the CAR of the present invention includes the antibody of the present invention. Further exemplarily, in addition to SEQ ID NO: 6, the CAR of the present invention can also be used with other antibodies of the present invention, such as Lead2, Lead3, Lead4, Lead5, Lead6, Lead7, Lead8, Lead9, Lead10, The CAR obtained by replacing Lead1 in SEQ ID NO: 6 with Lead11, Lead12, Lead13, Lead14, Lead15, Lead16, Lead17, Lead18, or Lead19.

protein

"Protein", "polypeptide fragment" and "polypeptide" can be used interchangeably, unless otherwise stated, and according to conventional meaning, that is, used as an amino acid sequence. Proteins are not limited to a specific length. For example, they can include full-length protein sequences or fragments of full-length proteins, and can include post-translational modifications of polypeptides (such as glycosylation, acetylation, phosphorylation, etc.) and include naturally-occurring and non- Naturally occurring other modifications known in the art.

In various embodiments, the CAR polypeptide or protein of the present invention includes a signal (or leader region) sequence at the N-terminus of the protein that can direct protein transfer during or after translation. A variety of well-known recombinant and/or synthetic techniques can be used to prepare polypeptides. The polypeptide of the present invention specifically includes the CAR of the present disclosure, or has a sequence that deletes, adds, and/or replaces one or more (for example, 1-20, 1-10, or 1-5) amino acids of the CAR disclosed herein .

Nucleic Acid

As used herein, the term "polynucleotide" refers to mRNA, RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotide of the present invention includes at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% of any reference sequence described herein. , 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 polynucleotides or variants, usually wherein the variant retains at least one biological activity of the reference sequence.

Illustratively, a polynucleotide sequence encoding the binding domain of BCMA -CD8αhinge-CD8 ^TM -4-1BB-CD3ζ fusion proteins of the present invention is any DNA sequence capable of encoding any of the fusion protein, preferably, the sequence SEQ ID NO: 5 or its complementary sequence. On the other hand, -4-1BB-CD3ζ a polynucleotide sequence of the fusion protein may be under stringent conditions to SEQ ID NO encoded by the present invention, the BCMA binding domain -CD8αhinge-CD8 ^TM: 5 polynucleotide sequence of Hybridize and encode the polynucleotide or its complementary sequence of the fusion protein;

The "stringent conditions" described herein may be any of low stringency conditions, medium stringency conditions, and high stringency conditions, and preferably high stringency conditions. Exemplarily, "low stringency conditions" may be 30°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, 52% formamide; “medium stringency conditions” may be 40°C, 5×SSC, 5× The conditions of Denhardt solution, 0.5% SDS, 52% formamide; "high stringency conditions" can be 50°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, 52% formamide. Those skilled in the art should understand that the higher the temperature, the more highly homologous polynucleotides can be obtained. In addition, those skilled in the art can select a comprehensive result formed by multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration that affect the stringency of hybridization to achieve the corresponding stringency.

In addition, the hybridizable polynucleotide can also be exemplified by the same search software as FASTA, BLAST, etc., when calculated with default parameters set by the system, it has a value of about 70% with that of the polynucleotide encoding SEQ ID NO: 5. % 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 99.9% or more identical polynucleotides.

The identity of the nucleotide sequence can be determined using the algorithm rule BLAST of Karlin and Altschul (Proc.Natl.Acad.Sci.USA87:2264-2268,1990; Proc.Natl.Acad.Sci.USA90:5873,1993) . Programs BLASTN and BLASTX based on BLAST algorithm rules have been developed (AltschulSF, etal: J Mol Biol 215:403, 1990). When using BLASTN to analyze the base sequence, if the parameters are score=100, wordlength=12; in addition, when using BLASTX to analyze the amino acid sequence, if the parameters are score=50, wordlength=3; when using the BLAST and Gapped BLAST programs, use each The system of the program can set default parameter values.

Any of various mature techniques known and available in the art can be used to prepare, manipulate, and/or express polynucleotides. In order to express the desired polypeptide or protein, the nucleotide sequence encoding the polypeptide can 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)), bacteriophages (e.g. λ Phage or M13 phage) and animal viruses. Examples of animal virus vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses ( For example SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; Lenti4/V5-DESTTM, pLenti6/V5-DESTTM, and Lenti4/V5-DESTTM for lentivirus-mediated gene transfer and expression in mammalian cells pLenti6.2/V5-GW/lacZ (Invitrogen). In a particular embodiment, the coding sequence of the chimeric protein disclosed herein can be linked to such an expression vector for expressing the chimeric protein in mammalian cells. The "control element" or "regulatory sequence" present in the expression vector is the untranslated region of the vector (such as the origin of replication, promoter, enhancer, translation initiation signal (SD sequence or Kozak sequence) intron, polyadenosine Acidified sequences, 5'and 3'untranslated regions), which interact with host cell proteins for transcription and translation. The strength and specificity of such elements or sequences can vary. Depending on the vector system and host used, any number of suitable transcription and translation elements or sequences can be used, including ubiquitous expression promoters and inducible promoters.

About 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. Generally, ADC works by using antibodies to target cancer cells and then release toxic substances (i.e. drugs) in the cells, thereby triggering cell death. Because ADC technology allows drugs to be accurately delivered to target cancer cells and released under specific conditions, while minimizing collateral damage to healthy cells, ADC technology increases the efficacy of therapeutic or targeted antibodies and reduces the risk of adverse reactions risk.

The basic structure of the antibody-drug conjugate can be "antibody-linker-pharmaceutical active molecule" or "antibody-pharmaceutical active molecule" (no linker). For conjugates with a linker, the linker allows the drug to exhibit an effect on the target cancer cell, for example after separation from the antibody (e.g., by enzyme-mediated hydrolysis) and after the drug reaches the target cell. The linker also plays a functional role by connecting the antibody and the drug. The efficacy and toxicity of the antibody-drug conjugate thus depend in part on the stability of the linker, and therefore, the linker plays an important role in drug safety.

The linkers of the antibody-drug conjugates can be roughly classified as non-cleavable or cleavable. Many non-cleavable linkers are attached to antibodies using thioethers, which contain the cysteine of the antibody. Pendant drugs usually cannot be separated from the antibody 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 cause the drug to separate from the conjugate before or after it reaches the target cell. The cleavable linker can be, for example, hydrolyzed by a lysosomal enzyme. The cleavable linker may comprise a disulfide bond, for example including the cysteine of an antibody. The disulfide linker that is allowed to dissociate via the thiol exchange reaction relies to some extent on the uptake of the antibody-drug conjugate into the target cell and exposure of the disulfide to the cytosol as a reducing environment. However, because various types of thiols (such as albumin and glutathione) are present in the blood, the drug may be separated from the antibody before reaching its target.

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

Existing documents have reported new methods for preparing antibody-drug conjugates, for example, see U.S. Patent Publication No. 2012/0308584. Further improvements are possible.

The CAR of the present invention or the antibody of the present invention can still be conjugated with pharmacologically active molecules on the side of the antigen binding domain, such as erlotinib, lymphokines, botulinum toxin, affinity ligands, radiolabels, immunomodulatory compounds, anticancer Agents, ribozymes, etc.

Carrier

In a particular embodiment, a retroviral vector (e.g., a lentiviral vector) encoding a CAR is used to transduce cells (e.g., immune effector cells, such as T cells). For example, a carrier encoding CAR is used to transduce immune effector cells, the carrier comprising a humanized anti-BCMA antibody or antigen-binding fragment that binds to a BCMA polypeptide, and the humanized anti-BCMA antibody or antigen-binding fragment has a transmembrane domain and a cell. The internal signaling domain allows these transduced cells to trigger CAR-mediated cytotoxicity.

Retroviruses are a common tool for gene delivery. In a particular embodiment, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CAR) to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "protovirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules that encode structural proteins and enzymes required to produce new viral particles.

Exemplary retroviruses suitable for specific 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 Gland Tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), murine stem cell virus (MSCV) and Rouss sarcoma virus (RSV)) and lentivirus.

As used herein, the term "lentivirus" refers to a group (or genus) containing many retroviruses. Exemplary lentiviruses include but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedivirus (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 a particular embodiment, lentiviruses are used to deliver CAR-containing polynucleotides to cells.

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 usually linked to the carrier nucleic acid molecule, for example inserted into the carrier 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's DNA. Useful vectors include, for example, plasmids (such as 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 is obvious to those skilled in the art that the term "viral vector" is widely used to refer to nucleic acid molecules (such as transfer plasmids) or viral particles that mediate nucleic acid transfer. Nucleic acid molecules include virus-derived ones that generally promote the transfer or integration of nucleic acid molecules into the cell genome. Nucleic acid elements in. Virus particles usually include various viral components, and sometimes host cell components other than nucleic acids.

The term viral vector may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements mainly derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof mainly derived from retroviruses. The term "lentivirus" refers to the genus of the Retroviridae family, which can effectively infect acyclic and post-mitotic cells; they can transmit a significant amount of genetic information into the host cell's DNA, so that they are the best gene delivery vector. One of the effective methods.

Therefore, in a preferred embodiment, the present invention relates to a method of transfecting cells with an expression vector encoding CAR. For example, in some embodiments, the vector contains additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, poly-A signals, and/or one or more introns. In a preferred embodiment, the CAR coding sequence is flanked by a transposon sequence so that a transposase is present to allow the coding sequence to be integrated into the genome of the transfected cell.

In some embodiments, the genetically transformed cell is further transfected with a transposase that promotes the 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 expressible RNA or protein, so that the transposase does not undergo long-term expression in the transgenic cells. For example, in some embodiments, the transposase is provided as mRNA (e.g., mRNA comprising a cap and a poly-A tail). Any transposase system can be used according to embodiments of the present invention. However, in some embodiments, the transposase is a salmon-type Tel-like transposase (SB). In some embodiments, the transposase is an engineered enzyme with increased enzyme activity. Some specific examples of transposase include, but are not limited to, SB 10, SB 11, or SB 100X transposase (see, for example, Mates et al., 2009, Nat Genet. 41(6): 753-61 or US9228180, which is incorporated by reference Incorporated into this article). For example, the method may include electroporating cells with mRNA encoding SB 10, SB 11, or SB 100X transposase.

Sequence variants:

The claimed nucleic acid, protein, antibody, antibody fragment and/or CAR sequence variants (such as those defined by percent sequence identity) are also included within the scope of the present invention, which maintain similar binding properties of the present invention. These variants display alternative sequences, but retain substantially the same binding properties such as target specificity, because the specific sequences provided are known to be functional analogs or functional analogs. Sequence identity relates to the percentage of identical nucleotides or amino acids when the sequence is aligned.

The term "sequence identity" as used herein refers to the degree of nucleotide-nucleotide or amino acid-amino acid based sequence identity in the comparison window. Therefore, the “percentage of sequence identity” can be calculated by the following: compare the two best aligned sequences on the comparison window, and determine the presence of the same nucleic acid base (such as A, T, C, G, I) on the two sequences Or the position of 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) To generate the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to get the percentage of sequence identity. It includes at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% sequence identity of nucleotides or polypeptides, usually wherein the polypeptide variant retains at least one biological activity of the reference polypeptide.

Those of ordinary skill in the art will understand that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides or proteins as described herein. Some of these polynucleotides have minimal homology or sequence identity with the nucleotide sequence of any natural gene. Nevertheless, the present invention specifically considers polynucleotides that vary due to differences in codon usage. Deletions, substitutions and other changes in the sequence that fall into the sequence identity are also included in the present invention.

Modifications of protein sequences that can occur by substitution are also included within the scope of the present invention. A substitution as defined herein is a modification of the amino acid sequence of a protein, whereby one or more amino acids are replaced by the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from that of the primary protein. Substitutions can be made, which preferably do not significantly change the function of the protein. Like addition, replacement can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly changing the function of the protein. This is especially true when the modification involves a "conservative" amino acid substitution that replaces one amino acid with another amino acid of similar properties. Such "conservative" amino acids can be natural or synthetic amino acids, which can be replaced due to size, charge, polarity, and conformation, but do not significantly affect the structure and function of the protein. Generally, many amino acids can be replaced by conservative amino acids without adversely affecting the function of the protein.

Generally speaking, 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 The amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu represent the conservative 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 if they belong to different groups.

Substitution variants remove at least one amino acid residue in the antibody molecule and insert a different residue in its position. For the occurrence of alternative mutagenesis, the most interesting locations include the hypervariable region, but the FR changes are also considered. If such a substitution results in a change in biological activity, then a larger number of changes can be introduced and the product screened.

Genetically modified gene cells and immune cells

In a particular embodiment, the present invention considers cells genetically modified to express the CAR of the present invention for the treatment of B cell related disorders. 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, corrects or modifies gene expression, or is used to express therapeutic polypeptides (such as CAR Or ADC). In a particular embodiment, the CAR of the present invention is introduced into immune effector cells and expressed in immune effector cells in order to redirect their specificity for the target antigen of interest, such as the BCMA polypeptide.

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

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

Exemplary immune effector cells used with the CAR of the present invention include T lymphocytes. The term "T cell" or "T lymphocyte" is recognized in the art 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 usually CD3- and CD56-positive non-major histocompatibility complex (MHC), which are restricted natural killer (NK)-like T lymphocytes. The T cell may be a T helper cell (Th), such as T helper cell 1 (Th1) or T helper cell 2 (Th2). The T cell may be a helper T cell or a cytotoxic T cell or any other T cell subpopulation. Other exemplary T cell populations suitable for particular embodiments include naive T cells and memory T cells.

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

As the skilled person understands, other cells can also be used as immune effector cells with the CAR described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include effector cell progenitor cells, where such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro.

The present invention provides a method for preparing immune effector cells expressing the CAR of the present invention. In one embodiment, the method includes transfection or transduction of immune effector cells isolated from the individual so that the immune effector cells express one or more CARs as described herein. In certain embodiments, immune effector cells are isolated from the individual and genetically modified without further manipulation in vitro. These cells can then be re-administered 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 CAR. In this regard, immune effector cells can be cultured before and/or after genetic modification (ie, transduction or transfection to express the CAR of the invention).

In a particular embodiment, prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, a source of cells is obtained from the subject. In a particular embodiment, the CAR-modified immune effector cells comprise T cells. T cells can be obtained from many sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from blood units 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 an individual are obtained by a blood aspiration technique. Blood components usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by blood aspiration collection can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing. The cells can be washed with PBS or another suitable solution that does not contain calcium, magnesium and most divalent cations. As understood by those of ordinary skill in the art, the washing step can be performed by methods known to those skilled in the art, for example, by using a semi-automatic flow-through centrifuge (such as Cobe2991 cell processor, Baxter CytoMate, etc.). After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, undesired components of the blood sample can be removed from the cells directly resuspended in the culture medium.

In certain embodiments, T cells are separated from peripheral blood mononuclear cells (PBMC) by lysing red blood cells and depleting monocytes (eg, by PERCOLLTM gradient centrifugation). Specific T cell subpopulations can be further separated by positive or negative selection techniques. One method that can be used is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against the cell surface present on the negatively selected cells. landmark.

The method of the present invention can be used to directly genetically modify PBMC to express CAR. In some embodiments, after the isolation of PBMC, T lymphocytes are further separated, and in some embodiments, cytotoxic and helper T lymphocytes can be sorted as initial, cytotoxic and helper T lymphocytes before or after genetic modification and/or amplification. Memory and effector T cell subsets. CD8+ cells can be obtained by using standard methods. In some embodiments, 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 (eg T cells) can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) before genetic modification. In a particular embodiment, immune effector cells (such as T cells) are genetically modified with the chimeric antigen receptor of the present invention (for example, transduced with a viral vector containing a nucleic acid encoding a CAR), and then activated and amplified in vitro . In various embodiments, for example, the methods described in U.S. Patent Nos. 5,858,358; 6,905,681; 7,067,318; 7,232,566; 5,883,223; T cells are activated and expanded before or after expression of CAR.

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

In one embodiment, the present invention provides a method for storing immune effector cells expressing genetically modified mouse, human, or humanized CAR protein targeting BCMA protein, which includes cryopreserving the immune effector cells so that the cells are in Stay alive when thawed. A part of the immune effector cells expressing CAR protein can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with B-cell-related disorders. When needed, cryopreserved transformed immune effector cells can be thawed, grown and expanded to obtain more such cells.

Composition and formulation

The composition of the present invention may comprise one or more polypeptides, polynucleotides, vectors comprising the polynucleotides, genetically modified immune effector cells, etc. as considered herein. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution, which is administered to cells or animals alone or in combination with one or more other treatment modalities. It should also be understood that, if desired, the composition of the present invention can 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. There are virtually no restrictions on 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 being suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications and with reasonable Those compounds, materials, compositions and/or dosage forms that have a commensurate benefit/risk ratio.

As used herein, "pharmaceutically acceptable carriers, diluents or excipients" include but are not limited to any adjuvants and carriers that have been approved by the US Food and Drug Administration or China Food and Drug Administration for use in humans or livestock , Excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents , Surfactant or emulsifier. 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 derivatives thereof, such as sodium carboxymethyl cellulose, ethyl cellulose And cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, wax, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, Olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, For example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and any other compatible substances used in pharmaceutical preparations.

In a particular embodiment, the composition of the present invention contains a certain amount of the immune effector cells expressing the CAR of the present invention. As used herein, the term "amount" refers to an "effective amount" of genetically modified therapeutic cells (eg, T cells) that achieve beneficial or desired preventive or therapeutic results (including clinical results).

"Prophylactically effective amount" refers to the amount of genetically modified therapeutic cells effective to achieve the desired preventive result. Usually, but not necessary, because the preventive dose is used in the subject before or in the early stage of the disease, the preventive effective amount is less than the therapeutically effective amount. The term prevention does not necessarily mean the complete prohibition or prevention of specific medical conditions. The term prevention also refers to reducing the risk of a certain medical condition or worsening symptoms.

The "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on various factors such as disease state, age, sex, and individual body weight, as well as the ability of stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effect exceeds 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 (eg, a patient). When the therapeutic amount is indicated, the doctor can determine the precise amount of the composition of the present invention to be administered in consideration of individual differences in age, weight, tumor size, degree of infection or metastasis, and patient (subject) condition. Can usually provide that a pharmaceutical composition comprising the T cells described herein may be 10 ² to 10 ¹⁰ cells / kg body weight, dose is preferably ¹⁰⁵ to ¹⁰⁶ cells / kg body weight (including all those within the scope of Integer value) to apply. 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 generally 1 L or less in volume, and can be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically greater than 10 ⁶ cells / ml, usually greater than 10 ⁷ cells / ml, usually ¹⁰⁸ cells / ml or more. The clinically relevant number of immune cells can be allocated to multiple infusions, and the multiple infusions accumulate equal to or exceed 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. In some embodiments of the invention, especially because all infused cells will be redirected to a specific target antigen, a lower number of cells can be administered. The CAR-expressing cell composition can be administered multiple times at doses within these ranges. For patients receiving treatment, the cells can be allogeneic, syngeneic, allogeneic, or autologous.

Generally, a composition comprising cells activated and expanded as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the composition comprising the CAR-modified T cell of the present invention is used to treat B cell malignancies. The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with carriers, diluents, excipients, and/or other components (such as IL-2) or other cytokines or cell populations. In a particular embodiment, the pharmaceutical composition of the present invention contains a certain amount of genetically modified T cells, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

The pharmaceutical composition of the present invention comprising a population of immune effector cells (such as T cells) expressing CAR may include: buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran Sugar, mannitol; protein; polypeptides or amino acids (for example, glycine); antioxidants; chelating agents (for example, EDTA) or glutathione; adjuvants, such as aluminum hydroxide; and preservatives. The composition of the invention is preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

The liquid pharmaceutical composition, whether in a solution, suspension or other similar form, may include one or more of the following: sterile diluent (for example, water for injection), saline solution (preferably normal saline, Ringer's solution, isotonic Sodium chloride), fixed oils (such as synthetic monoglycerides or diglycerides that can be used as solvents or suspension media), polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or p-hydroxybenzoic acid Methyl ester; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and buffers, such as acetate, citrate, or phosphate, and agents that regulate osmotic pressure, such as chlorination Sodium or glucose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

In a particular embodiment, the composition of the present invention contains an effective amount of immune effector cells expressing CAR alone, or in combination with one or more therapeutic agents. Therefore, the CAR-expressing immune effector cell composition can be administered alone or in combination with other known cancer treatments such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy Wait. The composition can also be administered in combination with antibiotics. 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, radiotherapeutics, therapeutic antibodies or other active and auxiliary agents.

treatment method

The genetically modified immune effector cells of the present invention provide an improved method of adoptive immunotherapy for the treatment of B cell-related disorders, including but not limited to immunomodulatory disorders and hematological malignancies.

In a particular embodiment, the composition comprising immune effector cells containing the CAR of the present invention is used to treat disorders related to abnormal B cell activity, also referred to as "medical disorders related to the presence of pathogenic B cells".

As used herein, "medical conditions associated with the presence of pathogenic B cells" or "B-cell malignancies" refer to medical conditions formed in B cells, such as cancer. In a particular embodiment, the composition comprising CAR-modified T cells of the present invention is used to treat hematological malignancies, including but not limited to B-cell malignancies, such as multiple myeloma (MM), acute myeloid leukemia And non-Hodgkin's lymphoma (NHL).

In another aspect of the present invention, the CAR and CAR-T according to the present invention as described herein are provided for the treatment of B cell-mediated or plasma cell-mediated diseases or antibody-mediated diseases selected from the group consisting of Diseases or conditions of: multiple myeloma (MM), chronic lymphocytic leukemia (CLL), nonsecretory multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy of unknown significance (MGUS), isolated Plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), plasma cell leukemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome /Sclerosing myeloma, type I and type II cryoglobulinemia, light chain deposition disease, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders, And epidermolysis bullosa; or any non-Hodgkin’s lymphoma B-cell leukemia or Hodgkin’s lymphoma (HL) with BCMA expression or any disease in which the patient produces neutralizing antibodies against recombinant protein replacement therapy, where The method includes administering to the patient a therapeutically effective amount of a CAR or CAR-T as described herein.

Multiple myeloma is a B-cell malignant tumor of mature plasma cell morphology, which is characterized by tumor transformation of a single clone of these types of cells. These plasma cells proliferate in the BM and may invade adjacent bones, sometimes invading the blood. Variant forms of multiple myeloma include dominant multiple myeloma (overt multiple myeloma), smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasma Cell tumor and extramedullary plasmacytoma.

Non-Hodgkin's lymphoma includes a large group of lymphocytic carcinomas (white blood cells). Non-Hodgkin's lymphoma can occur at any age and is usually marked by larger lymph nodes, fever, and weight loss than normal. Non-Hodgkin's lymphoma can also exist in extranodal sites, such as the central nervous system, mucosal tissues, including lungs, intestines, colons, and internal organs. There are many different types of non-Hodgkin's lymphoma. For example, non-Hodgkin's lymphoma can be divided into aggressive (rapid growth) and indolent (chronic growth) types. Although non-Hodgkin's lymphoma can be derived from B cells and T cells, as used herein, the terms "non-Hodgkin's lymphoma" and "B-cell non-Hodgkin's 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, immunogen Cellular large cell lymphoma, precursor B lymphoblastic lymphoma, and mantle cell lymphoma. Lymphoma that occurs after bone marrow or stem cell transplantation is usually B-cell non-Hodgkin's lymphoma.

As used herein, the terms "individual" and "subject" are generally used interchangeably, and refer to a disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein Symptoms of any animal. In a preferred embodiment, subjects include those exhibiting symptoms of diseases, disorders, or conditions of the hematopoietic system (such as B cell malignancies) that can be treated with gene therapy vectors, cell-based therapeutics and methods disclosed elsewhere herein Any animal. 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 who have B-cell malignancies, have been diagnosed with B-cell malignancies, or are at risk of having B-cell malignancies.

As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of the disease or pathological condition, and may include even minimal reduction of one or more measurable markers of the disease or condition being treated . The treatment may optionally involve the reduction or improvement of the symptoms of the disease or disorder, or the delay in the progression of the disease or disorder. "Treatment" does not necessarily mean the complete eradication or cure of a disease or condition or its related symptoms.

As used herein, "prevention" means a method of preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevent" and similar words also include reducing the intensity, impact, symptoms, and/or burden of the disease or condition before the onset or recurrence of the disease or condition.

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

The administration of the composition of the present invention can be carried out in any convenient way, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation or transplantation. In a preferred embodiment, the composition is administered parenterally. As used herein, the phrase "parenteral administration" refers to administration methods other than enteral and topical administration, usually by injection, including but not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, or orbital , Intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine and intrasternal injections and infusions. In one embodiment, the composition of the present invention is administered to the subject by direct injection into the tumor, lymph node, or infection site.

English names appearing in this article are not case-sensitive; BCMA CAR-T means CAR-T cells that can express BCMA specific binding domain; CD8 ^TM means transmembrane domain.

Compared with NK cells (including CAR-NK), the present invention provides a more detailed description of T cells (including CAR-T cells), but in general, these descriptions of T cells are also applicable to NK cells. Here, the description of "T cell" or its synonyms in the description of T cells is replaced with "NK cell" or its synonyms. This is very necessary from the perspective of narrative conciseness. If it is judged that the replacement is inappropriate in some cases according to the prior art, the replacement is not performed.

The terms and expressions used herein are used as descriptive rather than restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or parts thereof, but various modifications should be recognized It is possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can adopt modifications and variations of the concepts disclosed herein, and such modifications and variations are deemed to be as defined by the appended claims. Defined within the scope of the invention.

Description of the drawings

Figure 1 The test results of the affinity between the Lead 1 VHH-FC fusion protein (anti-BCMA antibody, VHH-IgG1Fc) and the human BCMA antigen recombinant protein using the ELISA method.

Figure 2 shows the results of the detection of the affinity between the Lead 1 VHH-FC fusion protein and the BCMA overexpression cell line RPMI8226 using flow cytometry.

Fig. 3 is a schematic structural diagram of CAR in the BCMA-CART structure provided by an embodiment of the present invention, in which the CD8a hinge-transmembrane (TM) domain and the 4-1BB co-stimulatory domain are fused with the CD3ζ signaling region.

Figure 4 CAR expression results in CAR-T cells detected by flow cytometry.

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

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

Figure 7 shows the in vitro killing effect of BCMA-PE24 recombinant immunotoxins (RITs) on multiple myeloma cell lines.

In order to illustrate the present invention more clearly, a detailed description will now be given in conjunction with the following embodiments, but these embodiments are merely exemplary descriptions of the present invention and cannot be construed as limiting the present application.

Example 1: Alpaca (Alpaca) immunization and antiserum titer detection

1. Immunization method

Multi-point injection of human BCMA antigen recombinant protein into multiple masses subcutaneously and intramuscularly on the back of the neck, follow up the absorption of subcutaneous injection masses to confirm the correct immunization.

2. Immune cycle

(1) First immunization: Use 0.5 mg of antigen and Freund's complete adjuvant to mix 1:1, emulsify and inject, with a volume of 1 ml per alpaca; (2) Second immunization: 3 weeks after the first immunization, use 0.25 mg of antigen Mix with Freund's incomplete adjuvant 1:1, emulsify and inject, with an injection volume of 1ml per alpaca; (3) Third immunization: 3 weeks after the second immunization, use 0.25mg antigen and Freund's incomplete adjuvant Mix 1:1, emulsify and inject, with injection volume of 1ml per alpaca; (4) Fourth immunization: 3 weeks after three immunizations, mix 1:1 with 0.25mg antigen and Freund’s incomplete adjuvant, after emulsification Injection, the injection volume is 1ml per alpaca.

3. Serum processing and antiserum titer detection: one week after the fourth immunization, 50ml of peripheral blood was collected, and serum and lymphocytes were separated. The antigen was coated in an ELISA 96-well plate, and the antibody titer in the serum was measured by the ELISA method. The titer of antiserum is shown in the table below.

Example 2: Construction and screening of phage display immune antibody library.

Because the titer of the fourth serum was >1:32000, it indicated that there were high-affinity antibodies against human BCMA in the serum. The phage display immune antibody library was further constructed, and a positive monoclonal antibody against human BCMA Nanobody was obtained through biological screening.

1. Collect the blood of the alpaca after the fourth immunization, separate lymphocyte PBMC; take 2×10 ⁷ PBMC, use RNA extraction kit to extract total RNA; take appropriate amount of RNA (such as 3-5ug), reverse by RT-PCR Transcription kit to obtain cDNA.

2. Obtain IgG2 and IgG3 heavy chain variable region sequences step by step (Nanobody heavy chain variable region VHH) by nested PCR or obtain IgG1, IgG2 and IgG3 heavy chain variable region sequences (routine The heavy chain variable region VH of antibody IgG1 and the heavy chain variable region VHH of Nanobody IgG2, IgG3).

3. Insert the heavy chain variable region sequence into the linearized phagemid vector pShort treated by restriction enzyme digestion by homologous recombination or restriction enzyme ligation to obtain the recombinant vector; after purification and recovery, transform super competent SS320 cells (Contains helper phage M13K07); the transformed bacterial solution is resuspended in SOC medium and activated for 1 hour; a small amount of bacterial solution is diluted by a 10-fold ratio, and the appropriate dilution titer is selected, which is used in LB/tet ₁₀ and LB/Carb _{Spread the 50} culture plate and place it in a 37℃ biochemical incubator overnight. The next day it will be used for the calculation of storage capacity; the remaining bacterial liquid will be transferred to a large volume of 2YT/Carb ₅₀ /Kan ₂₅ liquid medium and placed on a 37℃ shaker , Culture overnight, harvest the supernatant the next day, add 1/4 times the volume of PEG/NaCl solution, after precipitation of the phage, take an appropriate amount of PBT solution to resuspend and dilute to the required concentration to obtain the phage display immune antibody library (-80℃ Save it for later).

4. Count the _{number of clones on the LB/Carb 50} plate, and calculate the storage capacity as: library 1 (nested PCR): 2.25×10 ⁸ ; library 2 (one-step method): 1.63×10 ⁸ . Twenty single clones were randomly picked from the plate and sequenced, all of which were clones with correct insertion of VHH.

Example 3: Screening of antibody library

1. Add 10μg/mL human BCMA antigen recombinant protein to a 96-well plate and coat overnight at 4°C; grow NEB5αF' on a 2YT/Tet ₁₀ plate streak, and cultivate overnight at 37°C in an incubator;

2. On the second day, _{pick NEB5αF' monoclonal from the overnight 2YT/Tet 10} plate, add it to 3ml 2YT/Tet ₁₀ liquid medium, and grow the bacteria at 37°C to OD ₆₀₀ = 0.8;

3. At the same time, remove the antigen supernatant of the 96-well plate, add 200μL of 1% BSA to each well to block, and add 200μL of 1% BSA to the blank well as a negative control well, and place it on a 3D rotary shaker at room temperature for 2 hours; After that, remove the supernatants of the protein wells and control wells, wash with 200uL PT, add 100uL of phage antibody library to each, place in a 3D rotary shaker at room temperature for 2 hours; remove the supernatants of the protein wells and control wells, and use Wash with 200uL PT; add 100μL 100mM HCl to the well and leave it at room temperature for 5 minutes; aspirate the supernatant, add it to a 1.5ml centrifuge tube, and neutralize with 1M Tris-HCl.

4. Add the mixture obtained in step 3 to a centrifuge tube containing 1 mL of NEB5αF' bacteria, incubate at 37°C on a shaker for 1 hour; take 20 μL of the culture solution in the centrifuge tube and dilute it at an appropriate multiple and place it on an LB/Carb ₅₀ culture plate Coat the plate and place it in a 37°C biochemical incubator overnight. The next day is used for the calculation of titer and enrichment degree; 1μL helper phage M13K07 (final concentration of 10 ¹⁰ /mL) is added to the remaining culture solution, and the shaker is 37°C , Incubate for 1 hour; transfer the above-mentioned culture medium to 35mL 2YT/Carb ₅₀ /Kan ₂₅ culture medium, place in a shaker, and incubate at 37°C overnight, and collect phage to form an antibody library for each round.

5. Repeat the above operation for 3-5 rounds until phage enrichment occurs. If the number of colonies in the antigen-binding well on the LB/Carb ₅₀ culture plate is more than 10 times that of the negative control well, the enrichment is considered successful. In this experiment, after the third round of screening, the number of colonies in the antigen-binding wells was 100 times that of the negative control wells, indicating that the enrichment was successful, then enter Phage-ELISA to select high-affinity positive clones.

Example 4: Phage-ELISA identification of positive clones and sequencing

1. In a 96 deep well plate, add 400μL of 2YT/Carb ₅₀ /Kan ₂₅ /M13K07 medium to each well; pick a single clone from the enriched LB/Carb ₅₀ culture plate, transfer it to the 96 deep well plate, and place Place it on a shaker at 200 rpm and 37°C overnight, and centrifuge the next day. The supernatant is the phage produced by each single clone.

2. Dilute the recombinant human BCMA antigen protein to 1μg/mL at the same time, add 50μL/well to an ELISA 96-well plate, and place in a refrigerator at 4°C overnight;

3. On the second day, turn the ELISA plate upside down to remove the supernatant, and then add 100 μL 1% BSA to each well to block; add 100 μL 1% BSA to the blank well as a negative control well; incubate at room temperature for 1 hour. After the blocking is completed, the antigen wells and negative control wells are washed with PT solution, add 50μL of 96 deep-well plate supernatant and incubate at room temperature for 2 hours; add one antigen well and one negative for each supernatant obtained from each monoclonal Control hole.

4. After the binding is completed, wash the ELISA plate with PT solution, add 50μL of HRP-M13 antibody, and incubate at room temperature for 1 hour; after washing with PT solution and PBS solution, add 50μL of TMB, incubate at room temperature for 5 minutes, and then add 50μL of 1M phosphoric acid to terminate Reaction; Measure the absorbance value at 450nm with a microplate reader. The results of Phage-ELISA are shown in the table below.

Note: When the OD value>3 exceeds the reading range of the microplate reader, the microplate reader will automatically set the reading to "overflow".

The monoclonal corresponding to the antigen well OD value> 0.5 (including overflow wells) and the negative control well OD value <0.2 are identified as positive clones with higher affinity, and the monoclonal DNA sequencing is performed; the corresponding sequences are shown in the table below.

The monoclonal corresponding to the antigen well OD value> 0.5 (including overflow wells) and the negative control well OD value <0.2 are identified as positive clones with higher affinity, and the monoclonal DNA sequencing is performed; the corresponding sequences are shown in the table below (CDR regions) The definition of using the North method, Lead1-Lead3 are marked with italics and/or underline; the CDRs of Lead4-Lead19 are shown in Table 1).

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

Eukaryotic expression was performed on the selected Lead 1 sequence, and its affinity with antigen recombinant protein and antigen overexpression cell line was studied.

1. Amplify the VHH fragment of Lead 1 by PCR, insert the fragment into a partial fragment of human IgG1 (hinge+CH2+CH3, and the amino acid sequence of the fusion protein after fusion with VHH, such as SEQ ID) using homologous recombination or restriction enzyme ligation. As described in NO: 4, the nucleotide sequence is as described in SEQ ID NO: 3) in the eukaryotic expression vector pFcIG; electrotransformed into E. coli trans5α host bacteria, screened by ampicillin, and obtained the correct recombination by sequencing the monoclonal Plasmids; then expand the culture of host bacteria containing recombinant plasmids, and use endotoxin-removing kits to obtain sterile endotoxin-free plasmids;

2. Cultivate 293F cells with serum-free medium; use polyplus suspension cell transfection reagent to transfer the recombinant expression plasmid into 293F cells for expression. Feed was added 24 and 72 hours after transfection, and the supernatant was collected on the 5th day for purification. The calculation shows that: the yield of Lead 1's VHH-FC fusion protein is 95 mg/L; SDS-PAGE electrophoresis identified the lead 1 VHH-FC band size is normal, and the purity is >95%.

3. Using the ELISA method, determine the affinity between the Lead 1 VHH-FC fusion protein and the human BCMA antigen recombinant protein: 1) Add the human BCMA antigen recombinant protein to the ELISA 96-well plate and coat it overnight at 4°C; 2) Put the VHH- The Fc fusion protein was diluted to different concentrations and reacted with the antigen by ELISA, and the absorbance value at 450nm was measured with a microplate reader; the results are shown in Figure 1. The experimental results show that the BCMA Lead 1 VHH-FC fusion protein can bind to human BCMA antigen recombinant protein, and its EC ₅₀ =4.2nM.

4. Using a flow cytometer, test the affinity of the Lead 1 VHH-FC fusion protein and the BCMA overexpression cell line RPMI8226: 1) Take 0.3×10 ⁶ RPMI8226 cells, wash them twice with PBS, and then resuspend them in 100uL PBS; 2) Incubate with 1ug/ml Lead 1 VHH-FC fusion protein for 1 hour; 3) After washing the cells with PBS 3 times, resuspend the cells with 100uL PBS, add 0.2ug/ml FITC-labeled Anti-human FC antibody, and incubate 1 Hours; 4) After washing the cells with PBS 3 times, resuspend the cells with 300 uL PBS, and use a flow cytometer to detect the fluorescence. The result is shown in Figure 2. The experimental results show that the BCMA Lead 1 VHH-FC fusion protein can bind to the RPMI8226 cell line. Compared with the blank cell line, the binding curve of BCMA Lead 1 VHH-FC shifts to the right.

Example 6: Study on 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 1 VHH sequence to study the killing effect of BCMA Lead 1 VHH CAR-T on multiple myeloma (MM) cells .

1. Preparation of lentiviral vectors: 1) Gene synthesis BCMA lead 1VHH-CD8TM-4-1BB-CD3ζ fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 6, and the DNA sequence is shown in SEQ ID NO: 5. The schematic diagram of its structure is shown in Figure 3); 2) Use homologous recombination or restriction enzyme digestion to insert the fragment into the PWPXLD-kana vector; transform the recombinant vector into E. coli strain Stbl3, screen by kanamycin, and sequence the monoclonal Obtain the correct recombinant plasmid; then expand the culture of the host bacteria containing the recombinant plasmid, and use the endotoxin-removing kit to obtain a sterile endotoxin-free plasmid, that is, the PWPXLD plasmid vector containing the CAR gene fragment; 3) At the same time, the lentivirus packaging auxiliary plasmid psPax2 And PMD2.0G were transformed into DH5α, ampicillin screened, and plasmids were extracted.

2, the CAR expressing lentivirus prepared (Lenti-CAR): 1) 3x10 ⁶ 293T cells were seeded in petri dishes; 2) after 24 hours, the viral plasmid (CAR-PWPXLD: 9μg, psPax2 : 9μg, and PMD2. 0G:4.5μg), add 0.45mL of sterile water and 50μL of 2.5M CaCl ₂ solution, then add 500μL of 2×BBS (50mM BES, 280mM NaCl, 1.5mM Na ₂ HPO4) dropwise, keep the solution vortex mixed ; Place at room temperature for 30 minutes; then add the mixture to the 293T medium and mix gently; 3) After 18 hours, change the medium to DMEM medium containing 2% FBS; 4) Collect the medium after 48 hours The supernatant was centrifuged to remove cell debris, and the supernatant was filtered with a 0.45 μm filter; then one-third volume of TAKARA Lentivirus Concentration Reagent (Lenti-X ^TM Concentrator, product number 631231) was added, and the mixture was mixed and allowed to stand overnight at 4°C. Centrifuge at 1500g for 45 minutes at 4°C, and resuspend the pellet in PBS to obtain the virus solution, which is aliquoted and stored at -80°C.

3. Preparation of BCMA VHH CAR-T cells: 1) On Day 0, collect peripheral blood, separate lymphocytes, PBMC and plasma; sort out CD3+ T cells from PBMC; adjust the cell suspension to a concentration of 1×10 ⁶ cells /ml, culture in a 12-well plate; add dynabeads magnetic beads (Thermo Fisher) for stimulation; 2) On Day 0, add fibronectin solution (5μg/cm ² ) to a 12-well plate and coat overnight at 4°C 3) On Day 1, discard the fibronectin solution in the 12-well plate, and block it with 2% BSA for 30 minutes; remove the blocking solution, ^{add the virus solution at 750μL/4.5cm 2} and place it in the 37°C incubator, and let it stand for 4-6 hours; after stimulation of T cells were collected, taking ¹⁰⁶ added to each well, placed in an incubator at 37 ℃, 5% CO ₂ culture; 4) at Day 3, regulatory T cells to a concentration of ⁵ × 10 5 / ml , Replace all the fresh medium; 5) On Day 5, adjust the T cell concentration to 5×10 ⁵ /ml, and use flow cytometry to detect the CAR expression of CAR-T cells (Figure 4).

4. LDH experiment detects the killing effect of CAR-T on RPMI 8226 cells in vitro: 1) Configure RPMI 8226 cells with high BCMA expression or K562 and Raji cells with low BCMA expression to 2×10 ⁵ /mL, Day5 CAR-T The cell configuration is 4×10 ⁵ /mL; set the following groups: blank group (200 μL medium), spontaneous group A (100 μL CAR T cells + 100 μL medium), spontaneous group B (100 μL target cells + 100 μL medium), Experimental group (100μL CAR T cells+100μL target cells); another set 200μL medium as the volume correction group, target cells 100μL+100μL medium as the maximum release group; placed in a 96-well U-shaped plate and cultured at 37℃ Incubate with 5% CO ₂ for 24 hours; 2) On the second day, add 20 microliters of LDH maximum release reagent to each well in the maximum release group and volume correction group, place in a 37°C incubator, and _{incubate with 5% CO 2} for 45 3) At the same time, take an ELISA test 96-well plate, add 50 positive groups/well of LDH test reagent in the dark; take out the 96-well U-shaped plate, mix wells, centrifuge at 500g for 3 minutes, take 50μL and add phase Corresponding ELISA test 96-well plate; put the ELISA test 96-well plate into the microplate reader, shake and mix for 30 minutes; add 50μL stop reagent, detect the wavelength of 492nm; calculate the killing rate, the 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. It can be clearly seen from Fig. 5 that the CAR-T of the present invention can treat medulla Leukemia has a significant curative effect or inhibitory effect, and has no significant curative effect or inhibitory effect on tumor cells with low BCMA expression (K562 and Raji).

5. Animal experiments to detect the killing effect of CAR-T on RPMI 8226 cells in vivo: Purchase 4-6 weeks old B-NDG severe immunodeficiency mice, adapt to the breeding environment for one week and inoculate 2×10 ^{6 subcutaneously on the forelimb flanks.} RPMI 8226 multiple myeloma tumor cells. Observe and measure the tumor size. After 3-4 weeks, when the tumor grows to 80-100 mm ³ , the mice are randomly divided into 6 groups, and CAR-T cells cultured in vitro for 7-8 days are injected into the tail vein for two consecutive days. The dose of each dose was 2×10 ⁶ /head. Continue to observe and measure the tumor size, and the results are shown in Figure 6, where the tumor size calculation method = (1/2) * long diameter * short diameter square. It can be seen from Figure 6 that the CAR-T prepared in the examples of this application has a specific and significant curative effect or inhibitory effect on human multiple myeloma and acute myeloid leukemia.

Example 7: In vitro killing effect of BCMA-PE24 recombinant immunotoxins (RITs) on RPMI 8226 cells

Recombinant immunotoxins (RITs) are targeted biopharmaceuticals formed by linking highly specific monoclonal antibodies with biotoxin molecules with powerful killing effects. Pseudomonas extoxin (PE) is one of the commonly used biological toxins, among which PE24 only retains the third domain (Domain III) of PE toxin, so it greatly retains the toxicity of PE while reducing the drug Immunogenicity. Previous studies have shown that scFv-PE24RITs prepared using BCMA single-domain antibodies linked to PE24 have strong killing ability on multiple myeloma cells (see: https://academic.oup.com/abt/article/1/1/ 19/5076366 ). The Lead 1 VHH sequence obtained by screening was fused and expressed with PE24 to study the in vitro killing effect of the recombinant immunotoxin on RPMI8226 cells.

1. Artificially synthesize the DNA sequence of BCMA lead1 VHH-PE24 (SEQ ID NO: 13, the corresponding amino acid sequence is: SEQ ID NO: 14), and then use homologous recombination or enzyme digestion and ligation methods to insert the fragment into pet25b( +) In the prokaryotic expression vector, and introduce his tag at the C-terminus.

2. Transfer the recombinantly constructed plasmid into the rosetta2 host bacteria, and _{culture it overnight at 37°C in LB/Amp 100} /Chl ₁₅ medium; the next day, transfer the bacteria cultured overnight to LB/Amp _{100 at a ratio of 1:100} Chl ₁₅ medium, cultured at 37°C for 2-3 hours, when OD ₆₀₀ =0.6-1, 1mm IPTG was added, and expression was induced overnight at 25°C.

3. Centrifuge, collect the bacteria, and wash with PBS; resuspend the bacteria in 10ml PBS, sonicate and collect the supernatant; the protein is obtained by Ni column adsorption and elution. SDS-PAGE electrophoresis shows that the obtained protein has a single band and the position is correct.

4. According to 1×10 ⁴ /well, inoculate RPMI 8226 cells into a 96-well plate, and add BCMA VHH-PE24 biotoxin protein at the concentration of 100ng/ml, 30ng/ml, 10ng/ml, 3ng/ml, 1ng /ml, 0.3ng/ml, 0.1ng/ml, and 0ng/ml (blank); 96 hours later, CCK8 detection kit was used to detect the killing effect, and the results are shown in Figure 7. The results show that the BCMA lead1 VHH-PE24 prepared in the examples of this application has a specific and significant curative effect or inhibitory effect on human multiple myeloma.

The foregoing descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. within.

Claims

An anti-BCMA antibody or antigen-binding fragment thereof that can bind to human BCMA polypeptide, which can competitively bind to one of SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 19, or SEQ ID NO: 23-38 The human BCMA epitope bound by the single heavy chain variable region (VHH) shown, preferably, includes SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 19, or SEQ ID NO: 23-38 The heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 of a single heavy chain variable region (VHH) shown in one of the sequences or humanized variant sequences thereof or by adding, deleting or replacing 1, 2, or 3 Variants of HCDR1, HCDR2 and HCDR3 derived from amino acids, for example, said HCDR1, HCDR2 and HCDR3 comprise HCDR1, HCDR2 and HCDR3 shown in Table 1 of the specification, preferably said HCDR1, HCDR2 and HCDR3 comprise: SEQ ID NO: one, two or more CDRs shown in any one of 10-12;

SEQ ID NO: one, two or more CDRs shown in any one of 16-18; or

SEQ ID NO: One, two or more CDRs shown in any one of 20-22.
The antibody or antigen-binding fragment of claim 1, which comprises the heavy chain shown in one of SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 19, or SEQ ID NO: 23-38. Variable region sequence or its humanized sequence.
The antibody or antigen-binding fragment of claim 1 or 2, which is selected from camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' 2 fragment, F(ab)' 3 fragment, Fv, scFv , Double-scFv, (scFv) 2 , mini-antibody, double-chain antibody, tri-chain antibody, four-chain antibody, disulfide bond stabilized Fv protein and single domain antibody (sdAb, Nanobody), bispecific antibody or three Specific antibodies.
A fusion protein comprising the antibody or antigen-binding fragment of any one of claims 1-3.
The fusion protein of claim 4, which further comprises a tag sequence (such as Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an additional epitope (such as for human BCMA Other epitopes) or additional antibody active fragments (such as antibodies or antibody active fragments directed against other epitopes of human BCMA or the same epitope, or ligands capable of binding to human BCMA), preferably, the fusion protein has SEQ ID NO: the sequence shown in 4.
An antibody-drug conjugate comprising the antibody or antigen-binding fragment of any one of claims 1-3, preferably, the conjugate is conjugated with pseudomonas extoxin (PE) , More preferably PE24, and most preferably, the conjugate has the sequence of SEQ ID NO: 14.
The antibody-drug conjugate of claim 6, wherein the drug is selected from the group consisting of radiolabels, 32 P, 35 S, fluorescent dyes, electron densification reagents, enzymes, biotin, streptavidin , Digoxigenin, hapten, immunogenic protein, nucleic acid molecule having a sequence complementary to the target, or any combination of the foregoing; or immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents and Antiparasitic agent, or any combination of the foregoing.
A chimeric antigen receptor (CAR) comprising: (1) comprising the antibody or antigen-binding fragment according to any one of claims 1-3, and comprising the fusion according to any one of claims 4-5 A protein or an extracellular antigen binding domain comprising the antibody-drug conjugate of any one of claims 6-7; optionally, the chimeric antigen receptor further comprises (2) a transmembrane domain; and ( 3) Intracellular signal transduction domain.
The CAR according to claim 8, wherein the transmembrane domain is derived from α, β, or ζ chains selected from the group consisting of T cell receptors, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33 , CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154 and ICOS one or more transmembrane domains.
The CAR according to claim 8, wherein the intracellular signal transduction domain comprises a costimulatory signal transduction domain and is derived from: CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD7, CD22, CD27, CD28, CD30, One or more of CD40, CD66d, CD79a, CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C and B7-H3.
The CAR of any one of claims 8-10, further comprising a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.
The CAR of claim 11, wherein the hinge domain is derived from CD8α.
The CAR according to any one of claims 8-10, wherein the antibody or antigen-binding fragment is conjugated with a drug as defined in claim 6 or 7, or the antibody or antigen-binding fragment is further directly or indirectly ( For example, a peptide linker is used to connect a ligand or antibody capable of binding to human BCMA (for example, a single domain antibody against human BCMA).
The CAR according to claim 8, which has the sequence shown in SEQ ID NO: 6.
A polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1, the fusion protein of claim 4, or the CAR of claim 8,

Preferably, the polynucleotide encoding the antibody or antigen-binding fragment of claim 1 is shown in SEQ ID NO: 1 or its degenerate sequence or complementary sequence;

The polynucleotide encoding the fusion protein of claim 4 is shown in SEQ ID NO: 3 or its degenerate sequence or complementary sequence; or

The polynucleotide encoding the CAR of claim 8 is shown in SEQ ID NO: 5 or its degenerate sequence or complementary sequence.
An isolated CAR-T cell or CAR-NK cell, characterized in that the CAR-T cell or CAR-NK cell can express the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; CAR-T cells or CAR-NK cells can express the fusion protein of any one of claims 4-5; the CAR-T cells or CAR-NK cells can express the fusion protein of any one of claims 6-7 The antibody-drug conjugate of; the CAR-T cell or CAR-NK cell can express the CAR of any one of claims 8-14; the CAR-T cell or CAR-NK cell comprises claim 15 The polynucleotide described in.
A vector comprising the polynucleotide according to claim 15.
The vector according to claim 17, wherein 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 1 (HIV-1), and human immunodeficiency virus 1 (HIV-1). Deficiency virus 2 (HIV-2), Wisner-Medie virus (VMV) virus, goat arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), cattle Immunodeficiency virus (BIV) and Simian Immunodeficiency Virus (SIV).
An immune effector cell, which comprises the CAR according to any one of claims 8-14, or comprises the polynucleotide according to claim 15, or comprises the vector according to claim 17 or 18.
The immune effector cell of claim 19, wherein the immune effector cell is a T lymphocyte or a natural killer cell.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-3, comprising the fusion protein according to any one of claims 4-5, and comprising any one of claims 6-7 The antibody-drug conjugate according to item 1 comprises the CAR-T cell or CAR-NK cell according to claim 16, or comprises the immune effector cell according to claim 19 or 20, and optionally, pharmaceutically Acceptable carrier.
A method for preparing the CAR-T cell or CAR-NK cell according to claim 16, or comprising the immune effector cell according to claim 19 or 20, which comprises introducing the vector of claim 17 or 18 into T lymphocytes or natural Killer cells.
The antibody or antigen-binding fragment thereof according to any one of claims 1-3, the fusion protein according to any one of claims 4-5, the antibody-drug conjugate according to any one of claims 6-7 The compound expressing the CAR according to any one of claims 8-14, or the CAR-T cell or CAR-NK cell according to claim 16, or the immune effector cell according to claim 19 or 20 in the preparation of therapeutic And/or the use in drugs for preventing cancer, exemplarily, the cancer is multiple myeloma and acute myeloid leukemia, preferably recurrent multiple myeloma.