Bispecific chimeric antigen receptors targeting bcma-cd19 and uses thereof

Abstract

This invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application, wherein the bispecific chimeric antigen receptor comprises an extracellular antigen recognition domain; wherein: the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-CD19 extracellular antigen recognition domain comprises CD19VH and CD19VL, which are selected from one of groups 1) to 8).

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a bispecific chimeric antigen receptor targeting BCMA-CD19 and its applications. Background Technology

[0002] Cell therapy is an emerging medical technology. Its main principle is to utilize the patient's own immune cells, culturing and modifying them in vitro to give them the specific ability to kill tumor cells. These modified immune cells are then reinfused into the patient to treat tumors. Chimeric antigen receptor T-cell (CAR-T) technology is an important cell therapy technique. It uses genetic engineering to make T cells express a specific antibody, enabling them to specifically recognize and kill tumor cells expressing the corresponding antigen. Tumor immunotherapy is a treatment method that utilizes the body's own immune system to fight tumors. Its main goal is to enhance the body's immune system's ability to recognize and kill tumor cells. CAR-T cell therapy is an important tumor immunotherapy method that directly introduces T cells with specific killing capabilities into the patient, thereby achieving specific killing of tumor cells. Genetic engineering technology is an important component of modern biotechnology. It achieves genetic modification of organisms through operations such as gene cutting, ligation, and transfer. In CAR-T cell therapy, genetic engineering technology is widely used to modify T cells, giving them specific anti-tumor capabilities.

[0003] Current CAR-T cell therapies primarily target single tumor antigens, such as CD19 or BCMA. By designing and constructing CAR-T cells targeting specific tumor antigens, specific killing of corresponding tumor cells can be achieved. However, this single-target CAR-T cell therapy has some problems. For example, if the expression level of the antigen on the surface of tumor cells is low, or if the tumor cells escape antigens, the CAR-T cells may not be able to effectively kill the tumor cells. 3) Existing technological problems: Current single-target CAR-T cell therapies have some problems and limitations. First, due to the heterogeneity and complexity of tumor cells, a single tumor antigen may not meet all treatment needs. Second, tumor cells can evade CAR-T cell attacks by altering antigen expression or exhibiting antigen escape. In addition, single-target CAR-T cell therapy may also lead to increased drug resistance in tumor cells, thereby affecting the treatment effect. Therefore, how to construct CAR-T cells that simultaneously target multiple tumor antigens to improve treatment efficacy is a significant challenge currently facing cell therapy technology.

[0004] Autoimmune diseases (AIDs) are a group of diseases caused by the body's own tissues being damaged due to an immune response between pathogenic autoantibodies and autoantigens. Currently, they include more than 80 diseases, affecting approximately 7.6% to 9.4% of the world's population. The potential advantages of CAR-T therapy in autoimmune diseases such as systemic lupus erythematosus include: 1) the opportunity for immune memory remodeling through deep clearance of B cells. T cells can enter tissues and clear memory B cells. 2) long-term remission with a single treatment, avoiding continuous drug administration.

[0005] Autoantibodies and autoreactive B cells are directly associated with autoimmune diseases. These pathogenic B cells recognize self-antigens and produce autoantibodies, leading to the development of autoimmune diseases. B cells are the source of pathogenic antibodies. B cells can differentiate into plasmablasts and long-lived plasma cells, among which... B cells, memory B cells, and plasmablasts express CD19, while long-lived plasma cells express BCMA but not CD19. Therefore, CAR-T therapy that simultaneously expresses CD19 and BCMA holds promise for better therapeutic efficacy and broader indications. Summary of the Invention

[0006] This application provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application. Based on existing CD19 murine antibody sequences, the inventors designed eight humanized CD19 antibodies to reduce their heterologousity. However, during the humanization process, the antibody's functionality (e.g., CAR positivity rate, in vitro cytotoxic activity, cytokine release, etc.) may be altered. Therefore, obtaining humanized antibodies with reduced immunogenicity but without loss of functionality is of practical significance. This invention screened two humanized CD19 antibodies with good efficacy and, together with BCMA antibodies, constructed multiple bispecific chimeric antigen receptor expression vectors targeting BCMA-CD19, and prepared bispecific CAR-T cells targeting BCMA-CD19. Furthermore, the cellular level was verified to demonstrate that the BCMA-CD19 bispecific CAR-T cells possess good tumor-suppressive function.

[0007] This invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19, 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-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, selected from one of the following:

[0008] 1) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0009] 2) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0010] 3) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or

[0011] 4) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or

[0012] 5) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or

[0013] 6) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:9; or

[0014] 7) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or

[0015] 8) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11.

[0016] In a preferred embodiment of the present invention, the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or

[0017] The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11.

[0018] In a preferred embodiment of the present invention, the BCMA VH sequence comprises the amino acid sequence shown in SEQ ID NO:12, and the BCMA VL sequence comprises the amino acid sequence shown in SEQ ID NO:13.

[0019] In some embodiments, this application further includes the substitution, deletion, addition, and / or insertion of one or more amino acids in the amino acid sequence of any of the aforementioned bispecific chimeric antigen receptors, and such substitution results in an activity equivalent to any of the aforementioned chimeric antigen receptors. Those skilled in the art will understand that during humanization, amino acids in the FR region of the VH and VL sequences can be substituted so that the CDR region of the modified antibody retains a suitable antigen-binding site. Therefore, this application naturally includes different amino acid sequences obtained by humanizing the FR region of the VH and VL sequences based on the aforementioned CDR. Furthermore, those skilled in the art will also understand that during humanization, in order to ensure that the CDR region of the modified antibody retains a suitable antigen-binding site, if necessary, one, two, three, or no more than 10% of the amino acid sequence in the CDR may be substituted, deleted, added, and / or inserted; these are also included in this application.

[0020] In a preferred embodiment of the present invention, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor is a scFv antibody, a sc(Fv)2 antibody, or a [sc(Fv)2]2 antibody.

[0021] In a preferred embodiment of the present invention, the scFv antibody comprises any one of the following structures: CD19 VL sequence - first linker sequence - BCMA VL sequence - second linker sequence - BCMA VH sequence - third linker sequence - CD19VH sequence, BCMA VL sequence - fourth linker sequence - CD19 VL sequence - fifth linker sequence - CD19 VH sequence - sixth linker sequence - BCMA VH sequence, CD19 VL sequence - seventh linker sequence - CD19 VH sequence - eighth linker sequence - BCMA VL sequence - ninth linker sequence - BCMA VH sequence, BCMA VL sequence - tenth linker sequence - BCMA VH sequence - eleventh linker sequence - CD19VL sequence - twelfth linker sequence - CD19 VH sequence;

[0022] Optionally, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes any one of the following structures: CD19 VL sequence-first linker sequence-BCMA VL sequence-second linker sequence-BCMA VH sequence-third linker sequence-CD19 VH sequence and BCMA VL sequence-fourth linker sequence-CD19 VL sequence-fifth linker sequence-CD19 VH sequence-sixth linker sequence-BCMA VH sequence;

[0023] Further optionally, the first linking sequence, the second linking sequence, the third linking sequence, the fourth linking sequence, the fifth linking sequence, the sixth linking sequence, the seventh linking sequence, the eighth linking sequence, the ninth linking sequence, the tenth linking sequence, the eleventh linking sequence, and the twelfth linking sequence are independently selected from one or more of the following sequences: SEQ ID NO:14 and SEQ ID NO:15.

[0024] In a preferred embodiment of the present invention, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes an amino acid sequence as shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

[0025] In a preferred embodiment of the present invention, 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:29; and / or

[0026] The transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, 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:30.

[0027] In a preferred embodiment of the present invention, the intracellular domain comprises an intracellular signal transduction region; optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signal transduction region is derived from CD3ζ; even further optionally, the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:32.

[0028] In a preferred embodiment of the present invention, the intracellular domain further includes a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; further optionally, the co-stimulatory signal transduction region is derived from 4-1BB; and even more optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:31.

[0029] In a preferred embodiment of the present invention, the bispecific chimeric antigen receptor further comprises a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide 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:33.

[0030] In a preferred embodiment of the present invention, the bispecific chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37.

[0031] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned bispecific chimeric antigen receptor;

[0032] Optionally, the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises nucleotide sequences encoding CD19 VH and CD19 VL and nucleotide sequences encoding BCMA VH and BCMA VL, wherein the nucleotide sequences encoding CD19 VH and CD19 VL are selected from one group of the following:

[0033] 1) A nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:38; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or

[0034] 2) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or

[0035] 3) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:5, as shown in SEQ ID NO:42; and / or

[0036] 4) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:6, as shown in SEQ ID NO:43; and / or

[0037] 5) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:7, as shown in SEQ ID NO:44; and / or

[0038] 6) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:9, as shown in SEQ ID NO:46; and / or

[0039] 7) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:10, as shown in SEQ ID NO:47; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:48; and / or

[0040] 8) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:48;

[0041] The nucleotide sequences encoding BCMA VH and BCMA VL are as follows:

[0042] Nucleotide sequences encoding the BCMA VH amino acid sequence as shown in SEQ ID NO:12, as shown in SEQ ID NO:49; and nucleotide sequences encoding the BCMA VL amino acid sequence as shown in SEQ ID NO:13, as shown in SEQ ID NO:50.

[0043] The present invention also provides a carrier comprising the above-isolated nucleic acid molecules;

[0044] Optionally, the carrier is an expression carrier;

[0045] Further, optionally, the vector is a viral vector;

[0046] Alternatively, the vector may be a lentiviral vector.

[0047] The present invention also provides an engineered immune effector cell comprising the chimeric antigen receptor described above, the isolated nucleic acid described above, or the carrier described above.

[0048] In a preferred embodiment of the present invention, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells.

[0049] Optionally, the engineered immune effector cells are T lymphocytes;

[0050] Further, optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes.

[0051] In some embodiments, the surface of the engineered immune effector cells may express or have expressed the chimeric antigen receptor described in this application.

[0052] The present invention also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include one or more of the following: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

[0053] Optionally, pharmaceutically acceptable excipients include cryoprotectants; alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0054] In a preferred embodiment of the present invention, the pharmaceutical composition is a cell suspension or frozen cells thereof.

[0055] In a preferred embodiment of the present invention, the pharmaceutical composition is an intravenous injection.

[0056] The present invention also provides the use of the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid, the above-mentioned vector or the above-mentioned engineered immune effector cells in the preparation of a drug for treating diseases or conditions related to BCMA expression.

[0057] In a preferred embodiment of the invention, the disease or condition associated with BCMA expression is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma.

[0058] In a preferred embodiment of the present invention, the disease or condition associated with BCMA expression may be an autoimmune disease; optionally, the autoimmune disease may be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0059] On the other hand, this application also provides a method for treating diseases or conditions related to BCMA expression, the method comprising administering an effective dose of the chimeric antigen receptor, the isolated nucleic acid molecule, the vector and / or the engineered immune effector cells to a subject in need of treatment for diseases or conditions related to BCMA expression.

[0060] In some embodiments, the administration can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration. For example, administration can be given to the subject via intravenous injection. In some embodiments, an effective dose of engineered immune effector cells or pharmaceutical composition can be administered to the subject in a single dose or in multiple doses over a 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.

[0061] In some implementation schemes, the dosage may vary depending on the indication; the dosage may also vary depending on the severity of the patient's condition. The dosage range may be 1×10⁻⁶. 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, 0.5×10 6 CAR-positive T cells / kg, 0.6×10 6 CAR-positive T cells / kg, 0.7×10 6 CAR-positive T cells / kg, 0.8×10 6 CAR-positive T cells / kg, 0.9 × 10 6 CAR-positive T cells / kg, 1.0×10 6 CAR-positive T cells / kg, 1.1×10 6 CAR-positive T cells / kg, 1.2 × 10 6 CAR-positive T cells / kg, 1.3×10 6 CAR-positive T cells / kg, 1.4 × 10 6 CAR-positive T cells / kg, 1.5 × 10 6 CAR-positive T cells / kg, 1.6 × 10 6 CAR-positive T cells / kg, 1.7 × 10 6 CAR-positive T cells / kg, 1.8 × 10 6 CAR-positive T cells / kg, 1.9 × 10 6 CAR-positive T cells / kg, 2.0 × 10 6 CAR-positive T cells / kg.

[0062] In some implementations, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

[0063] Compared with the prior art, the bispecific chimeric antigen receptor targeting BCMA-CD19 of the present invention has the following beneficial effects:

[0064] The dual-target humanized CD19 / BCMACAR-T of the present invention has the same efficacy as single-target CD19 CAR-T and single-target BCMACAR-T, and the dual-target humanized CD19 / BCMACAR-T has lower immunogenicity than single-target murine CD19 CAR-T, thus having better clinical efficacy and safety. Attached Figure Description

[0065] Figure 1 : Graph showing the positive rate of CD19-CAR in cells at different time points.

[0066] Figure 2 Statistical graph of CD19-CAR MFI in cells at different time points.

[0067] Figure 3 : In vitro killing activity against positive target cells Nalm6.

[0068] Figure 4: Detection of cytokine release during Nalm6 killing of target cells.

[0069] Figure 5 : CAR-T cell expansion fold during multiple rounds of Nalm6 stimulation of target cells.

[0070] Figure 6 Day 9: CD19-CAR positivity rate and BCMA-CAR positivity rate in different groups.

[0071] Figure 7 Scoring results of CD19 humanized sequences using three biological computer evaluation tools (T20 score, Hscore, QASis).

[0072] Figure 8 The immunogenicity risk prediction results of two immunogenicity prediction tools (AlphaMHC v2 and CD4 T cell Immunogenicity prediction tool) for CD19 humanized sequences.

[0073] Figure 9 : In vitro killing activity against target cells Nalm6.

[0074] Figure 10: In vitro killing activity against target cells K562-CD19.

[0075] Figure 11 : In vitro killing activity against target cells MM.1S.

[0076] Figure 12 : In vitro killing activity against target cells K562-BCMA.

[0077] Figure 13: Detection of IL-2 release during killing of target cells Nalm6 and MM.1S.

[0078] Figure 14: Detection of IFN-γ cytokine release during killing of target cells Nalm6 and MM.1S.

[0079] Figure 15 : CAR-T cell expansion fold during multiple rounds of Nalm6 stimulation of target cells.

[0080] Figure 16 : CAR-T cell expansion fold during multiple rounds of stimulation with target cells RPMI-8226.

[0081] Figure 17 Animal models bearing Nalm6 tumors: Imaging fluorescence statistics of mice in different groups.

[0082] Figure 18 Animal model of tumor-bearing MM.1S, imaging fluorescence statistics of mice in different groups.

[0083] exist Figure 1-2 In the diagram, each bar, from left to right, represents CNCT19, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22. Figure 3 In Figure -4, each column, from left to right, represents UTD (untransduced CAR T cells), CNCT19, Q11, Q14, Q33, Q54, GS32, GS21, and GS22. Figure 5 In the diagram, each bar, from left to right, represents CNCT19, Q11, Q14, Q33, Q54, GS32, GS21, and GS22. Figure 9-1 In Figure 4, each column, from left to right, represents UTD (untransduced CAR T cells), CNCT19, Q33, GS21, O2G, O2G-Q33, Q33-O2G, O2G-GS21, and GS21-O2G. Figure 15-16 In the diagram, each column represents CNCT19, Q33, 02G, and 02G-Q33 from left to right. Detailed Implementation

[0084] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0085] The following further describes this application: In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0086] In this application, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain (e.g., a portion that binds to tumor-associated antigens (TAAs)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising approaches to combating cancer. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without antigen presentation, thereby activating and functionally affecting T cells.

[0087] In this application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (ARD). CAR cell therapy products (such as CAR-T cells) rely on extracellular antigen recognition domains to specifically recognize and / or bind to target antigens expressed by tumor cells. To date, antigen recognition domains are derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). The most common source to date is the scFv segment of antibodies. The scFv includes the antibody heavy chain variable region and the light chain variable region, linked by a peptide chain, such as the 18-amino acid linker sequence GSTGSGSGKPGSGEGSTKG. In antibody analysis, common CDR (Continuous Derivative) rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When applying websites that execute these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of this application covers combinations of CDR sequences obtained through analysis using different rules.

[0088] In this application, the term "hinge region" refers to the connecting segment that acts between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by providing a certain range of motion to the antigen recognition domain. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, typical hinge regions also contain residues that participate in CAR dimerization, which helps to enhance antigen sensitivity.

[0089] In this application, "transmembrane region" refers to a transmembrane domain connecting the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect CAR expression and stability to some extent, but do not directly participate in signal transduction; however, they can enhance downstream signal transduction through interactions. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0090] In this application, the term "intracellular domain" includes intracellular signal transduction regions and may also include co-stimulatory signal transduction regions.

[0091] In this application, the term "intracellular signal transduction region" refers to the activation of at least one normal effector function of an immune effector cell responsible for expressing CAR. The intracellular signal transduction region may originate from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0092] In this application, the term "co-stimulatory signal transduction region" is used because, in addition to antigen-specific signal stimulation, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. In some embodiments, the CAR may further include one or more co-stimulatory signal transduction regions, wherein the co-stimulatory signal transduction regions may be derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0093] In this application, the term "scFv" has the conventional meaning in the art and refers to a single chain variable fragment (scFv), which is an antibody composed of antibody heavy chain variable fragments and light chain variable fragments linked by a short peptide (linker).

[0094] In this application, terms such as “Sc(Fv)2” and “[Sc(Fv)2]2” that are not specifically explained also have their conventional meanings in the art.

[0095] In this application, the term "separated" generally refers to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" substance, it may be due to a change in its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated substance. The term "separated" does not exclude substances obtained artificially from their natural state and then synthesized, nor does it exclude the presence of other impurities that do not affect the substance's activity.

[0096] In this application, the term "guide peptide" refers to a short peptide preceding an extracellular antigen recognition domain (such as the scFv sequence), which guides the export of intracellularly synthesized recombinant proteins to the extracellular space. Commonly used guide peptides include the human CD8α signal peptide or the human GM-CSF receptor α signal peptide.

[0097] In this application, one of the key factors determining the efficacy of CAR-immunotherapy is the selection of tumor target antigens. In this application, 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 can be a suitable tumor antigen target for immunotherapeutic agents targeting multiple myeloma. However, due to the heterogeneity of specific antigens on the surface of multiple myeloma cells, the selection of antigen targets is not necessarily singular. By selecting appropriate targets, the anti-tumor activity of CAR-T cells can be optimized. The "CD19" molecule is currently a major target for the treatment of B-lymphocyte-derived hematologic malignancies and is a hot topic in CAR-T cell therapy research. Most B-cell-derived malignant tumor cells express CD19 molecules on their surface. Multiple myeloma, as a B-cell lineage tumor, generally does not express CD19 molecules; therefore, CD19 is usually not used as a target for multiple myeloma treatment. However, some literature suggests that some trace amounts of drug-resistant and relapsed multiple myeloma clones also possess CD19. + Phenotype. Meanwhile, compared to preparing CAR-immune cells targeting different targets separately and using them together, dual-target CAR-immune cells have the following advantages: 1. Fewer immune cells are required, making preparation easier and more cost-effective; 2. In terms of medication, the safety and operability of administering one product are significantly higher than administering two products.

[0098] In this application, the term "humanized antibody" also refers to a humanized antibody. Methods for humanization are known (e.g., WO96 / 02576). The purpose of humanization is to reduce heterology while essentially preserving the affinity and specificity of the parent antibody. For example, when the CDR is derived from a mouse antibody, primer 25 (the corresponding primer can be obtained by referring to the method described in WO98 / 13388) can be synthesized and used to link the CDR of the mouse antibody to the frame region (FR) of the human antibody.

[0099] In this application, the term "linking sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure / region of the chimeric antigen receptor of the present invention. The linking sequence may consist of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linking sequences may be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0100] In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide of any length, which may be isolated from its natural environment or an analogue synthesized artificially.

[0101] In this application, the gene transduction / transfection methods for CAR gene transduction / transfection and target gene expression mainly include viral and non-viral methods. These include: gamma retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

[0102] The term "vector" generally refers to a nucleic acid delivery vehicle that inserts a polynucleotide encoding a protein into itself, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cell. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances. The term "transposon" refers to a discontinuous segment of DNA capable of migrating between chromosomal loci and carrying genetic information, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

[0103] In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Immune effector cells may be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells.

[0104] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives. In some embodiments, pharmaceutically acceptable excipients include protectants, such as cell cryopreservation solutions. In some embodiments, the pharmaceutical composition of this application is a cell suspension or its cryopreserved cells.

[0105] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cattle, sheep, or monkeys.

[0106] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0107] In this application, the term "about" generally refers to a range of fluctuations acceptable to a person skilled in the art above or below a specified value, such as a variation within ±0.5% to 10%, for example, a variation within a range of 0.5%, 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% above or below a specified value.

[0108] On the other hand, this application also provides the chimeric antigen receptor, the isolated nucleic acid molecule, the vector, and / or the engineered immune effector cells, which can be used to treat diseases or conditions associated with BCMA expression.

[0109] In some embodiments, the disease or condition associated with BCMA expression may include a non-solid tumor, optionally a hematologic malignancy.

[0110] In some embodiments, the disease or condition associated with BCMA expression may include multiple myeloma.

[0111] In some embodiments, the multiple myeloma is a relapsed or refractory multiple myeloma.

[0112] Not intended to be limited by any theory, the embodiments described below are merely illustrative of the chimeric antigen receptor, engineered immune effector cells, preparation methods, and uses of this application, and are not intended to limit the scope of the invention. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been 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.

[0113] The mouse-derived antibody and humanized antibody scFv of the present invention

[0114] >HI19a(CNCT19) scFv

[0115] DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTLTVSS(SEQ ID NO:51)

[0116] >Q11 scFv

[0117] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:16)

[0118] The Q11 scFv contains Q11 VH and Q11 VL, and the amino acid sequence of Q11 VH is shown below:

[0119] QVQLVQSGAEVKKPGASVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARK TISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:1)

[0120] The nucleotide sequence encoding Q11 VH is shown below:

[0121] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCTCCGTGAAGGTGAGCTGCAAAGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATGGGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATGACAAGAGACACAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAAGACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:38)

[0122] The amino acid sequence of Q11 VL is shown below:

[0123] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLE IK(SEQ ID NO:2)

[0124] The nucleotide sequence encoding Q11 VL is shown below:

[0125] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCCCCTGATCTACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTTGGCGGGGGCACAAAGCTGGAGATTAAG(SEQ ID NO:39)

[0126] >Q14 scFv

[0127] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:17)

[0128] The Q14 scFv comprises Q14 VH and Q14 VL, and the amino acid sequence of Q14 VH is shown below:

[0129] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYFCARKT ISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:3)

[0130] The nucleotide sequence encoding Q14 VH is shown below:

[0131] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGTGAAGGTGAGCTGCAAAGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATGGGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATCACAGCAGACAAAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAAGACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:40)

[0132] The amino acid sequence of Q14 VL is shown below:

[0133] DIQLTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLE IK(SEQ ID NO:2)

[0134] The nucleotide sequence encoding Q14 VL is shown below (SEQ ID NO:39):

[0135] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCCCCTGATCTACAGCGCCACCTACAGA AACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTTGGCGGGGGCACAAAGCTGGAGATTAAG(SEQ ID NO:39)

[0136] >Q23 scFv

[0137] DIQLTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKSPKPLIYSATYRNSGVPSRFSGSGSGTDFTLTISSLQPKDFATYFCQQYNRYPYTSGGGTKLEIKGGGGSGGGGSGGGGSEV QLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:18)

[0138] Q23 scFv contains Q23 VH and Q23 VL, where the amino acid sequence of Q23 VH is shown below:

[0139] EVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWM GQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTI SSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:4)

[0140] The nucleotide sequence encoding Q23 VH is shown below:

[0141] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAAGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGG CAAGTTCAAGGGCCAAGTGACCCTGAGCGCCGACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCCCGACACCGCCATGTACTTCTGCGCTAGAAAGACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:41)

[0142] The amino acid sequence of Q23 VL is shown below:

[0143] DIQLTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKSPKPLIYSA TYRNSGVPSRFSGSGSGTDFTLTISSLQPKDFATYFCQQYNRYPYTSGGGTKLE IK(SEQ ID NO:5)

[0144] The nucleotide sequence encoding Q23 VL is shown below:

[0145] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTGCAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGAGCCCCAAGCCCCTGATCTACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCAAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATCCGGGGGCGGCACCAAGCTGGAAATCAAG(SEQ ID NO:42)

[0146] >Q33 scFv

[0147] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNRYPYTfGGGTKLEIKGGGGSGGGGSGGGGSEVQLvQSGAEvkkPGeSlKISCKASGYAFSSYWMNWVrQmPGkGLEWmGQIYPGDGDTNYNGKFKGQvTLsADKSiSTAYlQwSsLkasDtAmYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:19)

[0148] The Q33 scFv contains Q33 VH and Q33 VL, and the amino acid sequence of Q33 VH is shown below:

[0149] EVQLvQSGAEvkkPGeSlKISCKASGYAFSSYWMNWVrQmPGkGLEWmGQI YPGDGDTNYNGKFKGQvTLsADKSiSTAYlQwSsLkasDtAmYFCARKTISSVVDF YFDYWGQGTTVTVSS(SEQ ID NO:4)

[0150] The nucleotide sequence encoding Q33 VH is shown below:

[0151] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAAGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCCAAGTGACCCTGAGCGCCGACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCCGACACCGCCATGTACTTCTGCGCTAGAAAGACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:41)

[0152] The amino acid sequence of Q33 VL is shown below:

[0153] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYS ATYRNSGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNRYPYTfGGGTKLE IK(SEQ ID NO:6)

[0154] The nucleotide sequence encoding Q33 VL is shown below:

[0155] GAAATCGTGATGACCCAGTCCCCTGCTACACTGAGCGTGTCCCCAGGCGAGCGGGCCACACTGTCTTGCAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGGCCCCTGATCTACAGCGCCACCTACAGAAACAGCGGCATCCCTGCCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGTCCGAGGACTTCGCCGTCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTCGGCGGGGGGACCAAGCTGGAGATCAAA(SEQ ID NO:43)

[0156] >Q54 scFv

[0157] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLvQSGAEvkkPGSSVKvSCKASGYAFSSYWMNWVrQaPGQGLEWmGQIYPGDGDTNYNGKFKGrvTiTADKStSTAYMeLSsLrSEDtAVYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:20)

[0158] The Q54 scFv contains Q54 VH and Q54 VL, and the amino acid sequence of Q54 VH is shown below:

[0159] QVQLvQSGAEvkkPGSSVKvSCKASGYAFSSYWMNWVrQaPGQGLEWmGQIYPGDGDTNYNGKFKGrvTiTADKStSTAYMeLSsLrSEDtAVYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:3)

[0160] The nucleotide sequence encoding Q54 VH is shown below: ​​​​The amino acid sequence of Q54 VL is shown below:

[0163] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKVE IK(SEQ ID NO:7)

[0164] The nucleotide sequence encoding Q54 VL is shown below:

[0165] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTGTAAGGCCAGCCAGAACGTGGGCACCAATGTGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATCTACTCCGCTACATATCGG AACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCCTGGAACCGAGTTCACCCTGACCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTTCGGAGGCGGCACCAAGGTGGAGATTAAG(SEQ ID NO:44)

[0166] >GS32 scFv

[0167] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFADYFCQQYNRYPYTFGGGTKVEIKRGGGGSGGGGSGGGGSQ VQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWIGQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCARKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:21)

[0168] GS32 scFv contains GS32 VH and GS32 VL, where the amino acid sequence of GS32 VH is shown below:

[0169] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWI GQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCARKTIS SVVDFYFDYWGQGTLVTVSS(SEQ ID NO:8)

[0170] The nucleotide sequence encoding GS32 VH is shown below:

[0171] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGTAAAGCTTCCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATCGGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGG CAAGTTCAAGGGCAGAGCCACCATCACCGCCGACAAGAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAAGACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCAGC(SEQ IDNO:45)

[0172] The amino acid sequence of GS32 VL is shown below:

[0173] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFADYFCQQYNRYPYTFGGGTKVE IKR(SEQ ID NO:9)

[0174] The nucleotide sequence encoding GS32 VL is shown below:

[0175] GACATTCAGCTGACACAGAGCCCTAGCTTCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTGCAAGGCTTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAACCCGGCAAGGCCCCCAAGCCCCTGATCTACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACAATCAGCAGCCTGCAGCCCGAGGACTTCGCCGACTACTTCTGTCAGCAGTACAACAGATACCCCTACACCTTCGGCGGGGGCACCAAGGTGGAGATCAAGCGG(SEQ ID NO:46)

[0176] >GS21 scFv

[0177] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:22)

[0178] The GS21 scFv contains GS21 VH and GS21 VL, and the amino acid sequence of GS21 VH is shown as follows:

[0179] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARK TISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:10)

[0180] The nucleotide sequence encoding GS21 VH is shown as follows:

[0181] CAAGTGCAGCTGGTCCAGAGCGGCGCCGAGGTGAAGAAACCTGGCTCTAGCGTGAAAGTGTCCTGCAAGGCCTCTGGCTACGCCTTTTCCAGCTATTGGATGAACTGGGTGCGGCAGGCTCCTGGCCAGGGCCTGGAATGGATGGGCCAGATCTACCCCGGAGATGGCGATACCAACTACAATGGCAAGTTCAAGGGCAGAGTGACCATCACCGCTGACAAGTCTACAAGCACAGCCTACATGGAGCTGTCCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAAGACCATCTCCTCTGTGGTGGACTTCTACTTCGACTACTGGGGACAGGGCACGCTGGTGACCGTGTCCTCT(SEQ IDNO:47)

[0182] The amino acid sequence of GS21 VL is shown below:

[0183] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVE IKR(SEQ ID NO:11)

[0184] The nucleotide sequence encoding GS21 VL is shown below:

[0185] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTGTAAGGCCAGCCAGAACGTGGGCACCAATGTGGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATCTACTCCGCTACATATCGGAACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCTCTGGAACCGAGTTCACCCTGACCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTCCGGAGGCGGCACCAAGGTGGAAATCAAGCGG(SEQ ID NO:48)

[0186] >GS22 scFv

[0187] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWIGQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCARKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:23)

[0188] The GS22 scFv contains GS22 VH and GS22 VL, and the amino acid sequence of GS22 VH is shown below:

[0189] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWI GQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCARKTIS SVVDFYFDYWGQGTLVTVSS(SEQ ID NO:8)

[0190] The nucleotide sequence encoding GS22 VH is shown below:

[0191] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGATCTAGCGTGAAAGTGTCCTGCAAGGCCTCCGGCTACGCCTTCAGCTCCTATTGGATGAACTGGGTGCGGCAGGCTCCTGGACAAGGCCTGGAATGGATCGGCCAGATCTACCCTGGCGATGGCGATACCAACTACAATGGCAAGTTCAAGGGCAGAGCCACCATCACCGCCGACAAGTCCACATCTACCGCCTACATGGAGCTGAGCAGCCTGAGATCCGAGGACACCGCTGTGTACTTTTGTGCCAGAAAGACCATCAGCTCCGTGGTGGACTTCTACTTCGACTACTGGGGCCAGGGCACCCTGGTCACAGTGTCTTCT(SEQ IDNO:45)

[0192] The amino acid sequence of GS22 VL is shown below:

[0193] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVE IKR(SEQ ID NO:11)

[0194] The nucleotide sequence encoding GS22 VL is shown below:

[0195] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTGTAAGGCCAGCCAGAACGTGGGCACCAATGTGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATCTACTCCGCTACATATCGGAA CAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCCTCTGGAACCGAGTTCACCCTGACCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTCCGGAGGCGGCACCAAGGTGGAAATCAAGCGG(SEQ ID NO:48)

[0196] >02G scFv

[0197] EIVMTQSPSTLSASSVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSS(SEQ ID NO:24)

[0198] 02G scFv contains 02G VH and 02G VL, where the amino acid sequence of 02G VH is shown below:

[0199] EVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIG VISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVI DLWGPGTLVTVSS(SEQ ID NO:12)

[0200] The nucleotide sequence encoding 02G VH is shown below:

[0201] GAAGTGCAGCTGGTGGAGTCCGGCGGTGGACTGGTGCAACCGGGAGGCTCACTCAGATTGTCATGCACCGCCTCTGGCTTTAGTCTCTCCACCTATCATATGACTTGGGTGAGGCAGGCACCCGGCAAGGGCCTGGAATGGATCGGCGTGATCTCTTCCAGCGGTAGCACCTATTA CGCCTCTTGGGCGAAGGGCAGGTTTACCATCAGCCGCGACAACAGCAAGAATACCGTTTACCTGCGATGAATAGCCTGAGGGCCGAAGACACGGCGGTCTATTTCTGTGCACGGGACCTTGACTACGTTATTGACCTGTGGGGCCCTGGGACCCTCGTAACTGTGAGCAGC(SEQ ID NO:49)

[0202] The amino acid sequence of 02G VL is shown below:

[0203] EIVMTQSPSTLSSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYE TSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGT KLTVL(SEQ ID NO:13)

[0204] The nucleotide sequence encoding 02G VL is shown below:

[0205] GAGATCGTGATGACCCAGTCCCCAAGTACACTGAGCGCCTCCGTGGGCGACCGCGTGATCATAAACTGTCAAAGCTCACCCTCTGTTTACAACAATTACCTGTCTTGGTATCAACAGAAGCCCGGTAAGGCCCCCAAACTGCTCATTTACGAGACATCCACCCTGGCATCCGGGGTGCCAAGCCGCTTCTCCGGGAGTGGGTCTGGCGCCGAGTTCACCCTGACCATATCTTCCCTGCAGCCCGACGACTTCGCAACGTACTATTGCGCCGGAACCTATGTAAGTGGGGATAGACGCGCCTTCGGGCAGGGCACGAAGTTGACCGTGCTG(SEQ ID NO:50)

[0206] Dual-target scFv

[0207] 02G-Q33 scFv

[0208] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGGTKLEIKGGGGSEIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSSGGGGSEVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:25)

[0209] The 02G-Q33 scFv contains 02G VH, 02G VL, Q3VH, and Q33 VL

[0210] The structure of 02G-Q33 scFv is as follows:

[0211] Q33 VL-linked sequence (GGGGS, SEQ ID NO:14)-02G VL-linked sequence (GGGGSGGGGSGGGGS, SEQ ID NO:15)-02G VH-linked sequence (GGGGS, SEQ ID NO:14)-Q33 VH

[0212] Q33-02G scFv

[0213] EIVMTQSPSTTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSEIVMTQ SPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLV QSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGG SEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSS(SEQ ID NO:26)

[0214] Q33-02G scFv includes Q33 VH, Q33 VL and 02G VH, 02G VL

[0215] The structure of Q33-02G scFv is as follows:

[0216] 02G VL-linked sequence (GGGGS, SEQ ID NO:14)-Q33 VL-linked sequence (GGGGSGGGGSGGGGS, SEQ ID NO:15)-Q33 VH--linked sequence (GGGGS, SEQ ID NO:14)-02G VH

[0217] 02G-GS21 scFv

[0218] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSEIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:27)

[0219] The 02G-GS21 scFv contains 02G VH, 02G VL, GS21 VH, and GS21 VL

[0220] The structure of 02G-GS21 scFv is as follows:

[0221] GS21 VL - Linker sequence (GGGGS, SEQ ID NO:14) - 02G VL - Linker sequence (GGGGSGGGGSGGGGS, SEQ ID NO:15) - 02G VH - Linker sequence (GGGGS, SEQ ID NO:14) - GS21 VH

[0222] GS21-02G scFv

[0223] EIVMTQSPSTSLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSDIQLTQ SPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQL VQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKTISSVVDFYFDYWGQGTLVTVSSGGG GSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSS(SEQ ID NO:28)

[0224] GS21-02G scFv includes GS21 VH, GS21 VL and 02G VH, 02G VL

[0225] The structure of GS21-02G scFv is as follows:

[0226] 02G VL-linked sequence (GGGGS, SEQ ID NO:14)-GS21 VL-linked sequence (GGGGSGGGGSGGGGS, SEQ ID NO:15)-GS21 VH--linked sequence (GGGGS, SEQ ID NO:14)-02G VH

[0227] The amino acid sequence of the CD8 hinge region:

[0228] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:29)

[0229] The amino acid sequence of the CD8 transmembrane region:

[0230] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:30)

[0231] The amino acid sequence of the 4-1BB intracellular signal transduction region:

[0232] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR(SEQ ID NO:31)

[0233] The amino acid sequence of the CD3ζ co-stimulatory signal transduction region:

[0234] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:32)

[0235] The amino acid sequence of the CD8α guide peptide:

[0236] MALPPTALL PLALLLHAARP(SEQ ID NO:33)

[0237] The amino acid sequence of the first CD19 / BCMA-CAR (which sequentially contains the CD8α guide strand, O2G-Q33 scFv, CD8 hinge region, CD8 transmembrane region, 4-1BB intracellular domain, and CD3ζ) is as follows:

[0238] MALPVTALLLPLALLLHAARPEIVMTQSPATLSVSPGERATLSCKASQNV

[0239] GTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDF

[0240] AVYFCQQYNRYPYTFGGGTKLEIKGGGGSEIVMTQSPSTLSSASVGDRVIINCQS

[0241] SPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQ

[0242] PDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVES

[0243] GGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYA

[0244] SWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLV

[0245] TVSSGGGGSEVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGK

[0246] GLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYF

[0247] CARKTISSVVDFYFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACR

[0248] PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK

[0249] QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN

[0250] ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

[0251] IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:34)

[0252] The amino acid sequence of the second CD19 / BCMA-CAR (which contains, in sequence, the CD8α guide strand, Q33-02G scFv, CD8 hinge region, CD8 transmembrane region, 4-1BB intracellular domain, and CD3ζ) is as follows:

[0253] MALPVTALLLPLALLLHAARPEIVMTQSPSTLSASSVGDRVIINCQSSPSVYN

[0254] NYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFA

[0255] TYYCAGTYVSGDRRAFGQGTKLTVLGGGGSEIVMTQSPATLSVSPGERATLSC

[0256] KASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISS

[0257] LQSEDFAVYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVQS

[0258] GAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDT

[0259] NYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTISSVVDFYFDY

[0260] WGQGTTVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTW

[0261] VRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAED

[0262] TAVYFCARDLDYVIDLWGPGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC

[0263] RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

[0264] KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY

[0265] NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

[0266] EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:35)

[0267] The amino acid sequence of the third type of CD19 / BCMA-CAR (which contains, in sequence, the CD8α guide strand, O2G-GS21 scFv, CD8 hinge region, CD8 transmembrane region, 4-1BB intracellular domain, and CD3ζ) is as follows:

[0268] MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVG

[0269] TNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFA

[0270] TYFCQQYNRYPYTSGGGTKVEIKRGGGGSEIVMTQSPSTLSASVGDRVIINCQS

[0271] SPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQ

[0272] PDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVES

[0273] GGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYA

[0274] SWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLV

[0275] TVSSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPG

[0276] QGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVY

[0277] YCARKTISSVVDFYFDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC

[0278] RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

[0279] KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY

[0280] NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

[0281] EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:36)

[0282] The amino acid sequence of the fourth CD19 / BCMA-CAR (successively including the CD8α leader chain, GS21-02G scFv, CD8 hinge region, CD8 transmembrane region, the intracellular domain of 4-1BB, and CD3ζ) is as follows:

[0283] MALPVTALLLPLALLLHAARPEIVMTQSPSTLSASVGDRVIINCQSSPSVYN

[0284] NYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFA

[0285] TYYCAGTYVSGDRRAFGQGTKLTVLGGGGSDIQLTQSPSFLSASVGDRVTITC

[0286] KASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISS

[0287] LQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQLV

[0288] QSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDG

[0289] DTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKTISSVVDFYFD

[0290] YWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMT

[0291] WVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAE

[0292] DTAVYFCARDLDYVIDLWGPGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEA

[0293] CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

[0294] FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL

[0295] YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY

[0296] SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:37)

[0297] Example 1: Preparation of humanized CD19 CAR-T cells

[0298] I. Humanization Screening of Anti-CD19 Mutual Antibodies

[0299] 1. Construction and lentiviral packaging of humanized anti-CD19-CAR

[0300] Humanization was performed on the murine antibody HI19a (named CNCT19 in this patent, with its scFv amino acid sequence as shown in SEQ ID NO:51). The humanized antibody sequences are numbered as follows: Q11 (with scFv amino acid sequence as shown in SEQ ID NO:16, VH amino acid sequence as shown in SEQ ID NO:1, and VL amino acid sequence as shown in SEQ ID NO:2), Q14 (with scFv amino acid sequence as shown in SEQ ID NO:17, VH amino acid sequence as shown in SEQ ID NO:3, and VL amino acid sequence as shown in SEQ ID NO:2), Q23 (with scFv amino acid sequence as shown in SEQ ID NO:18, VH amino acid sequence as shown in SEQ ID NO:4, and VL amino acid sequence as shown in SEQ ID NO:5), Q33 (with scFv amino acid sequence as shown in SEQ ID NO:19, VH amino acid sequence as shown in SEQ ID NO:4, and VL amino acid sequence as shown in SEQ ID NO:6), and Q54 (with scFv amino acid sequence as shown in SEQ ID NO:16). NO:20, the amino acid sequence of VH is shown in SEQ ID NO:3, the amino acid sequence of VL is shown in SEQ ID NO:7), GS32 (the amino acid sequence of its scFv is shown in SEQ ID NO:21, the amino acid sequence of VH is shown in SEQ ID NO:8, the amino acid sequence of VL is shown in SEQ ID NO:9), GS21 (the amino acid sequence of its scFv is shown in SEQ ID NO:22, the amino acid sequence of VH is shown in SEQ ID NO:10, the amino acid sequence of VL is shown in SEQ ID NO:11), GS22 (the amino acid sequence of its scFv is shown in SEQ ID NO:23, the amino acid sequence of VH is shown in SEQ ID NO:8, the amino acid sequence of VL is shown in SEQ ID NO:11).Different anti-CD19 humanized scFvs were combined with CD8α guide chain signal peptide (as shown in SEQ ID NO:33), CD8 hinge region (as shown in SEQ ID NO:29), CD8 transmembrane region (as shown in SEQ ID NO:30), 4-1BB intracellular domain (as shown in SEQ ID NO:31), and CD3ζ (as shown in SEQ ID NO:30). CAR genes were constructed by combining different anti-CD19 CAR genes into different lentiviral master plasmids (manufacturer: SBI, catalog number: CD500-CD800, as described in Example 1 of WO2021 / 121227 for routine resistance modification) to obtain CAR expression vectors. The master plasmids were co-transfected with three packaging plasmids (pMD2.G (purchased from Biovector, product number Biovector012259), pMDLg / pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253)) into 293T cells. Lentiviral viruses containing different anti-CD19-CARs were collected for infecting T cells.

[0301] 2. Preparation of different humanized anti-CD19 CAR-T cells

[0302] The T cell culture medium is Optimizer basal medium supplemented with OpTmizer amplification additive, ISR and GlutaMAX (commercially available), and cytokines IL-7 (10 ng / mL) and IL-15 (5 ng / mL).

[0303] PBMC cell resuscitation was performed on day -1.

[0304] On day 0, T cells in PBMCs were sorted using CD4 and CD8 magnetic beads, and T cells were activated using Thermo CD3 / CD28 activation magnetic beads.

[0305] On day 1, T cells were infected with different humanized anti-CD19-CAR lentiviruses in the CNCT19, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 groups.

[0306] T cell culture medium was added every 2-3 days according to the culture progress. Cell viability and CD19-CAR positivity were measured by sampling and counting. After 9 days of culture, CAR-T cells (humanized Q11 CAR-T cells, Q14 CAR-T cells, Q23 CAR-T cells, Q33 CAR-T cells, Q54 CAR-T cells, GS32 CAR-T cells, GS21 CAR-T cells, GS22 CAR-T cells and one type of murine CNCT19 CAR-T cell) were harvested and subjected to in vitro functional tests.

[0307] After humanizing the CNCT19 sequence, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 were selected for a series of functional verification experiments. The experimental results are shown below. Figures 1-5 .

[0308] 1. Detection of CAR-positive proportion in CAR-T cells

[0309] Eight types of humanized CAR-T cells and one type of mouse-derived CNCT19 CAR-T cells obtained in Example 1 were mixed with CD19 antigen-conjugated PE fluorescein and incubated at room temperature in the dark for 15 minutes. The supernatant was removed by centrifugation, and the CAR molecule positivity rate of each cell type was detected by flow cytometry at different days after transduction (Day 3, Day 6, and Day 9). The results are as follows: Figure 1 and Figure 2 As shown: except for the poor CAR positivity rate of the Q23 structure (Q23 CAR-T cells), the expression of other structures is normal; except for the low MFI (mean fluorescence intensity) of CAR expression in the Q23 structure (Q23 CAR-T cells), the expression of other structures is normal.

[0310] 2. In vitro killing of CAR-T cells (short-term)

[0311] 1. Target cell plate

[0312] 1.1 Target cell preparation:

[0313] 1.1.1 The target cells are in good logarithmic growth phase with a cell viability of over 85%.

[0314] 1.1.2 Count the target cells according to standard cell counting procedures, and take 2 × 10⁻⁶ cells. 6 Add target cells (Nalm6, K562-CD19, MM.1S, K562-BCMA) to a 15ml centrifuge tube, centrifuge at 300g for 8 minutes, remove the supernatant, and resuspend the target cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁻⁶ cells / mL. 5 per ml.

[0315] 1.1.3 Carefully transfer the diluted target cells into a sterile sample loading tray, and use a pipette to add the target cells into a black, transparent, flat-bottomed 96-well plate at a rate of 50 μL / well.

[0316] 1.2 Effector cell plate

[0317] 1.2.1 Effector cell preparation: Based on effector-to-target ratios of 9:1, 3:1, and 1:1, the cell dosage was 9 × 10⁶ cells / year. 4 1, 3×10 4 1×10 4 Count the effector cells, take the required number of CAR-T cell samples into a 15ml centrifuge tube, centrifuge at 300g for 8min, and discard the supernatant.

[0318] 1.2.2 Resuspend CAR-T cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) and adjust the effector cell density according to the effector-target ratio.

[0319] 1.2.3 Add effector cells to the well plates at ratios of 9:1, 3:1, and 1:1, 50 μL / well.

[0320] 1.3 Setting up the target cell control group

[0321] Add 50 μL of target cells and 50 μL of T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group to make the total culture volume of the target cell control group consistent with the total culture volume of the co-culture experimental group.

[0322] 2. Detect luc value

[0323] 2.1 Cell co-culture: Place the 96-well plate with the added samples in a carbon dioxide incubator and co-culture for 4 hours.

[0324] 2.2 Adding the detection reagent: Melt steadyglo luciferase (commercially available) at 4°C or room temperature in the dark. Dilute the steadyglo required for the experiment with PBS 3 times and mix well. Add 50 μL to each well and place on a 96-well plate shaker. Shake at 100 rpm for 15 min.

[0325] 2.3 Instrumental Detection: Turn on the multi-mode microplate reader and computer software. Place the shaken 96-well plate into the multi-mode microplate reader and detect the luciferase fluorescence intensity. Remove the 96-well plate and turn off the instrument and computer.

[0326] 3. Calculation of cell killing activity

[0327] After obtaining the luc values ​​of all wells, calculate the cytotoxic activity using the following formula.

[0328] Cell-killing activity = (average luc value of target cells alone - luc value of co-culture wells) / average luc value of target cells alone × 100%

[0329] Test results as follows Figure 3 As shown, there was no significant difference in the killing activity of CAR-T cells against target cells Nalm6 among all groups.

[0330] 3. Cytokine detection

[0331] 1. CAR-T cells and target cells are mixed at an effector-to-target ratio of 1:1.

[0332] 2. Incubate in carbon dioxide medium for 24 hours

[0333] 3. After incubation, centrifuge at 500g for 10 minutes and collect the supernatant.

[0334] 4. Use the Human Th1 Panel (5-Plex) with Filter Plate V02 kit to detect the cytokine concentration in the supernatant.

[0335] Test results as follows Figure 4A and 4B As shown, the IL-2 release of CAR-T cells from humanized structures Q11, Q14, Q33, Q54, GS32, and GS21 was higher than that of the murine CNCT19 cell line. The IFN-γ release of CAR-T cells from humanized structures Q11, Q33, GS32, and GS21 was also higher than that of the murine CNCT19 cell line. These experimental results indicate that the humanization modification of the murine antibody in this application improved the antibody affinity, thereby resulting in higher IL-2 and IFN-γ release in the humanized CAR-T cells compared to the murine CNCT19 cell line.

[0336] 4. Multiple rounds of stimulation

[0337] 1. CAR-T cells and target cells were prepared at an effector-to-target ratio of 1:1, with a cell count of 0.5 × 10⁻⁶. 6 0.5×10 6 Mix

[0338] 2. Detect the number of CAR-T cells every two days, and at the same time take 0.5 × 10⁻⁶ cells. 6 CAR-T cells, added 0.5×10 6 target cells

[0339] 3. After four rounds of target cell stimulation, the expansion fold of CAR-T cells was calculated.

[0340] Test results as follows Figure 5As shown: After four rounds of target cell stimulation, the CAR-T amplification fold of humanized structures Q11, Q33, and GS21 was consistent with that of the mouse structure CNCT19.

[0341] In addition, the humanization scores of the VH and VL chains of CD19 were assessed using three biocomputation tools: T20 score, HS score, and QASis. The scores from the three tools were consistent, with Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 showing higher humanization scores compared to murine CNCT19 (see [link to relevant documentation]). Figure 6 Two immunogenicity prediction tools, Alpha MHCv2 and CD4 T cell Immunogenicity Prediction Tool, were used to predict the immunogenicity of humanized antibodies. The prediction results were largely consistent, showing that Q11, Q14, Q23, Q33, Q54, and GS21 had the lowest immunogenicity risk (see [link to study]). Figure 7 ).

[0342] The immunogenicity of antibody drugs may trigger an anti-drug immune response, thereby inducing the production of anti-drug antibodies (ADAs). The evaluation of antibody immunogenicity primarily utilizes ADA analysis. The immunogenicity of CAR-T cells induces an anti-CAR immune response, which in turn destroys and eliminates CAR-T cells, contributing to CAR-T therapy failure. The scFvs used in CAR-T therapy contain murine sequences, a crucial factor in generating anti-CAR immune responses. Humanizing murine scFvs to circumvent these murine-derived anti-CAR immune responses is a vital strategy; however, the success of this strategy hinges on whether antibody functional degradation or loss can be avoided during the modification process.

[0343] Based on in vitro functional data of different humanized CD19 CAR-T cells, combined with antibody humanization scores and antibody immunogenicity prediction data, the Q33 and GS21 sequences showed good CAR positivity rates, high in vitro cytokine activity and cytokine release, and low immunogenicity. We selected the Q33 and GS21 sequences to construct a dual-target CAR-T for further validation.

[0344] Example 2: Preparation and Screening Validation of BCMA-CD19 Bispecific CAR-T Cells

[0345] 1. Construction and lentiviral packaging of humanized anti-CD19 / BCMA-CAR

[0346] Different scFvs were constructed using humanized anti-CD19 sequences (Q33, GS21) and humanized anti-BCMA sequences (02G, whose scFv amino acid sequence is shown in SEQ ID NO:24, VH amino acid sequence is shown in SEQ ID NO:12, and VL amino acid sequence is shown in SEQ ID NO:13). The dual-target scFv antibody sequences are 02G-Q33 (whose scFv amino acid sequence is shown in SEQ ID NO:25), Q33-02G (whose scFv amino acid sequence is shown in SEQ ID NO:26), 02G-GS21 (whose scFv amino acid sequence is shown in SEQ ID NO:27), and GS21-02G (whose scFv amino acid sequence is shown in SEQ ID NO:28). Four dual-target scFvs were combined with the CD8α guide chain signal peptide (as shown in SEQ ID NO:33), CD8 hinge region (as shown in SEQ ID NO:29), CD8 transmembrane region (as shown in SEQ ID NO:30), 41BB intracellular domain (as shown in SEQ ID NO:31), and CD3ζ (as shown in SEQ ID NO:32) to construct the CD19 / BCMA-CAR gene. Different anti-CD19 / BCMA-CAR genes were constructed into different lentiviral master plasmids. The master plasmids were co-transfected with three packaging plasmids (pMD2.G (purchased from Biovector, product number Biovector012259), pMDLg / pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253)) into 293T cells. Lentiviral viruses containing different anti-CD19 / BCMA-CARs were collected for T cell infection.

[0347] 2. Preparation of different anti-CD19 / BCMACAR-T agents

[0348] The T cell culture medium is Optimizer basal medium supplemented with OpTmizer amplification additive and ISR (commercially available), and also supplemented with a certain concentration of cytokines IL-7 (10 ng / mL) and IL-15 (5 ng / mL).

[0349] PBMC cell resuscitation was performed on day -1.

[0350] On day 0, T cells in PBMCs were sorted using CD4 and CD8 magnetic beads, and T cells were activated using Thermo CD3 / CD28 activation magnetic beads.

[0351] On day 1, T cells were infected with the corresponding anti-CD19 / BCMA-CAR lentivirus in the 02G, 02G-Q33, Q33-02G, 02G-GS21, and GS21-02G groups.

[0352] T-cell culture medium was added every 2-3 days according to the culture progress. After culturing to 9 cells, CAR-T cells (02GCAR-T cells, 02G-Q33 CAR-T cells, Q33-02G CAR-T cells, 02G-GS21 CAR-T cells, and GS21-02GCAR-T cells, respectively) were harvested. Samples were taken to detect the positive rates of CD19-CAR and BCMA-CAR, and in vitro functional tests were performed. The experimental results are shown in [link to experimental results]. Figures 8-18 .

[0353] 1. Detection of CAR-positive proportion in CAR-T cells

[0354] CNCT19 CAR-T cells, Q33 CAR-T cells, GS21 CAR-T cells obtained in Example 1, and 02G CAR-T cells, 02G-Q33 CAR-T cells, Q33-02G CAR-T cells, 02G-GS21 CAR-T cells, and GS21-02G CAR-T cells or UTD (representing untransduced T cells) obtained in Example 2 were mixed with CD19 antigen-conjugated PE fluorescein or BCMA antigen-conjugated FITC fluorescein and incubated at room temperature in the dark for 15 minutes. The supernatant was removed by centrifugation, and the CAR molecule positivity rate of each cell type was detected by flow cytometry. The results are as follows: Figure 8 As shown, among the four dual-target structures, the 02G-Q33 structure showed the best CAR positivity rate.

[0355] 2. In vitro killing of CAR-T cells (short-term)

[0356] 1. Target cell plate

[0357] 1.1 Target cell preparation:

[0358] 1.1.1 The target cells are in good logarithmic growth phase with a cell viability of over 85%.

[0359] 1.1.2 Count the target cells according to standard cell counting procedures, and take 2 × 10⁻⁶ cells. 6 Add target cells (Nalm6, K562-CD19, MM.1S, K562-BCMA) to a 15ml centrifuge tube, centrifuge at 300g for 8 minutes, remove the supernatant, and resuspend the target cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁻⁶ cells / mL. 5 per ml.

[0360] 1.1.3 Carefully transfer the diluted target cells into a sterile sample loading tray, and use a pipette to add the target cells into a black, transparent, flat-bottomed 96-well plate at a rate of 50 μL / well.

[0361] 1.2 Effector cell plate

[0362] 1.2.1 Effector cell preparation: Based on effector-to-target ratios of 9:1, 3:1, and 1:1, the cell dosage was calculated to be 9 × 10⁻⁶ cells / year. 4 1, 3×10 4 1×10 4 Count the effector cells, take the required number of CAR-T cell samples into a 15ml centrifuge tube, centrifuge at 300g for 8min, and discard the supernatant.

[0363] 1.2.2 Resuspend CAR-T cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) and adjust the effector cell density according to the effector-target ratio.

[0364] 1.2.3 Add effector cells to the well plates at ratios of 9:1, 3:1, and 1:1, 50 μL / well.

[0365] 1.3 Setting up the target cell control group

[0366] Add 50 μL of target cells and 50 μL of T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group to make the total culture volume of the target cell control group consistent with the total culture volume of the co-culture experimental group.

[0367] 2. Detect luc value

[0368] 2.1 Cell co-culture: Place the 96-well plate with the added samples in a carbon dioxide incubator and co-culture for 4 hours.

[0369] 2.2 Adding the test reagents: Melt steadyglo luciferase (commercially available) at 4°C or room temperature in the dark. Dilute the steadyglo required for the experiment with PBS 3 times and mix well. Add 50 μL to each well and place on a 96-well plate shaker. Shake at 100 rpm for 15 min.

[0370] 2.3 Instrumental Detection: Turn on the multi-mode microplate reader and computer software. Place the shaken 96-well plate into the multi-mode microplate reader and detect the luciferase fluorescence intensity. Remove the 96-well plate and turn off the instrument and computer.

[0371] 3. Calculation of cell killing activity

[0372] After obtaining the luc values ​​of all wells, calculate the cytotoxic activity using the following formula.

[0373] Cell-killing activity = (average luc value of target cells alone - luc value of co-culture wells) / average luc value of target cells alone × 100%

[0374] Test results as follows Figure 9 As shown: In killing CD19+ target cells Nalm6, the killing activities of the four dual-target CD19 / BCMA CAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) were consistent with those of the single-target CD19 CAR-T (CNCT19, Q33, GS21).

[0375] Test results as follows Figure 10 As shown: In killing CD19+ target cells K562-CD19, the killing activity of the four dual-target CD19 / BCMACAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) was consistent with that of the single-target CD19CAR-T (CNCT19, Q33, GS21).

[0376] Test results as follows Figure 11 As shown, in killing BCMA+ target cells MM.1S, the killing activity of the four dual-target CD19 / BCMA CAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) was higher than that of the single-target BCMA CAR-T (02G).

[0377] Test results as follows Figure 12 As shown: In killing BCMA+ target cells K562-BCMA, the dual-target CD19 / BCMACAR-T (Q33-02G, GS21-02G) exhibited higher killing activity than the single-target BCMA CAR-T (02G). In killing BCMA+ target cells K562-BCMA, the dual-target CD19 / BCMA CAR-T (02G-Q33, 02G-GS21) showed consistent killing activity with the single-target BCMACAR-T (02G).

[0378] 3. Cytokine detection

[0379] 1. CAR-T cells and target cells are mixed at an effector-to-target ratio of 1:1.

[0380] 2. Incubate in carbon dioxide medium for 24 hours

[0381] 3. After incubation, centrifuge at 500g for 10 minutes and collect the supernatant.

[0382] 4. Use the Human Th1 Panel (5-Plex) with Filter Plate V02 kit to detect the cytokine concentration in the supernatant.

[0383] Test results as follows Figure 13A and 13B As shown, in killing CD19+ target cells Nalm6, the IL-2 release level was highest among the four dual-target structures, 02G-Q33. In killing BCMA+ target cells MM.1S, the IL-2 release levels of the four dual-target CD19 / BCMACAR-T structures (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) were lower than those of the single-target BCMACAR-T (02G).

[0384] Test results as follows Figure 14A and 14B As shown, in killing CD19+ target cells Nalm6, the IFN-γ release levels of the four dual-target CD19 / BCMACAR-T cells (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) were higher than those of the single-target BCMACAR-T cell (02G). In killing BCMA+ target cells MM.1S, the IFN-γ release levels of the four dual-target CD19 / BCMACAR-T cells (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) were lower than those of the single-target BCMACAR-T cell (02G).

[0385] 4. Multiple rounds of stimulation

[0386] 1. CAR-T cells were used in combination with CD19+ target cells (Nalm6) or BCMA+ target cells (RPMI-8226) at an effector-to-target ratio of 1:1 and a cell count of 0.5 × 10⁻⁶. 6 0.5×10 6 Mix

[0387] 2. Detect the number of CAR-T cells every two days, and at the same time take 0.5 × 10⁻⁶ cells. 6 CAR-T cells, added 0.5×10 6 target cells

[0388] 3. After four rounds of target cell stimulation, the expansion fold of CAR-T cells was calculated.

[0389] Test results as follows Figure 15 As shown, after four rounds of target cell stimulation, the CAR-T amplification fold of the dual-target structure O2G-Q33 was higher than that of the single-target CNCT19 and Q33.

[0390] Test results as follows Figure 16 As shown: After four rounds of target cell stimulation, the CAR-T amplification fold of the dual-target structure 02G-Q33 was consistent with that of the single-target structure 02G.

[0391] 4. Animal experiments and in vivo efficacy experiments

[0392] 1. Inject 5×10⁵ mg / L of the drug into the tail vein of NSG mice. 5 One CD19+ target cell Nalm6-luc or 2×10 6 BCMA+ target cells MM.1S-luc.

[0393] 2. Three days after tumor implantation, a tail vein injection was performed to detect 3×10⁻⁶ tumor cells. 6 One CAR-T cell.

[0394] 3. Perform fluorescence imaging on mice at time points (Day 0, Day 3, Day 7, Day 10, Day 14, Day 21, Day 28) and record the fluorescence intensity.

[0395] Test results as follows Figure 17 As shown, the efficacy of 02G-Q33 dual-target CD19 / BCMACAR-T is the same as that of Q33 single-target CD19 CAR-T.

[0396] Test results as follows Figure 18 As shown, the efficacy of 02G-Q33 dual-target CD19 / BCMACAR-T is the same as that of 02G single-target BCMACAR-T.

[0397] In the validation of CAR-T cells constructed with four dual-target structures (02G-Q33, Q33-02G, 02G-GS21, and GS21-02G), based on data including the expression of CD19-CAR and BCMA-CAR positive rates, in vitro killing activity, and cytokine release, the 02G-Q33 group showed the best expression and function.

[0398] Based on multi-round stimulation amplification data and in vivo efficacy data from animal experiments, the dual-target CD19 / BCMA CAR-T (02G-Q33) has the same efficacy as the single-target CD19 CAR-T and the single-target BCMA CAR-T.

Claims

1. A bispecific chimeric antigen receptor targeting BCMA-CD19, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; wherein: The extracellular antigen recognition domain includes an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19VL, which are selected from one of the following: 1) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 2) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 3) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 4) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or 5) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or 6) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:9; or 7) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or 8) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:

11.

2. The bispecific chimeric antigen receptor of claim 1, wherein, The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:

11.

3. The bispecific chimeric antigen receptor of claim 1 or 2, wherein, The BCMA VH sequence comprises the amino acid sequence shown in SEQ ID NO:12, and the BCMA VL sequence comprises the amino acid sequence shown in SEQ ID NO:

13.

4. The bispecific chimeric antigen receptor of claim 1 or 2, wherein, The extracellular antigen recognition domain of the bispecific chimeric antigen receptor is scFv antibody, sc(Fv)2 antibody or [sc(Fv)2]2 antibody.

5. The bispecific chimeric antigen receptor of claim 4, wherein, The scFv antibody comprises any one of the following structures: CD19 VL sequence - 1st linker sequence - BCMA VL sequence - 2nd linker sequence - BCMA VH sequence - 3rd linker sequence - CD19 VH sequence, BCMA VL sequence - 4th linker sequence - CD19 VL sequence - 5th linker sequence - CD19 VH sequence - 6th linker sequence - BCMA VH sequence, CD19 VL sequence - 7th linker sequence - CD19 VH sequence - 8th linker sequence - 9th linker sequence - BCMA VH sequence, BCMA VL sequence - 10th linker sequence - BCMA VH sequence - 11th linker sequence - CD19 VL sequence - 12th linker sequence - CD19 VH sequence; Optionally, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes any one of the following structures: CD19 VL sequence-first linker sequence-BCMA VL sequence-second linker sequence-BCMA VH sequence-third linker sequence-CD19 VH sequence and BCMA VL sequence-fourth linker sequence-CD19 VL sequence-fifth linker sequence-CD19 VH sequence-sixth linker sequence-BCMA VH sequence; Further optionally, the first linking sequence, the second linking sequence, the third linking sequence, the fourth linking sequence, the fifth linking sequence, the sixth linking sequence, the seventh linking sequence, the eighth linking sequence, the ninth linking sequence, the tenth linking sequence, the eleventh linking sequence, and the twelfth linking sequence are independently selected from one or more of the following sequences: SEQ ID NO:14 and SEQ ID NO:

15.

6. The bispecific chimeric antigen receptor of claim 5, wherein, The extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:

28.

7. The bispecific chimeric antigen receptor according to claim 1, wherein, 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:29; and / or The transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, 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:

30.

8. The bispecific chimeric antigen receptor according to claim 1, wherein, The intracellular domain includes an intracellular signal transduction region; optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signal transduction region is derived from CD3ζ; even further optionally, the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:

32.

9. The bispecific chimeric antigen receptor according to claim 1, wherein, The intracellular domain further includes a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; further optionally, the co-stimulatory signal transduction region is derived from 4-1BB; even further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:

31.

10. The bispecific chimeric antigen receptor according to any one of claims 1-9, further comprising a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide 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:

33.

11. The bispecific chimeric antigen receptor according to any one of claims 1-9, wherein, The bispecific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:

37.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific chimeric antigen receptor as described in any one of claims 1-11; Optionally, the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises nucleotide sequences encoding CD19 VH and CD19 VL and nucleotide sequences encoding BCMA VH and BCMA VL, wherein the nucleotide sequences encoding CD19 VH and CD19 VL are selected from one group of the following: 1) A nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:38; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or 2) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or 3) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:5, as shown in SEQ ID NO:42; and / or 4) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:6, as shown in SEQ ID NO:43; and / or 5) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:7, as shown in SEQ ID NO:44; and / or 6) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:9, as shown in SEQ ID NO:46; and / or 7) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:10, as shown in SEQ ID NO:47; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:48; and / or 8) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:48; The nucleotide sequences encoding BCMA VH and BCMA VL are as follows: Nucleotide sequences encoding the BCMA VH amino acid sequence as shown in SEQ ID NO:12, as shown in SEQ ID NO:49; and nucleotide sequences encoding the BCMA VL amino acid sequence as shown in SEQ ID NO:13, as shown in SEQ ID NO:

50.

13. A carrier comprising the isolated nucleic acid molecule of claim 12; Optionally, the carrier is an expression carrier; Further, optionally, the vector is a viral vector; Alternatively, the vector may be a lentiviral vector.

14. An engineered immune effector cell comprising any one of claims 1-11, the isolated nucleic acid of claim 12, or the vector of claim 13.

15. The engineered immune effector cells according to claim 14, wherein, The engineered immune effector cells are selected from one or more of the following: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells. Optionally, the engineered immune effector cells are T lymphocytes; Further, optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes.

16. A pharmaceutical composition comprising engineered immune effector cells as described in claim 14 or 15 and pharmaceutically acceptable excipients; optionally, the pharmaceutically acceptable excipients comprise protectants; optionally, the pharmaceutically acceptable excipients comprise cell cryopreservation solutions.

17. The pharmaceutical composition according to claim 16, wherein, The pharmaceutical composition is a cell suspension or frozen cells thereof; or the pharmaceutical composition is an intravenous injection.

18. The use of the chimeric antigen receptor of any one of claims 1-11, the isolated nucleic acid of claim 12, the vector of claim 13, or the engineered immune effector cell of claim 14 in the preparation of a medicament for treating diseases or conditions associated with BCMA expression.

19. The use according to claim 18, wherein, The disease or condition associated with BCMA expression is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma.

20. The use according to claim 19, wherein, The disease or condition associated with BCMA expression may be an autoimmune disease; optionally, the autoimmune disease may be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

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