WO2021057866A1 - Single domain antibody and chimeric antigen receptor comprising antibody structure - Google Patents

Abstract

An antigen binding protein that targets BCMA, in particular a chimeric antigen receptor comprising the antigen binding protein, and an application thereof. The chimeric antigen receptor comprises an extracellular domain that targets BCMA, a transmembrane domain and an intracellular domain. The chimeric antigen receptor has a good killing effect on tumor cells in CAR-T therapy that uses BCMA as a target.

Description

Single domain antibody and chimeric antigen receptor containing antibody structure Technical field

This application relates to the field of biomedicine, and in particular to a binding domain targeting BCMA and a chimeric antigen receptor containing the binding domain targeting BCMA.

Background technique

In recent years, with the development of tumor immunotherapy and clinical technology, Chimeric Antigen Receptor-T cells (CAR-T) immunotherapy is currently one of the most promising tumor immunotherapies. CAR-T therapy is a type of genetically modified cell therapy. By transferring chimeric antigen receptor (CAR) genes into T cells, T cells are given the ability to recognize and kill target cells. CAR is an artificial receptor that mimics the function of TCR and is the core component of CAR-T. The structure of CAR mainly includes: an antigen binding region (usually derived from the scFv segment of the antigen binding region of monoclonal antibodies), hinge region, transmembrane domain, Co-stimulatory domain and activation domain. After the extracellular scFv domain binds to the target protein expressed on the surface of the target cell, it activates the costimulatory domain and the activation domain of the CAR structure. CAR-T has both an activation signal and a co-activation signal, so it can effectively amplify while killing tumors. The effectiveness of CAR-T cell therapy depends on the specificity of the antibody that recognizes the antigen and the affinity of antigen binding.

B-cell maturation antigen (BCMA), also known as CD269 or TNFRSF13 (tumornecrosis factor receptor superfamily member17), is a member of the tumor necrosis factor receptor (TNF) superfamily, consisting of 185 amino acid residues Type Ⅲ transmembrane protein. BCMA can bind to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). After BCMA binds to its ligand, it can activate the proliferation and survival of B cells. As an extremely important B cell biomarker, it is mainly expressed on the surface of mature B lymphocytes and plasma cells. BCMA RNA is generally detected in MM cells, and BCMA protein can generally be detected on the surface of plasma cells in patients with multiple myeloma. According to reports, in normal cells, BCMA is mainly expressed by plasma cells and some mature B cells, but not expressed on most B cells and other organs. Therefore, BCMA is very suitable as a target for CAR-T treatment of multiple myeloma. CAR-T therapy with BCMA as the target has been undergoing clinical research and has achieved good clinical efficacy (Noopur Raje et al. N Engl J Med. 380:1726-1737 (2019); Jie Xu et al. PNAS .116(19):9543-9551(2019)).

Current CAR T therapy usually uses scFv fragments derived from the antigen binding region of monoclonal antibodies as the antigen binding region. The extracellular scFv domain can activate the costimulatory domain and activation domain of the CAR structure after binding to the target protein expressed on the surface of the target cell. However, scFv has a relatively large molecular weight and is easy to form multimers, which affects the function of CAR. Therefore, a CAR containing a new structure of the antigen binding region is required.

Summary of the invention

This application develops a specific single domain antibody against BCMA; and this application uses genetic engineering methods to design a specific single domain antibody as the antigen binding region of CAR for CAR modification and CAR-T cell therapy, and proposes a method based on The specific chimeric antigen receptor (CAR) of a single domain antibody, which includes a target binding domain (extracellular domain), a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain, wherein The extracellular domain is an antigen-binding fragment capable of binding to human BCMA. Although single-domain antibodies have a simple structure, they can still achieve a binding affinity to specific antigens that is comparable to or even higher than that of traditional antibodies. Compared with traditional antibodies, single-domain antibodies have the advantages of small molecular weight, strong stability, and ease of recombinant expression. The single domain antibody single heavy chain antibody variable region (VHH) is a single functional domain antibody fragment that can completely bind to the antigen, and can have high affinity to the antigen without the help of the light chain. VHH has a simple structure and is called the smallest functional antigen-binding fragment. Due to its high specificity, high affinity, low immunogenicity, and good permeability, it has access to relatively hidden targets that cannot be contacted by conventional antibodies during tumor treatment. The possibility. BCMA single domain high affinity antibodies of the present disclosure, the affinity of the antibody portion of the level reached ^10-11. CAR-T cells engineered and constructed from these high-affinity single-domain antibodies have stronger binding ability to target cells. In addition, the high-affinity BCMA CAR-T cells of the present application have a better killing effect on tumor cells than control CAR-T cells constructed using scFv.

In one aspect, the present application provides a chimeric antigen receptor (CAR) comprising a targeting moiety, wherein the targeting moiety comprises complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3(CDR3), wherein the CDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 23, 26, 29, 32, 35, and 38.

In some embodiments, the CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 24, 27, 30, 33, 36, and 39.

In some embodiments, the CDR3 includes the amino acid sequence shown in any one of SEQ ID NO: 25, 28, 31, 34, 37, and 40.

In some embodiments, the CDR1, CDR2, and CDR3 comprise a sequence selected from any one of the following groups:

a) CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, and CDR3: SEQ ID NO: 25;

b) CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, and CDR3: SEQ ID NO: 28;

c) CDR1: SEQ ID NO: 29, CDR2: SEQ ID NO: 30, and CDR3: SEQ ID NO: 31;

d) CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34;

e) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, and CDR3: SEQ ID NO: 37; and,

f) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, and CDR3: SEQ ID NO: 40.

In certain embodiments, the targeting moiety includes VHH.

In some embodiments, the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 1-6 and 41-46.

In some embodiments, the chimeric antigen receptor includes a transmembrane domain, the transmembrane domain comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4 -1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM , DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.

In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD8.

In some embodiments, the chimeric antigen receptor includes an intracellular costimulatory signal transduction domain, and the intracellular costimulatory signal transduction domain comprises one or more proteins selected from the group consisting of The intracellular costimulatory signaling domain: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, Ligands for GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40 and MyD88.

In certain embodiments, the intracellular costimulatory signaling domain comprises a costimulatory signaling domain derived from 4-1BB.

In some embodiments, the chimeric antigen receptor includes an intracellular signal transduction domain, and the intracellular signal transduction domain comprises a cell derived from one or more proteins selected from the group consisting of Internal signal transduction domain: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes Virus (HSKV), DAP10, DAP-12 and at least one ITAM domain.

In certain embodiments, the intracellular signal transduction domain comprises a signal transduction domain derived from CD3.

In some embodiments, the chimeric antigen receptor includes a hinge region between the targeting moiety and the transmembrane domain, and the hinge region includes a hinge derived from one or more proteins selected from the group consisting of Zone: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8alpha, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

In certain embodiments, the hinge region comprises a hinge region derived from CD8.

In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 7-12 and 53-58.

On the other hand, the present application provides an isolated antigen binding protein, which comprises complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3), wherein the CDR1 comprises SEQ ID NO : The amino acid sequence shown in any one of 23, 26, 29, 32, 35 and 38.

In some embodiments, the CDR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 24, 27, 30, 33, 36, and 39.

In some embodiments, the CDR3 includes the amino acid sequence shown in any one of SEQ ID NO: 25, 28, 31, 34, 37, and 40.

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

In some embodiments, the antigen-binding fragments include Fab, Fab', Fv fragments, F(ab') ₂ , scFv, di-scFv, VHH and/or sdAb.

In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In some embodiments, the CDR1, CDR2, and CDR3 comprise a sequence selected from any one of the following groups:

a) CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, and CDR3: SEQ ID NO: 25;

b) CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, and CDR3: SEQ ID NO: 28;

c) CDR1: SEQ ID NO: 29, CDR2: SEQ ID NO: 30, and CDR3: SEQ ID NO: 31;

d) CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34;

e) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, and CDR3: SEQ ID NO: 37; and,

f) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, and CDR3: SEQ ID NO: 40.

In certain embodiments, the isolated antigen binding protein includes VHH.

In some embodiments, the antigen binding protein includes the amino acid sequence shown in any one of SEQ ID NO: 1-6 and 41-46.

In some embodiments, the isolated antigen binding protein includes an Fc sequence.

In some embodiments, the isolated antigen binding protein includes a single domain antibody.

In another aspect, this application provides one or more isolated nucleic acid molecules that encode the chimeric antigen receptor and/or the antigen binding protein.

In some embodiments, the isolated nucleic acid molecule encoding the antigen binding protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 47-52.

In some embodiments, the isolated nucleic acid molecule encoding the chimeric antigen receptor comprises the nucleotide sequence shown in any one of SEQ ID NOs: 14-19 and 59-64.

In another aspect, the present application provides a vector, which contains the isolated nucleic acid molecule.

In some embodiments, the vector is a viral vector.

In some embodiments, the vector is a lentiviral vector.

In another aspect, the present application provides a cell, which comprises the chimeric antigen receptor, the antigen binding protein, the isolated nucleic acid molecule, and/or the carrier.

In some embodiments, the cells include immune cells.

In certain embodiments, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / Or peripheral blood mononuclear cells.

In another aspect, the present application provides a pharmaceutical composition comprising the chimeric antigen receptor, the antigen binding protein, the isolated nucleic acid molecule, the carrier, the cell and/or A pharmaceutically acceptable carrier.

In another aspect, this application provides the use of the chimeric antigen receptor, the antigen binding protein, the isolated nucleic acid molecule, the carrier, and/or the cell in the preparation of medicines The drug is used to treat diseases or disorders related to the expression of BCMA.

In certain embodiments, the disease or condition associated with the expression of BCMA includes multiple myeloma.

On the other hand, the present application provides a method for preventing or treating diseases or disorders related to the expression of BCMA, which comprises administering an effective amount of the chimeric antigen receptor to a subject in need, and the antigen binding Protein, said isolated nucleic acid molecule, said vector, and/or said cell.

In certain embodiments, the disease or condition associated with the expression of BCMA includes multiple myeloma.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Correspondingly, the drawings and descriptions in the specification of the present application are merely exemplary, rather than restrictive.

Description of the drawings

The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and the accompanying drawings described in detail below. A brief description of the drawings is as follows:

Figure 1 shows a schematic diagram of the basic structure of the chimeric antigen receptor described in the present application. The CAR contains a signal peptide, an anti-BCMA single domain antibody, a hinge region, a transmembrane domain, and an intracellular region connected in sequence.

Figure 2 shows the detection of IL-2 secretion in the supernatant after BCMA-CAR-T is co-cultured with target cells U266, K562.BCMA, K562 and 293T.

Figure 3 shows the detection of IFN-γ secretion in the supernatant after BCMA-CAR-T is co-cultured with target cells U266, K562.BCMA, K562 and 293T.

Figure 4 shows the secretion of IL-2 cytokines after anti-BCMA CAR-T cells recognize tumor cells

Figure 5 shows the secretion of IFN-γ cytokines after anti-BCMA CAR-T cells recognize tumor cells.

Figure 6 shows the anti-tumor activity of anti-BCMA CAR-T cells in animals.

detailed description

The following specific examples illustrate the implementation of the invention of the present application. Those familiar with the technology can easily understand the other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "BCMA" can be used interchangeably with "CD269", "BCM" and "TNFRSF17", and usually refers to B cell maturation antigen. For example, human BCMA is usually a 184 amino acid long protein encoded by a 994 nucleotide long primary mRNA transcript (NM_001192.2). The amino acid sequence of human BCMA is represented by UniProtKB accession number Q02223. In the present application, the term "BCMA" may include proteins containing mutations, for example, may include proteins including point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type BCMA. In this application, the term "BCMA" can also include a part of the complete BCMA protein, as long as the relevant biological activity is retained.

In the present application, the term "antigen-binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4 . The variable regions of the heavy and light chains contain binding domains that interact with antigens. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', Fv fragments, F(ab') ₂ , scFv, di-scFv and/or dAb), immunoconjugates, multispecific antibodies (E.g. bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

In this application, the term "antibody" generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds, and includes any molecule comprising an antigen binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), And antibody fragments that bind to the antigen (e.g., Fab, Fab' and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described in this application. Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain can be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

In the present application, the term "monoclonal antibody" generally refers to a group of substantially homologous antibodies, that is, each antibody contained in the group is identical except for possible naturally occurring mutations in trace amounts. Monoclonal antibodies can be highly specific, directed against a single antigenic site. The monoclonal antibodies can be prepared by hybridoma technology or by using recombinant DNA methods to produce monoclonal antibodies in bacteria, eukaryotic animals or plant cells, and can also be obtained from a phage antibody library, using, for example, Clackson et al., Nature, 352:624 -628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

In this application, the term "single-chain antibody" (scFv) generally refers to a molecule comprising the variable region of the heavy chain and the variable region of the light chain of an antibody. For example, the scFv may be formed by linking the variable region of the heavy chain of the antibody and the variable region of the light chain through a linker (for example, a linker)

In the present application, the term "chimeric antibody" generally refers to an antibody in which a part of the amino acid sequence of the heavy or light chain is homologous to the corresponding amino acid sequence of the antibody from a specific species or both belong to a certain category, and the chain The other part of is homologous to the corresponding sequence in another species. For example, the variable regions of the light chain and the heavy chain may both be derived from the variable region of an antibody from one animal species (such as mouse, rat, etc.), while the constant part is the same as the sequence of an antibody from another species (such as human). source. For example, in order to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, and the constant regions combined with them are derived from humans. Since the constant region of a chimeric antibody can be derived from humans, the possibility of a chimeric antibody eliciting an immune response when injected is lower than that of an antibody whose constant region is non-human origin.

In this application, the term "humanized antibody" generally refers to an antibody that contains fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity of the heterogeneous antibody when it is introduced into humans. For example, CDR grafting (Jones et al., Nature 321:522 (1986)) and its variants can be used; including "reshaping" (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann ,et al.,1988 Nature 332:323-337; Tempest,et al.,Bio/Technol1991 9:266-271), "hyperchimerization" (Queen,et al.,1989Proc Natl Acad Sci USA86: 10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88: 2869-2873; Co, et al., 1992J Immunol 148: 1149-1154) and "veneering" (Mark, et al., "Derivation of therapeutically active humanized and Veneered anti-CD18 antibodies." In:Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291 U.S. Patent US5639641) and other technical means to humanize non-human binding domains. If other regions, such as the hinge region and constant region domains, are also derived from non-human sources, these regions can also be humanized.

In this application, the term "fully human antibody" generally refers to a fully human antibody, that is, both the constant region and the variable region of the antibody are derived from humans. The fully human antibody can be realized by phage antibody library technology, transgenic mouse preparation of human antibody, ribosome display technology, EBV transformed B cell cloning technology, single B cell cloning and other technologies.

In this application, the term "single domain antibody (sdAb)" generally refers to an antibody whose target binding activity (such as BCMA binding activity) is located in a single domain, which is different from, for example, antibodies and single-chain antibodies (antibodies and single-chain antibodies are generally composed of Both the heavy chain variable domain and the light chain variable domain contribute to antigen binding activity). Single domain antibodies may include VHHs capable of recognizing or binding antigens. Single domain antibodies may also include other effector moieties, such as the Fc region. The Fc region (fragment crystallizable region), also known as the constant region, usually refers to the antibody region corresponding to the antigen binding region of the antibody. The Fc region can generally be kept constant by itself and is not responsible for binding to antigens, but it can bind to Fc receptors and complement and exert the biological functions of antibodies. The Fc region of the present application may be the Fc of a human antibody.

In this application, the term "VHH" usually refers to an antibody fragment composed of the variable region of a heavy chain antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490), also known as nano Antibody (Nanobody). In the present application, the single domain antibody may be an alpaca single domain antibody. For example, the VHH may be a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.

In this application, the term "CDR" generally refers to the complementarity determining region, and CDR is mainly responsible for binding to an epitope. The CDRs of the heavy chain are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The three CDR fragments and the framework region together constitute a VHH segment, and the VHH segment and the constant region (Fc) together constitute a full-length single domain antibody. There are many rules for the definition of CDR regions in the art, such as Chothia rule, Kabat rule and so on. In this application, the CDR can be divided by IMGT definition rules. The framework region refers to the four regions with constant sequence in the variable region of an antibody. The framework region occupies approximately 85% of the antibody variable region. The function of the framework region is to act as a scaffold for the CDR region, helping the CDR region to perform the function of specifically binding antigen. The framework regions of the present application can be derived from human antibodies or single domain antibodies derived from alpaca and camel. The Fc region (fragment crystallizable region), also known as the constant region, refers to the antibody region corresponding to the antigen binding region of an antibody. The Fc region itself generally remains constant and is not responsible for binding to antigens, but it can bind to Fc receptors and complement and exert the biological functions of antibodies.

In this application, the term "transmembrane domain" generally refers to a sequence of cell surface proteins that spans the cell membrane, which may include a hydrophobic alpha helix. The transmembrane domain can be connected to the intracellular signal transduction domain and play a role in transmitting signals. In this application, the transmembrane domain can be derived from any type I, type II or type III transmembrane protein.

In this application, the term "Chimeric Antigen Receptor (CAR)" generally refers to a fusion protein containing an extracellular domain capable of binding antigen and at least one intracellular domain. CAR is the core component of chimeric antigen receptor T cells (CAR-T), which may include antigen (for example, tumor-specific antigen and/or tumor-associated antigen) binding domain, transmembrane domain, costimulatory domain, and Intracellular signal domain. CAR is an engineered receptor, and any specific receptor can be implanted on immune effector cells, especially T cells. In CAR, scFv fragments or VHH fragments of monoclonal antibodies that specifically recognize tumor antigens can be implanted on T cells or NK cells. For example, retroviral vectors can be used to introduce CAR-encoding nucleic acids into T cells, NK cells, or NK T cells. In this way, a large number of cancer-specific T cells, NK cells, or NK T cells can be generated for adoptive cell transfer. In the present application, the CAR can be combined with the T cell receptor activation intracellular domain based on the antigen (for example, BCMA) specificity of the antibody. Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing target antigens. For descriptions of CAR and CAR-T cells, see, for example, Sadelain M, Brentjens R, Rivi`ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013; 3(4): 388-398; Turtle CJ, Hudecek M, Jensen MC, Riddell SR. Engineered T cells for anti-cancer therapy. Curr Opin Immunol. 2012; 24(5): 633-639; Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev. 2014; 257(1):107-126; and WO2013154760, WO2016014789.

In the present application, the term "costimulatory domain" generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens.

In this application, the term "hinge region" generally refers to the junction region between the extracellular domain (for example, the BCMA targeting moiety) and the transmembrane region. In the present application, the CAR may include one or more hinge regions between the BCMA targeting moiety and the transmembrane domain.

In this application, the term "signal transduction domain" generally refers to a domain located inside a cell capable of transducing signals. In the present application, the intracellular signal transduction domain can transmit signals into the cell. Generally, a signal transduction domain is any continuous amino acid sequence used to guide a protein to find a target. In some cases, the signaling domain can be derived from CD3ζ. CD3ζ can form a T cell receptor-CD3 complex with T cell receptor subunits and CD3-gamma, -delta, and -epsilon. CD3ζ contains three ITAM motifs, and the ITAM sequence mediates the activation of TCR intracellular signals. The ζ chain is a receptor-activated protein tyrosine kinase substrate. When the TCR receptor is bound to the polypeptide MHC complex, the ζ chain can quickly undergo tyrosine phosphorylation and participate in the transduction of lymphocyte activation signals. Therefore, CD3ζ plays a key role in antigen recognition and TCR signal transduction.

In this application, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and/or additives that can be used in the formulations described herein include: for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, Protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium) and so on.

In this application, the term "immune cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. The term also includes engineered immune cells, such as immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

In this application, the "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, and is used to transfer the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation. The carrier also includes a carrier having a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product. The vector can cover additional features besides the transgene insert sequence and main chain: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) can be used to express transgenes in target cells and usually have control sequences. The vector described in this application can be an expression vector, including viral vectors (lentiviral vectors and/or retroviral vectors), phage vectors, phagemids, cosmids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), Bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) and/or plasmids.

In this application, the term "treatment" generally refers to: (i) preventing the disease, disorder, or condition from appearing in patients who may be susceptible to, but have not yet been diagnosed with, the disease; (ii) inhibiting the disease , Disease or condition, that is, curb its development; and (iii) alleviate the disease, disease, or condition, that is, make the disease, condition, and/or condition and/or symptoms associated with the disease, condition, and/or condition Subside.

In this application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to polymers of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and can be interrupted by non-amino acids. These terms also cover amino acid polymers that have been modified. These modifications can include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.

In this application, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to nucleosides of any length. The polymerized form of an acid, such as deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one loci) defined by linkage analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be carried out before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

In addition to the specific proteins and nucleotides mentioned herein, the application may also include functional variants, derivatives, analogs, homologs and fragments thereof.

The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a naturally-occurring sequence or is encoded by substantially the same nucleotide sequence and is capable of having one or more activities of the naturally-occurring sequence. In the context of this application, a variant of any given sequence means that the specific sequence of residues (whether amino acid or nucleotide residues) has been modified so that the polypeptide or polynucleotide essentially retains at least one A sequence of endogenous functions. The variant sequence can be obtained by the addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in the naturally-occurring protein and/or polynucleotide, as long as the The original functional activity is sufficient.

In the present application, the term "derivative" generally refers to any substitution, variation, modification, substitution, deletion, and deletion of one (or more) amino acid residues of the self/pair sequence in the polypeptide or polynucleotide of the present application. /Or addition, as long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

In this application, the term "analog" generally refers to a polypeptide or polynucleotide, including any mimic of a polypeptide or polynucleotide, that is, possessing at least one endogenous function of the polypeptide or polynucleotide mimicked by the mimic. Chemical compounds.

Generally, amino acid substitutions can be made, for example, at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions, as long as the modified sequence basically maintains the requirements The activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

The protein or polypeptide used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Intentional amino acid substitutions can be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or similarity of the amphoteric properties of the residues, as long as the endogenous function is retained. For example, the negatively charged amino acids include aspartic acid and glutamic acid; the positively charged amino acids include lysine and arginine; and the amino acids with similar hydrophilicity values without an electrical head group include glutamic acid. Paraffin, glutamine, serine, threonine and tyrosine.

In this application, the term "homolog" generally refers to an amino acid sequence or a nucleotide sequence that has a certain degree of homology with the wild-type amino acid sequence and the wild-type nucleotide sequence. The term "homology" can be equated with sequence "identity". The homologous sequence can include an amino acid sequence that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence . Generally, the homologue will contain the same active site as the subject amino acid sequence, etc. Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), and homology can also be expressed in terms of sequence identity. In this application, any one of the SEQ ID NO of the mentioned amino acid sequence or nucleotide sequence has a percent identity sequence refers to a sequence having the percentage identity over the entire length of the mentioned SEQ ID NO the sequence of.

In order to determine sequence identity, sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.

In this application, the term "and/or" should be understood to mean any one of the optional items or two of the optional items.

In this application, the term "comprising" generally refers to the inclusion of explicitly specified features, but not excluding other elements.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

Detailed description of the invention

On the one hand, this application provides the following implementation solutions:

1. A chimeric antigen receptor comprising: a BCMA binding domain, a transmembrane domain, an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain; wherein the BCMA binding domain comprises a complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3), the BCMA binding domain is selected from one of the following a)-f) sequences:

a) Containing the 3 CDR fragments shown in the sequence GFTLDYYA, the sequence IGGSDGST and the sequence ATACEGAGDGDY;

b) 3 CDR fragments including the sequence GLTFSEYT, the sequence ISGGGIP and the sequence AADARAVMTVTPNY;

c) 3 CDR fragments including the sequence GRTFNLYA, the sequence IGWNDDTT and the sequence AADENFEMPYDY;

d) Containing the 3 CDR fragments shown in the sequence GRTFSDYV, the sequence IMWPTGTP and the sequence AAARVGASGYEY;

e) 3 CDR fragments including the sequence GSIVSIDY, the sequence INSGGST and the sequence AARQWSSVDFAS; and

f) Containing the 3 CDR fragments shown in the sequence GRTFSSYA, the sequence ISWIGGLP and the sequence AAHDCSGYVCYDKEPL YEYDY.

2. The chimeric antigen receptor according to embodiment 1, wherein the BCMA binding domain is the VHH structure of a single domain antibody.

3. The chimeric antigen receptor of embodiment 1 or 2, wherein the BCMA binding domain comprises or has one of the following sequences: the sequence shown in SEQ ID No: 1, SEQ ID No: The sequence shown in 2, the sequence shown in SEQ ID No: 3, the sequence shown in SEQ ID No: 4, the sequence shown in SEQ ID No: 5, and the sequence shown in SEQ ID No: 6, or variations thereof body.

4. The chimeric antigen receptor according to any one of the preceding embodiments, wherein a hinge region is further comprised between the BCMA binding domain and the transmembrane domain.

5. The chimeric antigen receptor of embodiment 4, wherein the hinge region comprises at least one of the following: the hinge region of CD8, CD28, 4-1BB, CD4, CD27, CD7 and PD-1.

6. The chimeric antigen receptor according to any one of the preceding embodiments, wherein the transmembrane domain comprises at least one of the following: CD8 transmembrane domain, CD28 transmembrane domain, 4-1BB transmembrane domain, CD4 transmembrane domain Membrane domain, transmembrane domain of CD27, transmembrane domain of CD7, transmembrane domain of PD-1, transmembrane domain of TRAC, and transmembrane domain of TRBC.

7. The chimeric antigen receptor according to any one of the preceding embodiments, wherein the intracellular costimulatory signal transduction domain comprises at least one of the following: CD28, 4-1BB, CD40L, TIM1, CD226, DR3, SLAM, ICOS, Co-stimulatory signal transduction regions in OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT and DAP12, or costimulatory molecules composed of a combination.

8. The chimeric antigen receptor according to any one of the preceding embodiments, wherein the intracellular signal transduction domain comprises at least one of the following: CD3zeta activation region, CD3delta activation region, CD3gamma activation region, FcεRIγ activation region, FcεRIβ activation Area, immunoglobulin alpha activation area, immunoglobulin beta activation area, bovine leukemia virus gp30 activation area, Epstein-Barr virus LMP2A activation area, simian immunodeficiency virus PBj14Nef activation area, Kaposi sarcoma herpes virus activation area, DAP-12 activation Region, including at least one ITAM domain, and a domain formed by a combination of the above domains.

9. The chimeric antigen receptor according to any one of the preceding embodiments, which comprises or has one of the following sequences: the sequence shown in SEQ ID No: 7, the sequence shown in SEQ ID No: 8, The sequence shown in SEQ ID No: 9, the sequence shown in SEQ ID No: 10, the sequence shown in SEQ ID No: 11, and the sequence shown in SEQ ID No: 12, or a variant thereof.

10. An isolated nucleic acid molecule encoding the chimeric antigen receptor of any one of embodiments 1-8.

11. The nucleic acid molecule of embodiment 10, which comprises or has one of the nucleic acid sequences shown in SEQ ID NO: 14-19.

12. A vector comprising the nucleic acid molecule according to embodiment 10 or 11.

13. An immune effector cell comprising the chimeric antigen receptor of any one of embodiments 1-8, the nucleic acid molecule of embodiment 10 or 11, or the vector of embodiment 11.

14. The immune effector cell of embodiment 13, wherein the immune effector cell is a lymphocyte, preferably the lymphocyte is T cell, B cell, monocyte, macrophage, granulocyte or NK cell.

15. The immune effector cell of embodiment 13 or 14, which is a T cell.

16. A single domain antibody comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3), the antibody is selected from one of the following a)-f) sequences Species:

a) Containing 3 CDR fragments shown in the sequence GFTLDYYA, the sequence IGGSDGST and the sequence ATACEGAGDGDY;

b) 3 CDR fragments including the sequence GLTFSEYT, the sequence ISGGGIP and the sequence AADARAVMTVTPNY;

c) 3 CDR fragments including the sequence GRTFNLYA, the sequence IGWNDDTT and the sequence AADENFEMPYDY;

d) Containing the 3 CDR fragments shown in the sequence GRTFSDYV, the sequence IMWPTGTP and the sequence AAARVGASGYEY;

e) 3 CDR fragments including the sequence GSIVSIDY, the sequence INSGGST and the sequence AARQWSSVDFAS; and

f) Containing the 3 CDR fragments shown in the sequence GRTFSSYA, the sequence ISWIGGLP and the sequence AAHDCSGYVCYDKEPL YEYDY.

17. The single domain antibody of embodiment 16, wherein the antibody comprises or has one of the following VHH sequences: the sequence shown in SEQ ID No: 1, the sequence shown in SEQ ID No: 2, The sequence shown in SEQ ID No: 3, the sequence shown in SEQ ID No: 4, the sequence shown in SEQ ID No: 5, and the sequence shown in SEQ ID No: 6, or variants thereof.

18. The single domain antibody of embodiment 17, which is formed by combining the VHH sequence and the human Fc sequence.

19. A composition comprising the immune effector cell according to any one of embodiments 13-15, or comprising the antibody according to any one of embodiments 15-17.

20. The composition of embodiment 19, wherein the composition is a composition for treating diseases or disorders related to the expression of BCMA.

21. The chimeric antigen receptor according to any one of embodiments 1-9, the immune effector cell according to any one of embodiments 12-14, or the antibody according to any one of embodiments 15-17 in use Use in the preparation of a medicament, wherein the medicament is used to treat diseases or disorders related to the expression of BCMA.

22. The use of embodiment 21, wherein the disease or disorder related to the expression of BCMA is multiple myeloma.

In one aspect, the application provides an isolated antigen binding protein. In the present application, the isolated antigen binding protein may include a targeting moiety that can specifically bind to BCMA. In another aspect, the present application provides a chimeric antigen receptor (CAR) comprising the isolated antigen binding protein (for example, the targeting moiety that can specifically bind to BCMA).

The isolated antigen binding protein described in this application may comprise complementarity determining region 1 (CDR1). For example, the CDR1 may include the amino acid sequence shown in any one of SEQ ID NOs: 23, 26, 29, 32, 35, and 38. For example, the CDR1 may comprise an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 91%, 92%) shown in any one of SEQ ID NOs: 23, 26, 29, 32, 35, and 38. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.

In this application, the isolated antigen binding protein may comprise complementarity determining region 2 (CDR2). For example, the complementarity determining region 2 (CDR2) may include the amino acid sequence shown in any one of SEQ ID NOs: 24, 27, 30, 33, 36, and 39. For example, the CDR2 may comprise an amino acid sequence with at least 80% (e.g., at least 85%, 90%, 91%, 92%) shown in any one of SEQ ID NO: 24, 27, 30, 33, 36, and 39. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.

In this application, the isolated antigen binding protein may comprise complementarity determining region 3 (CDR3). For example, the complementarity determining region 3 (CDR3) may include the amino acid sequence shown in any one of SEQ ID NOs: 25, 28, 31, 34, 37, and 40. For example, the CDR2 may comprise an amino acid sequence with at least 80% (e.g., at least 85%, 90%, 91%, 92%) shown in any one of SEQ ID NO: 25, 28, 31, 34, 37, and 40. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.

In this application, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3. For example, the CDR1, CDR2, and CDR3 may comprise an amino acid sequence selected from any group of the following group, or the isolated antigen binding protein may comprise an amino acid sequence selected from any group of the following group having at least 80% ( For example, an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology:

a) CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, and CDR3: SEQ ID NO: 25;

b) CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, and CDR3: SEQ ID NO: 28;

c) CDR1: SEQ ID NO: 29, CDR2: SEQ ID NO: 30, and CDR3: SEQ ID NO: 31;

d) CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34;

e) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, and CDR3: SEQ ID NO: 37;

f) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, and CDR3: SEQ ID NO: 40.

In this application, the antigen-binding protein also includes a framework region, which can be derived from human antibodies, or from a single domain antibody of species such as alpaca, camel, etc.; in some cases, the framework region The framework regions of the antigen binding protein can be derived from humans.

In the present application, the antigen binding protein may include an Fc region. In some cases, the antigen binding protein may include a single domain antibody.

In some cases, the antigen binding protein may include VHH.

In some cases, the targeting moiety of the CAR may comprise VHH.

For example, the VHH may include the amino acid sequence shown in any one of SEQ ID NO: 1-6 and 41-46. For example, the VHH may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence shown in any one of SEQ ID NO: 1-6 and 41-46. , 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.

In the present application, the CAR includes an extracellular BCMA targeting moiety, and may also include an intracellular domain.

In some cases, the CAR may include an intracellular costimulatory signaling domain, which may provide stimulation signals. The costimulatory signal transduction domain may include, but is not limited to, the following group: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88 Co-stimulatory molecules composed of costimulatory signal transduction regions and their combinations. For example, the costimulatory domain may be a costimulatory domain from human 4-1BB.

In some cases, the CAR may comprise an intracellular signal transduction domain, which may comprise a domain with at least one ITAM motif. The intracellular signal transduction domain can transmit the activation signal to the inside of the cell. Exemplary signal transduction domains can be derived from signal transduction domains selected from the group consisting of but not limited to CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30 activation region, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10 and DAP-12, and variants of the above.

The term "zeta", "ζ" or "ζ chain", "CD3-ζ" or "TCR-ζ" refers to the protein provided by the sequence Genebank: NM_198053.2 or the equivalent residues of species close to humans. CD3ζ forms a T cell receptor-CD3 complex with T cell receptor subunits and CD3-gamma, -delta, and -epsilon. CD3ζ contains three ITAM motifs, and the ITAM sequence mediates the activation of TCR intracellular signals. The zeta chain is a receptor-activated protein tyrosine kinase substrate. When the TCR receptor binds to the polypeptide MHC complex, the zeta chain quickly undergoes tyrosine phosphorylation and participates in the transduction of lymphocyte activation signals. Therefore, CD3ζ plays a key role in antigen recognition and TCR signal transduction. For example, the intracellular signal transduction domain may be a signal transduction domain from CD3.

In some cases, the CAR may include a transmembrane domain, which is a sequence of a cell surface protein that spans the cell membrane, and it may include a hydrophobic alpha helix. The transmembrane domain can be derived from CD28 and has good stability. The transmembrane domain can be derived from any type I transmembrane protein. The transmembrane domain can be a synthetic sequence predicted to form a hydrophobic helix. The transmembrane domain may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM. For example, the transmembrane domain may be a transmembrane domain from CD8.

In some cases, the CAR may include a hinge region, and the hinge region may be located between the extracellular targeting moiety and the transmembrane domain. The hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8alpha, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

In the present application, the CAR may include a BCMA targeting moiety (for example, the antigen binding protein, for example, the VHH), the hinge region, the transmembrane domain, the costimulatory signal transduction region, and The signal transduction domain. In some cases, the CAR may also include a signal peptide in the N segment of the BCMA, for example, a human CD8 signal peptide.

For example, the CAR may comprise the amino acid sequence shown in any one of SEQ ID NO: 7-12 and 53-58. For another example, the CAR may be included in the amino acid sequence shown in any one of SEQ ID NO: 7-12 and 53-58 with at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%). %, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.

The application also provides a nucleic acid molecule encoding the CAR. For example, the nucleic acid molecule encoding the CAR may comprise the amino acid sequence shown in any one of SEQ ID NO: 7-12 and 53-58. For another example, the nucleic acid molecule encoding the CAR may comprise an amino acid sequence shown in any one of SEQ ID NO: 7-12 and 53-58 with at least 80% (e.g., at least 85%, 90%, 91%). , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.

The nucleic acid molecules described in this application can be obtained by recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard The technology is directly isolated from the cells and tissues containing the gene, or the nucleic acid molecule can be obtained synthetically.

The application also provides a vector containing the nucleic acid molecule. In a brief summary, the expression of the natural or synthetic nucleic acid encoding the polypeptide of interest can usually be achieved by operably linking the nucleic acid encoding the polypeptide of interest or part thereof to the downstream of the promoter, and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotic cells. A typical vector may contain transcription and translation terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

The nucleic acid molecules described in this application can also be linked to many types of vectors. For example, the nucleic acid can be linked to, including but not limited to, plasmids, phagemids, phages, viruses, and/or cosmids. Specific vectors of interest can include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

The viral vector may be directly administered to the patient (in vivo) or may be in an indirect form, for example, the virus is used to treat cells in vitro, and then the treated cells are administered to the patient (ex vivo). Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Conventional virus-based systems can include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transfer. In some cases, retroviruses, lentiviruses, and adeno-associated viruses can be used to transfer genes into the host genome to allow long-term expression of the inserted genes. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce higher viral titers. The lentiviral vector may include a long terminal repeat sequence 5'LTR and a truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and/or post-translational regulatory element (WPRE). The vectors described in this application can be introduced into cells.

cell

In another aspect, the present application provides a cell, which may contain the chimeric antigen receptor, the antigen binding protein, the isolated nucleic acid molecule, and/or the carrier. The cell can be a prokaryotic cell (e.g., bacteria) or a eukaryotic cell (e.g., yeast, mammalian cells)

The cells may include immune cells. In some cases, the cells may include immune cells. For example, the cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells. cell.

In some cases, the cells may include T lymphocytes. The T lymphocytes may include thymocytes, natural T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The T cell may be a helper T cell (Th), such as a helper T cell 1 (Th1) or a helper T cell 2 (Th2) cell. The T lymphocytes may be CD4 ⁺ helper T cells (HTL; CD4 ⁺ T cells), cytotoxic T cells (CTL; CD8 ⁺ T cells), tumor infiltrating cytotoxic T cells (TIL; CD8 ⁺ T cells), CD4 ⁺ / CD8 ⁺ T cells, CD4 ^- / CD8 ^- T cells or any other subtypes of T lymphocytes. In some cases, the modified T cell is a human T cell. Before expanding and genetically modifying the cells of the present application, a source of cells can be obtained from a subject, such as a patient, by various non-limiting methods. T cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, any number of T cell lines available and known to those skilled in the art can be used. In other cases, the cells may be derived from a healthy donor, from a patient diagnosed with cancer, or obtained from a patient diagnosed with an infection. In other cases, the cells are part of a mixed population of cells with different phenotypic characteristics.

In some cases, the cells may include B cells. In some cases, the B cells may include effector B cells (plasma cells) and memory B cells. The B cells may include B2 cells, B1 cells, marginal zone B cells, follicular B cells, and regulatory B cells. In some cases, the immune cells may include macrophages. The B cells may include type I macrophages (M1) and type II macrophages (such as M2a, M2B, M2c).

In some cases, the cells may include NK cells. In some cases, the NK cells may include CD56bright and CD56dim. In some cases, the NK cells may include NK1 and NK2. In some cases, the NK cells may include A-NK and NA-NK.

In some cases, the cells may include leukocytes. White blood cells usually refer to a nucleated blood cell that has active mobility and can migrate from inside blood vessels to outside blood vessels, or from tissues outside blood vessels to inside blood vessels. In addition to blood, white blood cells can also be found in the lymphatic system, spleen, tonsils, and other tissues of the body. In this application, the white blood cells may include granulocytes (such as neutrophils, eosinophils, basophils), agranulocytes (such as lymphocytes, monocytes, macrophages, phagocytes, Mast cells).

In some cases, the cells may include lymphocytes, and the lymphocytes may include any monocytes, non-phagocytic leukocytes found in blood, lymph and lymphatic tissues, for example, B lymphocytes, T lymphocytes, natural Killer (NK) cells.

In some cases, the cells may include peripheral blood mononuclear cells, which may include any cell that has a mononucleus in peripheral blood. For example, in the present application, the peripheral blood mononuclear cells may include T cells, B cells, NK cells, lymphocytes, monocytes, and dendritic cells.

In some cases, the cells may include macrophages. Macrophages are a kind of substances that can swallow and digest cell debris, microorganisms, cancer cells, and all other substances that lack the surface markers expressed on the surface of normal cells. This process is called phagocytosis. Macrophages are found in almost all tissues, looking for possible pathogens through the movement of amoeba. In addition to playing an important role in the non-specific innate immune response, they can also help initiate adaptive immunity by recruiting other immune cell types, such as lymphocytes.

Pharmaceutical composition

In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition may include the modified immune cells described in this application, and a pharmaceutically acceptable carrier. In this application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, antibacterial agents that are compatible with the administration of immune cells and/or cell populations of this application. Agents and antifungal agents, isotonic agents and absorption delay agents. Unless it is incompatible with the immune cells of the present application and/or the cell population of the present application, any conventional medium or reagent can be considered for use in the pharmaceutical composition of the present application.

The pharmaceutical composition can be used to treat diseases or disorders related to the expression of BCMA, for example, to treat myeloma (eg, multiple myeloma) and other diseases caused by abnormal proliferation of B cells.

Uses and methods

In another aspect, this application provides the use of the chimeric antigen receptor, the antigen binding protein, the isolated nucleic acid molecule, the carrier, and/or the cell in the preparation of medicines The drug is used to treat diseases or disorders related to the expression of BCMA.

On the other hand, the present application provides a method for preventing or treating diseases or disorders related to the expression of BCMA, which comprises administering an effective amount of the chimeric antigen receptor to a subject in need, and the antigen binding Protein, said isolated nucleic acid molecule, said vector, and/or said cell.

In some cases, the diseases or disorders related to the expression of BCMA described in this application include myeloma (eg, multiple myeloma).

In another aspect, this application provides a composition comprising the chimeric antigen receptor, antigen binding protein and/or cell described in this application. The composition can be used to treat diseases or disorders related to the expression of BCMA, for example, to treat multiple myeloma and other diseases caused by abnormal proliferation of B cells.

The compositions and methods described in this application can be used in combination with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like. The compositions and methods described in this application can be used for other disease conditions that rely on immune response, such as inflammation, immune disease, and infectious disease.

Without intending to be limited by any theory, the following examples are only to illustrate the chimeric antigen receptor, antigen binding protein, preparation method and use, etc. of the present application, and are not used to limit the scope of the present application.

Example

Example 1: Acquisition of VHH gene sequence of BCMA single domain antibody

1.1 BCMA single domain antibody library construction:

The recombinant human BCMA antigen (BC7-H5254, ACRO Biosystems) with a C-terminal human IgGFc tag was used for alpaca immunization. A total of four immunizations, with an interval of about 3 weeks. For the first immunization, 0.5 mg of antigen and CFA (Complete Freund's Adjuvant) were mixed and emulsified at 1:1 and then injected subcutaneously, which was counted as the first day. In the next three immunizations, 0.25 mg of antigen and IFA (incomplete Freund's adjuvant) were mixed and emulsified at a ratio of 1:1, and then injected subcutaneously. During the period, the immune response effect was evaluated by blood sampling. On the 70th day, peripheral blood was taken to separate lymphocytes by gradient centrifugation, lymphocyte RNA was extracted and reverse transcribed into cDNA, and the variable alpaca heavy chain immunoglobulin was amplified by two rounds of polymerase chain reaction (PCR) The region fragments the VHH sequence, inserts the amplified VHH into a phage display vector, and transfers the product carrying the single domain antibody gene fragment into competent E. coli through electrotransformation, thereby obtaining a single domain antibody immune library.

1.2 Screening of BCMA specific antibody sequence:

Use phage display technology to display single-domain antibody molecules on the surface of phage, and then screen out antigen-specific single-domain antibodies, coat BCMA protein (BCA-H82E4, ACROBiosystems) into 96-well plates, and screen 3-5 by Elisa experiment In the next round, BCMA-specific phages are gradually enriched. A large number of positive clones were selected for Elisa detection and positive clones were screened and sequenced. The unique clones were determined according to sequence comparison and their sequences were divided into framework region FR and complementarity determining region CDR. Through the above methods, a total of 6 strains of BCMA single domain antibodies with higher affinity were obtained. These 6 antibodies were named BCMA-sdAb-4, BCMA-sdAb-15, BCMA-sdAb-20, BCMA-sdAb-52, BCMA-sdAb-19B, BCMA-sdAb-35B. The CDR regions and VHH amino acid sequences corresponding to different antibodies are shown in Table 1.

Example 2: Design, construction and cloning of BCMA chimeric antigen receptor molecules into lentiviral vectors

In order to screen out more effective chimeric antigen receptors targeting BCMA, all 6 sdAb strains were constructed on lentiviral vectors, and one CAR sequence that had been patented and published was selected as a positive control (SEQ ID in CN108004259A). No: 31).

2.1 Synthesize the gene sequence of the chimeric antigen receptor targeting BCMA containing different sdAbs:

The chimeric antigen receptor targeting BCMA contains human CD8 signal peptide, anti-human BCMA single domain antibody VHH sequence, human CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory signal transduction domain and CD3 signal transduction domain , They are connected in series in sequence (Figure 1). The corresponding amino acid sequences of different chimeric antigen receptor molecules (CAR molecules) are shown in Table 1, and the corresponding DNA sequences are shown in Table 1. The chimeric antigen receptor gene sequence targeting BCMA was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and cloned into pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.). When synthesizing genes, restriction enzymes BamHI (NEB: R3136S) and SalI (NEB: R3138S) restriction sites were added at the 5'end and 3'end of the gene, respectively.

2.2 Construction of a lentiviral vector expressing the BCMA chimeric antigen receptor:

The gene sequences of BCMA chimeric antigen receptor BCMA-CAR-4, BCMA-CAR-15, BCMA-CAR-20, BCMA-CAR-52, BCMA-CAR-19B, BCMA-CAR-35B and a positive control (BCMA-J22.9-CAR) (see Table 3) were digested from pUC57 vector, and the digested bands were detected by agarose gel electrophoresis and then gel recovery and purification were performed. (QIAGEN: 28706) The BCMA chimeric antigen receptor DNA fragment was obtained. The BCMA chimeric antigen receptor DNA fragment recovered by restriction digestion was cloned into a lentiviral vector (Addgene; catalog number: 12252) by T4 ligase (NEB: M0202S) to obtain 7 recombinant plasmids expressing the BCMA chimeric antigen receptor: p-lenti-BCMA-CAR-4, p-lenti-BCMA-CAR-15, p-lenti-BCMA-CAR-20, p-lenti-BCMA-CAR-52, p-lenti-BCMA-CAR-19B, p-lenti-BCMA-CAR-35B, p-lenti-BCMA-J22.9-CAR. The 7 lentiviral vectors were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing and verification. The sequencing primers were:

Lenti-For: TCAAGCCTCAGACAGTGGTTC (SEQ ID No: 21)

Lenti-Rev: CCTCATAAAGAGACAGCAACCAGG (SEQ ID No: 22)

Sequencing verified that all 7 lentiviral vectors were constructed correctly.

Table 1 Chimeric antigen receptor molecules (CAR molecules) and their corresponding amino acid sequences, DNA sequences, BCMA binding domains and CDR sequences

Example 3: Preparation of 7 BCMA chimeric antigen receptor molecules lentivirus

3.1 Large-scale plasmid:

The recombinant plasmid verified by sequencing was retransformed into Escherichia coli stbl3 (purchased from Beijing Cresbo). Pick a single clone from the transformed plate into a 3ml shaker tube of liquid LB medium containing ampicillin at 37°C at 220 rpm, shake culture for 8 hours on a shaker. Extract 500 μl from the activated bacterial solution and inoculate it into 250 ml of liquid LB medium containing ampicillin at 37° C., 220 rpm, and shake culture for 12-16 hours on a shaker. Use Qiagen HiSpeed Plasmid Maxi Kit kit (article number: 12662) to carry out plasmid extraction according to the experimental procedure provided by the kit. After the plasmid was extracted, Nanodrop (Thermo Fisher Scientific) was used to determine the plasmid concentration, and the supercoiled plasmid content was detected by DNA agarose gel.

3.2 Cultivating 293T cells:

After the frozen 293T cells (purchased from ATCC) were taken out of liquid nitrogen, they were continuously shaken in a 37°C water bath to promote their thawing. After wiping the mouth of the tube with medical alcohol, move it to a 15ml centrifuge tube containing 10ml of preheated DMEM complete medium (90% DMEM + 10% FBS + 1% penicillin/streptomycin) in advance, and blow gently, 1000 rpm Centrifuge for 3 min, aspirate and discard the supernatant. Add 10ml of DMEM complete medium, gently blow evenly, and then inoculate it into a 10cm culture dish, and then culture it in a cell culture incubator containing 5% CO2 at 37°C. On the second day, when the cell density reaches 80%-90%, the cells are subcultured, aspirate the medium and wash with 10ml PBS once, add 3ml trypsin containing 0.25% EDTA, and put it in the incubator for 1-2min (during this period, Take it out and observe under the microscope whether the cells are rounded). After the cells become round, add 1ml of DMEM complete medium to stop trypsinization, transfer to a 15ml centrifuge tube, centrifuge at 1000rpm for 3min, aspirate and discard the supernatant. According to the needs of the experiment, pass the passage at a ratio of 1:3 or 1:5, and inoculate it into a new 10cm petri dish to continue culturing or cryopreservation;

3.3 Preparation of lentivirus:

1) On the first day, inoculate 293T cells: Inoculate cells according to about 1.7×10 ⁷ cells/T175 flask (cultivation in 30 ml culture medium), so that they can be transfected when the cell density reaches 80-90% on the second day.

2) The next day, plasmid transfection: Before transfection, the medium needs to be changed to DMEM medium with 10% FBS but no double antibodies. First prepare the plasmid complex separately: add the following plasmids to 1.5ml Opti-MEM (Thermo Fisher Scientific; 31985-070) and mix well: 9μg of psPAX2 plasmid (Addgene; catalog number: 12260), 18μg of pMD2.G plasmid (Addgene; Product No.: 12259), 18 μg of viral vector plasmid. There are 7 kinds of viral vector plasmids: p-lenti-BCMA-CAR-4, p-lenti-BCMA-CAR-15, p-lenti-BCMA-CAR-20, p-lenti-BCMA-CAR-52, p-lenti -BCMA-CAR-19B, p-lenti-BCMA-CAR-35B, p-lenti-BCMA-CAR-J22.9. Then prepare the transfection reagent complex: According to the mass ratio of plasmid to PEI 1:3, add 67.5μl (2mg/ml) PEI (polysciences: 24765) to 1.5ml Opti-MEM and mix well, and let stand at room temperature for 5 minutes; Add the transfection reagent complex to the plasmid complex, mix well and let stand for 20 min. Finally, the transfection complex was slowly dropped into the 293T cell culture dish, mixed gently, and cultured in a cell incubator _{containing 5% CO 2 at 37°C.}

3) On the fourth day, the virus was collected: the culture supernatant was collected for 48 hours after transfection, and the cell debris was removed by centrifugation at 2000 rpm for 10 minutes. Use a 0.45μm filter membrane to filter the supernatant, and transfer the filtrate to a special centrifuge tube for balancing. Use an ultracentrifuge at 25000rpm to ultracentrifuge for 2h. After discarding the supernatant, resuspend the lentivirus in X-VIVO-15 medium, and store the lentivirus in an ultra-low temperature refrigerator at -80℃. Prepare BCMA-CAR-4, BCMA-CAR-15, BCMA-CAR-20, BCMA-CAR-52, BCMA-CAR-19B, BCMA-CAR-35B, BCMA-J22.9-CAR according to this process. Lentivirus with DNA sequence (see Table 1).

Example 4: Isolation and activation of human primary T cells, and transfection of T cells with lentivirus

Dilute human peripheral blood mononuclear cells (PBMCs, purchased from Shanghai Miaoshun Biotech) to 2×10 ⁶ /ml, and use anti-human CD3/CD28 magnetic beads (Thermo Fisher Scientific) according to the ratio of cells to magnetic beads of 1:3 To activate T cells, 300IU/ml IL-2 (PeproTech: 200-02) was added to the medium at the same time. After T cells are activated for 24 hours, count the T cells and adjust their cell concentration to 1×10 ⁶ /ml, and inoculate 500 μl per well into 24 wells, which will contain BCMA-CAR-4, BCMA-CAR-15, and BCMA. -CAR-20, BCMA-CAR-52, BCMA-CAR-19B, BCMA-CAR-35B, BCMA-J22.9-CAR DNA sequence lentiviruses were added to T cell culture wells to transfect T cells, but not transfected As a negative control (control T), different chimeric antigen receptor T cells that can express the targeted human BCMA antigen were obtained, named: BCMA-CAR-T-4, BCMA-CAR-T-15, BCMA -CAR-T-20, BCMA-CAR-T-52, BCMA-CAR-T-19B, BCMA-CAR-T-35B, BCMA-J22.9-CAR-T. After transfection, continue to culture the transfected T cells to a resting state.

Example 5: Detection of the expression of BCMA chimeric antigen receptor in cells

The intracellular expression of the BCMA chimeric antigen receptor molecule prepared in Example 4 was detected by fluorescent antibody staining and flow cytometry. The basic steps are as follows: BCMA-CAR-T cells infected with the lentivirus for 48 hours and the slow virus were collected by centrifugation. Virus-uninfected T cells (control T) were added with biotinylated human BCMA protein (100 μg/ml) (Acrobiosystems: BCA-H82E4), incubated at 4°C for 30 minutes in the dark, and washed once with PBS. Resuspend in an appropriate volume of PBS and add the secondary antibody PE-Streptavidin (BD Biosciences: 554061), incubate at 4°C for 20 minutes in the dark, wash with PBS once, then resuspend in an appropriate amount of PBS, and finally use flow cytometry to detect BCMA- The expression efficiency of CAR molecules on the surface of T cells.

Results: After 48 hours of infection of T cells with the lentivirus prepared in Example 3, BCMA-CAR can be well expressed on the surface of T cells. The experimental results are shown in Table 2:

Table 2 CAR-T cell expression efficiency

CAR-T cell Positive rate (number of positive cells/number of total cells%) Control T cell 0.18% BCMA-J22.9-CAR-T 42.60% BCMA-CAR-T-4 28.80% BCMA-CAR-T-15 27.50% BCMA-CAR-T-20 23.50% BCMA-CAR-T-52 33.70% BCMA-CAR-T-19B 24.30% BCMA-CAR-T-35B 23.30%

Example 6: Detection of IL-2 and IFN-γ secretion after BCMA chimeric antigen receptor is co-cultured with different target cells

Cell co-cultivation: After counting CAR-T cells and control T cells, adjust the concentration to 1×10 ⁶ /ml, and then inoculate 100ul per well into a flat-bottomed 96-well plate. Place the inoculated CAR-T and T cells temporarily at 37°C for incubation. Target cell U266 (purchased from the Cell Bank of the Chinese Academy of Sciences), K562.BCMA expressing BCMA protein, negative cells K562 (Cell Bank of the Chinese Academy of Sciences) and 293T (ATCC) that do not express BCMA protein according to the effective target ratio of 1:1, which is the cell concentration 1×10 ⁶ /ml, 100μl/well, added to CAR-T cells and control T cells for co-culture. K562 cells were transfected with lentivirus with BCMA gene (Genebank: NM_001192.3) to prepare K562.BCMA cell line expressing BCMA protein. See Example 3 for the lentivirus preparation process.

The supernatant after 24 hours of co-cultivation of the above cells was transferred to a new 96-well plate, and the secretion of IL-2 and IFN-γ cytokines of T cells was detected using an ELISA kit (Thermo Fisher Scientific; article number 88-7316). Plate preparation and supernatant cytokine detection follow the standard procedures provided by the kit.

Results: After co-incubating the prepared BCMA CAR T cells with target cells for 24 hours, the levels of IL-2 and IFN-γ in the supernatant were detected by ELISA. The results are shown in Figure 2 and Figure 3. It can be seen from the figure that BCMA-CAR-T cells and BCMA-positive cells (U266 and K562.BCMA) were co-cultured to release high levels of IL-2 and IFN-γ. However, BCMA-CAR-T cells and BCMA-negative cells (K562 and 293T) have almost no secretion of cytokines after co-culture, and the negative control T cells do not induce cytokine when co-cultured with BCMA-positive cells and BCMA-negative cells. Release, verifying that the cytokine release in the co-culture assay is CAR-dependent and BCMA-specific.

Example 7: BCMA chimeric antigen receptor was co-cultured with different target cells to detect its killing ability

Cell plating: U266 cells, K562 cells, A549 cells and 293T cells were transfected with lentivirus with luciferase (GenBank: AAR29591.1) to prepare cell lines labeled with luciferase. See Example 3 for the lentivirus preparation process. The cell line U266.luc labeled with luciferase, K562.BCMA.luc labeled with luciferase expressing BCMA, A549.luc labeled with luciferase, and K562.luc labeled with luciferase not expressing BCMA. , 293T.luc labeled with luciferase, according to the cell concentration of 1×10 ⁵ /ml, 50μl/well spread to 96-well flat-bottomed opaque white board. A total of 4 effective target ratios of 10:1, 5:1, 2.5:1 and 1:1 were set to co-culture CAR-T cells and T cells with target cells. Place the 96-well plate in a 37°C 5% CO ₂ cell incubator for culture.

After the cells were co-cultured for 24 hours, the remaining luciferase activity (relative light unit, RLU) of the target cells was measured to detect the killing ability of each CAR-T on different target cells. The specific steps are: centrifugation of the co-cultured cells at 800 rpm for 5 minutes, aspirate the supernatant and add 100 μl of D-luciferin substrate (Thermo Fisher Scientific: 88293), mix well and avoid light for 5 minutes, and detect in chemiluminescence mode on the microplate reader The fluorescence intensity. Since luciferase is only expressed in target cells, the remaining luciferase activity in the well is directly related to the number of live target cells in the well. In the absence of effector cells, the maximum luciferase activity was obtained by adding medium to the target cells as a control.

Results: After the CAR-T cells prepared in Example 4 were co-cultured with target cells for 24 hours, the killing results are shown in Table 3 to Table 7, and BCMA-J22.9-CAR-T was used as a positive control. The data in Table 3 and Table 4 confirm that the CAR-T cells of BCMA have different levels of killing ability against multiple myeloma cells U266.luc and the stable cell line K562.BCMA.luc expressing BCMA, and CAR-T has the ability to kill U266. The dose-dependent killing effect of .luc and K562.BCMA.luc cells, in the case of an effective target ratio of 5:1, more than 60% of the clones have a killing efficiency of more than 50%, and the effective target ratio of 2.5:1 and 1:1 Its killing efficiency began to decrease. Furthermore, comparing the killing rate of CAR T cells against U266.luc and K562.BCMA.luc under different effective target ratios, it was found that CAR T cells showed a potent cell killing effect against multiple myeloma cells U266.luc. At the same time, the data in Table 5, Table 6 and Table 7 confirm that when CAR T cells are co-cultured with BCMA-negative cell lines A549.luc, K562.luc, and 293T.luc, no luciferase activity is detected under the three effective target ratios. Significantly lower, that is, its killing rate did not increase significantly, indicating that CAR T cells do not have a dose-dependent killing effect on BCMA-negative cell lines A549.luc, K562.luc, and 293T.luc. The above data proves that the killing effect of BCMA CAR T cells against tumor cells is BCMA specific.

Table 3 After culturing BCMA-CAR-T and target cell U266.luc, the killing effect on tumor cells at the effective target ratio of 10:1, 5:1, 2.5:1 and 1:1

Table 4 After culturing BCMA-CAR-T with target cell K562.BCMA.luc, the killing effect on tumor cells at the effective target ratio of 10:1, 5:1, 2.5:1 and 1:1

Table 5 After culturing BCMA-CAR-T and target cell K562.luc, the killing effect on tumor cells at the effective target ratio of 10:1, 5:1, 2.5:1 and 1:1

Table 6 After culturing BCMA-CAR-T with target cell A549.luc, the killing effect on tumor cells at the effective target ratio of 10:1, 5:1, 2.5:1 and 1:1

Table 7 After culturing BCMA-CAR-T with target cell 293T.luc, the killing effect on tumor cells at the effective target ratio of 10:1, 5:1, 2.5:1 and 1:1

Example 8: BCMA antibody affinity detection

1. The VHH sequences in the 6 CARs selected were cloned into the expression vector containing the human IgG1Fc fragment (hIgG1Fc) sequence, and the BCMA antibody-Fc fusion protein was recombinantly expressed. The plasmid was transiently transfected into HEK293 suspension cells (from ATCC) for expression, and then the recombinant antibody was purified using a Protein A affinity chromatography column. The obtained recombinant antibody is used for affinity detection.

2. Use Biacore T200 (GE Healthcare) to determine the affinity of each antibody to BCMA:

The basic process is: 1) Antigen dilution gradient: According to the relative molecular mass of the antigen and the corresponding antigen concentration, the amount of the substance corresponding to the antigen is calculated. In this experiment, we chose to dilute from 40nM and dilute six gradients in a one-to-two manner. 2) Antibody dilution: Dilute the sample to be tested with HBS solution to 1 μg/ml. 3. Parameter setting: Set the sample to be tested, namely the antibody, to be separately captured on the chip surface fc2-fc4 flow cell for 30 seconds at a flow rate of 10μl/min. Use Protein A chip (GE, Cat#29127556) for antibody capture. The gradient antigen was set to flow through the four flow cells fc1-fc4 at a flow rate of 30μl/min, with a binding time of 180s and a dissociation time of 300s. The antibody affinities measured using this procedure are shown in Table 8.

Results: It can be seen from the determination of the dissociation constant KD of the antibodies in Table 8 that the 6 antibodies obtained by screening all have high affinity for BCMA, and the affinities are between 10 ^-11 M and 10 ^-10 M. Among them, the antibody The KD value of BCMA-sdAb-19B can reach 10 ^-11 M, and the KD value of antibody BCMA-sdAb-20 can reach 10 ^-10 M.

Table 8 Recombinant protein affinity detection

Antibodies detected ka(1/Ms) kd(1/s) KD(M) BCMA-sdAb-4 1.22E+07 3.45E-03 2.83E-10 BCMA-sdAb-15 1.25E+07 7.78E-04 6.21E-11 BCMA-sdAb-20 1.63E+06 7.79E-04 4.77E-10 BCMA-sdAb-52 1.23E+07 4.27E-03 3.48E-10 BCMA-sdAb-19B 9.99E+06 3.91E-04 3.92E-11 BCMA-sdAb-35B 7.24E+06 3.42E-04 4.72E-11

Example 9: Humanization of BCMA single domain antibody

Antibody Modeling，采用同源建模方法选取5-10个最优模拟结构，Loop区域一般使用同源建模方法建模。使用PDB BLAST调取数据库中序列最接近的10个抗体晶体结构模型。对比自动建模模型，选取最优的结构模型。使用IgBLAST将抗体序列与人Germline序列比对，找出驼源位点和同源性最高的人源化设计模 板。将驼源序列进行人源化突变。将设计好的人源化序列与人源Germline序列进行比对，计算出每个部分及全长抗体的人源化程度百分比。 Use Discovery Studio and

Antibody Modeling uses the homology modeling method to select 5-10 optimal simulation structures, and the Loop area is generally modeled by the homology modeling method. Use PDB BLAST to retrieve the 10 antibody crystal structure models with the closest sequence in the database. Compare the automatic modeling model and select the optimal structural model. Use IgBLAST to align the antibody sequence with the human Germline sequence to find the humanized design template with the highest camel-origin site and homology. The camel-derived sequence was humanized and mutated. Align the designed humanized sequence with the human Germline sequence, and calculate the percentage of humanization of each partial and full-length antibody.

Detect the affinity of humanized antibody and BCMA protein:

The basic process is: 1) The original single domain antibody and the designed humanized single domain antibody are expressed as a fusion protein, and the fusion tag is human IgG1Fc. 2) Use ExpiCHO-s expression system to transiently transfect, express and purify. 3) Use the ELISA method to detect the affinity of the humanized single domain antibody and the BCMA protein. 4) Select the group with the highest affinity among humanized single domain antibodies to obtain the corresponding antibodies BCMA-sdAb-4, BCMA-sdAb-15, BCMA-sdAb-20, BCMA-sdAb-52, BCMA-sdAb-19B, BCMA- The six humanized antibody sequences of sdAb-35B are named: sdAb hu-4-2 (amino acid sequence SEQ ID No: 41, DNA sequence SEQ ID No: 47), sdAb hu-15-2 (amino acid sequence SEQ ID No: 42, DNA sequence SEQ ID No: 48), sdAb hu-20-2 (amino acid sequence SEQ ID No: 43, DNA sequence SEQ ID No: 49), sdAb hu-52-2 (amino acid sequence SEQ ID No : 44, DNA sequence SEQ ID No: 50), sdAb hu-19B-2 (amino acid sequence SEQ ID No: 45, DNA sequence SEQ ID No: 51), sdAb hu-35B-2 (amino acid sequence SEQ ID No: 46 , DNA sequence SEQ ID No: 52).

Example 10: Evaluation of in vitro activity of humanized CAR-T cells

10.1 Preparation of humanized CAR-T cells:

The CARs modified by the above six humanized single domain antibodies are named: CAR-hu-4-2 (amino acid sequence SEQ ID No: 53, DNA sequence SEQ ID No: 59), CAR-hu-15-2 (amino acid sequence Sequence SEQ ID No: 54, DNA sequence SEQ ID No: 60), CAR-hu-20-2 (amino acid sequence SEQ ID No: 55, DNA sequence SEQ ID No: 61), CAR-hu-52-2 (amino acid sequence Sequence SEQ ID No: 56, DNA sequence SEQ ID No: 62), CAR-hu-19B-2 (amino acid sequence SEQ ID No: 57, DNA sequence SEQ ID No: 63), CAR-hu-35B-2 (amino acid sequence Sequence SEQ ID No: 58, DNA sequence SEQ ID No: 64). Obtain the positive control CAR humanized anti-BCMA-10-CAR nucleic acid sequence (SEQ ID NO 30 in US20170226216; amino acid sequence SEQ ID NO: 67, DNA sequence SEQ ID NO: 68) from published patents, through gene synthesis Preparation of lentiviral vector containing humanized BCMA-CAR and positive control CAR by method and molecular cloning, and prepare the corresponding lentivirus. Refer to Example 3 to prepare humanized CAR-T, and name the prepared humanized CAR-T as: CAR-T-hu-4-2, CAR-T-hu-15-2, CAR-T hu -20-2, CAR-T hu-52-2, CAR-T-hu-19B-2, CAR-T-hu-35B-2 and positive control BCMA-10-CAR-T. T cells without transfection were named control T cells. Referring to Example 5, the expression efficiency of CAR was tested.

Results: 48 hours after transfection of the humanized BCMA CAR-T cells prepared above, the CAR expression status is shown in Table 9: Humanized BCMA-CAR can be well expressed on the surface of T cells, and the CAR expression efficiency is 50% about.

Table 9 Humanized CAR-T cell expression efficiency

CAR-T Positive rate (positive cells/total cells%) Control T cell 0.30% BCMA-10-CAR-T 53.00% CAR-T-hu4-2 66.10% CAR-T-hu15-2 65.50% CAR-T-hu20-2 40.30% CAR-T-hu52-2 45.40% CAR-T-hu19B-2 55.40% CAR-T-hu35B-2 49.80%

10.2 Detection of IL-2 and IFN-γ secretion after co-cultivation of humanized BCMA chimeric antigen receptor with different target cells:

Refer to Example 6 to detect the secretion of humanized CAR-T cytokines IL-2 and IFN-γ using the ELISA method.

Results: As shown in Figure 4 and Figure 5, it can be seen from the figure that the humanized BCMA-CAR-T can secrete a large amount of cytokines IL-2 and IFN-γ after stimulation of target cells (MM.1S and K562-BCMA) However, humanized BCMA-CAR-T and BCMA-negative cells (K562 and 293T) were co-cultured with almost no secreted factors. Except for CAR-T-hu20-2, the secretion of humanized CAR-T cytokines IL-2 and IFN-γ is greater than that of the positive control CAR-T cells.

10.3 The killing ability of humanized BCMA-CAR-T is tested after co-cultivation with different target cells:

Refer to Example 7 for cell killing experiments.

Result: The killing result is shown in Table 10. The results show that humanized CAR-T can efficiently kill target cells in vitro. In general, the killing rate of humanized CAR-T at different effective target ratios is greater than or close to that of the positive control CAR-T cells.

Table 10 After culturing BCMA-CAR-T and target cell MM.1S, the ratio of effector cell to target cell (efficiency-target ratio) is 10:1, 5:1 and 2.5:1 on tumor cell killing effect

Example 11: In vivo activity detection of humanized CAR-T animals

MM.1S tumor cells are fluorescently labeled human myeloma cells. After routine resuscitation, they are expanded and cultured. After at least two passages, the cells in the logarithmic growth phase are harvested and resuspended to 1×10 in serum-free medium. ⁷ pcs/mL. Take 30 male NPG mice (Beijing Weitongda Biotechnology Co., Ltd.), and inoculate 0.2 mL of cell suspension in the tail vein of all animals under sterile conditions. 17 days after tail vein inoculation with MM.1S cells, 24 animals were screened according to the intensity of tumor signals and randomly divided into 5 groups: buffer group, control T cell group, CAR-T-hu-15-2 group, CAR-T-hu -19B-2 group and BCMA-10-CAR-T group, 5 mice in each group. Prepare the pre-warmed 1640 complete medium in advance and transfer it to a 15mL centrifuge tube, 5mL per tube. Take the cryopreservation tube out of the low-temperature storage place and quickly place it in a 37°C water bath. After the water bath is dissolved, transfer the cells to a 15mL centrifuge tube and mix with the culture medium. Take an appropriate amount of cells and count the cell density and viability with trypan blue. The cells were centrifuged at 1800 rpm for 5 min, and the cell density of the test substance was adjusted with PBS. The test article was administered to each group of animals at ^{a concentration of 0.30×10 7} T cells per animal (approximately 0.15×10 ⁷ CAR-positive cells), and the administration was carried out by adjusting the administration volume.

On D7, D14, D21, and D28 days, all animals were intraperitoneally injected with 150 mg/kg of D-luciferin potassium (Thermo Fisher Scientific). The chemiluminescence signal was taken with Bruker small animal imager under isoflurane anesthesia 10-15min after injection, and the imaging time was 180s.

Result: The imaging result is shown in Figure 6. The figure can show that under the experimental conditions, CAR-T-hu15-2 and CAR-T-hu-19B-2 can obviously inhibit the proliferation of tumor cells. Compared with the positive control BCMA-10-CAR-T, CAR-T-hu15-2 and CAR-T-hu-19B-2 can quickly make the tumor regress, and control the growth of the tumor very well, showing a good Anti-tumor effect.

Claims (41)

A chimeric antigen receptor (CAR), which comprises a targeting portion, wherein the targeting portion comprises complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3), and said CDR1 comprises SEQ ID NO: the amino acid sequence shown in any one of 23, 26, 29, 32, 35, and 38. The chimeric antigen receptor according to claim 1, wherein the CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 27, 30, 33, 36, and 39. The chimeric antigen receptor according to any one of claims 1-2, wherein the CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 25, 28, 31, 34, 37 and 40. The chimeric antigen receptor according to any one of claims 1-3, wherein the CDR1, CDR2, and CDR3 comprise an amino acid sequence selected from any one of the following groups: a) CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, and CDR3: SEQ ID NO: 25; b) CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, and CDR3: SEQ ID NO: 28; c) CDR1: SEQ ID NO: 29, CDR2: SEQ ID NO: 30, and CDR3: SEQ ID NO: 31; d) CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34; e) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, and CDR3: SEQ ID NO: 37; and f) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, and CDR3: SEQ ID NO: 40. The chimeric antigen receptor of any one of claims 1-4, wherein the targeting moiety comprises VHH. The chimeric antigen receptor according to any one of claims 1-5, wherein the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 1-6 and 41-46. The chimeric antigen receptor according to any one of claims 1-6, comprising a transmembrane domain comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM. The chimeric antigen receptor according to claim 7, wherein the transmembrane domain comprises a transmembrane domain derived from CD8. The chimeric antigen receptor according to any one of claims 1-8, which comprises an intracellular costimulatory signal transduction domain, the intracellular costimulatory signal transduction domain comprising one derived from one selected from the group consisting of The intracellular costimulatory signal transduction domains of one or more proteins: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4 , FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligands, CD40 and MyD88. The chimeric antigen receptor according to claim 9, wherein the intracellular costimulatory signaling domain is derived from the costimulatory signaling domain of 4-1BB. The chimeric antigen receptor according to any one of claims 1-10, comprising an intracellular signal transduction domain, the intracellular signal transduction domain comprising one or The intracellular signal transduction domains of a variety of proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef , Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and at least one ITAM-containing domain. The chimeric antigen receptor of claim 11, wherein the intracellular signal transduction domain comprises a signal transduction domain derived from CD3. The chimeric antigen receptor according to any one of claims 7-12, which comprises a hinge region between the targeting moiety and the transmembrane domain, the hinge region comprising one or The hinge region of a variety of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1 , CD226, SLAM, CD30 and LIGHT. The chimeric antigen receptor of claim 13, wherein the hinge region comprises a hinge region derived from CD8. The chimeric antigen receptor according to any one of claims 1-14, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-12 and 53-58. An isolated antigen binding protein comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3), wherein said CDR1 comprises SEQ ID NO: 23, 26, 29, 32, 35 The amino acid sequence shown in any one of and 38. The isolated antigen binding protein of claim 16, wherein the CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 27, 30, 33, 36, and 39. The isolated antigen binding protein of any one of claims 16-17, wherein the CDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 25, 28, 31, 34, 37, and 40. The isolated antigen-binding protein of any one of claims 16-18, which comprises an antibody or an antigen-binding fragment thereof. The isolated antigen binding protein of claim 19, wherein the antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab') ₂ , scFv, di-scFv, VHH and/or dAb. The isolated antigen binding protein of any one of claims 19-20, wherein the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. The isolated antigen binding protein of any one of claims 16-21, wherein the CDR1, CDR2, and CDR3 comprise a sequence selected from any one of the following groups: a) CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, and CDR3: SEQ ID NO: 25; b) CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, and CDR3: SEQ ID NO: 28; c) CDR1: SEQ ID NO: 29, CDR2: SEQ ID NO: 30, and CDR3: SEQ ID NO: 31; d) CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34; e) CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, and CDR3: SEQ ID NO: 37; and, f) CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, and CDR3: SEQ ID NO: 40. The isolated antigen binding protein of any one of claims 16-22, which comprises VHH. The isolated antigen binding protein according to any one of claims 16-23, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-6 and 41-46. The isolated antigen binding protein of any one of claims 16-24, which comprises an Fc sequence. The isolated antigen binding protein of any one of claims 16-25, which comprises a single domain antibody. One or more isolated nucleic acid molecules encoding the chimeric antigen receptor of any one of claims 1-15 and/or the isolated antigen binding protein of any one of claims 16-26. The isolated nucleic acid molecule of claim 27, which comprises the nucleotide sequence shown in any one of SEQ ID NOs: 14-19, 47-52, and 59-64. A vector comprising the isolated nucleic acid molecule of any one of claims 27-28. The vector according to claim 29, which is a viral vector. The vector according to any one of claims 29-30, which is a lentiviral vector. A cell comprising the chimeric antigen receptor according to any one of claims 1-15, the isolated antigen binding protein according to any one of claims 16-26, and any one of claims 27-28 The isolated nucleic acid molecule, and/or the vector of any one of claims 29-31. The cell of claim 32, which comprises immune cells. The cell according to any one of claims 32-33, which comprises T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, Lymphocytes, white blood cells and/or peripheral blood mononuclear cells. A pharmaceutical composition comprising the chimeric antigen receptor according to any one of claims 1-15, the isolated antigen binding protein according to any one of claims 16-26, any one of claims 27-28 The isolated nucleic acid molecule according to item, the vector according to any one of claims 29-31, the cell according to any one of claims 32-34, and/or a pharmaceutically acceptable carrier. The antigen chimeric receptor of any one of claims 1-15, the isolated antigen binding protein of any one of claims 16-25, the isolated antigen binding protein of any one of claims 27-28 Nucleic acid molecule, the vector according to any one of claims 29-31, the cell according to any one of claims 32-34, and/or the pharmaceutical composition according to claim 35, which is used for therapy and BCMA The expression of related diseases or disorders. The use according to claim 36, wherein the disease or disorder related to the expression of BCMA includes multiple myeloma. The chimeric antigen receptor of any one of claims 1-15, the isolated antigen binding protein of any one of claims 16-26, the isolated antigen binding protein of any one of claims 27-28 Nucleic acid molecule, the vector according to any one of claims 29-31, the cell according to any one of claims 32-34, and/or the use of the pharmaceutical composition according to claim 35 in the preparation of medicines The drug is used to treat diseases or disorders related to the expression of BCMA. The use according to claim 38, wherein the disease or disorder related to the expression of BCMA includes multiple myeloma. A method for preventing or treating a disease or disorder related to the expression of BCMA, which comprises administering to a subject in need an effective amount of the chimeric antigen receptor of any one of claims 1-15, claims 16- The antigen binding protein of any one of 26, the isolated nucleic acid molecule of any one of claims 27-28, the vector of any one of claims 29-31, any of claims 32-34 The cell according to one item, and/or the pharmaceutical composition according to claim 35. The method of claim 40, wherein the disease or condition associated with the expression of BCMA includes multiple myeloma.

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