WO2023125766A1

WO2023125766A1 - Chimeric antigen receptor targeting cs1, bispecific chimeric antigen receptor targeting bcma/cs1 and use thereof

Info

Publication number: WO2023125766A1
Application number: PCT/CN2022/143236
Authority: WO
Inventors: 张超; 丁伟; 张其猛; 吕璐璐; 周立; 王永增; 张云龙; 白大勇; 路佳兴
Original assignee: 合源生物科技(天津)有限公司
Priority date: 2021-12-31
Filing date: 2022-12-29
Publication date: 2023-07-06
Also published as: CN116410331A; CN116410331B

Abstract

The present invention provides a chimeric antigen receptor targeting CS1, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain, wherein the extracellular antigen recognition domain comprises an scFv antibody against CS1; amino acid sequences of VH complementarity determining regions CDR1, CDR2 and CDR3 of the scFv antibody respectively comprise amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and amino acid sequences of VL complementarity determining regions CDR1, CDR2 and CDR3 of the scFv antibody respectively comprise amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

Description

Chimeric antigen receptors targeting CS1, bispecific chimeric antigen receptors targeting BCMA/CS1 and applications thereof

technical field

The present application relates to the field of biomedicine, in particular to a chimeric antigen receptor targeting CS1, a bispecific chimeric antigen receptor targeting BCMA/CS1 and applications thereof.

Background technique

Multiple myeloma, defined as the malignant proliferation of plasma cells in the bone marrow, is the second most common hematological malignancy, accounting for 1% of all cancers. Studies have shown that multiple myeloma has a high incidence in the elderly over 60 years old and the incidence has increased steadily in recent years. For most patients, multiple myeloma is incurable and will eventually develop into relapsed/refractory multiple myeloma. The survival period of patients with relapsed/refractory multiple myeloma who are ineffective with existing multiple myeloma treatments (such as immunomodulators, proteasome inhibitors, antibody drugs) is only about 13 months.

CS1, also known as SLAMF7, CRACC or CD319, is a glycoprotein expressed on the surface of myeloma cells, which is a strong marker between normal plasma cells and malignant plasma cells in multiple myeloma. Chu et al. developed two second-generation CARs for the treatment of multiple myeloma, one target antigen is BCMA, and the other target antigen is CS1; in vitro experiments, although two CAR-Ts with different targets showed Similar killing activity, but in vivo experiments in mice, CS1 CAR-T cells have stronger anti-tumor activity.

A chimeric antigen receptor (Chimeric Antigen Receptor, CAR) is the core component of a CAR cell therapy drug, which may include a targeting moiety (for example, a part that binds a tumor-associated antigen (Tumor-Associated Antigen, TAA)), a hinge region, a spanning Membrane domains and intracellular domains. CAR-T cell immunotherapy is considered to be one of the most promising means to overcome tumors. CAR-T cells use genetic modification to enable T cells to express CAR proteins. This CAR protein has the ability to recognize the intact protein on the membrane surface without relying on antigen presentation, thereby causing the activation and functional effects of T cells.

In 2021, Bristol-Myers Squibb and Bluebird Bio jointly announced that the U.S. Food and Drug Administration (FDA) has approved its BCMA-targeted CAR-T cell therapy (bb2121) for use after 4 lines of treatment (including immunomodulators, proteases, etc.) Adult patients with relapsed or refractory multiple myeloma treated with body inhibitors and antibody drugs), but no CS1-targeting CAR-T cell therapy has been marketed. It is of practical significance to develop better cell therapy methods targeting CS1.

Contents of the invention

The present application provides a chimeric antigen receptor targeting CS1, a bispecific chimeric antigen receptor targeting BCMA/CS1 and applications thereof. The inventors used multiple CS1-targeting scFv antibodies to construct chimeric antigen receptor expression vectors and prepare CS1-targeting CAR-T cells, and also verified that CS1 CAR-T cells have good tumor-suppressing functions and Determination of the optimal chimeric antigen receptor targeting CS1.

A chimeric antigen receptor targeting CS1, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain comprises an anti-CS1 scFv antibody, The amino acid sequences of the VH complementarity-determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the VL complementarity-determining regions of the scFv antibody The amino acid sequences of regions CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

In certain embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 7, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 8. amino acid sequence.

In certain embodiments of the above-mentioned chimeric antigen receptor, the scFv antibody is a humanized antibody.

In certain embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 9, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 10. amino acid sequence.

In certain embodiments of the above-mentioned chimeric antigen receptor, the scFv antibody is a rabbit-derived antibody.

In certain embodiments of the above-mentioned chimeric antigen receptor, there is a connecting region between VH and VL in the scFv antibody, and the connecting region is selected from one or more of the following: SEQ ID NOs: 59-61.

In some embodiments of the above-mentioned chimeric antigen receptor, the sequence of the scFv antibody is shown in SEQ ID NO: 11 or SEQ ID NO: 12.

In certain embodiments of the above-mentioned chimeric antigen receptor, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α.

In some embodiments, the above-mentioned chimeric antigen receptor, the transmembrane region is derived from one of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70 or Various.

In some embodiments of the above-mentioned chimeric antigen receptor, wherein the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a co-stimulatory signal transduction region.

In some embodiments, the above-mentioned chimeric antigen receptor, wherein the intracellular signaling region is derived from CD3δ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, Syk one or more of.

In certain embodiments of the above-mentioned chimeric antigen receptor, wherein the co-stimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1 One, two or more of , ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, MyD88.

In some embodiments, the above chimeric antigen receptor further comprises a leader peptide located at the N-terminal of the amino acid sequence of the chimeric antigen receptor; optionally, the leader peptide is derived from CD8α.

In certain embodiments of the above chimeric antigen receptor, the extracellular antigen recognition domain further comprises scFv antibodies against one of the following targets: CD138, NKG2D, CD38, BCMA, CD19, CD70, CD44v6, Lewis Y.

In certain embodiments of the above-mentioned chimeric antigen receptor, the extracellular antigen recognition domain sequentially comprises anti-BCMA scFv VL, anti-CS1 scFv VL, anti-CS1 scFv VH and anti-BCMA scFv VH, the anti- The amino acid sequences of the scFv VH complementarity-determining regions CDR1, CDR2, and CDR3 of CS1 include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively, and the scFv VL complementarity-determining region of the anti-CS1 The amino acid sequences of CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acids of the scFv VH complementarity determining regions CDR1, CDR2, and CDR3 of the anti-BCMA The sequences include the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively, and the amino acid sequences of the anti-BCMA scFv VL complementarity determining regions CDR1, CDR2, and CDR3 include SEQ ID NO: 54. The amino acid sequence shown in SEQ ID NO:55, SEQ ID NO:56.

In certain embodiments, the VH sequence of the anti-CS1 scFv antibody includes the amino acid sequence shown in SEQ ID NO: 7, and the VL sequence of the anti-CS1 scFv antibody includes SEQ ID Amino acid sequence shown in NO:8.

Above-mentioned chimeric antigen receptor In some embodiments, the VH sequence of the scFv antibody against BCMA comprises the amino acid sequence shown in SEQ ID NO: 57, and the VL sequence of the scFv antibody against BCMA comprises the sequence of SEQ ID NO: The amino acid sequence shown in NO:58.

In some embodiments of the above-mentioned chimeric antigen receptor, the extracellular antigen recognition domain includes the amino acid sequence shown in SEQ ID NO:50.

The present application also provides an isolated nucleic acid molecule comprising the nucleotide sequence encoding the above-mentioned chimeric antigen receptor.

The present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

In some embodiments, the above-mentioned vector is an expression vector; in some embodiments, the vector is a viral vector; in some embodiments, it is a lentiviral vector.

The present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned carrier.

In some embodiments, the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and differentiated pluripotent stem cells. T cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC- NK), one or more of embryonic stem cells.

In some embodiments, the above-mentioned immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

The present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable auxiliary materials.

In some embodiments of the above pharmaceutical composition, the pharmaceutically acceptable excipients include protective agents.

In some embodiments of the above pharmaceutical composition, the pharmaceutically acceptable adjuvant includes cell cryopreservation solution.

In some embodiments, the above-mentioned pharmaceutical composition is an intravenous injection.

The present application also provides the use of the above-mentioned chimeric antigen receptor, nucleic acid molecule, carrier or immune effector cell in the preparation of medicines for treating diseases or conditions related to the expression of CS1.

In some embodiments, the above-mentioned use, the disease or disease associated with the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or recurrent multiple myeloma.

The above uses In certain embodiments, the disease or disorder associated with the expression of CS1 may be an autoimmune disease.

In some embodiments of the above use, the autoimmune disease may be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune hemolytic anemia.

The above use In some embodiments, the drug is an intravenous injection.

The present application also provides a method for treating diseases or disorders related to the expression of CS1, comprising the following steps: administering an effective amount of the above-mentioned immune effector cells or the pharmaceutical composition to a disease or disorder associated with the expression of CS1. subjects in need.

In some embodiments of the above method, the disease or disorder associated with the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or recurrent multiple myeloma.

The above methods In certain embodiments, the disease or condition associated with the expression of CS1 may be an autoimmune disease.

In some embodiments of the above method, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.

In some embodiments of the above methods, the administration is by intravenous injection.

In some embodiments of the above method, the administering method is to administer an effective amount of the immune effector cells or the pharmaceutical composition to the subject in a single injection.

In some embodiments of the above method, the effective amount of immune effector cells or the pharmaceutical composition is 1×10 ⁵ to 1×10 ⁷ cells/kg.

The present application also provides the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition for treating diseases or diseases related to the expression of CS1.

In some embodiments, the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition, the disease or disease related to the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is Refractory or relapsed multiple myeloma.

In certain embodiments of the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition, the disease or disorder associated with the expression of CS1 may be an autoimmune disease.

The above-mentioned immune effector cells or the above-mentioned pharmaceutical composition In some embodiments, the autoimmune disease can be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune Immune hemolytic anemia.

Description of drawings

Figure 1 shows a schematic diagram of the CS1 CAR structure in Example 1 of the present application.

Figure 2 shows the staining of UTD cells (T cells not transduced with CAR), CS1 CAR-T cells 21G-27G, and positive control CS1 CAR-T cells LUC90V2 with PE fluorescence-labeled CS1 antigen in Example 2 of the present application. The results of detecting the percentage of CAR molecules expressed on the surface.

Figure 3 shows the results of staining UTD cells, CS1 CAR-T cells 21G-27G, and positive control CAR-T cells 02G with fluorescently-labeled anti-human IgG antibodies in Example 2 of the present application to detect the percentage of CAR molecules expressed on their surfaces picture.

Figure 4 shows the release of IL-2 after CS1 CAR-T cells were activated by CS1 positive target cells detected in Example 3 of the present application. For K562 cells and K562-CS1 cells, from left to right are UTD, 21G cells, The release of IL-2 from 22G cells, 23G cells, 24G cells, 25G cells and 26G cells.

Figure 5 shows the killing effect of CS1 CAR-T cells on CS1 positive target cells in Example 3 of the present application. For K562 cells and K562-CS1 cells, from left to right are the killing effects of UTD, 21G cells, 22G cells, 23G cells, 24G cells, 25G cells, 26G cells and positive control LUC90V2 cells on target cells.

Figure 6A shows the continuous proliferation of CD3+ cells after CS1 CAR-T cells are activated by multiple rounds of antigen stimulation in Example 4 of the present application; Figure 6B shows the activation of CS1 CAR-T cells by multiple rounds of antigen stimulation in Example 4 of the present application , Continuous proliferation of CAR+ cells.

Fig. 7 shows a schematic diagram of the construction of the BCMA-CS1 bispecific CAR in Example 5 of the present application.

Figure 8 shows the results of flow cytometry for the detection of the positive rate of BCMA-CS1 bispecific CAR-T cells in each group in Example 6 of the present application.

Figure 9A, Figure 9B, and Figure 9C show the effects of BCMA-CS1 bispecific CAR-T cells, negative control cells, and positive control cells in Example 7 of the present application on double-negative target cells K562 and exogenous BCMA-positive cells K562- BCMA, exogenous CS1 positive cell K562-CS1 cell killing experiment results.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

The application is further described as follows: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In this application, the term "Chimeric Antigen Receptor" (Chimeric Antigen Receptor, CAR) is the core component of CAR cell therapy drugs, which may include extracellular antigen recognition domains (for example, binding tumor-associated antigen (Tumor-Associated Antigen) , part of TAA), hinge region, transmembrane region and intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered to be one of the most promising means to overcome tumors. CAR-T cells use genetic modification to enable T cells to express CAR proteins. This CAR protein has the ability to recognize the intact protein on the membrane surface without relying on antigen presentation, thereby causing the activation and functional effects of T cells.

In this application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (Antigen Recognition Domain, ARD). The ability of CAR cell therapy products (such as CAR-T cells) to specifically recognize and/or bind to target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. Chain variable region (Single Chain Variable Fragment, abbreviated as scFv), or derived from receptor ligand interaction, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR). By far the most common source is the scFv fragment of an antibody. Unless otherwise specified in this application, the scFv antibody refers to the scFv antibody targeting CS1. scFv antibodies can include one, two or more antibody heavy chain variable regions (VH) and one, two or more light chain variable regions (VL), connected by a peptide chain , such as: the connecting sequence GSTSGSGKPGSGEGSTKG composed of 18 amino acids. In the variable region of the antibody, there are a small number of amino acid residues that change particularly strongly, and the residue composition and sequence of these amino acids are more likely to change. The regions are called hypervariable regions (HVR); in the V regions of the L chain and H chain Each has three hypervariable regions, which are also called complementarity determining regions (CDRs) because they can form precise complementarity with antigenic determinants in terms of spatial structure. In antibodies, common CDR division rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying the website that implements these rules, just input the VH and VL sequences and select the corresponding rules. CDR sequences according to different rules can be obtained. Those skilled in the art should understand that the protection scope of the present application covers the combination of CDR sequences obtained by analysis using different rules. In this application, the CDR is divided according to the IMGT rule.

In this application, the term "specific recognition and/or binding" refers to the recognition and/or binding between CAR and a specific target, with greater affinity, avidity, easier, and/or bind the target for a greater duration.

In the present application, the term "humanized antibody" is also referred to as a humanized engineered antibody, which is obtained by combining the complementarity-determining regions (CDRs) of non-human mammalian antibodies, such as mouse antibodies, rat antibodies, and rabbit antibodies. ) are transplanted into the CDRs of human antibodies for preparation, and conventional recombinant DNA techniques for preparing humanized antibodies are known (eg WO96/02576). For example, when the CDRs are obtained from a rabbit antibody, primers can be synthesized (corresponding primers can be obtained by referring to the method described in WO98/13388) and used to link the CDRs of the rabbit antibody to the framework regions (FRs) of the human antibody. As for human antibody FRs linked to CDRs, those are selected such that the CDR regions form good antigen-binding sites.

In this application, the term "hinge region" refers to the link between the extracellular antigen recognition domain and the transmembrane domain. This region allows CAR to recognize antigen by giving the antigen recognition domain a certain range of motion. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, the typical hinge region also contains residues that are involved in CAR dimerization and contribute to enhanced antigen sensitivity.

In this application, "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but they are not directly involved in signal transmission, and the downstream signal transmission can be improved through interaction. The transmembrane region can be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

Within this application, the term "intracellular domain" includes intracellular signaling regions and may also include co-stimulatory signaling regions.

In the present application, the term "intracellular signaling region" refers to the activation of at least one normal effector function of an immune effector cell responsible for the expression of CAR. The intracellular signaling region can be derived from one or more of CD3δ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

In this application, the term "co-stimulatory signaling domain" exists because, in addition to the stimulation of antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, and activation of cell activation. Effector function. In some embodiments, the CAR may also include one or more co-stimulatory signaling regions, wherein the co-stimulatory signaling regions may be derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4- One, two or more of 1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, MyD88.

In the present application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude artificial or synthetic substances obtained from the natural state by artificial means, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

In this application, the term "guide peptide" refers to a short peptide before the extracellular antigen recognition domain (such as scFv sequence), whose function is to guide the export of the recombinant protein synthesized in the cell to the outside of the cell. Commonly used guide peptides are human CD8α signal peptide, or human GM-CSF receptor α signal peptide.

In this application, one of the key factors determining the efficacy of CAR-immune cell therapy is the selection of tumor target antigens. In this application, the term "CS1" is also called SLAMF7 (signaling-lymphocyte-activating molecule F7) or CD319, which is a glycoprotein on the cell surface, expressed in plasma cells, NK cells, CD8+ T cells, activated In normal tissues such as B cells and dendritic cells, but not expressed in hematopoietic stem cells and non-hematopoietic organs, it can participate in the mutual adhesion between myeloma cells and bone marrow stromal cells. The term "BCMA" refers to B cell maturation antigen, a member of the tumor necrosis factor receptor superfamily. Human BCMA is expressed almost exclusively in plasma cells and multiple myeloma cells. BCMA may be a suitable tumor antigen target for immunotherapeutics against multiple myeloma. However, due to the heterogeneity of specific antigens on the surface of multiple myeloma cells, the selection of its antigen target is not necessarily single. By selecting appropriate targets, the anti-tumor activity of CAR-T cells can be optimized.

In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, which may be isolated from its natural environment or a synthetic analogue .

In this application, when referring to CAR gene transduction/transfection and target gene expression, gene transduction/transfection methods mainly include viral and non-viral methods. Such as: through γ-retroviral vectors, lentiviral vectors, adeno-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

The term "vector" generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect the host cell, so that the genetic material elements carried by it can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes or protein coats, but not only. The term "transposon" refers to a discontinuous DNA segment that has the ability to migrate between chromosomal sites and carry genetic information, such as: Sleeping Beauty SB system and PB system derived from Lepidoptera insects. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, eg, promotes an immune effector response. Immune effector cells may be selected from the group consisting of: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells, induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), embryonic stem cells one or more species.

In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to patients, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as : one or more of carrier, protective agent, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative. In some embodiments, the pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solution. In some embodiments, the pharmaceutical composition of the present application is a cell suspension or frozen cells thereof.

In this application, the term "subject" generally refers to human or non-human animals, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cows, sheep or monkeys.

In this application, the term "comprising" generally means including specifically specified features, but not excluding other elements.

In the present application, the term "about" usually refers to the fluctuation range acceptable to those skilled in the art above or below the specified value, such as: within the range of ±0.5%-10%, for example, 0.5% above or below the specified value %, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% range.

Chimeric antigen receptors, nucleic acids, vectors, immune effector cells, pharmaceutical compositions

In one aspect, the present application provides a chimeric antigen receptor targeting CS1, which comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain comprises Anti-CS1 scFv antibody, the amino acid sequences of the VH complementarity determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively, and the The amino acid sequences of the VL complementarity determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

In some embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:7, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:8.

In some embodiments, the scFv antibody is a humanized antibody.

In some embodiments, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:9, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO:10.

In some embodiments, the scFv antibody is a rabbit antibody.

In some embodiments, there is a connecting region between VH and VL in the scFv antibody, and the connecting region is selected from one or more of the following: SEQ ID NOs: 59-61.

In some embodiments, the sequence of the scFv antibody is shown in SEQ ID NO: 11 or SEQ ID NO: 12.

In some embodiments, the present application also includes substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence of any one of the above chimeric antigen receptors, and it has the equivalent of any one of the above Chimeric antigen receptor activity; optionally, the substitutions are conservative substitutions. Those skilled in the art know that during the process of humanization, amino acids in the scFv FR region can be substituted so that the CDR region of the engineered antibody can retain a suitable antigen-binding site. Therefore, this application is certainly included in this application. In the case of the above CDR, the humanized transformation of the FR region in the scFv is different from the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 . And those skilled in the art also know that in order to make the CDR region of the modified antibody retain a suitable antigen-binding site during the process of humanization, if necessary, one, two, three or none of the CDRs More than 10% of the amino acid sequence may be substituted, deleted, added and/or inserted, and these are also included in this application.

In some embodiments, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the amino acid sequence of the hinge region is derived from CD8α; further Optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO: 13.

In some embodiments, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70; alternatively, The amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:14.

In some embodiments, wherein the intracellular domain comprises an intracellular signaling region; optionally, also includes a co-stimulatory signaling region; further optionally, wherein the intracellular signaling region is derived from CD3δ, CD3γ , one or more of CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, Syk; still further optionally, the intracellular signaling region is derived from CD3δ, For example: the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:15.

In some embodiments, wherein the co-stimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, One, two, or more than three of NKG2D, DAP10, B7-H3, and MyD88; optionally, the costimulatory signal transduction region is derived from CD28 or 4-1BB; further optionally, the costimulatory signal transduction region The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:16.

In some embodiments, the chimeric antigen receptor further comprises a guide peptide located at the N-terminal of the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8α; further optionally, The amino acid sequence of the leader peptide comprises the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the chimeric antigen receptor of the present invention comprises the amino acid sequence shown in SEQ ID NO:32.

In some embodiments, the extracellular antigen recognition domain only comprises a scFv antibody targeting a single CS1 target.

In some embodiments, the extracellular antigen recognition domain further comprises scFv antibodies against any of the following targets: CD138, NKG2D, CD38, BCMA, CD19, CD70, CD44v6, Lewis Y.

In some embodiments, the extracellular antigen recognition domain sequentially comprises anti-BCMA scFv VL, anti-CS1 scFv VL, anti-CS1 scFv VH and anti-BCMA scFv VH, and the anti-CS1 scFv VH complementarity determining region The amino acid sequences of CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acids of the scFv VL complementarity determining regions CDR1, CDR2, and CDR3 of the anti-CS1 The sequences include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and the amino acid sequences of the anti-BCMA scFv VH complementarity determining regions CDR1, CDR2, and CDR3 include SEQ ID NO: 51. The amino acid sequences shown in SEQ ID NO:52 and SEQ ID NO:53, the amino acid sequences of the anti-BCMA scFv VL complementarity determining regions CDR1, CDR2, and CDR3 respectively include SEQ ID NO:54, SEQ ID NO:55 , the amino acid sequence shown in SEQ ID NO:56.

In some embodiments, the VH sequence of the anti-CS1 scFv antibody includes the amino acid sequence shown in SEQ ID NO:7, and the VL sequence of the anti-CS1 scFv antibody includes the amino acid sequence shown in SEQ ID NO:8 sequence.

In some embodiments, the VH sequence of the anti-BCMA scFv antibody includes the amino acid sequence shown in SEQ ID NO:57, and the VL sequence of the anti-BCMA scFv antibody includes the amino acid sequence shown in SEQ ID NO:58 sequence.

In some embodiments, the extracellular antigen recognition domain includes the amino acid sequence shown in SEQ ID NO:50.

On the other hand, the present application also provides an isolated nucleic acid molecule comprising the nucleotide sequence encoding the above-mentioned chimeric antigen receptor.

In another aspect, the application provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, which is selected from one of the following nucleic acid molecules: comprising the nucleotide sequence shown in SEQ ID NO: 18, or Nucleic acid molecule that is similar to the nucleotide sequence shown in :18 and encodes the same chimeric antigen receptor nucleotide sequence.

In some embodiments, the similarity to the nucleotide sequence shown in SEQ ID NO: 18 refers to having at least 70%, 75%, 80%, 85% of the nucleotide sequence shown in SEQ ID NO: 18 , 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some embodiments, the nucleotide sequence shown in SEQ ID NO: 18 is similar to that shown in SEQ ID NO: 18 due to the wobble (degeneracy) of the third base of the nucleic acid codon. Nucleotide sequences are different, but they encode the same chimeric antigen receptor nucleic acid molecule. Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:39.

On the other hand, the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule. Vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc. . Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).

In some embodiments, the vector is an expression vector; optionally, the vector is a viral vector; further optionally, a lentiviral vector.

On the other hand, the present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned carrier.

In some embodiments, the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, pluripotent NK cells differentiated from stem cells, induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), embryonic stem cells one or more of.

In some embodiments, the immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

In some embodiments, the surface of the immune effector cells may express the chimeric antigen receptors described herein.

On the other hand, the present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable auxiliary materials. The pharmaceutically acceptable auxiliary materials include: one or more of carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

In some embodiments, the pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solution.

In some embodiments, the pharmaceutical composition is a cell suspension or frozen cells thereof.

In some embodiments, the pharmaceutical composition is an intravenous injection.

Preparation method and use

On the other hand, the present application also provides a method for preparing immune effector cells, which includes the following steps: transducing the vector described in the present application into the immune effector cells.

On the other hand, the present application also provides the use of the chimeric antigen receptor described in the present application, the nucleic acid molecule, the carrier and/or the immune effector cell for the preparation of a drug, wherein the drug For use in the treatment of a disease or condition associated with the expression of CS1.

On the other hand, the present application also provides a method for treating a disease or disorder associated with the expression of CS1, the method comprising administering an effective dose of the present invention to a subject in need of treating a disease or disorder associated with the expression of CS1. The chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the immune effector cell.

In some embodiments, the administration can be performed by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. For example, the mode of administration may be administered to a subject by intravenous injection. In some embodiments, the effective dose of immune effector cells or the pharmaceutical composition can be administered to the subject once, or dividedly administered to the subject within a certain period of time, such as once a week, once every two weeks, Once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

In some embodiments, for different indications, the dosage may be different; for patients with different disease severity, the dosage may also be different. The administered dose may range from 1×10 ⁵ CAR-positive T cells/kg to 1×10 ⁷ CAR-positive T cells/kg, for example, 1×10 ⁵ CAR-positive T cells/kg to 1×10 ⁶ CAR-positive T cells/kg, 1×10 ⁶ CAR-positive T cells/kg to 1×10 ⁷ CAR-positive T cells/kg. The administered dose can also be measured by the total administered dose, for example: the total administered dose does not exceed 5×10 ⁸ CAR-positive T cells. In this application, the administration dose is based on the count of CAR-positive T cells, and details will not be repeated in the specific examples.

In some embodiments, the subjects can include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cows, sheep, rabbits or monkeys.

On the other hand, the present application also provides the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the immune effector cell, which can be used to treat diseases related to the expression of CS1 or illness.

In some embodiments, the disease or condition associated with the expression of CS1 may include non-solid tumors, and optionally, the non-solid tumors are hematological tumors.

In some embodiments, the disease or disorder associated with the expression of CS1 may include multiple myeloma.

In some embodiments, the multiple myeloma is relapsed or refractory multiple myeloma.

In some embodiments, the disease or disorder associated with expression of BCMA may be an autoimmune disease.

In some embodiments, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.

Without intending to be limited by any theory, the following examples are only for explaining the chimeric antigen receptor, immune effector cell, preparation method and application of the present application, and are not intended to limit the scope of the present invention. The Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, to insert genes encoding proteins into such vectors and plasmids, or to introduce plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold Spring Harbor Laboratory Press.

Example 1. Acquisition of CS1 CAR-T cells

We have 7 CS1-specific humanized scFv sequences (the amino acid sequences of these scFv are respectively as SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22. As shown in SEQ ID NO: 23 and SEQ ID NO: 24, the amino acid sequences of these seven scFvs are respectively referred to as

numbers

21G, 22G, 23G, 24G, 25G, 26G, and 27G in this application. The above-mentioned 21G～ The nucleotide sequences of 27G scFv are as SEQ ID NO:25, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 shown). Since the functional verification of scFv at the protein level cannot reflect its function at the cellular level, we chose to screen candidate CS1 scFv sequences on the second-generation CAR structure. The schematic diagram of the CAR structure is shown in Figure 1. We used the CD8α guide chain as the signal peptide (its amino acid sequence is shown in SEQ ID NO: 17), CS1scFv as the extracellular tumor antigen recognition region, and CD8α as the hinge region and transmembrane region. Structure (the amino acid sequence is shown in SEQ ID NO:13 and SEQ ID NO:14), 4-1BB is the intracellular co-stimulatory signal (the amino acid sequence is shown in SEQ ID NO:16), and CD3δ is the T cell Activation signal (its amino acid sequence is shown in SEQ ID NO: 15).

1. Construction of lentiviral vector

Artificially synthesize CAR structure fragments containing 21G-27G scFv in this application (the amino acid sequences of these CAR structures are respectively as SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID As shown in NO:35, SEQ ID NO:36, and SEQ ID NO:37, the nucleotide sequences encoding the above CAR structure are respectively as SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO : 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44), and respectively constructed to the transformed empty lentiviral vector (manufacturer: SBI company, article number: CD500-CD800, such as WO2021/ 121227 The CAR expression vector was obtained from the method described in Example 1, and then the CAR expression vector and three packaging plasmids were transfected into 293T cells, and a functional lentiviral vector was obtained after collection and purification. The three packaging plasmids are pMD2.0G (purchased from Biovector, product number Biovector012259), pMDLg-/pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253).

2. Preparation of corresponding 7 kinds of CS1 CAR-T cells by lentiviral transduction

The transduction experiment was carried out according to conventional methods known to those skilled in the art, and the transduction steps are briefly described as follows:

1) Sorting T cells

Peripheral blood mononuclear cells (PBMC) were isolated from human single blood cells, and then T cells were sorted from PBMC cells.

2) Activate T cells

The isolated T cells were treated with complete lymphocyte culture medium (X-VIVO15 medium+5%FBS+300IU/ml IL-2 or X-VIVO15 medium+5%FBS+5ng/ml IL-15+10ng/ml IL -7) Resuspend to make the final concentration (1～2)×10 ⁶ cells/ml, and add 5～10 μl of CD3/CD28 magnetic beads to stimulate, mix well and place in the incubator for culture, the culture condition is 37 °C + 5% CO ₂ , the incubation time is at least 24 hours.

3) Lentiviral transduction of T cells

Take out the activated T cells, add polybrene (polybrene) with a final concentration of 8 μg/ml, mix well, and slowly add lentiviral vector according to MOI=2, put it in a centrifuge after mixing, and centrifuge at 1500rpm 1.5 hours. Then place it in an incubator for cultivation, the cultivation condition is 37° C.+5% CO ₂ , and the cultivation time is at least 24 hours.

4) Expansion and culture of T cells after transduction

The transduced cells were taken out, and the cell density was monitored to maintain the cells at (0.5-1)×10 ⁶ cells/ml for use in subsequent examples.

The T cells obtained after infecting T cells with lentiviruses containing 21G-27G scFv were named CS1 CAR-T cells 21G-27G, respectively. Next, we screened seven CAR structures at the cellular level to finally determine an optimal candidate scFv sequence.

Example 2. Detection of CAR molecules expressed on the surface of CS1 CAR-T cells 21G-27G

The CAR protein molecules expressed on the surface of the 21G-27G CS1 CAR-T cells obtained in Example 1 were detected. We used PE fluorescence-labeled CS1 antigen (manufacturer: ACRO Biosystems, product number: SL7-HP2H3) to stain 7 kinds of CS1 CAR-T cells, UTD cells (T cells not transduced with CAR), LUC90V2 CAR-T cells (LUC90V2 is Positive control CS1 CAR sequence, the CAR sequence is shown in SEQ ID NO: 63, the scFv in the CAR sequence is a mouse sequence, the method for obtaining the CAR-T cells is also the method in Example 1), by flow cytometry Cytometry was used to detect and analyze the positive ratio of CAR. The results are shown in Figure 2: the CAR-positive ratios of CS1 CAR-

T cells

22G, 26G, 21G, and 23G were high, reaching 96.70%, 82.61%, 77.61%, and 68.99%, respectively, but the CS1 CAR-

T cells

24G, 25G, and 27G hardly detected CAR protein expression. In addition, since the CS1 antigen was used for staining in the experiment, the above results also suggested that the ability of these CAR proteins to specifically recognize the CS1 antigen was different.

In the above experiment of staining CS1 CAR-T cells with PE fluorescently labeled CS1 antigen, almost no CAR protein expression was detected in CS1 CAR-

T cells

24G, 25G, and 27G. Whether CS1 CAR is not expressed or CS1 CAR is expressed but cannot recognize CS1 antigen, we further use fluorescently labeled anti-human IgG antibody (manufacturer: invitrogen, product number: A11013) to treat 7 kinds of CS1 CAR-T cells, UTD cells, Positive control BCMA CAR-T cell 02G staining (because scFv 21G ~ 27G are humanized antibodies, which contain human FR regions, and human T cells basically do not express IgG, so use anti-human IgG antibody Staining can clarify whether CS1 CAR-

T cells

24G, 25G, and 27G do not express CS1 CAR on the surface or they cannot recognize CS1 antigen), and the CAR positive ratio is detected and analyzed by flow cytometry. In addition, since the scFv in the LUC90V2 CAR sequence is of mouse origin and does not contain human FR regions, BCMA CAR-T cell 02G containing another humanized scFv was used as a positive control in this method (the sequence of BCMA 02G CAR is as follows: shown in SEQ ID NO:62). The results are shown in Figure 3: In this method, the CAR-positive ratios of CS1 CAR-

T cells

22G, 26G, 21G, and 23G were still high, reaching 85.14%, 79.76%, 79.95%, and 79.55%, respectively; CS1 CAR-T cells Both

cells

24G and 25G can clearly detect CAR-positive cells, indicating that the CAR protein on the surface of CS1 CAR-

T cells

24G and 25G can be expressed, but it may not be able to recognize the CS1 antigen, while CS1 CAR-T cell 27G still does not detect CAR protein Express.

Example 3, CS1 CAR-T cells were subjected to cytokine release experiments and cell killing experiments

1. Cytokine release experiment: The CS1 CAR-T cells 21G-26G obtained in Example 1 (because there is no CAR protein expression on the surface of CS1 CAR-T cells 27G, so this cell will not be involved in subsequent experiments) and target cells After co-cultivating in X-VIVO15 medium for 24 hours according to the condition that the effect-to-target ratio of CAR+ cells and target cells was 1:1, the concentration of IL-2 in the cell supernatant was detected by ELISA method (manufacturer: Dakowi, article number: 1110202). Among them: K562 is the CS1-negative target cell, K562-CS1 is the positive target cell expressing CS1 exogenously, and the UTD group is used as the negative control. The results of cytokine release experiments are shown in Figure 4: the IL-2 release levels of CS1 CAR-

T cells

21G, 22G, and 26G were significantly higher than those of CS1 CAR-

T cells

23G, 24G, 25G, and UTD groups.

2. Cell killing experiment: CS1 CAR-T cells 21G-26G obtained in Example 1 and target cells were co-cultured in X-VIVO15 medium for 48 hours according to the condition of CAR+ cells and target cell effect-to-target ratio of 1:1, Detect target cells by detecting stably expressed luciferase activity in target cells (construct a lentiviral expression vector containing green fluorescent protein GFP and luciferase Luc coding region, then package lentivirus, transduce K562-CS1 cells with lentivirus, Use the GFP signal to sort positive monoclonal cells by flow cytometry, through culture expansion and GFP expression identification, after confirming that they are monoclonal, the cell preparation is completed) the survival ratio (the detection reagent is purchased from Promega, product number: E2520), the results are as follows As shown in Figure 5: CS1 CAR-

T cells

21G, 22G, 23G, 26G and the positive control CS1 CAR-T cell LUC90V2 all had good killing effect on the positive target cell K562-CS1 expressing CS1 exogenously.

Example 4, Sustained proliferation of CS1 CAR-T cells

Antigen stimulation can activate CAR-T cells to proliferate CAR-T cells, and the continuous activation of T cells will lead to cell exhaustion, and the proliferation ability and effector function of exhausted T cells will decrease. We detected CS1 CAR-T cells 21G The proliferation of CD3+ cells (i.e. the proliferation of T cells, CD3 is a marker to distinguish whether they are T cells) and the proliferation of CAR+ cells after multiple rounds of antigen stimulation experiments of ~26G, to verify the continuous proliferation of CS1 CAR-T cells .

Before antigen stimulation, the CAR-positive ratio of CS1 CAR-T cells in each group was adjusted to a level consistent with that of the group of CAR-T cells with the lowest CAR-positive ratio using T cells that were not transduced with CAR. In multiple rounds of antigen stimulation experiments, CS1 CAR-T cells in each group were co-cultured with CS1 endogenous positive target cells MM.1S (human multiple myeloma cells) in a 24-well plate according to the effect-to-target ratio of 1:2. , 2ml X-VIVO15 medium per well, repeat 3 wells for each group of cells. After 3 to 4 days, 500 μl of cells were stained with fluorescently labeled CD3 antibody (manufacturer: BioLegend, product number: 317318) and CS1 antigen (same as Example 2) for CD3 and CAR, and detected and analyzed by flow cytometry, showing CD3 positive cells The proportion, number and fluorescence intensity of CAR-positive cells in the sample can also be calculated according to the conversion of the volume multiple, and the number of CAR-positive cells in the CD3-positive cells in the sample can be calculated, and then the CAR-T cells are taken out from each group according to the calculation results according to the effect-to-target ratio: 1: 2 Add MM.1S cells for a new round of stimulation, and repeat until CAR-T cell proliferation stagnates, ending antigen stimulation.

The results of sustained proliferation are shown in Figure 6A and Figure 6B: only CS1 CAR-

T cells

22G and 26G can maintain the expansion ability for 23 days under the condition of repeated antigen stimulation, among which CD3+ cells and CAR+ cells in CS1 CAR-T cell 22G Cell proliferation was the best and was significantly better than that of CD3+ cells and CAR+ cells in 26G.

In summary, in vitro pharmacodynamic tests show that the expression of 22G CAR on the surface of T cells is superior to other candidates; it is superior to other candidate CAR-T in terms of cytokine release, in vitro cell killing, and sustained proliferation of CAR-T. . CDR1, CDR2, and CDR3 of VH in 22G scFv are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and CDR1, CDR2, and CDR3 of VL in 22G scFv are shown in SEQ ID NO:4, Shown in SEQ ID NO:5 and SEQ ID NO:6.

Example 5, Preparation of BCMA-CS1 bispecific CAR-T cells

We chose to screen candidate BCMA-CS1 bispecific scFv sequences on the second-generation CAR construct. The 02G scFv selected for the BCMA target (the CDR1, CDR2, and CDR3 of its VH are shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively, and the CDR1, CDR2, and CDR3 of its VL are shown in Shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, the amino acid sequence of scFv VH is shown in SEQ ID NO:57, and the amino acid sequence of scFv VL is shown in SEQ ID NO:58), for After the 22G scFv was selected for the CS1 target, the construction of the BCMA-CS1 bispecific CAR began. Considering the changes in the sequence of target BCMA scFv and CS1 scFv, the sequence of VH and VL in each target scFv, the type of linker, and the different structures of tandem and loop, there are many structural designs for BCMA-CS1 bispecific CAR. One option, tried four tandem structures and two loop structures as shown in Figure 7, and built them into the second-generation CAR structure. We use CD8α guide chain as signal peptide, BCMA-CS1 bispecific scFv as extracellular tumor antigen recognition region, hinge region and transmembrane region as CD8α structure, 4-1BB as intracellular co-stimulatory signal, CD3δ as T Cell activation signal. In addition to replacing the CAR sequence, the method for preparing CS1 CAR-T cells in Example 1 was still used to prepare BCMA-CS1 bispecific CAR-

T cells

41A, 41B, 42A, 42B, 43A, 43B (the sequence of the corresponding scFv Respectively such as: SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50), the amino acid sequence of the components in the second generation CAR structure and Example 1 is the same.

Example 6. Positive rate detection of BCMA-CS1 bispecific CAR-T cells

BCMA-CS1 bispecific CAR-

T cells

41A, 41B, 42A, 42B, 43A, and 43B were tested for CAR positive rate, among which: UTD cells were used as negative controls, and BCMA CAR-T cells 02G (replacing the CAR sequence, still using Prepared by the method for preparing CS1 CAR-T cells in Example 1) and CS1 CAR-T cells 22G (prepared in Example 1) were used as positive controls, and FITC fluorescently labeled BCMA antigen (manufacturer: ACRO Biosystems, article number: BCA-HF254) and PE fluorescently labeled CS1 antigen (same as Example 2) were stained, and the results are shown in Figure 8: CAR-T cells with four tandem structures (41A, 41B, 42A, 42B) were almost not detected When it comes to CAR-positive cells, both 43A and 43B can clearly detect CAR-positive cells, but most of the positive cells of 43A only recognize BCMA antigens, while the positive cells of 43B can recognize both BCMA and CS1 antigens, indicating that different structural designs of scFv will Affects its own antigen-binding ability.

Example 7, BCMA-CS1 CAR-T cell killing experiment

In Example 6, since only BCMA-CS1 bispecific CAR-

T cells

43A and 43B clearly detected CAR-positive cells, 43A, 43B and BCMA CAR-T cells 02G, CS1 CAR-T cells The CAR positive rate of 22G was adjusted to be consistent. Since the positive rate of CAR in other groups was very low, the positive rate of CAR in all groups was not uniformly adjusted to the lowest level. In the end, there were no adjusted cells in each group (41A, 41B, 42A, 42B) Only the same number of T cells can be added in the experiment, but the same number of CAR+T cells cannot be satisfied. In the cell killing experiment, the obtained BCMA-CS1 bispecific CAR-

T cells

41A, 41B, 42A, 42B, 43A, 43B and target cells were placed in X-VIVO15 medium according to the conditions of different effect-to-target ratios between CAR+ cells and target cells. After co-cultivation for 48 hours, the survival rate of target cells was detected by detecting the stably expressed luciferase activity in the target cells (same as in Example 3). and the killing ability of target cells expressing exogenous CS1, and UTD cells were used as negative controls, and BCMA CAR-T cells 02G and CS1 CAR-T cells 22G were used as positive controls. The results of the killing test are shown in Figure 9A, Figure 9B and Figure 9C: It can be seen from the results that BCMA-CS1 dual-target CAR-

T cells

43A and 43B have a good ability to kill K562-BCMA cells, while K562 - In the killing experiment of CS1 cells, only 43B has obvious killing ability. Among the six structures screened, only 43B has complete dual-target killing activity of BCMA and CS1, and the other groups only have different degrees of ability to kill BCMA-positive target cells.

sequence description

SEQ ID NO: 1: GFSLSNYG, which is 22G scFv VH CDR1;

SEQ ID NO:2: IGTIGAT, which is 22G scFv VH CDR2;

SEQ ID NO: 3: ARGIYGDIYVYAFDI, which is 22G scFv VH CDR3;

SEQ ID NO:4: QSVRDNGD, which is 22G scFv VL CDR1;

SEQ ID NO:5: DVS, which is 22G scFv VL CDR2;

SEQ ID NO:6: AGGYIAGSDRWV, which is 22G scFv VL CDR3;

SEQ ID NO: 7: amino acid sequence of humanized 22G scFv VH;

SEQ ID NO: 8: amino acid sequence of humanized 22G scFv VL;

SEQ ID NO: 9: amino acid sequence of rabbit source 22G scFv VH;

SEQ ID NO: 10: amino acid sequence of rabbit source 22G scFv VL;

SEQ ID NO:11: amino acid sequence of humanized 22G scFv;

SEQ ID NO:12: Amino acid sequence of rabbit-derived 22G scFv;

SEQ ID NO:13: the amino acid sequence of the hinge region;

SEQ ID NO:14: the amino acid sequence of the transmembrane region;

SEQ ID NO:15: the amino acid sequence of the intracellular signal transduction region;

SEQ ID NO:16: the amino acid sequence of costimulatory signal transduction region;

SEQ ID NO:17: the amino acid sequence of the leader peptide (i.e. signal peptide);

SEQ ID NO: 18: nucleotide sequence encoding 22G scFv amino acid sequence;

SEQ ID NO: 19: 21G scFv amino acid sequence;

SEQ ID NO:20: 23G scFv amino acid sequence;

SEQ ID NO:21: 24G scFv amino acid sequence;

SEQ ID NO:22: 25G scFv amino acid sequence;

SEQ ID NO:23: 26G scFv amino acid sequence;

SEQ ID NO:24: 27G scFv amino acid sequence;

SEQ ID NO:25: nucleotide sequence encoding 21G scFv amino acid sequence;

SEQ ID NO:26: nucleotide sequence encoding 23G scFv amino acid sequence;

SEQ ID NO:27: nucleotide sequence encoding 24G scFv amino acid sequence;

SEQ ID NO:28: nucleotide sequence encoding 25G scFv amino acid sequence;

SEQ ID NO:29: nucleotide sequence encoding 26G scFv amino acid sequence;

SEQ ID NO: 30: nucleotide sequence encoding 27G scFv amino acid sequence;

SEQ ID NO:31: 21G CAR amino acid sequence;

SEQ ID NO:32: 22G CAR amino acid sequence;

SEQ ID NO:33: 23G CAR amino acid sequence;

SEQ ID NO:34: 24G CAR amino acid sequence;

SEQ ID NO:35: 25G CAR amino acid sequence;

SEQ ID NO:36: 26G CAR amino acid sequence;

SEQ ID NO:37: 27G CAR amino acid sequence;

SEQ ID NO:38: nucleotide sequence encoding 21G CAR amino acid sequence;

SEQ ID NO:39: nucleotide sequence encoding 22G CAR amino acid sequence;

SEQ ID NO:40: nucleotide sequence encoding 23G CAR amino acid sequence;

SEQ ID NO:41: nucleotide sequence encoding 24G CAR amino acid sequence;

SEQ ID NO:42: Nucleotide sequence encoding 25G CAR amino acid sequence;

SEQ ID NO:43: Nucleotide sequence encoding 26G CAR amino acid sequence;

SEQ ID NO:44: Nucleotide sequence encoding 27G CAR amino acid sequence;

SEQ ID NO:45: bispecific scFv 41A amino acid sequence;

SEQ ID NO:46: bispecific scFv 41B amino acid sequence;

SEQ ID NO:47: bispecific scFv 42A amino acid sequence;

SEQ ID NO:48: bispecific scFv 42B amino acid sequence;

SEQ ID NO:49: bispecific scFv 43A amino acid sequence;

SEQ ID NO:50: bispecific scFv 43B amino acid sequence;

SEQ ID NO:51: GFSLSTYH, which is 02G scFv VH CDR1;

SEQ ID NO:52: ISSSGST, which is 02G scFv VH CDR2;

SEQ ID NO:53: ARDLDYVIDL, which is 02G scFv VH CDR3;

SEQ ID NO:54: PSVYNNY, which is 02G scFv VL CDR1;

SEQ ID NO:55: ETS, which is 02G scFv VL CDR2;

SEQ ID NO:56: AGTYVSGDRRA, which is 02G scFv VL CDR3;

SEQ ID NO:57: VH amino acid sequence of humanized 02G scFv antibody;

SEQ ID NO:58: VL amino acid sequence of humanized 02G scFv antibody;

SEQ ID NOs:59-61: the amino acid sequence of the linking region connecting VH and VL;

SEQ ID NO:62: BCMA 02G CAR;

SEQ ID NO:63: LUC90V2 positive control CS1 CAR sequence.

Claims

A chimeric antigen receptor targeting CS1, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein the extracellular antigen recognition domain comprises an anti-CS1 scFv antibody, the The amino acid sequences of the VH complementarity-determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the VL complementarity-determining region of the scFv antibody The amino acid sequences of CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.
The chimeric antigen receptor according to claim 1, wherein the scFv antibody is a humanized antibody, optionally, the VH sequence of the scFv antibody comprises an amino acid sequence as shown in SEQ ID NO: 7, and its VL sequence Including the amino acid sequence shown in SEQ ID NO: 8;

Or, the scFv antibody is a rabbit-derived antibody, optionally, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 9, and its VL sequence includes the amino acid sequence shown in SEQ ID NO: 10 .
The chimeric antigen receptor according to claim 2, wherein there is a connecting region between VH and VL in the scFv antibody, and the connecting region is selected from one or more of the following: SEQ ID NOs: 59-61.
The chimeric antigen receptor according to claim 1, wherein the sequence of the scFv antibody is as shown in SEQ ID NO:11 or SEQ ID NO:12.
The chimeric antigen receptor according to any one of claims 1-4, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α; optionally , the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO: 13;

And/or, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70; optionally, the The amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 14;

And/or, the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a costimulatory signal transduction region.
The chimeric antigen receptor according to claim 5, wherein the intracellular signaling region is derived from CD3δ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, Syk One or more in; Optionally, the intracellular signaling region is derived from CD3δ; further optionally, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:15 ;

And/or, the co-stimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10 , B7-H3, MyD88, one, two or more; optionally, the co-stimulatory signal transduction region is derived from CD28 or 4-1BB; further optionally, the co-stimulatory signal transduction region The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 16.
The chimeric antigen receptor according to any one of claims 1-4, said chimeric antigen receptor further comprising a leader peptide located at the N-terminal of its amino acid sequence; optionally, wherein said leader peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:17.
The chimeric antigen receptor according to any one of claims 1-4, which comprises the amino acid sequence shown in SEQ ID NO:32.
The chimeric antigen receptor according to any one of claims 1-4, wherein the extracellular antigen recognition domain further comprises scFv antibodies against one of the following targets: CD138, NKG2D, CD38, BCMA, CD19, CD70, CD44v6, Lewis Y.
The chimeric antigen receptor according to claim 9, wherein the extracellular antigen recognition domain comprises anti-BCMA scFv VL, anti-CS1 scFv VL, anti-CS1 scFv VH and anti-BCMA scFvVH, said anti- The amino acid sequences of the scFv VH complementarity-determining regions CDR1, CDR2, and CDR3 of CS1 include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively, and the scFv VL complementarity-determining region of the anti-CS1 The amino acid sequences of CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acids of the scFv VH complementarity determining regions CDR1, CDR2, and CDR3 of the anti-BCMA The sequences include the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively, and the amino acid sequences of the anti-BCMA scFv VL complementarity determining regions CDR1, CDR2, and CDR3 include SEQ ID NO: 54. The amino acid sequence shown in SEQ ID NO:55, SEQ ID NO:56.
The chimeric antigen receptor according to claim 10, wherein the VH sequence of the scFv antibody against CS1 comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL sequence of the scFv antibody against CS1 comprises the sequence of SEQ ID NO:7 Amino acid sequence shown in ID NO:8; The VH sequence of the scFv antibody against BCMA includes the amino acid sequence shown in SEQ ID NO:57, and the VL sequence of the scFv antibody against BCMA includes SEQ ID NO:58 Amino acid sequence shown.
The chimeric antigen receptor according to claim 10 or 11, wherein the extracellular antigen recognition domain comprises the amino acid sequence shown in SEQ ID NO:50.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor described in any one of claims 1-12; alternatively, the nucleic acid molecule comprises SEQ ID NO: 18 Nucleotide sequence, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence shown in SEQ ID NO: 18 Sequence identity and the nucleotide sequence encoding the same chimeric antigen receptor; further optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:39.
A vector comprising the nucleic acid molecule of claim 13; optionally, the vector is an expression vector; further optionally, the vector is a viral vector; further optionally, the vector is a lentivirus carrier.
An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 1-12, the isolated nucleic acid molecule of claim 13, or the carrier of claim 14 .
The immune effector cell according to claim 15, which is selected from the group consisting of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, One or more of NK cells and embryonic stem cells that can be differentiated from stem cells; optionally, the immune effector cells are T lymphocytes; further optionally, the source of the T lymphocytes is autologous T lymphocytes or Allogeneic T lymphocytes.
A pharmaceutical composition comprising the engineered immune effector cells and pharmaceutically acceptable adjuvants according to claim 15 or 16; optionally, the pharmaceutically acceptable adjuvants include protective agents; further optional Preferably, the protective agent includes a cell cryopreservation solution.
The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is an intravenous injection.
The chimeric antigen receptor of any one of claims 1-12, the isolated nucleic acid molecule of claim 13, the vector of claim 14, or the engineered nucleic acid molecule of claim 15 or 16. Use of immune effector cells in the preparation of a medicament for treating a disease or condition associated with the expression of CS1.
The use according to claim 19, wherein the disease or disease associated with the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the multiple myeloma is refractory relapsed or relapsed multiple myeloma.
The use according to claim 19, wherein the disease or disease associated with the expression of CS1 is an autoimmune disease; optionally, the autoimmune disease may be selected from the following: systemic lupus erythematosus, rheumatoid arthritis , idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.
A method for treating a disease or disease related to the expression of CS1, comprising the steps of: injecting an effective amount of the engineered immune effector cell according to claim 15 or 16 or the pharmaceutical composition according to claim 17 or 18 Administration to a subject in need of treatment of a disease or condition associated with expression of CS1.
The method according to claim 22, wherein the disease or disease associated with the expression of CS1 is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma.
The method according to claim 22, wherein the disease or disease associated with the expression of CS1 is an autoimmune disease; optionally, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, Idiopathic thrombocytopenic purpura, myasthenia gravis, or autoimmune hemolytic anemia.
The method according to claim 22, wherein the mode of administration is intravenous injection; optionally, the mode of administration is an effective amount of the engineered immune effector cell or the right The pharmaceutical composition described in claim 17 or 18 is administered to the subject in a single injection; further optionally, the effective amount of the engineered immune effector cell described in claim 15 or 16 or claim The dosage of the pharmaceutical composition described in 17 or 18 is 1×10 5 to 1×10 7 cells/kg.
The engineered immune effector cell according to claim 15 or 16 or the pharmaceutical composition according to claim 17 or 18, for treating diseases or conditions related to the expression of CS1.
The engineered immune effector cell or pharmaceutical composition according to claim 26, for treating a disease or disorder associated with the expression of CS1, wherein the disease or disorder associated with the expression of CS1 is cancer; optionally , the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma.
The engineered immune effector cell or pharmaceutical composition according to claim 26, for treating a disease or disorder associated with the expression of CS1, wherein the disease or disorder associated with the expression of CS1 is an autoimmune disease; Optionally, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune hemolytic anemia.