WO2024051057A1 - Multi-target chimeric antigen receptor comprising cd38 targeting - Google Patents

Multi-target chimeric antigen receptor comprising cd38 targeting Download PDF

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WO2024051057A1
WO2024051057A1 PCT/CN2022/144325 CN2022144325W WO2024051057A1 WO 2024051057 A1 WO2024051057 A1 WO 2024051057A1 CN 2022144325 W CN2022144325 W CN 2022144325W WO 2024051057 A1 WO2024051057 A1 WO 2024051057A1
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linker
amino acid
binding domain
cells
acid sequence
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French (fr)
Chinese (zh)
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王建祥
王敏
陈兆琪
熊冬生
卢杨
饶青
田征
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中国医学科学院血液病医院(中国医学科学院血液学研究所)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of cell therapy, and in particular to a multi-target chimeric antigen receptor containing CD38.
  • CAR Chimeric Antigen Receptor
  • T cells can specifically kill tumor cells expressing specific tumor-associated antigens, and have received widespread attention and application in tumor treatment.
  • the structure of CAR generally consists of an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction region.
  • the extracellular antigen-binding region is composed of sequences derived from monoclonal antibodies (scFv).
  • scFv monoclonal antibodies
  • CARs are divided into first generation (without costimulatory molecules), second generation (with one costimulatory molecule) and third generation (with two costimulatory molecules) based on the number of costimulatory molecules added to the intracellular signal transduction region.
  • second-generation CAR-T cell therapy is more commonly used in the clinical treatment of hematological tumors.
  • CAR-T has been widely used in the treatment of blood tumors.
  • CD19 CAR-T has shown significant therapeutic effects on B-cell acute lymphoblastic leukemia (ALL) or lymphoma.
  • ALL B-cell acute lymphoblastic leukemia
  • lymphoma There are also studies exploring the therapeutic effect of CAR-T on multiple myeloma.
  • MM Multiple myeloma
  • the current treatment of choice for MM is still allogeneic hematopoietic stem cell transplantation.
  • traditional treatments can only delay disease progression and prolong patient survival.
  • MM is still considered to be completely incurable.
  • new treatments need to be explored.
  • BCMA (CD269) is specifically expressed on the surface of MM cells, while it is not expressed or has low expression on plasma cells and mature B cells. It is also not expressed on the surface of other peripheral blood mononuclear cells and CD34+ hematopoietic cells. Therefore, it is an ideal target for MM immunotherapy. point.
  • CD38 is highly expressed on the surface of various blood tumor cells such as MM, diffuse large B lymphoma, acute myeloid leukemia, etc.
  • Daratumumab targeting CD38 is widely used in the treatment of relapsed and refractory MM.
  • mice-derived monoclonal antibodies Commonly used antibodies for immunotherapy against MM are mouse-derived monoclonal antibodies.
  • mouse-derived antibodies have poor immunogenicity and can easily cause immune escape and lead to disease recurrence. Therefore, humanizing them has become a priority. the main means to solve this problem.
  • how to maintain the affinity of the antibody during the humanization process and how to achieve better efficacy of the humanized antibody for the application of CAR-T therapy have become urgent problems.
  • One aspect of the present invention is to target CD38+ tumors in the prior art, such as the mouse-derived monoclonal antibodies used in immunotherapy for multiple myeloma, which have poor immunogenicity and can easily cause immune escape and lead to disease recurrence.
  • the prior art To solve the problem of the lack of CAR-T therapy that utilizes humanized antibodies and has better efficacy, a multi-target chimeric antigen receptor containing CD38 is provided.
  • the chimeric antigen receptor includes an extracellular region, a transmembrane region and an intracellular region, the intracellular region includes an intracellular signal transduction region, and the The outer region includes a first binding domain that specifically binds to the CD38 molecule, and the first binding domain includes the CDR1, CDR2, and CDR3 amino acids of the heavy chain variable region (VH) as shown in SEQ ID No. 1-3.
  • having 85% to 99% identity means having 85% to 99% identity with the nucleotide and amino acid sequences described in the present invention, which can be 85%, 86%, or 87%. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity can be modified in a suitable manner. Certain sites of the above-mentioned nucleotide and amino acid sequences, such as certain conserved sites, are subject to random or engineered point mutations and other conservative sequence modifications.
  • the purpose may be, for example, to obtain better expression levels, affinity and/or dissociation properties, but retain the same biological function as the original sequence, these mutated nucleotide or amino acid sequences are also deemed to be included within the protection scope of the present invention.
  • the extracellular region of the chimeric antigen receptor may further comprise a second binding domain, which together with the first binding domain forms a binding domain capable of binding dual targets.
  • the second binding domain specifically binds to a protein molecule selected from BCMA, CD19, CD138, SLAMF7, GAL-3, GPRC5D, PD-L1, CD155, FCRL5 or ICAM1.
  • the second binding domain is a binding domain that specifically binds to BCMA protein molecules.
  • the second binding domain includes the CDR1, CDR2, CDR3 amino acid sequences of the heavy chain variable region (VH) shown in sequence as SEQ ID No. 7-9 and The CDR1, CDR2, and CDR3 amino acid sequences of the light chain variable region (VL) shown in SEQ ID No. 10-12, or have 85% to 99% identity with the amino acid sequence and have the same biological function. the sequence of.
  • point mutations can be made to the complementarity determining regions (CDRs), for example, to obtain better affinity and/or dissociation properties, to obtain better expression levels, etc. , as long as it can form a complementary binding structure with the antigenic determinant, the mutated amino acid sequence is also deemed to be included in the protection scope of the present invention.
  • CDRs complementarity determining regions
  • the first binding domain and the second binding domain can be constructed in the same chimeric antigen receptor expression vector; or ii) the first binding domain
  • the structural domain and the second binding domain can be constructed in different chimeric antigen receptor expression vectors respectively.
  • the different expression vectors can be transduced into the same isolated cell using conventional methods, so that It has the same or similar biological function as that after the expression vector in i) is transduced into the same isolated cell.
  • the first binding domain is an scFv that specifically binds to a CD38 molecule, the scFv comprising the amino acid sequence of the heavy chain variable region (VH) and the light chain variable region. Amino acid sequence of region (VL).
  • the amino acid sequence of the scFv is a humanized sequence.
  • the amino acid sequence of the heavy chain variable region (VH) of the scFv is shown in SEQ ID No. 13, and the light chain variable region of the scFv ( The amino acid sequence of VL) is as shown in SEQ ID No. 14, or a sequence that has 85% to 99% identity with the amino acid sequence and has the same biological function.
  • the scFv may generally comprise a single or repeated number of VL and VH sequences, and the VL and VH sequences comprise the CDRs.
  • the scFv that specifically binds to CD38 molecules is a monovalent antibody.
  • the number of repeat sequences of the light chain variable region (VL) and the heavy chain variable region (VH) may be 2 or 3.
  • the scFv that specifically binds to CD38 molecules forms a bivalent Diabody; when the number of repeat sequences is 3, the scFv that specifically binds to CD38 molecules forms a trivalent Triabody. form.
  • the light chain variable region (VL) and the heavy chain variable region (VH) can be connected in any suitable order.
  • the heavy chain variable region (VH) and The connection sequence of the light chain variable region (VL) is 5'-VL-first linker-VH-3' or 5'-VH-first linker-VL-3'.
  • the first linker is a linker composed of glycine (Gly) and serine (Ser). More preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is one or multiple GGGGS sequences in series; further preferably, in one embodiment of the present invention, the plurality of GGGGS sequences are Three; further preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is shown in SEQ ID No. 34.
  • the extracellular region of the chimeric antigen receptor further includes a second binding domain, and the second binding domain is an scFv that specifically binds to the protein molecule, and the scFv It includes the amino acid sequence of the heavy chain variable region (VH) and the amino acid sequence of the light chain variable region (VL).
  • the amino acid sequence of the scFv is a humanized sequence.
  • the protein molecule is a BCMA protein molecule.
  • the amino acid sequence of the heavy chain variable region (VH) of the scFv is shown in SEQ ID No. 15, and the light chain variable region of the scFv
  • the amino acid sequence of (VL) is as shown in SEQ ID No. 16, or a sequence that has 85% to 99% identity with the amino acid sequence and has the same biological function.
  • the scFv may generally comprise a single or repeated number of VL and VH sequences, and the VL and VH sequences comprise the CDRs.
  • the scFv that specifically binds to the protein molecule is a monovalent antibody.
  • the number of repeat sequences of the light chain variable region (VL) and the heavy chain variable region (VH) may be 2 or 3.
  • the scFv that specifically binds to the protein molecule forms a bivalent Diabody; when the number of repeat sequences is 3, the scFv that specifically binds to the protein molecule forms a bivalent Diabody. Trivalent Triabody form.
  • the light chain variable region (VL) and the heavy chain variable region (VH) can be connected in any suitable order.
  • the protein molecule is a BCMA protein molecule.
  • the connection sequence of the heavy chain variable region (VH) and the light chain variable region (VL) is 5'-VL-first linker-VH-3 'or 5'-VH-first linker-VL-3'.
  • the first linker is a linker composed of glycine (Gly) and serine (Ser). More preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is one or multiple GGGGS sequences in series; further preferably, in one embodiment of the present invention, the plurality of GGGGS sequences are three; further preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is shown in SEQ ID No. 34.
  • the first binding domain and the second binding domain are connected through a second linker.
  • Any suitable connector can achieve the purpose of the present invention.
  • the amino acid sequence of the second linker is one or multiple EAAAK sequences in series. More preferably, in one embodiment of the present invention, the plurality is five;; further preferably, in one embodiment of the present invention, the amino acid sequence of the second linker is such as SEQ ID No. 35 shown.
  • the binding domain of the extracellular region includes:
  • the protein molecule is a BCMA protein molecule.
  • the binding domain of the extracellular region includes:
  • the binding domain of the extracellular region includes the amino acid sequence shown in SEQ ID No. 17 or SEQ ID No. 18, or has 85% similarity with the amino acid sequence. A sequence that is ⁇ 99% identical and has the same biological function.
  • the VH and VL of the first binding domain and the VH and VL of the second binding domain can also be connected through a third linker and a fourth linker. Connect to form a loop.
  • the formation method may be that the binding domain of the extracellular region includes:
  • the protein molecule is a BCMA protein molecule.
  • the amino acid sequence of the third linker is shown in SEQ ID No. 36
  • the amino acid sequence of the fourth linker is shown in SEQ ID No. 37.
  • the binding domain of the extracellular region includes:
  • the binding domain of the extracellular region includes the amino acid sequence shown in SEQ ID No. 19 or SEQ ID No. 20, or has 85% similarity with the amino acid sequence. A sequence that is ⁇ 99% identical and has the same biological function.
  • BCMA + tumor cells and CD38 + tumor cells are combined with tanBCMA scFv-CD38 scFv (tanBC38 scFv), tanCD38 scFv-BCMA scFv (tan38BC scFv), loopBCMA scFv-CD38 scFv (loopBC38 scFv) or loopCD38
  • tanBCMA scFv-CD38 scFv tanBC38 scFv
  • tanCD38 scFv-BCMA scFv tanCD38 scFv
  • loopBCMA scFv-CD38 scFv loopBC38 scFv
  • loopBC38 scFv loopCD38
  • CAR-T cells prepared with tanBC38 scFv, tan38BC scFv, loopBC38 scFv and loop38BC scFv as antigen recognition regions can recognize and kill BCMA + and CD38 + tumor cells while not producing off-target effects on cells that do not express BCMA and CD38. .
  • the extracellular region may further comprise a signal peptide.
  • the signal peptide can guide the transfer of the antigen recognition region and hinge region to the outside of the cell. Any suitable signal peptide or combination of signal peptides can achieve the purpose of the present invention.
  • the extracellular region also includes a signal peptide constructed at the amino terminus of the chimeric antigen receptor or an amino acid sequence having more than 90% identity with the signal peptide. ;
  • the signal peptide is the signal peptide sequence in CD8 ⁇ or GM-CSF.
  • the signal peptide is the signal peptide shown in SEQ ID NO. 21.
  • the binding domain (C-terminus) comprised by the chimeric antigen receptor of the present invention is connected to the transmembrane region encoded by the hinge region.
  • Any suitable hinge region sequence may achieve the purposes of the present invention.
  • the hinge region is a hinge region sequence in CD8 ⁇ .
  • the chimeric antigen receptor further includes a transmembrane domain.
  • Any suitable transmembrane domain can achieve the purpose of the present invention.
  • the transmembrane region may be a transmembrane domain selected from the following proteins or an amino acid sequence having more than 90% identity with the protein: ⁇ of T cell receptor , ⁇ or ⁇ chain, CD2, CD3 ⁇ , CD4, CD7, CD8 ⁇ , CD8 ⁇ , CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD28, CD29, CD30, CD40, CD48, CD49a, CD49d, CD49f, CD66a, CD66b, CD66c, CD66d, CD66e, CD69, CD79A, CD79B, CD84, CD96, CD100, CD103, CD134, CD137, CD150, CD158A, CD158B1, CD158B2, CD158C, CD158D, CD158F1, CD
  • the transmembrane region is a transmembrane region sequence in CD8 ⁇ .
  • the intracellular region comprised by the chimeric antigen receptor further comprises a costimulatory factor
  • the costimulatory factor may be one of the functional signaling domains obtained by being selected from the following proteins or amino acid sequences having more than 90% identity with the proteins or Several: integrin, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, 4-1BB, B7-H3, CD278, GITR , BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49 ⁇ , IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD , CD11d, ITGAE, CD103, ITGAL, CD11 ⁇ , ITGAM, CD11b, ITGAX, CD11c
  • the costimulatory factor is CD28 or 4-1BB, or an amino acid sequence with more than 90% identity thereto.
  • the chimeric antigen receptor further comprises an intracellular signal transduction region.
  • the intracellular signal transduction region can be selected from the following proteins or an amino acid sequence having more than 90% homology with the protein: 4-1BB, B7-H3 , BAFFR, BLAME, BTLA, CD100, CD103, CD160, CD18, CD19, CD19a, CD2, CD247, CD27, CD276, CD28, CD29, CD3 ⁇ , CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84 , CD8alpha, CD8beta, CD96, CDS, CEACAM1, CRTAM, DAP-10, DNAM1, Fc gamma receptor, GADS, GITR, HVEM, IA4, ICAM-1, ICAM-1, Ig alpha, IL2R beta, IL2R gamma, IL7R alpha, integrin, IT
  • the intracellular signal transduction region is CD3 ⁇ .
  • the chimeric antigen receptor is based on anti-BCMA scFv and CD38 scFv antigen recognition regions, CD8 ⁇ hinge region and transmembrane region, and 4-1BB and CD3 ⁇ intracellular signaling structures
  • the structure formed by connecting the domains in series is the signaling domain, and its amino acid sequence is shown in SEQ ID NO. 22-25.
  • any peptide chain can be inserted as a spacer at an appropriate position between the antigen recognition region, hinge region, transmembrane region and intracellular signal region, and the peptide chain can be an oligopeptide or a polypeptide.
  • Another aspect of the present invention provides an isolated nucleic acid molecule encoding the amino acid sequence of the chimeric antigen receptor.
  • the sequence of the nucleic acid molecule is as shown in SEQ ID NO. 30-33, or a sequence that has more than 90% identity with it and has the same biological function.
  • the preparation method of the above-mentioned nucleic acid molecules can be based on the base sequences of the above-mentioned antigen recognition region, hinge region, transmembrane region, intracellular signal region and other structural domains, and can be prepared through known techniques such as chemical synthesis or PCR amplification. Generally, the codons encoding the amino acids of the above-described domains can be optimized to optimize their expression in the host cell. Information on the above base sequence can be obtained by searching known literature or databases such as NCBI (https://www.ncbi.nlm.nih.gov/).
  • Another aspect of the invention provides a vector comprising the above nucleic acid molecule.
  • the vector can be a linear vector or a circular vector. It may be a non-viral vector such as a plasmid, a viral vector, or a vector utilizing transposons.
  • the vector may contain regulatory sequences such as promoters and terminators, as well as marker sequences such as drug resistance genes and reporter genes.
  • the above-mentioned vector can also contain a sequence encoding a suicide gene, and the number of CAR-T cells in the body can be controlled by administering substances that activate the suicide gene according to the treatment process.
  • viral vector examples include plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and the like.
  • lentiviral expression vectors are used.
  • the vector contains an EF1 ⁇ promoter sequence.
  • Another aspect of the invention provides an isolated cell comprising the chimeric antigen receptor, the nucleic acid molecule or the vector.
  • the cells are i) immune response cells, preferably T cells, NK cells, NKT cells or CTL cells, more preferably T cells; or ii) induced pluripotent stem cells (iPSC).
  • immune response cells preferably T cells, NK cells, NKT cells or CTL cells, more preferably T cells; or ii) induced pluripotent stem cells (iPSC).
  • the cells are T cells of autologous or allogeneic origin.
  • the cells are autologous T cells.
  • the T cells are human T cells.
  • the T cells can come from body fluids such as blood and bone marrow, or from tissues such as spleen, thymus, lymph, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites, and can be obtained after separation and purification.
  • the T cells are autologous T cells.
  • the T cells can be CD4 + T cells, CD8 + T cells, ⁇ T cells or ⁇ T cells, or they can be a mixture of the above types of T cells.
  • the cells are T cells of allogeneic origin, and the T cells are universal chimeric antigen receptor T cells.
  • the T cells In order to obtain the universal chimeric antigen receptor T cells, preferably, in certain embodiments of the present invention, the T cells lack genes encoding TCR, HLA, CD52, PD-1 or CD7.
  • Deletion of the gene can be achieved by appropriate methods in the art, for example, gene knockout.
  • Another aspect of the present invention provides the use of the chimeric antigen receptor, the isolated nucleic acid molecule, the vector or the isolated cell in the preparation of drugs for treating tumors.
  • the tumor is a hematological tumor.
  • the hematological tumor is a CD38 + and/or BCMA + hematological tumor.
  • the hematological tumor is a CD38 + and/or BCMA + hematological tumor that relapses after treatment or is ineffective for immunotherapy targeting BCMA or CD38 alone.
  • the hematological tumor is multiple myeloma.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor, the isolated nucleic acid molecule, the vector or the isolated cell.
  • the pharmaceutical composition of the present invention may also contain any pharmaceutically acceptable additives, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol and their combinations, stabilizers, and surfactants. , preservatives, isotonic agents, etc.
  • pharmaceutically acceptable additives such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol and their combinations, stabilizers, and surfactants. , preservatives, isotonic agents, etc.
  • composition of the present invention can also be used in combination with other suitable anti-cancer agents.
  • suitable anti-cancer agents for example, vincristine, daunorubicin, asparaginase, cyclophosphamide, prednisone, etc.
  • Another aspect of the present invention provides a method for treating hematological tumors, which involves administering the above-mentioned antibody, the above-mentioned antigen-binding portion, the above-mentioned cells or the above-mentioned pharmaceutical composition to a patient.
  • Another aspect of the present invention provides the use of the above pharmaceutical composition in preparing drugs for treating tumors.
  • the tumor is a hematological tumor.
  • the hematological tumor is a CD38 + and/or BCMA + hematological tumor.
  • the hematological tumor is a CD38 + and/or BCMA + hematological tumor that relapses after treatment or is refractory to immunotherapy targeting BCMA or CD38 alone.
  • the hematological tumor is multiple myeloma.
  • the present invention constructs a T cell that expresses a chimeric antigen receptor targeting CD38 and is prepared using the chimeric antigen receptor. Detection of cytokines secreted by T cells through flow cytometry, degranulation analysis experiments, and ELISA proved that the CAR-T cells can effectively expand in vitro and have a strong killing effect on multiple myeloma cells expressing CD38. , has almost no killing effect on cells that do not express CD38, effectively preventing off-target effects.
  • the chimeric antigen receptor of the present invention can be used for the treatment of CD38 + cell hematological tumors, especially the treatment of BCMA + and CD38 + cell hematological tumors, as well as combined treatment with other CAR-T cells.
  • Figure 1 is a schematic diagram of the partial structure of the scFv of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, and loop38BC CAR constructed in the embodiment of the present invention
  • Figure 2 is a diagram showing the results of double-enzyme digestion with NheI and NotI to identify the dual-target CAR-T vector plasmid in the embodiment of the present invention.
  • the sizes of the two fragments after tanBC38 CAR and tan38BC CAR digestion are respectively 2215bp (CAR) and 7297bp (vector), the sizes of the two fragments after digestion of loopBC38 CAR and loop38BC CAR are 2140bp (CAR) and 7297bp (vector) respectively;
  • Figure 3 is a schematic diagram of the tanBC38 CAR lentiviral expression vector in the embodiment of the present invention, in which the counterclockwise sequence is the forward gene fragment and the clockwise sequence is the reverse gene fragment;
  • Figure 4 is a diagram showing the results of using flow cytometry to detect the expression of CAR molecules in tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR modified T cells constructed in the embodiment of the present invention, in which GFP is carried by the vector.
  • Expression of marker protein, F(ab') 2 is the expression of rabbit anti-mouse IgG labeled scFv on the surface of T cells;
  • Figure 5 shows the use of flow cytometry to detect multiple myeloma cell lines H929, MM.1S, ARP-1 and chronic myelogenous leukemia cell line K562 in the embodiment of the present invention through exogenous lentivirus overexpression.
  • the results of BCMA antigen K562 cells (referred to as K562-BCMA) and Burkitt lymphoma cell line Raji, where A is the positive rate of BCMA target antigen molecule expression, and B is the expression positive rate of CD38 target antigen molecule;
  • Figure 6 is a graph showing the results of using flow cytometry to detect the proportion of tumor cells remaining after co-culture of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR modified T cells and target cells in the embodiment of the present invention, wherein, VEC-T is the control group of T cells transfected with empty vector.
  • the CAR-Ts are BCMA CAR-T, CD38 CAR-T, tanBC38 CAR-T, tan38BC CAR-T, loopBC38 CAR-T, loop38BC CAR-T.
  • a and B are the results of the proportion of residual tumor cells after co-culture of T cells and BCMA+CD38+ H929 cell line at target ratios of 1:4, 1:2, and 1:1 for 24 hours (6A) and 48 hours (6B) respectively.
  • Figures: C and D respectively show the remaining residues after co-culture of T cells and BCMA+CD38-K562-BCMA cell line for 24 (6C) and 48 hours (6D) at the target ratio of 1:4, 1:2 and 1:1.
  • the results of the tumor cell ratio; E and F are the T cells and BCMA-CD38+ Raji cell line co-cultured at the effective target ratio of 1:4, 1:2, 1:1 for 24 hours (6E) and 48 hours (6F) respectively.
  • G and H are T cells and BCMA-CD38-K562 cell line co-cultured at target ratios of 1:4, 1:2, and 1:1 for 24 hours (6G) and 48 hours respectively.
  • Figure 7 is a graph showing the results of using flow cytometry to detect the degranulation level of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR-modified T cells after co-culture with target cells in the embodiment of the present invention, wherein, VEC-T is the control group of T cells transfected with empty vector.
  • the CAR-Ts are BCMA CAR-T, CD38 CAR-T, tanBC38 CAR-T, tan38BC CAR-T, loopBC38 CAR-T, and loop38BC CAR-T; T cells and K562, H929, K562-BCMA and Raji cells were mixed at 1:1 for 5 hours, and the level of T cell degranulation was detected by flow cytometry;
  • Figure 8 is a graph showing the results of cytokine release levels after co-culture of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR modified T cells and target cells in the embodiment of the present invention.
  • VEC-T is a T cell transfected with an empty vector.
  • CAR-T are BCMA CAR-T, CD38 CAR-T, tanBC38 CAR-T, tan38BC CAR-T, loopBC38 CAR-T, loop38BC CAR-T; T cells and K562, H929, K562-BCMA and After Raji cells were co-cultured for 24 hours at an effector-target ratio of 1:2, the cytokines IFN- ⁇ (A), TNF- ⁇ (B), IL-2 (C) and IL-6 released by T cells were detected by ELISA ( D);
  • FIG. 9 is a diagram showing the expression results of BCMA and CD38 target antigen molecules in bone marrow mononuclear cells (BMMNCs) of patients with multiple myeloma using flow cytometry in an embodiment of the present invention, in which P1 to P4 represent the numbers of 4 patients;
  • Figure 10 shows the results of the proportion of target cells remaining after co-culture of loopBC38 CAR-modified T cells and BMMNCs of 4 multiple myeloma patients for 24 hours at an effect-to-target ratio of 1:1 in the embodiment of the present invention, in which VEC-T is transformed
  • the control group of T cells stained with empty vector, CAR-T are BCMA CAR-T, CD38 CAR-T, loopBC38 CAR-T respectively
  • A is the proportion of bone marrow mononuclear cells measured after 24 hours of co-culture
  • B is the total
  • C is the proportion of CD38+ cells measured after 24 hours of co-culture;
  • FIG 11 is a diagram showing the evaluation results of the therapeutic effect of loopBC38 CAR and loop38BC CAR-modified T cells in mice in the embodiment of the present invention.
  • NOD/SCID female mice of 6 to 8 weeks were selected and irradiated with a semi-lethal dose (2Gy).
  • 2Gy semi-lethal dose
  • B is the bioluminescence imaging of mice on the 5th, 8th, 15th, 21st and 33rd days after transplantation of tumor cells
  • C is the survival curve.
  • SEQ ID No. 1 is the amino acid sequence of CDR1 of the heavy chain variable region of the first binding domain of the present invention
  • SEQ ID No. 2 is the amino acid sequence of CDR2 of the heavy chain variable region of the first binding domain of the present invention
  • SEQ ID No. 3 is the amino acid sequence of CDR3 of the heavy chain variable region of the first binding domain of the present invention.
  • SEQ ID No. 4 is the amino acid sequence of CDR1 of the light chain variable region of the first binding domain of the present invention.
  • SEQ ID No. 5 is the amino acid sequence of CDR2 of the light chain variable region of the first binding domain of the present invention.
  • SEQ ID No. 6 is the amino acid sequence of CDR3 of the light chain variable region of the first binding domain of the present invention.
  • SEQ ID No. 7 is the amino acid sequence of CDR1 of the heavy chain variable region of the second binding domain of the present invention.
  • SEQ ID No. 8 is the amino acid sequence of CDR2 of the heavy chain variable region of the second binding domain of the present invention.
  • SEQ ID No. 9 is the amino acid sequence of CDR3 of the heavy chain variable region of the second binding domain of the present invention.
  • SEQ ID No. 10 is the amino acid sequence of CDR1 of the light chain variable region of the second binding domain of the present invention.
  • SEQ ID No. 11 is the amino acid sequence of CDR2 of the light chain variable region of the second binding domain of the present invention.
  • SEQ ID No. 12 is the amino acid sequence of CDR3 of the light chain variable region of the second binding domain of the present invention.
  • SEQ ID No. 13 is the amino acid sequence of the heavy chain variable region of the first binding domain scFv of the present invention.
  • SEQ ID No. 14 is the amino acid sequence of the light chain variable region of the first binding domain scFv of the present invention.
  • SEQ ID No. 15 is the amino acid sequence of the heavy chain variable region of the second binding domain scFv of the present invention.
  • SEQ ID No. 16 is the amino acid sequence of the light chain variable region of the second binding domain scFv of the present invention.
  • SEQ ID No. 17 is the amino acid sequence of the extracellular region binding domain of tanBC38 CAR of the present invention.
  • SEQ ID No. 18 is the amino acid sequence of the extracellular region binding domain of tan38BC CAR of the present invention.
  • SEQ ID No. 19 is the amino acid sequence of the extracellular region binding domain of loopBC38 CAR of the present invention.
  • SEQ ID No. 20 is the amino acid sequence of the loop38BC CAR extracellular region binding domain of the present invention.
  • SEQ ID No. 21 is the amino acid sequence of the signal peptide of the present invention.
  • SEQ ID No. 22 is the amino acid sequence of tanBC38 CAR of the present invention.
  • SEQ ID No. 23 is the amino acid sequence of tan38BC CAR of the present invention.
  • SEQ ID No. 24 is the amino acid sequence of loopBC38 CAR of the present invention.
  • SEQ ID No. 25 is the amino acid sequence of loop38BC CAR of the present invention.
  • SEQ ID No. 26 is the nucleotide sequence of the extracellular region binding domain of tanBC38 CAR of the present invention.
  • SEQ ID No. 27 is the nucleotide sequence of the extracellular region binding domain of tan38BC CAR of the present invention.
  • SEQ ID No. 28 is the nucleotide sequence of the extracellular region binding domain of loopBC38 CAR of the present invention.
  • SEQ ID No. 29 is the nucleotide sequence of the loop38BC CAR extracellular region binding domain of the present invention.
  • SEQ ID No. 30 is the nucleotide sequence of tanBC38 CAR of the present invention.
  • SEQ ID No. 31 is the nucleotide sequence of tan38BC CAR of the present invention.
  • SEQ ID No. 32 is the nucleotide sequence of loopBC38 CAR of the present invention.
  • SEQ ID No. 33 is the nucleotide sequence of loop38BC CAR of the present invention.
  • SEQ ID No. 34 is the amino acid sequence of the first linker of the present invention.
  • SEQ ID No. 35 is the amino acid sequence of the second linker of the present invention.
  • SEQ ID No. 36 is the amino acid sequence of the third linker of the present invention.
  • SEQ ID No. 37 is the amino acid sequence of the fourth linker of the present invention.
  • SEQ ID No. 38 is the nucleotide sequence of the first binding domain scFv of the present invention.
  • SEQ ID No. 39 is the nucleotide sequence of the second binding domain scFv of the present invention.
  • the present invention discloses a multi-target chimeric antigen receptor containing CD38.
  • Those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention, and the relevant person can obviously make the same without departing from the content, spirit and scope of the present invention. Modifications or appropriate changes and combinations are made to the contents described in this article to implement and apply the technology of the present invention.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified into kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH 1, CH 2 and CH 3).
  • Each light chain consists of a light chain variable region (V L ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair respectively form the antibody binding site.
  • the term "antibody” is not limited to any particular method of producing the antibody. For example, this includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • the antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtypes
  • IgA1, IgA2, IgD, IgE or IgM antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
  • binding refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination.
  • variable regions of the heavy and light chains each consist of 4 framework regions (FR) linked by 3 complementarity determining regions (CDRs), also called hypervariable regions.
  • FR framework regions
  • CDRs complementarity determining regions
  • the CDRs in each chain are held tightly together by FRs, as well as from other chains, helping to form the antibody's antigen-binding site.
  • There are at least two techniques used to determine CDRs (1) methods based on cross-species sequence variability (i.e., Kabat et al.
  • a CDR may refer to a CDR defined by either method or by a combination of both methods.
  • costimulation refers to secondary signaling events that activate immune responses for immune cells; immune cells rely on costimulation to activate immune responses in the presence of antigen-presenting cells.
  • T cells two stimuli are needed to fully activate their immune response. , that is, during lymphocyte activation, costimulation is often crucial for the development of an effective immune response and is required in addition to antigen-specific signals from its antigen receptors.
  • the second signal saves activated T cells from anergy and causes the T cells to produce additional lymphokines necessary for T cell growth.
  • BCMA B-cell maturation antigen
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art. Conservative substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine acid, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine) acid, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • RNA from the mouse anti-human CD38 monoclonal antibody hybridoma cell line, reverse-transcribe and synthesize cDNA, and use the mouse antibody scFv gene amplification kit to amplify the mouse anti-human CD38 monoclonal antibody light chain (VL). and heavy chain (VH) gene fragments, connect VL and VH to the pMD19-simple T vector and sequence, and determine the nucleic acid sequences of VL and VH based on the sequencing results.
  • VL mouse anti-human CD38 monoclonal antibody light chain
  • VH heavy chain
  • the liquid is divided into three layers, the upper layer is a transparent water layer, RNA is dissolved in this layer, the middle layer is a semi-solid layer, and the lower layer is a yellow organic solvent layer;
  • the reverse transcription PCR reaction system is as follows (20 ⁇ l)
  • Reaction conditions 37°C for 60 minutes, 70°C for 10 minutes, 4°C ⁇ .
  • DNA agarose gel electrophoresis separates and recovers PCR products and gel recovery.
  • CD38 scFv Arrange the humanized sequence according to 5'-VL-first linker-VH-3', called CD38 scFv, add NheI restriction site at the 5' end of the sequence, and add EcoRII at the 3' end of the sequence. Enzyme cutting site for gene synthesis.
  • the enzyme digestion system is as follows (50 ⁇ l)
  • DNA gel electrophoresis observe the size of the PCR amplification band to determine whether it is a positive clone.
  • RNA iso Plus (Takara) to 5 ⁇ 10 6 cells and mix by pipetting. Add 200 ⁇ l chloroform, turn upside down, vortex and mix. Centrifuge at 4°C, 12000rpm for 5 minutes. Pipette the supernatant into a 1.5ml EP tube, add the same volume of isopropyl alcohol, and mix gently by inverting up and down. Centrifuge at 12000 rpm for 15 minutes at 4°C. Precipitate RNA with 75% ethanol pre-cooled at 4°C, and dissolve total RNA in 50 ⁇ l DEPC water.
  • Reverse transcription to synthesize the first strand of cDNA Prepare the PCR reaction system (20 ⁇ l) as follows: Oligo d(T)15 Primers: 2 ⁇ l; M-MLV (200u/ ⁇ l): 1 ⁇ l; dNTP (each 2.5mM): 1 ⁇ l; DTT (0.1M): 2 ⁇ l; First strand buffer (5 ⁇ ): 4 ⁇ l; BCMA-RNA: 2 ⁇ g; DEPC water: make up to 20 ⁇ l. Reaction conditions: 37°C, 60 minutes, 70°C, 10 minutes.
  • Amplify VL chain Prepare the PCR reaction system (50 ⁇ l) as follows: MVL mix: 45 ⁇ l; DNA polymerase: 0.3 ⁇ l; cDNA: 400ng; ddH 2 O: make up to 50 ⁇ l. Reaction conditions: pre-denaturation at 94°C for 3 minutes; repeat the following cycle 30 times: 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds; finally, extension at 72°C for 10 minutes;
  • Amplify VH chain Prepare the PCR reaction system (50 ⁇ l) as follows: MVH mix: 45 ⁇ l; DNA polymerase: 0.3 ⁇ l; cDNA: 400ng; ddH 2 O: make up to 50 ⁇ l. Reaction conditions: pre-denaturation at 94°C for 3 minutes; repeat the following cycle 30 times: 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds; finally, extension at 72°C for 10 minutes;
  • the enzyme digestion system is as follows (50 ⁇ l)
  • the BCMA scFv domain and the CD38 scFv domain are connected through the second linker to obtain tan BC38 CAR and tan38BC CAR respectively, that is, the structure of the tanBC38 CAR antigen-binding domain is BCMA VL-first linker-BCMA VH-second linker -CD38 VL-first linker-CD38VH, the structure of the tan38BC CAR antigen-binding domain is CD38 VL-first linker-CD38 VH-second linker-BCMA VL-first linker-BCMA VH.
  • primers When designing primers, add a vector fragment of about 15 bp and NheI restriction site at the 5' end, and add an EcoRI restriction site and a vector of about 15 bp at the 3' end. Fragment, add a second linker sequence through primers between the two ends of the scFv.
  • the pCDH vector containing the CD8 ⁇ -4-1BB-CD3 ⁇ fragment was double digested with NheI and EcoRI, and the vector fragment was recovered. The three fragments were then connected using a recombination method.
  • Use DNA ligase to insert the target fragment to obtain pCDH-EF1 ⁇ -tanBC38 CAR-T2A-copGFP and pCDH-EF1 ⁇ -tan38BC CAR-T2A-copGFP vectors.
  • EA38F and CZ38R, CZ38F and 38EAR as two pairs of primers respectively to amplify CD38 scFv to obtain two fragments, 38-1 and 38-2;
  • DNA agarose gel electrophoresis separates and recovers PCR products
  • Enzyme digestion conditions 37°C, 4 hours; 7728bp vector fragment was recovered after DNA electrophoresis;
  • the molar ratio of the vector to each fragment is 1:3; reaction conditions: 50°C, 15min;
  • the antigen-binding domain of loopBC38 CAR is BCMA VL-third linker-CD38 VH-fourth linker-CD38 VL-third linker-BCMA VH.
  • the antigen-binding domain of loop38BC CAR is CD38 VL-third linker.
  • the loopBC38 CAR and loop38BC CAR antigen binding domain (scFv) sequences were genetically synthesized, and Nhe I and EcoR I enzyme cutting sites were added to both ends of the sequence.
  • the target fragment was inserted into the pCDH vector to obtain pCDH-EF1 ⁇ -loopBC38 CAR- T2A-copGFP and pCDH-EF1 ⁇ -loop38BC CAR-T2A-copGFP vectors.
  • the enzyme digestion system is as follows (50 ⁇ l)
  • Enzyme digestion conditions 37°C, 4 hours;
  • connection system is as follows (10 ⁇ l)
  • the molar ratio of vector to fragment is 1:5, room temperature for 30 minutes;
  • FIG. 1 The schematic diagram of the antigen-binding part (scFv) of the BCMA and CD38 dual-target CAR is shown in Figure 1.
  • the DNA electrophoresis pattern of the vector after double enzyme digestion with NheI and NotI is shown in Figure 2.
  • the schematic diagram of the vector structure of the tanBC38 CAR is shown in Figure 2. 3 shown.
  • Example 2 Chimeric Antigen Receptor tanBC38 scFv-CD8 ⁇ -4-1BB-CD3 ⁇ ,tan38BC scFv- CD8 ⁇ -4-1BB-CD3 ⁇ ,loopBC38 scFv-CD8 ⁇ -4-1BB-CD3 ⁇ ,loop38BC scFv-CD8 ⁇ -4- Preparation of 1BB-CD3 ⁇ lentivirus modified T cells
  • Lentivirus-infected T cells and culture of T cells after infection Take the virus supernatant from -80°C and melt at room temperature. Add 100 ⁇ l of virus supernatant per 1 ⁇ 10 6 T cells, and add Polybrene to a final concentration of 8 ⁇ g. /ml. Centrifuge at 32°C, 1800rpm for 1.5 hours, transfer to 5% CO 2 , and incubate in a 37°C incubator.
  • K562, K562-BCMA, H929, Raji and ARP-1 cell lines were cultured in RMPI 1640 medium containing 10% FBS, MM.1S was cultured in high glucose 1640 medium containing 10% FBS, and centrifuged The conditions are all 800rpm 5min;
  • Label BCMA One tube uses 1 ⁇ l APC IgG2a, ⁇ isotype ctrl to label cells, the other tube uses APC anti-human BCMA antibody to label cells;
  • Label CD38 One tube uses 1 ⁇ l PE IgG1, ⁇ isotype ctrl to label cells, the other tube uses PE anti-human CD38 antibody marks cells;
  • Human IFN- ⁇ , IL-2, TNF ⁇ , IL6 ELISA detection kit (R&D Company) was used to detect the co-culture supernatant (see the ELISA detection kit instructions for specific steps).
  • CD38 CAR-T secreted a large amount of IL-6, presumably due to CD38 CAR-T killing target cells or its own CD38+ T cells.
  • the level of IL-6 secreted by BCMA CAR-T and four groups of dual-target CAR-T, especially the two groups of loop-structured CAR-T was significantly lower than that of CD38 CAR-T.
  • VEC-T There is no significant difference from VEC-T.
  • CAR-T cells in each group secreted a large amount of IL-6.
  • the sample is divided into three layers. Collect the white coat cell layer in the middle into a new 15mL centrifuge tube, add an equal volume of PBS, mix well, and centrifuge at 1500 rpm for 10 minutes;
  • Figure 9 shows the flow cytometry results of four patients. As shown in the figure, the CD38-positive cells of BMMNC in the four patients accounted for 30.63% to 53.65% of all cells, and the proportion of BCMA-positive cells was lower.
  • Figure 10 shows the proportion of all cells remaining after 24 h of co-culture.
  • Dual-target CAR-T could significantly reduce the proportion of BMMNC cells in 4 patients, and its effect was stronger than BCMA CAR-T and CD38 CAR-T.
  • Three types of CAR-T can almost completely kill BCMA-positive cells; CD38 CAR-T and loopBC38 CAR-T can kill CD38-positive cells, and the killing effect of CD38 CAR-T is slightly stronger than that of loopBC38 CAR-T.
  • the above results suggest that dual-target BCMA/CD38 CAR-T can effectively kill primary cells of MM patients.
  • mice were randomly divided into 5 groups according to the average fluorescence intensity of bioluminescence, namely VEC-T group and BCMA CAR-T group.
  • VEC-T group the average fluorescence intensity of bioluminescence
  • BCMA CAR-T group the average fluorescence intensity of bioluminescence
  • mice The tumor status in mice was evaluated by bioluminescence imaging on days 5, 8, 15, 21, and 33 after transplantation of tumor cells.
  • Figure 11 A is a mouse experiment flow chart.
  • Figure 11 B shows the tumor burden of mice in each group.
  • the average fluorescence intensity statistics at each time point show that CAR-T in all groups can significantly eliminate tumors in mice. tumor cells.
  • C the median survival period of mice in the VEC-T group was 35 days, which was significantly statistically different from all CAR-T groups (p ⁇ 0.01).

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Abstract

Disclosed is a multi-target chimeric antigen receptor comprising CD38 targeting. The CAR-T cells prepared by the present invention can be effectively expanded in vitro, have a very strong killing effect on multiple myeloma cells expressing CD38, and have almost no killing effect on cells not expressing CD38, thereby effectively preventing off-target effects. The chimeric antigen receptor of the present invention can be used for the therapy of CD38 + cellular hematologic malignancies, especially the therapy of BCMA + and CD38 + cellular hematologic malignancies, as well as the combination therapy with other CAR-T cells.

Description

包含靶向CD38的多靶点嵌合抗原受体Contains multi-target chimeric antigen receptors targeting CD38
交叉引用说明Cross reference instructions
本申请要求最早于2022年9月9日提交中国专利局、申请号为202211100848.2，发明名称为“包含靶向CD38的多靶点嵌合抗原受体”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application requests the priority of the Chinese patent application submitted to the China Patent Office on September 9, 2022 at the earliest, with the application number 202211100848.2, and the invention title is "Multi-target chimeric antigen receptor including CD38 targeting", and its entire content incorporated herein by reference.
技术领域Technical field
本发明涉及细胞治疗领域，特别涉及包含靶向CD38的多靶点嵌合抗原受体。The present invention relates to the field of cell therapy, and in particular to a multi-target chimeric antigen receptor containing CD38.
背景技术Background technique
嵌合抗原受体(Chimeric antigen receptor,CAR)修饰的T细胞作为一种免疫治疗策略，可以特异性的杀伤表达特异性肿瘤相关性抗原的肿瘤细胞，在肿瘤治疗中受到广泛的重视和应用。CAR的结构一般由胞外抗原结合区、跨膜区和胞内信号转导区组成，其中胞外抗原结合区由来源于单克隆抗体(scFv)的序列构成。目前根据胞内信号转导区加入共刺激分子的数量，将CAR分为一代(无共刺激分子)、二代(含一种共刺激分子)和三代(含两种共刺激分子)。目前在血液肿瘤的临床中应用更多的是第二代CAR-T细胞治疗。As an immunotherapy strategy, Chimeric Antigen Receptor (CAR)-modified T cells can specifically kill tumor cells expressing specific tumor-associated antigens, and have received widespread attention and application in tumor treatment. The structure of CAR generally consists of an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction region. The extracellular antigen-binding region is composed of sequences derived from monoclonal antibodies (scFv). Currently, CARs are divided into first generation (without costimulatory molecules), second generation (with one costimulatory molecule) and third generation (with two costimulatory molecules) based on the number of costimulatory molecules added to the intracellular signal transduction region. Currently, second-generation CAR-T cell therapy is more commonly used in the clinical treatment of hematological tumors.
近年来，CAR-T被广泛应用于血液肿瘤的治疗，CD19 CAR-T对B细胞急性淋巴细胞白血病(Acute lymphocytic leukemia,ALL)或淋巴瘤表现出显著的治疗效果。也有研究探索了CAR-T对多发性骨髓瘤的治疗作用。In recent years, CAR-T has been widely used in the treatment of blood tumors. CD19 CAR-T has shown significant therapeutic effects on B-cell acute lymphoblastic leukemia (ALL) or lymphoma. There are also studies exploring the therapeutic effect of CAR-T on multiple myeloma.
多发性骨髓瘤(Multiple myeloma,MM)是一种以骨骼破坏、肾功能衰竭、贫血和高钙血症为特征的浆细胞恶性肿瘤，其致死率是所有血液肿瘤的第二位。目前治疗MM的首选治疗措施仍是异基因造血干细胞移植。然而，传统疗法均只能延缓疾病进展，延长患者生存期。MM仍被认为是无法完全治愈的。面对MM患者的耐药和难治复发，需要探索新的治疗手段。Multiple myeloma (MM) is a plasma cell malignancy characterized by bone destruction, renal failure, anemia, and hypercalcemia. Its fatality rate is the second highest among all blood tumors. The current treatment of choice for MM is still allogeneic hematopoietic stem cell transplantation. However, traditional treatments can only delay disease progression and prolong patient survival. MM is still considered to be completely incurable. In the face of drug resistance and refractory relapse in MM patients, new treatments need to be explored.
BCMA(CD269)特异性表达于MM细胞表面，而浆细胞和成熟B细胞不表达或者低表达，在其他外周血单个核细胞、CD34+的造血细胞表面也不表达，因此是MM免疫治疗的理想靶点。CD38在MM、弥漫大B淋巴瘤、急性髓系 白血病等多种血液肿瘤细胞表面高表达，靶向CD38的达雷木单抗被广泛应用于复发难治MM的治疗。BCMA (CD269) is specifically expressed on the surface of MM cells, while it is not expressed or has low expression on plasma cells and mature B cells. It is also not expressed on the surface of other peripheral blood mononuclear cells and CD34+ hematopoietic cells. Therefore, it is an ideal target for MM immunotherapy. point. CD38 is highly expressed on the surface of various blood tumor cells such as MM, diffuse large B lymphoma, acute myeloid leukemia, etc. Daratumumab targeting CD38 is widely used in the treatment of relapsed and refractory MM.
常见的针对MM的免疫疗法所应用的抗体为鼠源性单克隆抗体，但鼠源抗体存在免疫原性差，容易造成免疫逃逸而导致疾病复发的缺陷，因此对其进行人源化处理就成为了解决该问题的主要手段。但在人源化过程中如何保持抗体亲和力能力，同时，针对CAR-T疗法的应用，该人源化抗体如何取得更好的疗效，成为了急需解决的问题。Commonly used antibodies for immunotherapy against MM are mouse-derived monoclonal antibodies. However, mouse-derived antibodies have poor immunogenicity and can easily cause immune escape and lead to disease recurrence. Therefore, humanizing them has become a priority. the main means to solve this problem. However, how to maintain the affinity of the antibody during the humanization process and how to achieve better efficacy of the humanized antibody for the application of CAR-T therapy have become urgent problems.
发明内容Contents of the invention
本发明的一个方面，是针对现有技术中针对CD38+的肿瘤，例如多发性骨髓瘤的免疫疗法所应用的鼠源性单抗免疫原性差，容易造成免疫逃逸而导致疾病复发，同时现有技术中缺少利用人源化抗体且疗效更优的CAR-T疗法的问题，提供了一种包含靶向CD38的多靶点嵌合抗原受体。One aspect of the present invention is to target CD38+ tumors in the prior art, such as the mouse-derived monoclonal antibodies used in immunotherapy for multiple myeloma, which have poor immunogenicity and can easily cause immune escape and lead to disease recurrence. At the same time, the prior art To solve the problem of the lack of CAR-T therapy that utilizes humanized antibodies and has better efficacy, a multi-target chimeric antigen receptor containing CD38 is provided.
本发明提供的技术方案为：The technical solution provided by the invention is:
包含靶向CD38的多靶点嵌合抗原受体，所述嵌合抗原受体包含胞外区、跨膜区和胞内区，所述胞内区包含胞内信号转导区，所述胞外区包括特异性结合CD38分子的第一结合结构域，所述第一结合结构域包括依次如SEQ ID No.1-3所示的重链可变区(VH)的CDR1、CDR2、CDR3氨基酸序列和依次如SEQ ID No.4-6所示的轻链可变区(VL)的CDR1、CDR2、CDR3氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。Comprising a multi-target chimeric antigen receptor targeting CD38, the chimeric antigen receptor includes an extracellular region, a transmembrane region and an intracellular region, the intracellular region includes an intracellular signal transduction region, and the The outer region includes a first binding domain that specifically binds to the CD38 molecule, and the first binding domain includes the CDR1, CDR2, and CDR3 amino acids of the heavy chain variable region (VH) as shown in SEQ ID No. 1-3. The sequence and the CDR1, CDR2, CDR3 amino acid sequence of the light chain variable region (VL) shown in sequence as SEQ ID No. 4-6, or have 85% to 99% identity with the amino acid sequence and have the same organism as it sequence of learning functions.
在本发明中，所述具有85％～99％同一性，是指与本发明中所述核苷酸、氨基酸序列具有85％～99％同一性，其可以为具有85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性，可以以合适的方式对本发明中所述核苷酸、氨基酸序列的某些位点，例如某些保守位点进行随机或者工程化的点突变等保守序列修饰，其目的可以为，例如，获得更好的表达水平、亲和力和/或解离性质，但其保留与原始序列相同的生物学功能，这些突变后的核苷酸或氨基酸序列也视为包含在本发明的保护范围之内。In the present invention, having 85% to 99% identity means having 85% to 99% identity with the nucleotide and amino acid sequences described in the present invention, which can be 85%, 86%, or 87%. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity can be modified in a suitable manner. Certain sites of the above-mentioned nucleotide and amino acid sequences, such as certain conserved sites, are subject to random or engineered point mutations and other conservative sequence modifications. The purpose may be, for example, to obtain better expression levels, affinity and/or dissociation properties, but retain the same biological function as the original sequence, these mutated nucleotide or amino acid sequences are also deemed to be included within the protection scope of the present invention.
在本发明中，所述嵌合抗原受体的胞外区还可以包含第二结合结构域，其与所述第一结合结构域形成能够结合双靶点的结合结构域。在本发明的某些实施方式中，所述第二结合结构域为特异性结合选自BCMA、CD19、CD138、SLAMF7、GAL-3、GPRC5D、PD-L1、CD155、FCRL5或ICAM1蛋白质分子中的一种。作为优选，在本发明的一个实施方式中，所述第二结合结构域为特异性结合 BCMA蛋白质分子的结合结构域。更优选地，在本发明的一个实施方式中，所述第二结合结构域包括依次如SEQ ID No.7-9所示的重链可变区(VH)的CDR1、CDR2、CDR3氨基酸序列和依次如SEQ ID No.10-12所示的轻链可变区(VL)的CDR1、CDR2、CDR3氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。In the present invention, the extracellular region of the chimeric antigen receptor may further comprise a second binding domain, which together with the first binding domain forms a binding domain capable of binding dual targets. In certain embodiments of the invention, the second binding domain specifically binds to a protein molecule selected from BCMA, CD19, CD138, SLAMF7, GAL-3, GPRC5D, PD-L1, CD155, FCRL5 or ICAM1. A sort of. Preferably, in one embodiment of the present invention, the second binding domain is a binding domain that specifically binds to BCMA protein molecules. More preferably, in one embodiment of the present invention, the second binding domain includes the CDR1, CDR2, CDR3 amino acid sequences of the heavy chain variable region (VH) shown in sequence as SEQ ID No. 7-9 and The CDR1, CDR2, and CDR3 amino acid sequences of the light chain variable region (VL) shown in SEQ ID No. 10-12, or have 85% to 99% identity with the amino acid sequence and have the same biological function. the sequence of.
在本发明的某些实施方式中，可以对所述互补决定区(CDRs)进行点突变，其目的可以为，例如获得更好的亲和力和/或解离性质，获得更好的表达水平，等，只要其能够与抗原决定簇形成互补结合的结构即可，该突变后的氨基酸序列也视为包含在本发明的保护范围之内。In certain embodiments of the invention, point mutations can be made to the complementarity determining regions (CDRs), for example, to obtain better affinity and/or dissociation properties, to obtain better expression levels, etc. , as long as it can form a complementary binding structure with the antigenic determinant, the mutated amino acid sequence is also deemed to be included in the protection scope of the present invention.
在本发明的某些实施方式中，i)所述第一结合结构域和所述第二结合结构域可以构建于同一个嵌合抗原受体的表达载体中；或ii)所述第一结合结构域和所述第二结合结构域可以分别构建于不同的嵌合抗原受体的表达载体中。当所述第一结合结构域和所述第二结合结构域分别构建于不同的嵌合抗原受体的表达载体中时，可以利用常规方法将不同的表达载体转导入同一分离的细胞，从而使其具有与将i)中的表达载体转导入同样的分离的细胞后相同或近似的生物学功能。In certain embodiments of the present invention, i) the first binding domain and the second binding domain can be constructed in the same chimeric antigen receptor expression vector; or ii) the first binding domain The structural domain and the second binding domain can be constructed in different chimeric antigen receptor expression vectors respectively. When the first binding domain and the second binding domain are constructed in different chimeric antigen receptor expression vectors, the different expression vectors can be transduced into the same isolated cell using conventional methods, so that It has the same or similar biological function as that after the expression vector in i) is transduced into the same isolated cell.
在本发明的某些实施方式中，所述第一结合结构域为特异性结合CD38分子的scFv，所述scFv包括所述重链可变区(VH)的氨基酸序列和所述轻链可变区(VL)的氨基酸序列。In certain embodiments of the invention, the first binding domain is an scFv that specifically binds to a CD38 molecule, the scFv comprising the amino acid sequence of the heavy chain variable region (VH) and the light chain variable region. Amino acid sequence of region (VL).
为了实现本发明的目的，在本发明的某些实施方式中，所述scFv的氨基酸序列为经过人源化的序列。作为优选，在本发明的一个实施方式中，所述scFv的所述重链可变区(VH)的氨基酸序列如SEQ ID No.13所示，所述scFv的所述轻链可变区(VL)的氨基酸序列如SEQ ID No.14所示，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。In order to achieve the purpose of the present invention, in certain embodiments of the present invention, the amino acid sequence of the scFv is a humanized sequence. Preferably, in one embodiment of the present invention, the amino acid sequence of the heavy chain variable region (VH) of the scFv is shown in SEQ ID No. 13, and the light chain variable region of the scFv ( The amino acid sequence of VL) is as shown in SEQ ID No. 14, or a sequence that has 85% to 99% identity with the amino acid sequence and has the same biological function.
在本发明中，所述scFv通常可以包含单个或重复个数的VL和VH序列，所述VL和VH序列包含所述CDRs。在本发明的某些实施方式中，所述特异性结合CD38分子的scFv为一价抗体。在本发明的另一些实施方式中，所述轻链可变区(VL)和重链可变区(VH)的重复序列数可以为2或3。当所述重复序列数为2时，所述特异性结合CD38分子的scFv形成二价Diabody的形式；当所述重复序列数为3时，所述特异性结合CD38分子的scFv形成三价Triabody的形式。所述轻链可变区(VL)和重链可变区(VH)可以以任意合适的顺序连接，作为优选，在本发明的一个实施方式中，所述重链可变区(VH)和所述轻 链可变区(VL)的连接顺序为5’-VL-第一连接体-VH-3’或5’-VH-第一连接体-VL-3’。In the present invention, the scFv may generally comprise a single or repeated number of VL and VH sequences, and the VL and VH sequences comprise the CDRs. In certain embodiments of the invention, the scFv that specifically binds to CD38 molecules is a monovalent antibody. In other embodiments of the present invention, the number of repeat sequences of the light chain variable region (VL) and the heavy chain variable region (VH) may be 2 or 3. When the number of repeat sequences is 2, the scFv that specifically binds to CD38 molecules forms a bivalent Diabody; when the number of repeat sequences is 3, the scFv that specifically binds to CD38 molecules forms a trivalent Triabody. form. The light chain variable region (VL) and the heavy chain variable region (VH) can be connected in any suitable order. Preferably, in one embodiment of the present invention, the heavy chain variable region (VH) and The connection sequence of the light chain variable region (VL) is 5'-VL-first linker-VH-3' or 5'-VH-first linker-VL-3'.
作为优选，在本发明的某些实施方式中，所述第一连接体为由甘氨酸(Gly)和丝氨酸(Ser)构成的连接体。更优选地，在本发明的一个实施方式中，所述第一连接体的氨基酸序列为一个或串联的多个GGGGS序列；进一步优选地，在本发明的一个实施方式中，所述多个为三个；进一步优选地，在本发明的一个实施方式中，所述第一连接体的氨基酸序列如SEQ ID No.34所示。Preferably, in certain embodiments of the present invention, the first linker is a linker composed of glycine (Gly) and serine (Ser). More preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is one or multiple GGGGS sequences in series; further preferably, in one embodiment of the present invention, the plurality of GGGGS sequences are Three; further preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is shown in SEQ ID No. 34.
在本发明的某些实施方式中，所述嵌合抗原受体的胞外区还包含第二结合结构域，所述第二结合结构域为特异性结合所述蛋白质分子的scFv，所述scFv包括所述重链可变区(VH)的氨基酸序列和所述轻链可变区(VL)的氨基酸序列。In certain embodiments of the invention, the extracellular region of the chimeric antigen receptor further includes a second binding domain, and the second binding domain is an scFv that specifically binds to the protein molecule, and the scFv It includes the amino acid sequence of the heavy chain variable region (VH) and the amino acid sequence of the light chain variable region (VL).
为了实现本发明的目的，在本发明的某些实施方式中，所述scFv的氨基酸序列为经过人源化的序列。作为优选，在本发明的某些实施方式中，所述蛋白质分子为BCMA蛋白质分子。更优选地，在本发明的一个实施方式中，所述scFv的所述重链可变区(VH)的氨基酸序列如SEQ ID No.15所示，所述scFv的所述轻链可变区(VL)的氨基酸序列如SEQ ID No.16所示，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。In order to achieve the purpose of the present invention, in certain embodiments of the present invention, the amino acid sequence of the scFv is a humanized sequence. Preferably, in certain embodiments of the invention, the protein molecule is a BCMA protein molecule. More preferably, in one embodiment of the present invention, the amino acid sequence of the heavy chain variable region (VH) of the scFv is shown in SEQ ID No. 15, and the light chain variable region of the scFv The amino acid sequence of (VL) is as shown in SEQ ID No. 16, or a sequence that has 85% to 99% identity with the amino acid sequence and has the same biological function.
在本发明中，所述scFv通常可以包含单个或重复个数的VL和VH序列，所述VL和VH序列包含所述CDRs。在本发明的某些实施方式中，所述特异性结合所述蛋白质分子的scFv为一价抗体。在本发明的另一些实施方式中，所述轻链可变区(VL)和重链可变区(VH)的重复序列数可以为2或3。当所述重复序列数为2时，所述特异性结合所述蛋白质分子的scFv形成二价Diabody的形式；当所述重复序列数为3时，所述特异性结合所述蛋白质分子的scFv形成三价Triabody的形式。所述轻链可变区(VL)和重链可变区(VH)可以以任意合适的顺序连接，作为优选，在本发明的某些实施方式中，所述蛋白质分子为BCMA蛋白质分子。作为优选，在本发明的一个实施方式中，所述重链可变区(VH)和所述轻链可变区(VL)的连接顺序为5’-VL-第一连接体-VH-3’或5’-VH-第一连接体-VL-3’。In the present invention, the scFv may generally comprise a single or repeated number of VL and VH sequences, and the VL and VH sequences comprise the CDRs. In certain embodiments of the invention, the scFv that specifically binds to the protein molecule is a monovalent antibody. In other embodiments of the present invention, the number of repeat sequences of the light chain variable region (VL) and the heavy chain variable region (VH) may be 2 or 3. When the number of repeat sequences is 2, the scFv that specifically binds to the protein molecule forms a bivalent Diabody; when the number of repeat sequences is 3, the scFv that specifically binds to the protein molecule forms a bivalent Diabody. Trivalent Triabody form. The light chain variable region (VL) and the heavy chain variable region (VH) can be connected in any suitable order. Preferably, in certain embodiments of the present invention, the protein molecule is a BCMA protein molecule. Preferably, in one embodiment of the present invention, the connection sequence of the heavy chain variable region (VH) and the light chain variable region (VL) is 5'-VL-first linker-VH-3 'or 5'-VH-first linker-VL-3'.
作为优选，在本发明的某些实施方式中，所述第一连接体为由甘氨酸(Gly)和丝氨酸(Ser)构成的连接体。更优选地，在本发明的一个实施方式中，所述第一连接体的氨基酸序列为一个或串联的多个GGGGS序列；进一步优选地，在本发明的一个实施方式中，所述多个为三个；；进一步优选地，在本发明的一个 实施方式中，所述第一连接体的氨基酸序列如SEQ ID No.34所示。Preferably, in certain embodiments of the present invention, the first linker is a linker composed of glycine (Gly) and serine (Ser). More preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is one or multiple GGGGS sequences in series; further preferably, in one embodiment of the present invention, the plurality of GGGGS sequences are three; further preferably, in one embodiment of the present invention, the amino acid sequence of the first linker is shown in SEQ ID No. 34.
在本发明的实施方式中，所述第一结合结构域和所述第二结合结构域之间通过第二连接体连接。任意合适的连接体均能实现本发明的目的。但作为优选，在本发明的某些实施方式中，所述第二连接体的氨基酸序列为一个或串联的多个EAAAK序列。更优选地，在本发明的一个实施方式中，所述多个为五个；；进一步优选地，在本发明的一个实施方式中，所述第二连接体的氨基酸序列如SEQ ID No.35所示。In an embodiment of the present invention, the first binding domain and the second binding domain are connected through a second linker. Any suitable connector can achieve the purpose of the present invention. However, preferably, in certain embodiments of the present invention, the amino acid sequence of the second linker is one or multiple EAAAK sequences in series. More preferably, in one embodiment of the present invention, the plurality is five;; further preferably, in one embodiment of the present invention, the amino acid sequence of the second linker is such as SEQ ID No. 35 shown.
更优选地，在本发明的某些实施方式中，所述胞外区的结合结构域包括：More preferably, in certain embodiments of the invention, the binding domain of the extracellular region includes:
i)5’-第二结合结构域的VL-第一连接体-第二结合结构域的VH-第二连接体-CD38 VL-第一连接体-CD38 VH-3’；或i) 5’-VL of the second binding domain-first linker-VH of the second binding domain-second linker-CD38 VL-first linker-CD38 VH-3’; or
ii)5’-CD38 VL-第一连接体-CD38 VH-第二连接体-第二结合结构域的VL-第一连接体-第二结合结构域的VH-3’。ii) 5’-CD38 VL-first linker-CD38 VH-second linker-VL of the second binding domain-first linker-VH-3’ of the second binding domain.
作为优选，在本发明的某些实施方式中，所述蛋白质分子为BCMA蛋白质分子。Preferably, in certain embodiments of the invention, the protein molecule is a BCMA protein molecule.
更优选地，在本发明的某些实施方式中，所述胞外区的结合结构域包括：More preferably, in certain embodiments of the invention, the binding domain of the extracellular region includes:
i)5’-BCMA VL-第一连接体-BCMA VH-第二连接体-CD38 VL-第一连接体-CD38 VH-3’；或i) 5’-BCMA VL-first linker-BCMA VH-second linker-CD38 VL-first linker-CD38 VH-3’; or
ii)5’-CD38 VL-第一连接体-CD38 VH-第二连接体-BCMA VL-第一连接体-BCMA VH-3’。ii) 5’-CD38 VL-first linker-CD38 VH-second linker-BCMA VL-first linker-BCMA VH-3’.
进一步优选地，在本发明的一个实施方式中，所述胞外区的结合结构域包括如SEQ ID No.17或SEQ ID No.18所示的氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。Further preferably, in one embodiment of the present invention, the binding domain of the extracellular region includes the amino acid sequence shown in SEQ ID No. 17 or SEQ ID No. 18, or has 85% similarity with the amino acid sequence. A sequence that is ~99% identical and has the same biological function.
为了进一步提高本发明的效果，在本发明中，所述第一结合结构域的VH、VL和所述第二结合结构域的VH、VL之间还可以通过第三连接体、第四连接体连接，形成loop的形式。在本发明的一个实施方式中，其形成方法可以为所述胞外区的结合结构域包括：In order to further improve the effect of the present invention, in the present invention, the VH and VL of the first binding domain and the VH and VL of the second binding domain can also be connected through a third linker and a fourth linker. Connect to form a loop. In one embodiment of the present invention, the formation method may be that the binding domain of the extracellular region includes:
i)5’-第二结合结构域的VL-第三连接体-CD38 VH-第四连接体-CD38 VL-第三连接体-第二结合结构域的VH-3’；或i) 5’-VL of the second binding domain-third linker-CD38 VH-fourth linker-CD38 VL-third linker-VH-3’ of the second binding domain; or
ii)5’-CD38 VL-第三连接体-第二结合结构域的VH-第四连接体-第二结合结构域的VL-第三连接体-CD38 VH-3’。ii) 5’-CD38 VL-third linker-VH of the second binding domain-fourth linker-VL of the second binding domain-third linker-CD38 VH-3’.
作为优选，在本发明的某些实施方式中，所述蛋白质分子为BCMA蛋白质分子。Preferably, in certain embodiments of the invention, the protein molecule is a BCMA protein molecule.
更优选地，在本发明的某些实施方式中，所述第三连接体的氨基酸序列如SEQ ID No.36所示，所述第四连接体的氨基酸序列如SEQ ID No.37所示。More preferably, in certain embodiments of the present invention, the amino acid sequence of the third linker is shown in SEQ ID No. 36, and the amino acid sequence of the fourth linker is shown in SEQ ID No. 37.
进一步优选地，在本发明的某些实施方式中，所述胞外区的结合结构域包括：Further preferably, in certain embodiments of the invention, the binding domain of the extracellular region includes:
i)5’-BCMA VL-第三连接体-CD38 VH-第四连接体-CD38 VL-第三连接体-BCMA VH-3’；或i) 5’-BCMA VL-third linker-CD38 VH-fourth linker-CD38 VL-third linker-BCMA VH-3’; or
ii)5’-CD38 VL-第三连接体-BCMA VH-第四连接体-BCMA VL-第三连接体-CD38 VH-3’。ii) 5’-CD38 VL-third linker-BCMA VH-fourth linker-BCMA VL-third linker-CD38 VH-3’.
进一步优选地，在本发明的一个实施方式中，所述胞外区的结合结构域包括如SEQ ID No.19或SEQ ID No.20所示的氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。Further preferably, in one embodiment of the present invention, the binding domain of the extracellular region includes the amino acid sequence shown in SEQ ID No. 19 or SEQ ID No. 20, or has 85% similarity with the amino acid sequence. A sequence that is ~99% identical and has the same biological function.
在本发明的一个实施方式中，BCMA +肿瘤细胞和CD38 +肿瘤细胞与tanBCMA scFv-CD38 scFv(tanBC38 scFv)、tanCD38 scFv-BCMA scFv(tan38BC scFv)、loopBCMA scFv-CD38 scFv(loopBC38 scFv)或loopCD38 scFv-BCMA scFv(loop38BC scFv)结合后可以激活相应的CAR-T细胞，产生细胞毒效应；而BCMA和CD38都不表达的细胞不能激活CAR-T细胞产生应答。因此，以tanBC38 scFv、tan38BC scFv、loopBC38 scFv和loop38BC scFv为抗原识别区制备的CAR-T细胞在识别并杀伤BCMA +和CD38 +肿瘤细胞的同时，对不表达BCMA和CD38的细胞不产生脱靶效应。 In one embodiment of the invention, BCMA + tumor cells and CD38 + tumor cells are combined with tanBCMA scFv-CD38 scFv (tanBC38 scFv), tanCD38 scFv-BCMA scFv (tan38BC scFv), loopBCMA scFv-CD38 scFv (loopBC38 scFv) or loopCD38 After scFv-BCMA scFv (loop38BC scFv) combines, it can activate the corresponding CAR-T cells and produce a cytotoxic effect; while cells that express neither BCMA nor CD38 cannot activate CAR-T cells to produce a response. Therefore, CAR-T cells prepared with tanBC38 scFv, tan38BC scFv, loopBC38 scFv and loop38BC scFv as antigen recognition regions can recognize and kill BCMA + and CD38 + tumor cells while not producing off-target effects on cells that do not express BCMA and CD38. .
在本发明的某些实施方式中，所述胞外区还可以包含信号肽。信号肽可引导抗原识别区及铰链区转移到胞外。任意合适的信号肽或信号肽的组合均可实现本发明的目的。In certain embodiments of the invention, the extracellular region may further comprise a signal peptide. The signal peptide can guide the transfer of the antigen recognition region and hinge region to the outside of the cell. Any suitable signal peptide or combination of signal peptides can achieve the purpose of the present invention.
作为优选，在本发明的某些实施方式中，所述胞外区还包含构建在所述的嵌合抗原受体氨基末端的信号肽或与所述信号肽具有90％以上同一性的氨基酸序列；Preferably, in certain embodiments of the present invention, the extracellular region also includes a signal peptide constructed at the amino terminus of the chimeric antigen receptor or an amino acid sequence having more than 90% identity with the signal peptide. ;
更优选地，在本发明的一个实施方式中，所述信号肽为CD8α中的信号肽序列或GM-CSF。More preferably, in one embodiment of the invention, the signal peptide is the signal peptide sequence in CD8α or GM-CSF.
进一步优选地，在本发明的一个实施方式中，所述信号肽为如SEQ ID NO.21所示的信号肽。Further preferably, in one embodiment of the present invention, the signal peptide is the signal peptide shown in SEQ ID NO. 21.
在本发明的某些实施方式中，本发明所述嵌合抗原受体包含的所述结合结构域(C末端)通过铰链区与其编码的所述跨膜区连接。任意合适的铰链区序列均可实现本发明的目的。作为优选，在本发明的一个实施方式中，所述铰链区为CD8α中的铰链区序列。In certain embodiments of the present invention, the binding domain (C-terminus) comprised by the chimeric antigen receptor of the present invention is connected to the transmembrane region encoded by the hinge region. Any suitable hinge region sequence may achieve the purposes of the present invention. Preferably, in one embodiment of the present invention, the hinge region is a hinge region sequence in CD8α.
在本发明的某些实施方式中，所述嵌合抗原受体还包括跨膜结构域。任意合适的跨膜结构域均能实现本发明的目的。作为优选，在本发明的某些实施方式中，所述跨膜区可以为选自以下蛋白质的跨膜结构域或与所述蛋白质具有90％以上同一性的氨基酸序列：T细胞受体的α、β或ζ链、CD2、CD3ε、CD4、CD7、CD8α、CD8β、CD11a、CD11b、CD11c、CD11d、CD18、CD19、CD27、CD28、CD29、CD30、CD40、CD48、CD49a、CD49d、CD49f、CD66a、CD66b、CD66c、CD66d、CD66e、CD69、CD79A、CD79B、CD84、CD96、CD100、CD103、CD134、CD137、CD150、CD158A、CD158B1、CD158B2、CD158C、CD158D、CD158F1、CD158F2、CD158K、CD160、CD162、CD226、CD229、CD244、CD247、CD258、CD268、CD270、CD272、CD276、CD279、CD314、CD319、CD335、CD336、CD337、CD352、CD353、CD355、CD357、LFA-1、NKG2C、DAP-10、ICAM-1、NKp80、IL-2R beta、IL-2Rgamma、IL-7R alpha、LFA-1、SLAMF9、LAT、GADS、SLP-76、PAG1/CBP、CD83配体、Fc gamma受体、整联蛋白、激活性NK细胞受体或Toll配体受体，或其组合。In certain embodiments of the invention, the chimeric antigen receptor further includes a transmembrane domain. Any suitable transmembrane domain can achieve the purpose of the present invention. Preferably, in certain embodiments of the present invention, the transmembrane region may be a transmembrane domain selected from the following proteins or an amino acid sequence having more than 90% identity with the protein: α of T cell receptor , β or ζ chain, CD2, CD3ε, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD28, CD29, CD30, CD40, CD48, CD49a, CD49d, CD49f, CD66a, CD66b, CD66c, CD66d, CD66e, CD69, CD79A, CD79B, CD84, CD96, CD100, CD103, CD134, CD137, CD150, CD158A, CD158B1, CD158B2, CD158C, CD158D, CD158F1, CD158F2, CD158K, CD160, CD162, CD226, CD229, CD244, CD247, CD258, CD268, CD270, CD272, CD276, CD279, CD314, CD319, CD335, CD336, CD337, CD352, CD353, CD355, CD357, LFA-1, NKG2C, DAP-10, ICAM-1, NKp80, IL-2R beta, IL-2Rgamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS, SLP-76, PAG1/CBP, CD83 ligand, Fc gamma receptor, integrin, activating NK Cellular receptor or Toll ligand receptor, or a combination thereof.
更优选地，在本发明的一个实施方式中，所述跨膜区为CD8α中的跨膜区序列。More preferably, in one embodiment of the present invention, the transmembrane region is a transmembrane region sequence in CD8α.
在本发明的某些实施方式中，所述嵌合抗原受体包含的所述胞内区还包含共刺激因子；In certain embodiments of the invention, the intracellular region comprised by the chimeric antigen receptor further comprises a costimulatory factor;
作为优选，在本发明的某些实施方式中，所述共刺激因子可以为通过选自以下蛋白质或与所述蛋白质具有90％以上同一性的氨基酸序列获得的功能性信号结构域的一种或几种：整联蛋白、BTLA、Toll配体受体、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CDS、ICAM-1、LFA-1、4-1BB、B7-H3、CD278、GITR、BAFFR、LIGHT、HVEM、KIRDS2、SLAMF7、NKp80、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49α、IA4、CD49D、ITGA6、VLA6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11α、ITGAM、CD11b、ITGAX、CD11c、CD29、ITGB1、ITGB2、CD18、ITGB7、NKG2D、NKG2C、TNFR2、CD226、CD84、CD96、CEACAM1、CRTAM、CD229、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、CD162、LTBR、LAT、GADS或SLP-76；Preferably, in certain embodiments of the present invention, the costimulatory factor may be one of the functional signaling domains obtained by being selected from the following proteins or amino acid sequences having more than 90% identity with the proteins or Several: integrin, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, 4-1BB, B7-H3, CD278, GITR , BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49α, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD , CD11d, ITGAE, CD103, ITGAL, CD11α, ITGAM, CD11b, ITGAX, CD11c, CD29, ITGB1, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, CD226, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1 , CD100, CD69, SLAMF6, SLAM, BLAME, CD162, LTBR, LAT, GADS or SLP-76;
更优选地，在本发明的一个实施方式中，所述共刺激因子为CD28或4-1BB，或与其具有90％以上同一性的氨基酸序列。More preferably, in one embodiment of the present invention, the costimulatory factor is CD28 or 4-1BB, or an amino acid sequence with more than 90% identity thereto.
在本发明的某些实施方式中，所述嵌合抗原受体还包含胞内信号转导区。作 为优选，在本发明的某些实施方式中，所述胞内信号转导区可以为选自以下蛋白质或与所述蛋白质具有90％以上同源性的氨基酸序列：4-1BB、B7-H3、BAFFR、BLAME、BTLA、CD100、CD103、CD160、CD18、CD19、CD19a、CD2、CD247、CD27、CD276、CD28、CD29、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8alpha、CD8beta、CD96、CDS、CEACAM1、CRTAM、DAP-10、DNAM1、Fc gamma受体、GADS、GITR、HVEM、IA4、ICAM-1、ICAM-1、Ig alpha、IL2R beta、IL2R gamma、IL7R alpha、整联蛋白、ITGA4、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB2、ITGB7、KIRDS2、LAT、LFA-1、LFA-1、LIGHT、LIGHT、LTBR、Ly9、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80、OX-40、PAG/Cbp、PD-1、PSGL1、SELPLG、SLAMF4、SLAMF6、SLAMF7、SLP-76、TNFR2、Toll配体受体、TRANCE/RANKL、VLA1或VLA-6，或其组合。In certain embodiments of the invention, the chimeric antigen receptor further comprises an intracellular signal transduction region. Preferably, in certain embodiments of the present invention, the intracellular signal transduction region can be selected from the following proteins or an amino acid sequence having more than 90% homology with the protein: 4-1BB, B7-H3 , BAFFR, BLAME, BTLA, CD100, CD103, CD160, CD18, CD19, CD19a, CD2, CD247, CD27, CD276, CD28, CD29, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84 , CD8alpha, CD8beta, CD96, CDS, CEACAM1, CRTAM, DAP-10, DNAM1, Fc gamma receptor, GADS, GITR, HVEM, IA4, ICAM-1, ICAM-1, Ig alpha, IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT, LTBR, Ly9, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, OX-40, PAG/Cbp, PD-1, PSGL1, SELPLG, SLAMF4, SLAMF6, SLAMF7, SLP-76, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA- 6, or a combination thereof.
更优选地，在本发明的一个实施方式中，所述胞内信号转导区为CD3ζ。More preferably, in one embodiment of the invention, the intracellular signal transduction region is CD3ζ.
作为优选，在本发明的某些实施方式中，所述嵌合抗原受体是以抗BCMA scFv和CD38 scFv抗原识别区、CD8α铰链区和跨膜区、以及4-1BB和CD3ζ胞内信号结构域串联而成的结构为信号传导结构域，其氨基酸序列如SEQ ID NO.22-25所示。Preferably, in certain embodiments of the invention, the chimeric antigen receptor is based on anti-BCMA scFv and CD38 scFv antigen recognition regions, CD8α hinge region and transmembrane region, and 4-1BB and CD3ζ intracellular signaling structures The structure formed by connecting the domains in series is the signaling domain, and its amino acid sequence is shown in SEQ ID NO. 22-25.
另外，在所述抗原识别区、铰链区、跨膜区以及胞内信号区之间合适的位置可***任意肽链作为间隔区，所述肽链可以为寡肽或多肽。In addition, any peptide chain can be inserted as a spacer at an appropriate position between the antigen recognition region, hinge region, transmembrane region and intracellular signal region, and the peptide chain can be an oligopeptide or a polypeptide.
本发明的另一个方面，是提供了一种分离的核酸分子，所述核酸分子编码所述的嵌合抗原受体的氨基酸序列。Another aspect of the present invention provides an isolated nucleic acid molecule encoding the amino acid sequence of the chimeric antigen receptor.
作为优选，在本发明的某些实施方式中，所述核酸分子的序列如SEQ ID NO.30-33所示，或与其具有90％以上同一性且与其具有相同生物学功能的序列。Preferably, in certain embodiments of the present invention, the sequence of the nucleic acid molecule is as shown in SEQ ID NO. 30-33, or a sequence that has more than 90% identity with it and has the same biological function.
对于上述核酸分子的制备方法，可基于上述抗原识别区、铰链区、跨膜区以及胞内信号区等结构域的碱基序列，通过化学合成或PCR扩增等已知技术制备。通常，可以对编码上述结构域的氨基酸的密码子进行优化，以优化其在宿主细胞中的表达。上述碱基序列的信息可通过检索已知文献或NCBI(https://www.ncbi.nlm.nih.gov/)等数据库来获得。The preparation method of the above-mentioned nucleic acid molecules can be based on the base sequences of the above-mentioned antigen recognition region, hinge region, transmembrane region, intracellular signal region and other structural domains, and can be prepared through known techniques such as chemical synthesis or PCR amplification. Generally, the codons encoding the amino acids of the above-described domains can be optimized to optimize their expression in the host cell. Information on the above base sequence can be obtained by searching known literature or databases such as NCBI (https://www.ncbi.nlm.nih.gov/).
本发明的另一个方面，是提供了一种载体，所述载体包含上述核酸分子。Another aspect of the invention provides a vector comprising the above nucleic acid molecule.
在本发明中，所述载体可以为直链载体，也可以为环状载体。可以为质粒等非病毒载体，也可以为病毒载体，还可以为利用转座子的载体。所述载体中可含有启动子、终止子等调控序列，以及耐药基因、报告基因等标记序列。另外，上 述载体也可包含编码***基因的序列，可根据治疗过程，通过给予激活***基因的物质，从而控制体内CAR-T细胞的数目。In the present invention, the vector can be a linear vector or a circular vector. It may be a non-viral vector such as a plasmid, a viral vector, or a vector utilizing transposons. The vector may contain regulatory sequences such as promoters and terminators, as well as marker sequences such as drug resistance genes and reporter genes. In addition, the above-mentioned vector can also contain a sequence encoding a suicide gene, and the number of CAR-T cells in the body can be controlled by administering substances that activate the suicide gene according to the treatment process.
作为上述病毒载体，可以为质粒载体、逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体等。在本发明的一个实施方式中，使用的是慢病毒表达载体。Examples of the viral vector include plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and the like. In one embodiment of the invention, lentiviral expression vectors are used.
作为优选，在本发明的一个实施方式，所述载体中包含EF1α启动子序列。Preferably, in one embodiment of the present invention, the vector contains an EF1α promoter sequence.
本发明的另一个方面，是提供了一种分离的细胞，所述细胞中包含所述嵌合抗原受体、所述核酸分子或所述载体。Another aspect of the invention provides an isolated cell comprising the chimeric antigen receptor, the nucleic acid molecule or the vector.
作为优选，在本发明的某些实施方式中，所述细胞为i)免疫应答细胞，优选为T细胞、NK细胞、NKT细胞或CTL细胞，更优选为T细胞；或ii)诱导多能干细胞(iPSC)。Preferably, in certain embodiments of the present invention, the cells are i) immune response cells, preferably T cells, NK cells, NKT cells or CTL cells, more preferably T cells; or ii) induced pluripotent stem cells (iPSC).
在本发明的某些实施方式中，所述细胞为自体或异体来源的T细胞。In certain embodiments of the invention, the cells are T cells of autologous or allogeneic origin.
作为优选，在本发明的一个实施方式，所述细胞为自体来源的T细胞。Preferably, in one embodiment of the present invention, the cells are autologous T cells.
在本发明的某些实施方式中，所述T细胞为人的T细胞。所述T细胞可以来自血液、骨髓等体液，也可以来自脾脏、胸腺、淋巴等组织，或者原发肿瘤、转移性肿瘤、癌性腹水等癌症组织，经分离、纯化后得到。作为优选，所述T细胞为自体T细胞。同时，所述T细胞可以为CD4 +T细胞、CD8 +T细胞、αβT细胞或γδT细胞，也可以为上述多种T细胞的混合物。 In certain embodiments of the invention, the T cells are human T cells. The T cells can come from body fluids such as blood and bone marrow, or from tissues such as spleen, thymus, lymph, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites, and can be obtained after separation and purification. Preferably, the T cells are autologous T cells. At the same time, the T cells can be CD4 + T cells, CD8 + T cells, αβ T cells or γδ T cells, or they can be a mixture of the above types of T cells.
在本发明的另一些实施方式中，所述细胞为异体来源的T细胞，所述T细胞为通用型嵌合抗原受体T细胞。In other embodiments of the invention, the cells are T cells of allogeneic origin, and the T cells are universal chimeric antigen receptor T cells.
为了得到所述通用型嵌合抗原受体T细胞，作为优选，在本发明的某些实施方式中，所述T细胞缺失编码TCR、HLA、CD52、PD-1或CD7的基因。In order to obtain the universal chimeric antigen receptor T cells, preferably, in certain embodiments of the present invention, the T cells lack genes encoding TCR, HLA, CD52, PD-1 or CD7.
所述基因的缺失可以通过现有技术中合适的方法实现，例如，基因敲除。Deletion of the gene can be achieved by appropriate methods in the art, for example, gene knockout.
本发明的另一个方面，是提供了所述嵌合抗原受体、所述分离的核酸分子、所述载体或所述分离的细胞在制备***的药物中的用途。Another aspect of the present invention provides the use of the chimeric antigen receptor, the isolated nucleic acid molecule, the vector or the isolated cell in the preparation of drugs for treating tumors.
作为优选，在本发明的某些实施方式中，所述肿瘤为血液肿瘤。Preferably, in certain embodiments of the invention, the tumor is a hematological tumor.
更优选地，在本发明的某些实施方式中，所述血液肿瘤为CD38 +和/或BCMA +的血液肿瘤。 More preferably, in certain embodiments of the invention, the hematological tumor is a CD38 + and/or BCMA + hematological tumor.
进一步优选地，在本发明的某些实施方式中，所述血液肿瘤为经治疗后复发，或对单独靶向BCMA或CD38的免疫治疗无效的CD38 +和/或BCMA +的血液肿瘤。 Further preferably, in certain embodiments of the present invention, the hematological tumor is a CD38 + and/or BCMA + hematological tumor that relapses after treatment or is ineffective for immunotherapy targeting BCMA or CD38 alone.
进一步优选地，在本发明的一个实施方式中，所述血液肿瘤为多发性骨髓瘤。Further preferably, in one embodiment of the present invention, the hematological tumor is multiple myeloma.
本发明的另一个方面，是提供了一种药物组合物，所述药物组合物包括所述嵌合抗原受体、所述分离的核酸分子、所述载体或所述分离的细胞。Another aspect of the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor, the isolated nucleic acid molecule, the vector or the isolated cell.
本发明药物组合物除包含上述成分以外，还可包含任意药学上允许的添加剂，例如，生理盐水、细胞培养基、葡萄糖、注射用水、甘油、乙醇以及它们的组合物、稳定剂、表面活性剂、防腐剂、等渗剂等。In addition to the above ingredients, the pharmaceutical composition of the present invention may also contain any pharmaceutically acceptable additives, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol and their combinations, stabilizers, and surfactants. , preservatives, isotonic agents, etc.
同样，本发明药物组合物也可以和其他合适的抗癌剂联合应用。例如，长春新碱、柔红霉素、门冬酰胺酶、环磷酰胺、***等。Likewise, the pharmaceutical composition of the present invention can also be used in combination with other suitable anti-cancer agents. For example, vincristine, daunorubicin, asparaginase, cyclophosphamide, prednisone, etc.
本发明的另一个方面，是提供了一种治疗血液肿瘤的方法，向患者施用上述抗体、上述抗原结合部分、上述细胞或上述药物组合物。Another aspect of the present invention provides a method for treating hematological tumors, which involves administering the above-mentioned antibody, the above-mentioned antigen-binding portion, the above-mentioned cells or the above-mentioned pharmaceutical composition to a patient.
本发明的另一个方面，是提供了上述药物组合物在制备***的药物中的用途。Another aspect of the present invention provides the use of the above pharmaceutical composition in preparing drugs for treating tumors.
作为优选，在本发明的某些实施方式中，所述肿瘤为血液肿瘤。Preferably, in certain embodiments of the invention, the tumor is a hematological tumor.
更优选地，在本发明的某些实施方式中，所述血液肿瘤为CD38 +和/或BCMA +的血液肿瘤。 More preferably, in certain embodiments of the invention, the hematological tumor is a CD38 + and/or BCMA + hematological tumor.
进一步优选地，在本发明的某些实施方式中，所述血液肿瘤为经治疗后复发或对单独靶向BCMA或CD38的免疫治疗无效的CD38 +和/或BCMA +的血液肿瘤。 Further preferably, in certain embodiments of the present invention, the hematological tumor is a CD38 + and/or BCMA + hematological tumor that relapses after treatment or is refractory to immunotherapy targeting BCMA or CD38 alone.
进一步优选地，在本发明的一个实施方式中，所述血液肿瘤为多发性骨髓瘤。Further preferably, in one embodiment of the present invention, the hematological tumor is multiple myeloma.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明构建了表达包含靶向CD38的嵌合抗原受体及用其制备的T细胞。通过流式细胞术、脱颗粒分析实验、ELISA检测T细胞分泌的细胞因子，证明该CAR-T细胞在体外可有效扩增，并可对表达CD38的多发性骨髓瘤细胞有很强的杀伤作用，对不表达CD38的细胞几乎没有杀伤作用，有效防止了脱靶效应。本发明嵌合抗原受体可用于CD38 +细胞血液肿瘤的治疗，特别是BCMA +和CD38 +细胞血液肿瘤的治疗，以及与其他CAR-T细胞的联合治疗。 The present invention constructs a T cell that expresses a chimeric antigen receptor targeting CD38 and is prepared using the chimeric antigen receptor. Detection of cytokines secreted by T cells through flow cytometry, degranulation analysis experiments, and ELISA proved that the CAR-T cells can effectively expand in vitro and have a strong killing effect on multiple myeloma cells expressing CD38. , has almost no killing effect on cells that do not express CD38, effectively preventing off-target effects. The chimeric antigen receptor of the present invention can be used for the treatment of CD38 + cell hematological tumors, especially the treatment of BCMA + and CD38 + cell hematological tumors, as well as combined treatment with other CAR-T cells.
附图说明Description of the drawings
图1为本发明实施例中构建的tanBC38 CAR、tan38BC CAR、loopBC38 CAR、loop38BC CAR的scFv部分结构示意图；Figure 1 is a schematic diagram of the partial structure of the scFv of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, and loop38BC CAR constructed in the embodiment of the present invention;
图2为本发明实施例中用NheⅠ和NotⅠ双酶切鉴定双靶点CAR-T载体质粒的结果图，其中，tanBC38 CAR和tan38BC CAR酶切后两个片段的大小分别为2215bp(CAR)和7297bp(载体)，loopBC38 CAR和loop38BC CAR酶切后两 个片段的大小分别为2140bp(CAR)和7297bp(载体)；Figure 2 is a diagram showing the results of double-enzyme digestion with NheI and NotI to identify the dual-target CAR-T vector plasmid in the embodiment of the present invention. The sizes of the two fragments after tanBC38 CAR and tan38BC CAR digestion are respectively 2215bp (CAR) and 7297bp (vector), the sizes of the two fragments after digestion of loopBC38 CAR and loop38BC CAR are 2140bp (CAR) and 7297bp (vector) respectively;
图3为本发明实施例中tanBC38 CAR慢病毒表达载体示意图，其中，逆时针序列是正向基因片段，顺时针序列为反向基因片段；Figure 3 is a schematic diagram of the tanBC38 CAR lentiviral expression vector in the embodiment of the present invention, in which the counterclockwise sequence is the forward gene fragment and the clockwise sequence is the reverse gene fragment;
图4为本发明实施例中利用流式细胞术检测本发明实施例中构建的tanBC38 CAR、tan38BC CAR、loopBC38 CAR、loop38BC CAR修饰T细胞中CAR分子的表达结果图，其中，GFP为载体携带的标志蛋白的表达，F(ab') 2为兔抗鼠IgG标记scFv在T细胞表面的表达； Figure 4 is a diagram showing the results of using flow cytometry to detect the expression of CAR molecules in tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR modified T cells constructed in the embodiment of the present invention, in which GFP is carried by the vector. Expression of marker protein, F(ab') 2 is the expression of rabbit anti-mouse IgG labeled scFv on the surface of T cells;
图5为本发明实施例中利用流式细胞术检测本发明实施例中多发性骨髓瘤细胞系H929、MM.1S、ARP-1，慢性粒细胞白血病细胞系K562，通过慢病毒外源过表达BCMA抗原的K562细胞(简称K562-BCMA)以及Burkitt淋巴瘤细胞系Raji的结果图，其中，A为BCMA靶抗原分子表达阳性率，B为CD38靶抗原分子的表达阳性率；Figure 5 shows the use of flow cytometry to detect multiple myeloma cell lines H929, MM.1S, ARP-1 and chronic myelogenous leukemia cell line K562 in the embodiment of the present invention through exogenous lentivirus overexpression. The results of BCMA antigen K562 cells (referred to as K562-BCMA) and Burkitt lymphoma cell line Raji, where A is the positive rate of BCMA target antigen molecule expression, and B is the expression positive rate of CD38 target antigen molecule;
图6为本发明实施例中利用流式细胞术检测本发明实施例中tanBC38 CAR、tan38BC CAR、loopBC38 CAR、loop38BC CAR修饰的T细胞与靶细胞共培养后残留的肿瘤细胞比例结果图，其中，VEC-T为转染空载体的T细胞的对照组，CAR-T分别为BCMA CAR-T,CD38 CAR-T,tanBC38 CAR-T,tan38BC CAR-T,loopBC38 CAR-T,loop38BC CAR-T，A、B分别为T细胞与BCMA+CD38+的H929细胞系按效靶比1:4、1:2、1:1共培养24小时(6A)、48小时(6B)后残留的肿瘤细胞比例结果图；C、D分别为T细胞与BCMA+CD38-的K562-BCMA细胞系按效靶比1:4、1:2、1:1共培养24(6C)、48小时(6D)小时后残留的肿瘤细胞比例结果图；E、F分别为T细胞与BCMA-CD38+的Raji细胞系按效靶比1:4、1:2、1:1共培养24小时(6E)、48小时(6F)后残留的肿瘤细胞比例结果图；G、H分别为T细胞与BCMA-CD38-的K562细胞系按效靶比1:4、1:2、1:1共培养24小时(6G)、48小时(6H)后残留的肿瘤细胞比例结果图；Figure 6 is a graph showing the results of using flow cytometry to detect the proportion of tumor cells remaining after co-culture of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR modified T cells and target cells in the embodiment of the present invention, wherein, VEC-T is the control group of T cells transfected with empty vector. The CAR-Ts are BCMA CAR-T, CD38 CAR-T, tanBC38 CAR-T, tan38BC CAR-T, loopBC38 CAR-T, loop38BC CAR-T. A and B are the results of the proportion of residual tumor cells after co-culture of T cells and BCMA+CD38+ H929 cell line at target ratios of 1:4, 1:2, and 1:1 for 24 hours (6A) and 48 hours (6B) respectively. Figures: C and D respectively show the remaining residues after co-culture of T cells and BCMA+CD38-K562-BCMA cell line for 24 (6C) and 48 hours (6D) at the target ratio of 1:4, 1:2 and 1:1. The results of the tumor cell ratio; E and F are the T cells and BCMA-CD38+ Raji cell line co-cultured at the effective target ratio of 1:4, 1:2, 1:1 for 24 hours (6E) and 48 hours (6F) respectively. Result of residual tumor cell ratio; G and H are T cells and BCMA-CD38-K562 cell line co-cultured at target ratios of 1:4, 1:2, and 1:1 for 24 hours (6G) and 48 hours respectively. Result chart of the proportion of tumor cells remaining after (6H);
图7为本发明实施例中利用流式细胞术检测本发明实施例中tanBC38 CAR、tan38BC CAR、loopBC38 CAR、loop38BC CAR修饰的T细胞与靶细胞共培养后的脱颗粒水平的结果图，其中，VEC-T为转染空载体的T细胞的对照组，CAR-T分别为BCMA CAR-T,CD38 CAR-T,tanBC38 CAR-T,tan38BC CAR-T,loopBC38 CAR-T,loop38BC CAR-T；T细胞和K562、H929、K562-BCMA及Raji细胞，以1:1混合5小时后，流式检测T细胞脱颗粒水平；Figure 7 is a graph showing the results of using flow cytometry to detect the degranulation level of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR-modified T cells after co-culture with target cells in the embodiment of the present invention, wherein, VEC-T is the control group of T cells transfected with empty vector. The CAR-Ts are BCMA CAR-T, CD38 CAR-T, tanBC38 CAR-T, tan38BC CAR-T, loopBC38 CAR-T, and loop38BC CAR-T; T cells and K562, H929, K562-BCMA and Raji cells were mixed at 1:1 for 5 hours, and the level of T cell degranulation was detected by flow cytometry;
图8为本发明实施例中tanBC38 CAR、tan38BC CAR、loopBC38 CAR、loop38BC CAR修饰的T细胞与靶细胞共培养后的细胞因子释放水平的结果图 其中，VEC-T为转染空载体的T细胞的对照组，CAR-T分别为BCMA CAR-T,CD38 CAR-T,tanBC38 CAR-T,tan38BC CAR-T,loopBC38 CAR-T,loop38BC CAR-T；T细胞和K562、H929、K562-BCMA及Raji细胞，以效靶比1:2共培养24小时后，通过ELISA检测T细胞释放的细胞因子IFN-γ(A)、TNF-α(B)、IL-2(C)和IL-6(D)；Figure 8 is a graph showing the results of cytokine release levels after co-culture of tanBC38 CAR, tan38BC CAR, loopBC38 CAR, loop38BC CAR modified T cells and target cells in the embodiment of the present invention. Among them, VEC-T is a T cell transfected with an empty vector. The control group, CAR-T are BCMA CAR-T, CD38 CAR-T, tanBC38 CAR-T, tan38BC CAR-T, loopBC38 CAR-T, loop38BC CAR-T; T cells and K562, H929, K562-BCMA and After Raji cells were co-cultured for 24 hours at an effector-target ratio of 1:2, the cytokines IFN-γ (A), TNF-α (B), IL-2 (C) and IL-6 released by T cells were detected by ELISA ( D);
图9为本发明实施例中利用流式细胞术检测多发性骨髓瘤患者骨髓单个核细胞(BMMNCs)BCMA和CD38靶抗原分子的表达结果图，其中P1～P4表示4例患者编号；Figure 9 is a diagram showing the expression results of BCMA and CD38 target antigen molecules in bone marrow mononuclear cells (BMMNCs) of patients with multiple myeloma using flow cytometry in an embodiment of the present invention, in which P1 to P4 represent the numbers of 4 patients;
图10为本发明实施例中loopBC38 CAR修饰的T细胞与4例多发性骨髓瘤患者BMMNCs按照效靶比为1:1共培养24小时后残留靶细胞比例结果图，其中，VEC-T为转染空载体的T细胞的对照组，CAR-T分别为BCMA CAR-T,CD38 CAR-T,loopBC38 CAR-T；A为共培养24小时后测得的骨髓单个核细胞的比例，B为共培养24小时后测得的BCMA+细胞的比例，C为共培养24小时后测得的CD38+细胞的比例；Figure 10 shows the results of the proportion of target cells remaining after co-culture of loopBC38 CAR-modified T cells and BMMNCs of 4 multiple myeloma patients for 24 hours at an effect-to-target ratio of 1:1 in the embodiment of the present invention, in which VEC-T is transformed The control group of T cells stained with empty vector, CAR-T are BCMA CAR-T, CD38 CAR-T, loopBC38 CAR-T respectively; A is the proportion of bone marrow mononuclear cells measured after 24 hours of co-culture, B is the total The proportion of BCMA+ cells measured after 24 hours of culture, C is the proportion of CD38+ cells measured after 24 hours of co-culture;
图11为本发明实施例中loopBC38 CAR、loop38BC CAR修饰的T细胞在小鼠体内治疗效果的评价结果图，选用6～8周的NOD/SCID雌性小鼠半致死量(2Gy)照射后，随机分为5组，每组5只小鼠，在第0天经尾静脉注射2×10 6的ARP-1-Fluc细胞，第7天、第10天和第14天分别经尾静脉注射1×10 7的T细胞(A)，B为小鼠在移植肿瘤细胞后第5天、第8天、第15天、第21天和第33天的生物发光成像图，C为生存曲线图。 Figure 11 is a diagram showing the evaluation results of the therapeutic effect of loopBC38 CAR and loop38BC CAR-modified T cells in mice in the embodiment of the present invention. NOD/SCID female mice of 6 to 8 weeks were selected and irradiated with a semi-lethal dose (2Gy). Divided into 5 groups, with 5 mice in each group, 2 × 10 6 ARP-1-Fluc cells were injected through the tail vein on day 0, and 1 × ARP-1-Fluc cells were injected through the tail vein on days 7, 10 and 14 respectively. 10 7 T cells (A), B is the bioluminescence imaging of mice on the 5th, 8th, 15th, 21st and 33rd days after transplantation of tumor cells, and C is the survival curve.
序列说明Sequence description
SEQ ID No.1为本发明第一结合结构域重链可变区的CDR1的氨基酸序列；SEQ ID No. 1 is the amino acid sequence of CDR1 of the heavy chain variable region of the first binding domain of the present invention;
SEQ ID No.2为本发明第一结合结构域重链可变区的CDR2的氨基酸序列；SEQ ID No. 2 is the amino acid sequence of CDR2 of the heavy chain variable region of the first binding domain of the present invention;
SEQ ID No.3为本发明第一结合结构域重链可变区的CDR3的氨基酸序列；SEQ ID No. 3 is the amino acid sequence of CDR3 of the heavy chain variable region of the first binding domain of the present invention;
SEQ ID No.4为本发明第一结合结构域轻链可变区的CDR1的氨基酸序列；SEQ ID No. 4 is the amino acid sequence of CDR1 of the light chain variable region of the first binding domain of the present invention;
SEQ ID No.5为本发明第一结合结构域轻链可变区的CDR2的氨基酸序列；SEQ ID No. 5 is the amino acid sequence of CDR2 of the light chain variable region of the first binding domain of the present invention;
SEQ ID No.6为本发明第一结合结构域轻链可变区的CDR3的氨基酸序列；SEQ ID No. 6 is the amino acid sequence of CDR3 of the light chain variable region of the first binding domain of the present invention;
SEQ ID No.7为本发明第二结合结构域重链可变区的CDR1的氨基酸序列；SEQ ID No. 7 is the amino acid sequence of CDR1 of the heavy chain variable region of the second binding domain of the present invention;
SEQ ID No.8为本发明第二结合结构域重链可变区的CDR2的氨基酸序列；SEQ ID No. 8 is the amino acid sequence of CDR2 of the heavy chain variable region of the second binding domain of the present invention;
SEQ ID No.9为本发明第二结合结构域重链可变区的CDR3的氨基酸序列；SEQ ID No. 9 is the amino acid sequence of CDR3 of the heavy chain variable region of the second binding domain of the present invention;
SEQ ID No.10为本发明第二结合结构域轻链可变区的CDR1的氨基酸序列；SEQ ID No. 10 is the amino acid sequence of CDR1 of the light chain variable region of the second binding domain of the present invention;
SEQ ID No.11为本发明第二结合结构域轻链可变区的CDR2的氨基酸序列；SEQ ID No. 11 is the amino acid sequence of CDR2 of the light chain variable region of the second binding domain of the present invention;
SEQ ID No.12为本发明第二结合结构域轻链可变区的CDR3的氨基酸序列；SEQ ID No. 12 is the amino acid sequence of CDR3 of the light chain variable region of the second binding domain of the present invention;
SEQ ID No.13为本发明第一结合结构域scFv的重链可变区的氨基酸序列；SEQ ID No. 13 is the amino acid sequence of the heavy chain variable region of the first binding domain scFv of the present invention;
SEQ ID No.14为本发明第一结合结构域scFv的轻链可变区的氨基酸序列；SEQ ID No. 14 is the amino acid sequence of the light chain variable region of the first binding domain scFv of the present invention;
SEQ ID No.15为本发明第二结合结构域scFv的重链可变区的氨基酸序列；SEQ ID No. 15 is the amino acid sequence of the heavy chain variable region of the second binding domain scFv of the present invention;
SEQ ID No.16为本发明第二结合结构域scFv的轻链可变区的氨基酸序列；SEQ ID No. 16 is the amino acid sequence of the light chain variable region of the second binding domain scFv of the present invention;
SEQ ID No.17为本发明tanBC38 CAR胞外区结合结构域的氨基酸序列；SEQ ID No. 17 is the amino acid sequence of the extracellular region binding domain of tanBC38 CAR of the present invention;
SEQ ID No.18为本发明tan38BC CAR胞外区结合结构域的氨基酸序列；SEQ ID No. 18 is the amino acid sequence of the extracellular region binding domain of tan38BC CAR of the present invention;
SEQ ID No.19为本发明loopBC38 CAR胞外区结合结构域的氨基酸序列；SEQ ID No. 19 is the amino acid sequence of the extracellular region binding domain of loopBC38 CAR of the present invention;
SEQ ID No.20为本发明loop38BC CAR胞外区结合结构域的氨基酸序列；SEQ ID No. 20 is the amino acid sequence of the loop38BC CAR extracellular region binding domain of the present invention;
SEQ ID No.21为本发明信号肽的氨基酸序列；SEQ ID No. 21 is the amino acid sequence of the signal peptide of the present invention;
SEQ ID No.22为本发明tanBC38 CAR的氨基酸序列；SEQ ID No. 22 is the amino acid sequence of tanBC38 CAR of the present invention;
SEQ ID No.23为本发明tan38BC CAR的氨基酸序列；SEQ ID No. 23 is the amino acid sequence of tan38BC CAR of the present invention;
SEQ ID No.24为本发明loopBC38 CAR的氨基酸序列；SEQ ID No. 24 is the amino acid sequence of loopBC38 CAR of the present invention;
SEQ ID No.25为本发明loop38BC CAR的氨基酸序列；SEQ ID No. 25 is the amino acid sequence of loop38BC CAR of the present invention;
SEQ ID No.26为本发明tanBC38 CAR胞外区结合结构域的核苷酸序列；SEQ ID No. 26 is the nucleotide sequence of the extracellular region binding domain of tanBC38 CAR of the present invention;
SEQ ID No.27为本发明tan38BC CAR胞外区结合结构域的核苷酸序列；SEQ ID No. 27 is the nucleotide sequence of the extracellular region binding domain of tan38BC CAR of the present invention;
SEQ ID No.28为本发明loopBC38 CAR胞外区结合结构域的核苷酸序列；SEQ ID No. 28 is the nucleotide sequence of the extracellular region binding domain of loopBC38 CAR of the present invention;
SEQ ID No.29为本发明loop38BC CAR胞外区结合结构域的核苷酸序列；SEQ ID No. 29 is the nucleotide sequence of the loop38BC CAR extracellular region binding domain of the present invention;
SEQ ID No.30为本发明tanBC38 CAR的核苷酸序列；SEQ ID No. 30 is the nucleotide sequence of tanBC38 CAR of the present invention;
SEQ ID No.31为本发明tan38BC CAR的核苷酸序列；SEQ ID No. 31 is the nucleotide sequence of tan38BC CAR of the present invention;
SEQ ID No.32为本发明loopBC38 CAR的核苷酸序列；SEQ ID No. 32 is the nucleotide sequence of loopBC38 CAR of the present invention;
SEQ ID No.33为本发明loop38BC CAR的核苷酸序列；SEQ ID No. 33 is the nucleotide sequence of loop38BC CAR of the present invention;
SEQ ID No.34为本发明第一连接体的氨基酸序列；SEQ ID No. 34 is the amino acid sequence of the first linker of the present invention;
SEQ ID No.35为本发明第二连接体的氨基酸序列；SEQ ID No. 35 is the amino acid sequence of the second linker of the present invention;
SEQ ID No.36为本发明第三连接体的氨基酸序列；SEQ ID No. 36 is the amino acid sequence of the third linker of the present invention;
SEQ ID No.37为本发明第四连接体的氨基酸序列；SEQ ID No. 37 is the amino acid sequence of the fourth linker of the present invention;
SEQ ID No.38为本发明第一结合结构域scFv的核苷酸序列；SEQ ID No. 38 is the nucleotide sequence of the first binding domain scFv of the present invention;
SEQ ID No.39为本发明第二结合结构域scFv的核苷酸序列。SEQ ID No. 39 is the nucleotide sequence of the second binding domain scFv of the present invention.
具体实施方式Detailed ways
本发明公开了一种包含靶向CD38的多靶点嵌合抗原受体，本领域技术人 员可以借鉴本文内容，适当改进工艺参数实现。需要特别指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明，并且相关人员明显能在不脱离本发明内容、精神和范围的基础上对本文所述内容进行改动或适当变更与组合，来实现和应用本发明技术。The present invention discloses a multi-target chimeric antigen receptor containing CD38. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention, and the relevant person can obviously make the same without departing from the content, spirit and scope of the present invention. Modifications or appropriate changes and combinations are made to the contents described in this article to implement and apply the technology of the present invention.
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。术语“如”、“例如”等旨在指示例性实施方案，而不意图限制本公开的范围。In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless expressly stated otherwise, throughout the specification and claims, the term "comprises" or its variations such as "comprises" or "comprising" will be understood to include the stated elements or components, and to Other elements or other components are not excluded. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the disclosure.
下面就本发明中出现的部分术语作以解释。Some terms appearing in the present invention are explained below.
术语“抗体”，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(C1q)的结合。V H和V L区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(V H和V L)分别形成抗体结合部位。。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。 The term "antibody" refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified into kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ). The heavy chain constant region consists of 3 domains ( CH 1, CH 2 and CH 3). Each light chain consists of a light chain variable region (V L ) and a light chain constant region ( CL ). The light chain constant region consists of one domain, CL . The constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions ( VH and VL ) of each heavy chain/light chain pair respectively form the antibody binding site. . The term "antibody" is not limited to any particular method of producing the antibody. For example, this includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。As used herein, when the term "antibody" is mentioned, it includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
术语“特异性结合”，是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.
术语抗体的“可变区”，是指抗体轻链的可变区或抗体重链的可变区，单独或 组合。如本领域已知的，重链和轻链的可变区各自由通过也称作高变区的3个互补决定区(CDR)连接的4个框架区(FR)组成。每条链中的CDR通过FR紧紧保持在一起，以及与来自其他链的保持在一起，有助于形成抗体的抗原结合位点。有至少两种技术用于确定CDR：(1)基于跨物种序列变异性的方法(即，Kabat et al.Sequences of Proteins of Immunological Interest,(5th ed.,1991,National Institutes of Health,Bethesda MD))；以及(2)基于抗原-抗体复合物的晶体学研究的方法(Al-lazikani et al.,1997,J.Molec.Biol.273:927-948)。如本文所用，CDR可以指通过任一种方法或通过两种方法的组合定义的CDR。The term "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of 4 framework regions (FR) linked by 3 complementarity determining regions (CDRs), also called hypervariable regions. The CDRs in each chain are held tightly together by FRs, as well as from other chains, helping to form the antibody's antigen-binding site. There are at least two techniques used to determine CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD) ); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR may refer to a CDR defined by either method or by a combination of both methods.
术语“共刺激”，是指为免疫细胞激活免疫应答的次级信号事件；免疫细胞在抗原呈递细胞存在下依靠共刺激来激活免疫应答，对于T细胞，需要两个刺激才能完全激活其免疫应答，就是说在淋巴细胞活化过程中，共刺激通常对有效免疫应答的发展至关重要，除了来自其抗原受体的抗原特异性信号之外，还需要共刺激。第二个信号使活化的T细胞免于无反应性，使T细胞产生额外T细胞生长所必需的淋巴因子。The term "costimulation" refers to secondary signaling events that activate immune responses for immune cells; immune cells rely on costimulation to activate immune responses in the presence of antigen-presenting cells. For T cells, two stimuli are needed to fully activate their immune response. , that is, during lymphocyte activation, costimulation is often crucial for the development of an effective immune response and is required in addition to antigen-specific signals from its antigen receptors. The second signal saves activated T cells from anergy and causes the T cells to produce additional lymphokines necessary for T cell growth.
术语“BCMA”，即B细胞表面成熟抗原(B-cell maturation antigen,BCMA)是一种浆细胞选择性的蛋白,最早发现于成熟的B淋巴细胞表面,在其他组织细胞中几乎不表达.其在恶性增殖的B淋巴细胞(例如骨髓瘤细胞、白血病细胞)中高度表达,同时其介导的下游信号通路,对细胞的存活、增殖、转移和耐药中起着关键性的作用。The term "BCMA", that is, B-cell maturation antigen (BCMA), is a plasma cell-selective protein that was first discovered on the surface of mature B lymphocytes and is rarely expressed in other tissue cells. It is highly expressed in malignantly proliferating B lymphocytes (such as myeloma cells, leukemia cells), and the downstream signaling pathways it mediates play a key role in cell survival, proliferation, metastasis and drug resistance.
术语“保守序列修饰”是指不显著影响或改变含有氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、添加和缺失。可以通过本领域已知的标准技术将修饰引入本发明的抗体或抗体片段中。保守取代是其中氨基酸残基被具有相似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基的家族已在本领域中进行了定义。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)以及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。The term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art. Conservative substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine acid, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine) acid, tryptophan, histidine) amino acids.
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实施例对本发明作进一步的详细说明。In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to specific embodiments.
实施例1：载体的构建Example 1: Construction of vector
1.pCDH-EF1α-CD38 CAR-T2A-copGFP载体的构建1. Construction of pCDH-EF1α-CD38 CAR-T2A-copGFP vector
i)提取小鼠抗人CD38单克隆抗体杂交瘤细胞株的总RNA，逆转录合成cDNA，使用小鼠抗体scFv基因扩增试剂盒，扩增小鼠抗人CD38单克隆抗体轻链(VL)和重链(VH)的基因片段，将VL、VH连接至pMD19-simple T载体并测序，根据测序结果确定VL、VH的核酸序列。用计算机软件进行人源化模拟，得到人源化的VL、VH核酸序列，按照5’-VL-第一连接体-VH-3’的顺序排列，得到CD38 scFv人源化序列，如SEQ ID No.38所示，并将其合成。i) Extract the total RNA from the mouse anti-human CD38 monoclonal antibody hybridoma cell line, reverse-transcribe and synthesize cDNA, and use the mouse antibody scFv gene amplification kit to amplify the mouse anti-human CD38 monoclonal antibody light chain (VL). and heavy chain (VH) gene fragments, connect VL and VH to the pMD19-simple T vector and sequence, and determine the nucleic acid sequences of VL and VH based on the sequencing results. Use computer software to perform humanization simulation to obtain humanized VL and VH nucleic acid sequences, arrange them in the order of 5'-VL-first linker-VH-3', and obtain the CD38 scFv humanized sequence, such as SEQ ID No.38 is shown and synthesized.
ii)将CD38 scFv人源化序列进行Nhe I、EcoR I双酶切，并酶切含有CD8α-4-1BB-CD3ζ片段的质粒，将两者链接，得到pCDH-EF1α-CD38 CAR-T2A-copGFP载体，具体实验步骤如下：ii) Double digest the CD38 scFv humanized sequence with Nhe I and EcoR I, and digest the plasmid containing the CD8α-4-1BB-CD3ζ fragment, and link the two to obtain pCDH-EF1α-CD38 CAR-T2A-copGFP carrier, the specific experimental steps are as follows:
1)提取分泌抗人CD38单克隆抗体的小鼠杂交瘤细胞(HIT2)总RNA1) Extract total RNA from mouse hybridoma cells (HIT2) secreting anti-human CD38 monoclonal antibodies
a)在5×10 6细胞中加入RNAiso Plus 1mL，迅速吹打混匀； a) Add 1mL of RNAiso Plus to 5×10 6 cells, and mix quickly by pipetting;
b)加入200μl三氯甲烷，上下颠倒、振荡混匀至溶液呈乳红色，静置5min；b) Add 200 μl chloroform, mix upside down and shake until the solution turns milky red, and let it sit for 5 minutes;
c)4℃，12000rpm，离心15分钟，液体分为三层，上层为透明水层，RNA溶解在此层中，中间层呈半固体层，下层为黄色的有机溶剂层；c) 4℃, 12000rpm, centrifuge for 15 minutes, the liquid is divided into three layers, the upper layer is a transparent water layer, RNA is dissolved in this layer, the middle layer is a semi-solid layer, and the lower layer is a yellow organic solvent layer;
d)吸取上清至1.5mL EP管，加入同体积异丙醇，上下颠倒混匀，静置10min；d) Pipet the supernatant into a 1.5mL EP tube, add the same volume of isopropyl alcohol, mix by inverting, and let stand for 10 minutes;
e)4℃，12000rpm，离心15分钟，小心弃去上清，加入4℃预冷的75％乙醇沉淀EP管底部的RNA；e) Centrifuge at 12,000 rpm at 4°C for 15 minutes, carefully discard the supernatant, and add 75% ethanol pre-cooled at 4°C to precipitate the RNA at the bottom of the EP tube;
f)小心弃净上清，放于通风橱内10min，使RNA沉淀尽量干燥；f) Carefully discard the supernatant and place it in a fume hood for 10 minutes to dry the RNA precipitation as much as possible;
g)加入50μl DEPC水溶解总RNA；g) Add 50μl DEPC water to dissolve total RNA;
h)测定RNA的浓度和纯度，放于-80℃冰箱储存。h) Determine the concentration and purity of the RNA and store it in a -80°C refrigerator.
2)将RNA逆转为cDNA2) Reverse RNA to cDNA
逆转录PCR反应体系如下(20μl)The reverse transcription PCR reaction system is as follows (20μl)
Figure PCTCN2022144325-appb-000001
Figure PCTCN2022144325-appb-000001
反应条件：37℃60分钟，70℃10分钟，4℃∞。Reaction conditions: 37°C for 60 minutes, 70°C for 10 minutes, 4°C∞.
3)PCR扩增小鼠抗人HIT2单克隆抗体的VH、VL基因片段3) PCR amplification of VH and VL gene fragments of mouse anti-human HIT2 monoclonal antibody
a)配制PCR反应体系(50μl)a) Prepare PCR reaction system (50μl)
扩增VHAmplified VH
Figure PCTCN2022144325-appb-000002
Figure PCTCN2022144325-appb-000002
扩增VLAmplified VL
Figure PCTCN2022144325-appb-000003
Figure PCTCN2022144325-appb-000003
b)反应条件b) Reaction conditions
Figure PCTCN2022144325-appb-000004
Figure PCTCN2022144325-appb-000004
4)DNA琼脂糖凝胶电泳分离并回收PCR产物，胶回收。4) DNA agarose gel electrophoresis separates and recovers PCR products and gel recovery.
5)分别将VH、VL片段连接至pMD19-simple T载体上5) Connect the VH and VL fragments to the pMD19-simple T vector respectively
a)VH连接体系(10μl)a)VH connection system (10μl)
Figure PCTCN2022144325-appb-000005
Figure PCTCN2022144325-appb-000005
VL连接体系(10μl)VL connection system (10μl)
Figure PCTCN2022144325-appb-000006
Figure PCTCN2022144325-appb-000006
载体(mol)：目的片段(mol)＝1:5，计算X值；Vector (mol): target fragment (mol) = 1:5, calculate the X value;
b)室温反应30min后，加入20μl冰上融化的JM109感受态细胞，轻轻混匀后冰上放置30min；b) After reacting at room temperature for 30 minutes, add 20 μl of JM109 competent cells melted on ice, mix gently and place on ice for 30 minutes;
c)42℃金属浴热激30s，冰上放置2～3min；c) Heat shock in a metal bath at 42°C for 30 seconds and place on ice for 2 to 3 minutes;
d)加入1mL SOC培养基，于37℃恒温振荡器中160rpm震荡60min；d) Add 1mL of SOC medium and shake in a constant temperature oscillator at 37°C at 160 rpm for 60 minutes;
e)3000rpm 3min离心菌液，剩余200μl上清重悬细菌团块，逐滴加入含有Amp的LB培养平板上，用无菌涂布器涂布均匀；e) Centrifuge the bacterial liquid at 3000 rpm for 3 minutes, resuspend the remaining 200 μl supernatant of the bacterial clump, add it drop by drop to the LB culture plate containing Amp, and spread evenly with a sterile spreader;
f)将平板倒置于37℃恒温孵箱中，培养12～16h。f) Place the plate upside down in a 37°C constant temperature incubator and incubate for 12 to 16 hours.
6)挑取20个菌落，放入500μl含有Amp的LB培养基中，在37℃摇床中200rpm振摇6～8h，吸取200μl菌液测序。6) Pick 20 colonies, put them into 500 μl of LB medium containing Amp, shake at 200 rpm in a 37°C shaker for 6 to 8 hours, and draw 200 μl of bacterial liquid for sequencing.
7)测序结果按照单克隆抗体序列原则进行比对，确定鼠源化HIT2VH和VL的序列，用计算机Discovery Studio软件进行人源化模拟。7) The sequencing results were compared according to the principles of monoclonal antibody sequences to determine the sequences of murine HIT2VH and VL, and computer Discovery Studio software was used to perform humanization simulations.
8)将人源化的序列按照5’-VL-第一连接体-VH-3’进行排列，称为CD38 scFv，在序列5’端加入NheⅠ酶切位点，在序列3’端加入EcoRⅠ酶切位点，进行基因合成。8) Arrange the humanized sequence according to 5'-VL-first linker-VH-3', called CD38 scFv, add NheⅠ restriction site at the 5' end of the sequence, and add EcoRIⅠ at the 3' end of the sequence. Enzyme cutting site for gene synthesis.
9)利用NheⅠ和EcoRⅠ对含有合成序列的质粒进行酶切9) Use NheⅠ and EcoRⅠ to digest the plasmid containing the synthetic sequence.
酶切体系如下(50μl)The enzyme digestion system is as follows (50μl)
Figure PCTCN2022144325-appb-000007
Figure PCTCN2022144325-appb-000007
37℃反应4小时后，加入5.5μl 10×Loading buffer，DNA电泳后回收条带大小为735bp的片段。After reacting at 37°C for 4 hours, add 5.5μl 10× Loading buffer, and recover a fragment with a band size of 735bp after DNA electrophoresis.
10)连接目的片段与载体片段10) Connect the target fragment and vector fragment
连接体系(10μl)Connection system (10μl)
Figure PCTCN2022144325-appb-000008
Figure PCTCN2022144325-appb-000008
载体(mol)：目的片段(mol)＝1:5，计算X值；室温静置30min。Vector (mol): target fragment (mol) = 1:5, calculate the X value; let stand at room temperature for 30 minutes.
11)转化JM109感受态细胞，步骤同前述；11) Transform JM109 competent cells, the steps are the same as mentioned above;
12)用枪尖蘸取10个左右菌落，分别加入1mL含有Amp的LB培养基中，在37℃200rpm条件下震荡培养，2h后吸取1μl菌液行菌落PCR鉴定目的片段是否连入载体；12) Use the tip of a gun to pick up about 10 colonies, add them to 1 mL of LB medium containing Amp, and culture them with shaking at 37°C and 200 rpm. After 2 hours, take 1 μl of bacterial liquid and perform colony PCR to identify whether the target fragment is connected to the vector;
菌液PCR反应体系Bacterial liquid PCR reaction system
Figure PCTCN2022144325-appb-000009
Figure PCTCN2022144325-appb-000009
反应条件Reaction conditions
Figure PCTCN2022144325-appb-000010
Figure PCTCN2022144325-appb-000010
DNA凝胶电泳，观察PCR扩增条带的大小，确定是否为阳性克隆。DNA gel electrophoresis, observe the size of the PCR amplification band to determine whether it is a positive clone.
13)将条带正确的克隆继续震摇培养6～8h，吸取200μl至公司测序，余下菌液放于4℃冰箱备用。13) Continue to culture the clones with correct bands for 6 to 8 hours with shaking, pipet 200 μl to the company for sequencing, and store the remaining bacterial solution in a 4°C refrigerator for later use.
2.pCDH-EF1α-BCMA CAR-T2A-copGFP载体的构建2. Construction of pCDH-EF1α-BCMA CAR-T2A-copGFP vector
i)根据SEQ ID No.15、16、26-29所示的序列商业化化学合成所述BCMA scFv，如SEQ ID No.39所示。i) Commercially chemically synthesize the BCMA scFv according to the sequences shown in SEQ ID No. 15, 16, 26-29, as shown in SEQ ID No. 39.
ii)BCMA scFv的克隆。具体步骤如下：ii) Cloning of BCMA scFv. Specific steps are as follows:
1)提取鼠抗人BCMA单克隆抗体杂交瘤细胞株的总RNA：在5×10 6细胞中加入RNA iso Plus(Takara)1ml，吹打混匀。加入200μl三氯甲烷，上下颠倒、涡旋振荡混匀。4℃，12000rpm，离心5分钟。吸取上清至1.5ml EP管，加入同体积异丙醇，轻轻上下颠倒混匀。4℃，12000rpm，离心15分钟。4℃预冷75％乙醇沉淀RNA，50μl DEPC水溶解总RNA。 1) Extract total RNA from mouse anti-human BCMA monoclonal antibody hybridoma cell lines: Add 1 ml of RNA iso Plus (Takara) to 5×10 6 cells and mix by pipetting. Add 200 μl chloroform, turn upside down, vortex and mix. Centrifuge at 4°C, 12000rpm for 5 minutes. Pipette the supernatant into a 1.5ml EP tube, add the same volume of isopropyl alcohol, and mix gently by inverting up and down. Centrifuge at 12000 rpm for 15 minutes at 4°C. Precipitate RNA with 75% ethanol pre-cooled at 4°C, and dissolve total RNA in 50 μl DEPC water.
2)逆转录合成cDNA第一链：配制PCR反应体系(20μl)如下：Oligo d(T)15 Primers:2μl；M-MLV(200u/μl)：1μl；dNTP(each 2.5mM)：1μl；DTT(0.1M)：2μl；First strand buffer(5×)：4μl；BCMA-RNA:2μg；DEPC水:补足至20μl。反应条件：37℃，60分钟，70℃10分钟。2) Reverse transcription to synthesize the first strand of cDNA: Prepare the PCR reaction system (20μl) as follows: Oligo d(T)15 Primers: 2μl; M-MLV (200u/μl): 1μl; dNTP (each 2.5mM): 1μl; DTT (0.1M): 2μl; First strand buffer (5×): 4μl; BCMA-RNA: 2μg; DEPC water: make up to 20μl. Reaction conditions: 37°C, 60 minutes, 70°C, 10 minutes.
3)使用“小鼠抗体scFv基因扩增试剂盒”(Public Protein/Plasmid Library)PCR扩增鼠抗人BCMA单克隆抗体轻链(VL)和重链(VH)的基因片段：3) Use the "Mouse Antibody scFv Gene Amplification Kit" (Public Protein/Plasmid Library) to PCR amplify the gene fragments of the mouse anti-human BCMA monoclonal antibody light chain (VL) and heavy chain (VH):
扩增VL链：配制PCR反应体系(50μl)如下：MVL mix：45μl；DNA polymerase：0.3μl；cDNA：400ng；ddH 2O：补足至50μl。反应条件：94℃预变性3分钟；重复如下循环30次：94℃30秒，56℃30秒，72℃45秒；最后， 72℃延伸10分钟； Amplify VL chain: Prepare the PCR reaction system (50 μl) as follows: MVL mix: 45 μl; DNA polymerase: 0.3 μl; cDNA: 400ng; ddH 2 O: make up to 50 μl. Reaction conditions: pre-denaturation at 94°C for 3 minutes; repeat the following cycle 30 times: 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds; finally, extension at 72°C for 10 minutes;
扩增VH链：配制PCR反应体系(50μl)如下：MVH mix：45μl；DNA polymerase：0.3μl；cDNA：400ng；ddH 2O：补足至50μl。反应条件：94℃预变性3分钟；重复如下循环30次：94℃30秒，56℃30秒，72℃45秒；最后，72℃延伸10分钟； Amplify VH chain: Prepare the PCR reaction system (50 μl) as follows: MVH mix: 45 μl; DNA polymerase: 0.3 μl; cDNA: 400ng; ddH 2 O: make up to 50 μl. Reaction conditions: pre-denaturation at 94°C for 3 minutes; repeat the following cycle 30 times: 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds; finally, extension at 72°C for 10 minutes;
琼脂糖凝胶电泳分离并回收VL、VH片段。Agarose gel electrophoresis separates and recovers VL and VH fragments.
4)将VL、VH连接至pMD19-simple T载体并测序，根据测序结果确定BCMA单克隆抗体VL、VH的核酸序列。而后用计算机软件进行人源化模拟，得到人源化的VL、VH核酸序列，并按照5’-VL-第一连接体-VH-3’的方向得到BCMA scFv人源化序列，在5’端加入Nhe Ⅰ酶切位点，在序列3’端加入EcoR Ⅰ酶切位点，将其合成。4) Connect VL and VH to the pMD19-simple T vector and sequence, and determine the nucleic acid sequences of BCMA monoclonal antibodies VL and VH based on the sequencing results. Then use computer software to perform humanization simulation to obtain the humanized VL and VH nucleic acid sequences, and obtain the BCMA scFv humanized sequence in the direction of 5'-VL-first linker-VH-3', at 5' Add Nhe Ⅰ enzyme cleavage site to the end, add EcoR Ⅰ enzyme cleavage site to the 3' end of the sequence, and synthesize it.
5)利用Nhe Ⅰ和EcoR Ⅰ对含有合成序列的质粒进行酶切5) Use Nhe Ⅰ and EcoR Ⅰ to digest the plasmid containing the synthetic sequence.
酶切体系如下(50μl)The enzyme digestion system is as follows (50μl)
Figure PCTCN2022144325-appb-000011
Figure PCTCN2022144325-appb-000011
37℃反应4小时后，加入5.5μl 10×Loading buffer，DNA电泳后回收条带大小为735bp的片段。After reacting at 37°C for 4 hours, add 5.5μl 10× Loading buffer, and recover a fragment with a band size of 735bp after DNA electrophoresis.
6)连接目的片段与载体片段6) Connect the target fragment and vector fragment
连接体系(10μl)Connection system (10μl)
Figure PCTCN2022144325-appb-000012
Figure PCTCN2022144325-appb-000012
载体(mol)：目的片段(mol)＝1:5，计算X值；室温静置30min。Vector (mol): target fragment (mol) = 1:5, calculate the X value; let stand at room temperature for 30 minutes.
7)转化JM109感受态细胞，菌落PCR鉴定将条带正确的克隆，余下菌液放于4℃冰箱备用。7) Transform JM109 competent cells, identify the clone with the correct band by colony PCR, and store the remaining bacterial solution in a 4°C refrigerator for later use.
3.双靶点BCMA/CD38 CAR载体的构建3. Construction of dual-target BCMA/CD38 CAR vector
1)pCDH-EF1α-tanBC38 CAR-T2A-copGFP和pCDH-EF1α-tan38BC CAR-T2A-copGFP载体的构建1) Construction of pCDH-EF1α-tanBC38 CAR-T2A-copGFP and pCDH-EF1α-tan38BC CAR-T2A-copGFP vectors
通过第二连接体连接BCMA scFv结构域和CD38 scFv结构域，分别得到 tan BC38 CAR和tan38BC CAR，即tanBC38 CAR抗原结合结构域的结构为BCMA VL-第一连接体-BCMA VH-第二连接体-CD38 VL-第一连接体-CD38VH，tan38BC CAR抗原结合结构域的结构为CD38 VL-第一连接体-CD38 VH-第二连接体-BCMA VL-第一连接体-BCMA VH。首先用PCR的方法分别扩增BCMA scFv和CD38 scFv，设计引物时，在5’端加入15bp左右的载体片段和NheⅠ酶切位点，在3’端加入EcoRⅠ酶切位点和15bp左右的载体片段，在两端scFv中间通过引物加入第二连接体序列。采用NheⅠ和EcoRⅠ对含有CD8α-4-1BB-CD3ζ片段的pCDH载体进行双酶切，回收载体片段。而后采用重组的方法将三个片段连接起来。用DNA连接酶***目的片段，得到pCDH-EF1α-tanBC38 CAR-T2A-copGFP和pCDH-EF1α-tan38BC CAR-T2A-copGFP载体。The BCMA scFv domain and the CD38 scFv domain are connected through the second linker to obtain tan BC38 CAR and tan38BC CAR respectively, that is, the structure of the tanBC38 CAR antigen-binding domain is BCMA VL-first linker-BCMA VH-second linker -CD38 VL-first linker-CD38VH, the structure of the tan38BC CAR antigen-binding domain is CD38 VL-first linker-CD38 VH-second linker-BCMA VL-first linker-BCMA VH. First, use PCR to amplify BCMA scFv and CD38 scFv respectively. When designing primers, add a vector fragment of about 15 bp and NheⅠ restriction site at the 5' end, and add an EcoRⅠ restriction site and a vector of about 15 bp at the 3' end. Fragment, add a second linker sequence through primers between the two ends of the scFv. The pCDH vector containing the CD8α-4-1BB-CD3ζ fragment was double digested with NheⅠ and EcoRI, and the vector fragment was recovered. The three fragments were then connected using a recombination method. Use DNA ligase to insert the target fragment to obtain pCDH-EF1α-tanBC38 CAR-T2A-copGFP and pCDH-EF1α-tan38BC CAR-T2A-copGFP vectors.
a.扩增BCMA scFv结构域和CD38 scFv结构域a. Amplify BCMA scFv domain and CD38 scFv domain
设计PCR引物如下Design PCR primers as follows
Figure PCTCN2022144325-appb-000013
Figure PCTCN2022144325-appb-000013
用CZBCF和BCEAR,EABCF和CZBCR分别作为两对引物，扩增BCMA scFv，得到BCMA-1和BCMA-2两个片段；Use CZBCF and BCEAR, EABCF and CZBCR as two pairs of primers respectively to amplify BCMA scFv to obtain two fragments, BCMA-1 and BCMA-2;
用EA38F和CZ38R,CZ38F和38EAR分别作为两对引物扩增CD38 scFv得到38-1和38-2两个片段；Use EA38F and CZ38R, CZ38F and 38EAR as two pairs of primers respectively to amplify CD38 scFv to obtain two fragments, 38-1 and 38-2;
配制PCR反应体系(50μl)Prepare PCR reaction system (50μl)
Figure PCTCN2022144325-appb-000014
Figure PCTCN2022144325-appb-000014
Figure PCTCN2022144325-appb-000015
Figure PCTCN2022144325-appb-000015
反应条件：Reaction conditions:
Figure PCTCN2022144325-appb-000016
Figure PCTCN2022144325-appb-000016
b.DNA琼脂糖凝胶电泳分离并回收PCR产物；b. DNA agarose gel electrophoresis separates and recovers PCR products;
c.酶切并回收含有CD8α-4-1BB-CD3ζ片段的pCDH载体c. Enzyme digestion and recovery of pCDH vector containing CD8α-4-1BB-CD3ζ fragment
酶切体系(50μl)Enzyme digestion system (50μl)
Figure PCTCN2022144325-appb-000017
Figure PCTCN2022144325-appb-000017
酶切条件：37℃，4小时；DNA电泳后回收7728bp的载体片段；Enzyme digestion conditions: 37°C, 4 hours; 7728bp vector fragment was recovered after DNA electrophoresis;
d.对三个片段进行重组d. Reorganize the three fragments
重组体系(10μl)Recombinant system (10μl)
Figure PCTCN2022144325-appb-000018
Figure PCTCN2022144325-appb-000018
载体与各个片段的摩尔比例为1:3；反应条件：50℃，15min；The molar ratio of the vector to each fragment is 1:3; reaction conditions: 50°C, 15min;
e.转化JM109感受态细胞，挑菌送测序。e. Transform JM109 competent cells, select bacteria and send them for sequencing.
2)pCDH-EF1α-loopBC38 CAR-T2A-copGFP和pCDH-EF1α-loop38BC CAR-T2A-copGFP载体的构建2) Construction of pCDH-EF1α-loopBC38 CAR-T2A-copGFP and pCDH-EF1α-loop38BC CAR-T2A-copGFP vectors
loopBC38 CAR的抗原结合结构域为BCMA VL-第三连接体-CD38 VH-第四连接体-CD38 VL-第三连接体-BCMA VH，loop38BC CAR的抗原结合结构域为CD38 VL-第三连接体-BCMA VH-第四连接体-BCMA VL-第三连接体-CD38 VH。基因合成loopBC38 CAR和loop38BC CAR抗原结合结构域(scFv)序列，序列 两端加入Nhe Ⅰ和EcoR Ⅰ酶切位点，酶切后，将目的片段***pCDH载体中，得到pCDH-EF1α-loopBC38 CAR-T2A-copGFP和pCDH-EF1α-loop38BC CAR-T2A-copGFP载体。The antigen-binding domain of loopBC38 CAR is BCMA VL-third linker-CD38 VH-fourth linker-CD38 VL-third linker-BCMA VH. The antigen-binding domain of loop38BC CAR is CD38 VL-third linker. -BCMA VH-Fourth linker-BCMA VL-Third linker-CD38 VH. The loopBC38 CAR and loop38BC CAR antigen binding domain (scFv) sequences were genetically synthesized, and Nhe Ⅰ and EcoR Ⅰ enzyme cutting sites were added to both ends of the sequence. After enzyme digestion, the target fragment was inserted into the pCDH vector to obtain pCDH-EF1α-loopBC38 CAR- T2A-copGFP and pCDH-EF1α-loop38BC CAR-T2A-copGFP vectors.
a.将含有loopBC38 CAR和loop 38BC CAR序列的两个质粒，连接在pUC57载体上，利用Nhe Ⅰ和EcoR Ⅰ对载体质粒进行酶切。a. Connect the two plasmids containing loopBC38 CAR and loop 38BC CAR sequences to the pUC57 vector, and use Nhe Ⅰ and EcoR Ⅰ to digest the vector plasmid.
酶切体系如下(50μl)The enzyme digestion system is as follows (50μl)
Figure PCTCN2022144325-appb-000019
Figure PCTCN2022144325-appb-000019
酶切条件：37℃，4小时；Enzyme digestion conditions: 37°C, 4 hours;
b.酶切体系加入5.5μl 10×loading buffer，DNA电泳后回收条带大小为1470bp的片段，步骤同前述；b. Add 5.5μl 10× loading buffer to the enzyme digestion system, and recover a fragment with a band size of 1470bp after DNA electrophoresis. The steps are the same as above;
c.连接目的片段与载体片段c. Connect the target fragment to the vector fragment
连接体系如下(10μl)The connection system is as follows (10μl)
Figure PCTCN2022144325-appb-000020
Figure PCTCN2022144325-appb-000020
载体与片段的摩尔比例为1:5，室温30min；The molar ratio of vector to fragment is 1:5, room temperature for 30 minutes;
d.转化JM109感受态细胞，挑菌送测序。d. Transform JM109 competent cells, select bacteria and send them for sequencing.
最终，BCMA和CD38双靶点CAR的抗原结合部分(scFv)示意图如图1所示，载体用NheⅠ和NotⅠ双酶切后的DNA电泳图如图2所示，tanBC38 CAR的载体结构示意图如图3所示。Finally, the schematic diagram of the antigen-binding part (scFv) of the BCMA and CD38 dual-target CAR is shown in Figure 1. The DNA electrophoresis pattern of the vector after double enzyme digestion with NheⅠ and NotⅠ is shown in Figure 2. The schematic diagram of the vector structure of the tanBC38 CAR is shown in Figure 2. 3 shown.
实施例2：嵌合抗原受体tanBC38 scFv-CD8α-4-1BB-CD3ζ,tan38BC scFv-Example 2: Chimeric Antigen Receptor tanBC38 scFv-CD8α-4-1BB-CD3ζ,tan38BC scFv- CD8α-4-1BB-CD3ζ,loopBC38 scFv-CD8α-4-1BB-CD3ζ,loop38BC scFv-CD8α-4-CD8α-4-1BB-CD3ζ,loopBC38 scFv-CD8α-4-1BB-CD3ζ,loop38BC scFv-CD8α-4- 1BB-CD3ζ慢病毒修饰T细胞的制备Preparation of 1BB-CD3ζ lentivirus modified T cells
1.采用EndoFree Plasmid Maxi质粒抽提试剂盒(QIAGEN公司)提取表达质粒和三种包装质粒：pMDLg/pRRE、pRes-Rev、pCMV-VSVG。四种质粒用3:1:1:1比例用PEI转染试剂(Polyscience公司)进行转染(具体方法见PEI转染试剂说明书)。转染后12小时更换新鲜培养液，之后24小时、48小时分别收取病毒上清，于4℃，3000rpm，离心15分钟，经0.45μm滤器过滤后，采用50000g，4℃，1.5小时超速离心后浓缩10倍，后转入-80℃保存。1. Use the EndoFree Plasmid Maxi Plasmid Extraction Kit (QIAGEN Company) to extract the expression plasmid and three packaging plasmids: pMDLg/pRRE, pRes-Rev, and pCMV-VSVG. The four plasmids were transfected with PEI transfection reagent (Polyscience Company) at a ratio of 3:1:1:1 (see the PEI transfection reagent instructions for specific methods). Replace the culture medium with fresh culture medium 12 hours after transfection. Collect the virus supernatant at 24 hours and 48 hours after transfection. Centrifuge at 4°C, 3000rpm for 15 minutes. After filtering with a 0.45μm filter, use 50000g, 4°C, and ultracentrifuge for 1.5 hours. Concentrate 10 times, then transfer to -80°C for storage.
2.T细胞的制备：取新鲜健康人外周血10ml，采用RosetteSep T cell enrichment Cocktail(Stem cell公司)和Ficoll样本密度分离液(旷博生物)提取T细胞(具体步骤按照RosetteSep T cell enrichment Cocktail说明书)。按细胞：磁珠＝1:1比例加入抗CD3/CD28磁珠(Gibco公司)，培养24小时即为转染前的T细胞。2. Preparation of T cells: Take 10 ml of fresh healthy human peripheral blood, and use RosetteSep T cell enrichment Cocktail (Stem cell company) and Ficoll sample density separation solution (Kuangbo Biotech) to extract T cells (specific steps follow the RosetteSep T cell enrichment Cocktail instructions) ). Add anti-CD3/CD28 magnetic beads (Gibco Company) at a ratio of cells: magnetic beads = 1:1, and culture for 24 hours to obtain T cells before transfection.
3.慢病毒感染T细胞及感染后T细胞的培养：从-80℃中取出病毒上清，室温下融化，按每1×10 6T细胞加入100μl病毒上清，加入Polybrene至终浓度为8μg/ml。32℃，1800rpm，离心1.5小时，转入5％CO 2，37℃孵箱培养。 3. Lentivirus-infected T cells and culture of T cells after infection: Take the virus supernatant from -80°C and melt at room temperature. Add 100 μl of virus supernatant per 1×10 6 T cells, and add Polybrene to a final concentration of 8 μg. /ml. Centrifuge at 32°C, 1800rpm for 1.5 hours, transfer to 5% CO 2 , and incubate in a 37°C incubator.
4.流式细胞术检测CAR修饰T细胞的阳性率：收集细胞，标记兔抗鼠IgG F(ab') 2抗体，流式细胞术分析T细胞F(ab') 2的表达。结果如图4所示，BCMA CAR-T的阳性率为76.29％，CD38 CAR-T的阳性率为95.28％，tanBC38 CAR-T的阳性率为87.87％，tan38BC CAR-T的阳性率为68.13％，loopBC38 CAR-T的阳性率为84.13％，loop38BC CAR-T的阳性率为68.14％；vector-T细胞带有绿色荧光蛋白，用GFP标记阳性率为95％。 4. Detect the positive rate of CAR-modified T cells by flow cytometry: collect cells, label rabbit anti-mouse IgG F(ab') 2 antibody, and analyze the expression of T cell F(ab') 2 by flow cytometry. The results are shown in Figure 4. The positive rate of BCMA CAR-T was 76.29%, the positive rate of CD38 CAR-T was 95.28%, the positive rate of tanBC38 CAR-T was 87.87%, and the positive rate of tan38BC CAR-T was 68.13%. , the positive rate of loopBC38 CAR-T is 84.13%, and the positive rate of loop38BC CAR-T is 68.14%; vector-T cells carry green fluorescent protein, and the positive rate of labeling with GFP is 95%.
实验例3：BCMA/CD38双靶点嵌合抗原受体慢病毒修饰T细胞对多发性Experimental Example 3: BCMA/CD38 dual-target chimeric antigen receptor lentivirus-modified T cells treat multiple 骨髓瘤细胞系的杀伤作用Killing effect of myeloma cell lines
1.流式细胞术检测细胞系BCMA和CD38的表达水平1. Flow cytometry to detect the expression levels of BCMA and CD38 in cell lines
(1)K562、K562-BCMA、H929、Raji及ARP-1细胞系均在含10％FBS的RMPI 1640培养基中培养，MM.1S在含10％FBS的高糖1640培养基中培养，离心条件均为800rpm 5min；(1) K562, K562-BCMA, H929, Raji and ARP-1 cell lines were cultured in RMPI 1640 medium containing 10% FBS, MM.1S was cultured in high glucose 1640 medium containing 10% FBS, and centrifuged The conditions are all 800rpm 5min;
(2)取部分上述细胞系于1.5mL EP中，分成4管，PBS离心洗涤1次；(2) Take some of the above cell lines in 1.5mL EP, divide into 4 tubes, and centrifuge and wash once with PBS;
(3)标记BCMA：一管用1μl APC IgG2a,κisotype ctrl标记细胞，另一管用APC anti-human BCMA antibody标记细胞；标记CD38：一管用1μl PE IgG1,κisotype ctrl标记细胞，另一管用PE anti-human CD38 antibody标记细胞；(3) Label BCMA: One tube uses 1μl APC IgG2a, κisotype ctrl to label cells, the other tube uses APC anti-human BCMA antibody to label cells; Label CD38: One tube uses 1μl PE IgG1, κisotype ctrl to label cells, the other tube uses PE anti-human CD38 antibody marks cells;
(4)4℃避光孵育30min后，加入1mL PBS洗涤一次；(4) After incubating for 30 minutes at 4°C in the dark, add 1 mL of PBS and wash once;
(5)用200μl PBS重悬细胞，流式细胞仪检测细胞表面BCMA或CD38的阳性率。(5) Resuspend the cells in 200 μl PBS, and detect the positive rate of BCMA or CD38 on the cell surface using flow cytometry.
流式细胞术测得MM细胞系H929、MM.1S及ARP-1表面BCMA和CD38阳性率均在85％以上。K562-RFP表面不表达BCMA或CD38，K562-BCMA细胞表面BCMA阳性率为95％，CD38阴性表达；Raji细胞表面BCMA阳性率为2.91％，CD38阳性率为99.3％(图5的A，图5的B)。Flow cytometry showed that the positive rates of BCMA and CD38 on the surface of MM cell lines H929, MM.1S and ARP-1 were all above 85%. The surface of K562-RFP does not express BCMA or CD38. The positive rate of BCMA on the surface of K562-BCMA cells is 95% and the negative expression of CD38. The positive rate of BCMA on the surface of Raji cells is 2.91% and the positive rate of CD38 is 99.3% (Figure 5A, Figure 5 B).
2.体外共培养实验检测BMCA/CD38双靶点CAR-T细胞的杀伤作用2. In vitro co-culture experiment to detect the killing effect of BMCA/CD38 dual-target CAR-T cells
(1)取感染后第7天的VEC-T和CAR-T细胞，1500rpm离心10min，用含5％FBS的KBM581培养基重悬细胞团快，细胞计数的同时流式检测CAR的感 染效率(GFP阳性率)，将细胞密度调整为1×106/mL，用没有转染的T细胞(Non-transduced control T cell，NC-T)把所有CAR-T细胞比例调整到一致，均调至所有CAR中感染效率最低一组，约为30％，作为效应细胞；(1) Take the VEC-T and CAR-T cells on the 7th day after infection, centrifuge at 1500 rpm for 10 minutes, resuspend the cell pellet in KBM581 medium containing 5% FBS, and measure the infection efficiency of CAR by flow cytometry while counting the cells ( GFP positive rate), adjust the cell density to 1×106/mL, use non-transduced control T cells (NC-T) to adjust the proportion of all CAR-T cells to the same The group with the lowest infection efficiency among CARs, about 30%, serves as effector cells;
(2)分别取K562，K562-BCMA，Raji，H929细胞系，800rpm离心5min后，用含5％FBS的KBM581培养基重悬，计数调整细胞密度到1×10 6/mL，作为靶细胞； (2) Take the K562, K562-BCMA, Raji, and H929 cell lines respectively, centrifuge them at 800 rpm for 5 minutes, resuspend them in KBM581 culture medium containing 5% FBS, count and adjust the cell density to 1×10 6 /mL as target cells;
(3)共培养体系:(3) Co-culture system:
1)将靶细胞固定为2×10 5个/孔，接种到24孔板里； 1) Fix the target cells at 2×10 5 cells/well and seed them into a 24-well plate;
2)按照不同效靶比(1:1，1:2，1:4)取相应体积的VEC-T或CAR-T细胞悬液加入到相应的孔中；2) Add the corresponding volume of VEC-T or CAR-T cell suspension into the corresponding well according to different effect-to-target ratios (1:1, 1:2, 1:4);
3)用含5％FBS的KBM581培养基将每个孔的体积补齐至1mL；3) Fill the volume of each well to 1 mL with KBM581 medium containing 5% FBS;
(4)分别于0h，24h，48h收集共培养后的细胞，离心后收集上清，保存于-80℃，用于ELISA测量细胞因子；(4) Collect co-cultured cells at 0h, 24h, and 48h respectively, collect the supernatant after centrifugation, and store it at -80°C for ELISA measurement of cytokines;
(5)PBS洗涤一次后用100μl PBS重悬细胞，加入1μl PE/Cy7 anti-human CD3 antibody,1μl APC anti-human BCMA antibody，1μl PE anti-human CD38 antibody标记细胞；混匀后4℃避光孵育30min；(5) After washing once with PBS, resuspend the cells in 100 μl PBS, add 1 μl PE/Cy7 anti-human CD3 antibody, 1 μl APC anti-human BCMA antibody, and 1 μl PE anti-human CD38 antibody to label the cells; mix well and protect from light at 4°C. Incubate for 30 minutes;
(6)PBS洗涤后加入200μl PBS重悬细胞，流式细胞仪检测不同细胞的比例。(6) After washing with PBS, add 200 μl PBS to resuspend the cells, and detect the ratio of different cells with a flow cytometer.
与H929细胞系共培养24h、48h后，所有CAR-T细胞对肿瘤细胞都存在杀伤作用，随着效靶比的升高和时间的延长杀伤作用更明显(图6的A，图6的B)。BCMA CAR-T及四种双靶点CAR-T都能够杀伤K562-BCMA细胞，而CD38CAR-T对CD38阴性的K562-BCMA细胞不发挥杀伤功能(图6的C，图6的D)。CD38 CAR-T和双靶点CAR-T能够杀伤CD38阳性的Raji细胞，而BCMA CAR-T则表现为极微弱的非特异性杀伤(图6的E，图6的F)。与K562共培养24h和48h后，所有CAR-T和VEC-T组残留靶细胞的比例没有明显区别，说明CAR-T的非特异性杀伤作用微弱(图6的G，图6的H)。After co-culture with the H929 cell line for 24 hours and 48 hours, all CAR-T cells had a killing effect on tumor cells, and the killing effect became more obvious with the increase of the effect-to-target ratio and the prolongation of time (Figure 6 A, Figure 6 B ). BCMA CAR-T and four dual-target CAR-T can kill K562-BCMA cells, while CD38 CAR-T does not exert a killing function on CD38-negative K562-BCMA cells (Figure 6 C, Figure 6 D). CD38 CAR-T and dual-target CAR-T can kill CD38-positive Raji cells, while BCMA CAR-T shows very weak non-specific killing (Figure 6 E, Figure 6 F). After co-culture with K562 for 24 hours and 48 hours, there was no significant difference in the proportion of residual target cells in all CAR-T and VEC-T groups, indicating that the non-specific killing effect of CAR-T was weak (Figure 6 G, Figure 6 H).
3.T细胞脱颗粒水平检测3. Detection of T cell degranulation level
(1)在96孔板种，加入步骤2中调整好密度的T细胞和靶细胞，每种细胞各100μl，每组设置3个复孔；(1) Seed in a 96-well plate, add the T cells and target cells whose density was adjusted in step 2, 100 μl of each type of cells, and set 3 duplicate wells in each group;
(2)每个孔加入0.5μl PE anti-human CD107a抗体和50IU/mL rhIL-2，放入CO 2孵箱孵育； (2) Add 0.5μl PE anti-human CD107a antibody and 50IU/mL rhIL-2 to each well, and incubate in a CO 2 incubator;
(3)培养1小时后每孔加入1μM莫能霉素(Monensin)，混匀后放回孵箱；(3) After culturing for 1 hour, add 1 μM monensin to each well, mix well and return to the incubator;
(4)培养4小时后取出，每孔加入1μl APC/Cy7anti-human CD3抗体；(4) Take it out after 4 hours of culture, and add 1μl APC/Cy7anti-human CD3 antibody to each well;
(5)4℃避光孵育30min后吸出细胞，用PBS洗涤一次，用200μl PBS重悬，流式细胞仪检测CD3+CD107a+细胞的比例。(5) After incubating at 4°C for 30 minutes in the dark, aspirate the cells, wash once with PBS, resuspend in 200 μl PBS, and detect the proportion of CD3+CD107a+ cells with a flow cytometer.
结果如图7表示，与H929细胞共培养后，和VEC-T相比，所有CAR-T都 能够发挥强烈的脱颗粒作用，而双靶点CAR-T的脱颗粒作用比单靶点CAR-T稍强。与K562-BCMA共培养后，CD38 CAR-T发挥的脱颗粒作用可能也是由于其对T细胞自身，而非针对靶细胞的细胞毒作用；与Raji共培养后BCMA CAR-T发挥的脱颗粒作用可能与淋巴瘤细胞系也很微弱的表达BCMA相关。与K562细胞共培养后，VEC-T、BCMA CAR-T、tan38BC CAR-T与tanBC38 CAR-T没有发挥脱颗粒作用，而CD38 CAR-T、loopBC38 CAR-T和loop38BC CAR-T产生了强烈的脱颗粒作用(p<0.0001)，而上述共培养实验结果说明这几组CAR-T细胞并不能降低靶细胞的比例，可以推测这种脱颗粒作用是由于CAR-T对表达CD38的T细胞发挥细胞毒作用而产生的。The results are shown in Figure 7. After co-culture with H929 cells, compared with VEC-T, all CAR-T can exert a strong degranulation effect, and the degranulation effect of dual-target CAR-T is higher than that of single-target CAR-T. T is slightly stronger. After co-culture with K562-BCMA, the degranulation effect of CD38 CAR-T may also be due to its cytotoxic effect on the T cells themselves rather than on the target cells; the degranulation effect of BCMA CAR-T after co-culture with Raji It may be related to the fact that lymphoma cell lines also express BCMA very weakly. After co-culture with K562 cells, VEC-T, BCMA CAR-T, tan38BC CAR-T and tanBC38 CAR-T did not exert a degranulation effect, while CD38 CAR-T, loopBC38 CAR-T and loop38BC CAR-T produced strong degranulation. Degranulation (p<0.0001), and the above co-culture experiment results show that these groups of CAR-T cells cannot reduce the proportion of target cells. It can be speculated that this degranulation is due to the effect of CAR-T on CD38-expressing T cells. Produced by cytotoxicity.
4.ELISA检测细胞因子释放水平4. ELISA to detect cytokine release levels
采用人IFN-γ、IL-2、TNFα、IL6 ELISA检测试剂盒(R&D公司)，对共培养上清进行检测(具体步骤见ELISA检测试剂盒说明书)。Human IFN-γ, IL-2, TNFα, IL6 ELISA detection kit (R&D Company) was used to detect the co-culture supernatant (see the ELISA detection kit instructions for specific steps).
结果如图8所示，与H929共培养后，与VEC-T相比，所有组别的CAR-T都能够明显分泌IFN-γ，组间没有差异；与K562-BCMA细胞共培养后CD38 CAR-T释放的IFN-γ可能因为自我杀伤作用；BCMA CAR-T和Raji共培养后也会在一定程度分泌细胞因子；与K562共培养后，CD38 CAR-T的IFN-γ分泌上升，其他组CAR-T和VEC-T的分泌情况没有明显区别。与各组靶细胞共培养后，CD38 CAR-T分泌大量的IL-6，推测是由于CD38 CAR-T对于靶细胞或自身的CD38+T细胞进行杀伤所造成的。与K562、H929、Raji细胞共培养后，BCMA CAR-T和四组双靶点CAR-T，尤其是两组loop结构的CAR-T，分泌的IL-6水平明显低于CD38 CAR-T，与VEC-T没有明显区别。与K562-BCMA细胞共培养后，相比于VEC-T，各组CAR-T细胞分泌大量IL-6。The results are shown in Figure 8. After co-culture with H929, compared with VEC-T, CAR-T in all groups can significantly secrete IFN-γ, with no difference between groups; after co-culture with K562-BCMA cells, CD38 CAR The IFN-γ released by -T may be due to self-killing effect; BCMA CAR-T will also secrete cytokines to a certain extent after co-culture with Raji; after co-culture with K562, the IFN-γ secretion of CD38 CAR-T increased, and other groups There is no significant difference in the secretion of CAR-T and VEC-T. After co-culture with each group of target cells, CD38 CAR-T secreted a large amount of IL-6, presumably due to CD38 CAR-T killing target cells or its own CD38+ T cells. After co-culture with K562, H929, and Raji cells, the level of IL-6 secreted by BCMA CAR-T and four groups of dual-target CAR-T, especially the two groups of loop-structured CAR-T, was significantly lower than that of CD38 CAR-T. There is no significant difference from VEC-T. After co-culture with K562-BCMA cells, compared with VEC-T, CAR-T cells in each group secreted a large amount of IL-6.
实验例4：BCMA/CD38双靶点嵌合抗原受体修饰T细胞对MM患者骨髓Experimental Example 4: BCMA/CD38 dual-target chimeric antigen receptor modified T cells on bone marrow of MM patients 原代细胞的体外杀伤作用In vitro killing of primary cells
1.流式细胞术检测MM的BMMNC细胞表面BCMA和CD38表达水平1. Detection of BCMA and CD38 expression levels on the surface of BMMNC cells in MM by flow cytometry
(1)将BMMNC与等体积的PBS均匀混合稀释，在15mL离心管中准备5mL Ficoll样本密度分离液，将稀释后的样本轻轻地沿着管壁加入分离液的上层，注意不要破坏液面，离心500g 15min；(1) Evenly mix and dilute BMMNC with an equal volume of PBS. Prepare 5 mL of Ficoll sample density separation solution in a 15 mL centrifuge tube. Gently add the diluted sample to the upper layer of the separation solution along the tube wall. Be careful not to damage the liquid surface. , centrifuge at 500g for 15min;
(2)样本分为三层，将中间的白膜细胞层收集到新的15mL离心管中，加入等体积的PBS混匀，离心1500rpm 10min；(2) The sample is divided into three layers. Collect the white coat cell layer in the middle into a new 15mL centrifuge tube, add an equal volume of PBS, mix well, and centrifuge at 1500 rpm for 10 minutes;
(3)用PBS重复洗涤一次；(3) Repeat washing once with PBS;
(4)用IMDM培养基重悬计数，流式检测表面的BCMA，CD38，CD138和CD56的表达情况，细胞冻存备用。(4) Resuspend in IMDM medium for counting, detect the expression of BCMA, CD38, CD138 and CD56 on the surface by flow cytometry, and freeze the cells for later use.
2.T细胞和原代细胞共培养2. Co-culture of T cells and primary cells
(1)进行共培养实验之前24小时复苏原代细胞，配制培养原代细胞的培养基(IMDM+15％FBS+100ng/mL hSCF+100ng/mL hFlt3-L+50ng/mL hTPO)，并以3×10 5/mL的密度进行培养； (1) Resuscitate the primary cells 24 hours before conducting the co-culture experiment, prepare the medium for culturing the primary cells (IMDM+15% FBS+100ng/mL hSCF+100ng/mL hFlt3-L+50ng/mL hTPO), and use Culture at a density of 3×10 5 /mL;
(2)取感染后第7天的T细胞，离心计数，调整细胞密度至1×10 6/mL，将CAR-T细胞的感染效率调成一致(约40％)； (2) Take T cells on the 7th day after infection, count them by centrifugation, adjust the cell density to 1×10 6 /mL, and adjust the infection efficiency of CAR-T cells to the same level (about 40%);
(3)将原代细胞离心1000rpm 5min；(3) Centrifuge the primary cells at 1000 rpm for 5 minutes;
1)DiD染料染色原代细胞1) DiD dye staining primary cells
2)将T细胞和靶细胞各200μl接种至24孔板中，补齐培养体系至1mL；2) Inoculate 200 μl each of T cells and target cells into a 24-well plate, and fill the culture system to 1 mL;
3)在0h和24h分别检测原代细胞的比例以及原代细胞中BCMA，CD38的表达情况。3) Detect the proportion of primary cells and the expression of BCMA and CD38 in primary cells at 0h and 24h respectively.
图9为四位患者的流式检测结果，如图所示四位患者BMMNC的CD38阳性细胞占所有细胞的30.63％到53.65％，BCMA阳性的细胞所占比例较低。Figure 9 shows the flow cytometry results of four patients. As shown in the figure, the CD38-positive cells of BMMNC in the four patients accounted for 30.63% to 53.65% of all cells, and the proportion of BCMA-positive cells was lower.
图10显示了共培养24h剩余的所有细胞的比例。双靶点CAR-T能够明显降低4例患者BMMNC细胞的比例，其作用强于BCMA CAR-T和CD38 CAR-T。三种CAR-T能够几乎完全杀伤BCMA阳性的细胞；CD38 CAR-T和loopBC38 CAR-T可以杀伤CD38阳性的细胞，且CD38 CAR-T的杀伤效果稍强于loopBC38 CAR-T。以上结果提示，双靶点BCMA/CD38 CAR-T可以有效杀伤MM病人原代细胞。Figure 10 shows the proportion of all cells remaining after 24 h of co-culture. Dual-target CAR-T could significantly reduce the proportion of BMMNC cells in 4 patients, and its effect was stronger than BCMA CAR-T and CD38 CAR-T. Three types of CAR-T can almost completely kill BCMA-positive cells; CD38 CAR-T and loopBC38 CAR-T can kill CD38-positive cells, and the killing effect of CD38 CAR-T is slightly stronger than that of loopBC38 CAR-T. The above results suggest that dual-target BCMA/CD38 CAR-T can effectively kill primary cells of MM patients.
实验例5：BCMA/CD38双靶点嵌合抗原受体修饰T细胞对小鼠移植瘤模Experimental Example 5: BCMA/CD38 dual-target chimeric antigen receptor modified T cells on mouse transplanted tumor model 型的体内治疗效果type of in vivo therapeutic effect
1.NOD/SCID小鼠MM模型的构建1.Construction of NOD/SCID mouse MM model
(1)6周龄的雌性NOD/SCID小鼠在SPF环境下饲养1～2周后，予以半致死量照射(200cGy)，4～24h内进行下一步操作；(1) After 6-week-old female NOD/SCID mice were raised in a SPF environment for 1 to 2 weeks, they were irradiated with a semi-lethal dose (200 cGy), and the next step was performed within 4 to 24 hours;
(2)取生长状态良好的ARP-1细胞系，将细胞密度调整到1×107/mL，分装，将细胞颠倒震荡混匀后，用1mL医用注射器吸取细胞，给小鼠尾静脉酒精消毒后，每只小鼠尾静脉注射200μl细胞悬液(2×10 6个细胞)。 (2) Take the ARP-1 cell line that is in good growth status, adjust the cell density to 1×107/mL, aliquot, mix the cells by inverting and shaking, use a 1mL medical syringe to absorb the cells, and disinfect the mouse tail vein with alcohol. Afterwards, 200 μl of cell suspension (2 × 10 6 cells) was injected into the tail vein of each mouse.
2.对ARP-1移植后的NOD/SCID鼠进行CAR-T治疗2. CAR-T treatment of NOD/SCID mice after ARP-1 transplantation
1)在移植肿瘤后的第5天，通过生物发光观察每只小鼠的肿瘤负荷，按照生物发光的平均荧光强度将小鼠随机分成5组，即VEC-T组，BCMA CAR-T组，CD38 CAR-T组，loop38BC CAR-T组和loopBC38 CAR-T组；1) On the 5th day after tumor transplantation, the tumor burden of each mouse was observed through bioluminescence. The mice were randomly divided into 5 groups according to the average fluorescence intensity of bioluminescence, namely VEC-T group and BCMA CAR-T group. CD38 CAR-T group, loop38BC CAR-T group and loopBC38 CAR-T group;
2)在肿瘤移植后第7天，离心、洗涤，将细胞密度调整到一致(3.33×107/mL)；2) On the 7th day after tumor transplantation, centrifuge and wash to adjust the cell density to a consistent level (3.33×107/mL);
3)给每只小鼠尾静脉注射300μl(约1×10 7/mL)相应组别的T细胞； 3) Inject 300 μl (approximately 1×10 7 /mL) of T cells from the corresponding group into the tail vein of each mouse;
4)在移植后第10天和第14天重复注射T细胞，细胞密度为1.66×107/mL，细胞数为5×10 6/mL。 4) Repeat the injection of T cells on the 10th and 14th days after transplantation, with a cell density of 1.66×107/mL and a cell number of 5× 106 /mL.
3.小鼠发病情况的监测3. Monitoring of the disease in mice
1)在移植肿瘤细胞后的第5、8、15、21、33天通过生物发光成像评价小鼠体内肿瘤情况。1) The tumor status in mice was evaluated by bioluminescence imaging on days 5, 8, 15, 21, and 33 after transplantation of tumor cells.
2)记录小鼠的生存期。2) Record the survival period of mice.
图11的A为小鼠实验流程图，图11的B显示了各组小鼠的肿瘤负荷，每个时间点的平均荧光强度统计示，所有组别CAR-T均可明显清除小鼠体内的肿瘤细胞。由图11的C可见，VEC-T组小鼠的中位生存期为35天，与所有各组CAR-T组之间都有明显的统计学差异(p<0.01)。Figure 11 A is a mouse experiment flow chart. Figure 11 B shows the tumor burden of mice in each group. The average fluorescence intensity statistics at each time point show that CAR-T in all groups can significantly eliminate tumors in mice. tumor cells. As can be seen from Figure 11, C, the median survival period of mice in the VEC-T group was 35 days, which was significantly statistically different from all CAR-T groups (p<0.01).
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (21)

  1. 包含靶向CD38的多靶点嵌合抗原受体，所述嵌合抗原受体包含胞外区、跨膜区和胞内区，所述胞内区包含胞内信号转导区，其特征在于，所述胞外区包括特异性结合CD38分子的第一结合结构域，所述第一结合结构域包括依次如SEQ ID No.1-3所示的重链可变区(VH)的CDR1、CDR2、CDR3氨基酸序列和依次如SEQ ID No.4-6所示的轻链可变区(VL)的CDR1、CDR2、CDR3氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。Comprising a multi-target chimeric antigen receptor targeting CD38, the chimeric antigen receptor includes an extracellular region, a transmembrane region and an intracellular region, and the intracellular region includes an intracellular signal transduction region, characterized by: , the extracellular region includes a first binding domain that specifically binds to the CD38 molecule, and the first binding domain includes the CDR1 of the heavy chain variable region (VH) shown in SEQ ID No. 1-3, The CDR2, CDR3 amino acid sequence and the CDR1, CDR2, CDR3 amino acid sequence of the light chain variable region (VL) shown in SEQ ID No. 4-6, or have 85% to 99% identity with the amino acid sequence and Sequences that have the same biological function.
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包括第二结合结构域，所述第二结合结构域为特异性结合选自BCMA、CD19、CD138、SLAMF7、GAL-3、GPRC5D、PD-L1、CD155、FCRL5或ICAM1蛋白质分子中的一种；The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor further includes a second binding domain, and the second binding domain is specifically selected from the group consisting of BCMA, CD19, and CD138. , one of SLAMF7, GAL-3, GPRC5D, PD-L1, CD155, FCRL5 or ICAM1 protein molecules;
    优选地，所述第二结合结构域为特异性结合BCMA蛋白质分子的结合结构域；Preferably, the second binding domain is a binding domain that specifically binds to BCMA protein molecules;
    更优选地，所述第二结合结构域包括依次如SEQ ID No.7-9所示的重链可变区(VH)的CDR1、CDR2、CDR3氨基酸序列和依次如SEQ ID No.10-12所示的轻链可变区(VL)的CDR1、CDR2、CDR3氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。More preferably, the second binding domain includes the CDR1, CDR2, and CDR3 amino acid sequences of the heavy chain variable region (VH) as shown in SEQ ID No. 7-9 in sequence and SEQ ID No. 10-12 in sequence The CDR1, CDR2, and CDR3 amino acid sequence of the light chain variable region (VL) shown, or a sequence that has 85% to 99% identity with the amino acid sequence and has the same biological function.
  3. 根据权利要求2所述的嵌合抗原受体，其特征在于，i)所述第一结合结构域和所述第二结合结构域构建于同一个嵌合抗原受体的表达载体中；或ii)所述第一结合结构域和所述第二结合结构域分别构建于不同的嵌合抗原受体的表达载体中。The chimeric antigen receptor according to claim 2, wherein i) the first binding domain and the second binding domain are constructed in the same expression vector of the chimeric antigen receptor; or ii ) The first binding domain and the second binding domain are respectively constructed in different expression vectors of chimeric antigen receptors.
  4. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述第一结合结构域为特异性结合CD38分子的scFv，所述scFv包括所述重链可变区(VH)的氨基酸序列和所述轻链可变区(VL)的氨基酸序列；The chimeric antigen receptor according to claim 1, wherein the first binding domain is an scFv that specifically binds to CD38 molecules, and the scFv includes the amino acid sequence of the heavy chain variable region (VH) and the amino acid sequence of the light chain variable region (VL);
    优选地，所述scFv的氨基酸序列为经过人源化的序列；Preferably, the amino acid sequence of the scFv is a humanized sequence;
    更优选地，所述scFv的所述重链可变区(VH)的氨基酸序列如SEQ ID No.13所示，所述scFv的所述轻链可变区(VL)的氨基酸序列如SEQ ID No.14所示，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。More preferably, the amino acid sequence of the heavy chain variable region (VH) of the scFv is as shown in SEQ ID No. 13, and the amino acid sequence of the light chain variable region (VL) of the scFv is as SEQ ID No. No. 14, or a sequence that has 85% to 99% identity with the amino acid sequence and has the same biological function.
  5. 根据权利要求4所述的嵌合抗原受体，其特征在于，所述重链可变区(VH)和所述轻链可变区(VL)的连接顺序为5’-VL-第一连接体-VH-3’或5’-VH-第一 连接体-VL-3’；优选地，所述第一连接体为由甘氨酸(Gly)和丝氨酸(Ser)构成的连接体；The chimeric antigen receptor according to claim 4, wherein the connection sequence of the heavy chain variable region (VH) and the light chain variable region (VL) is 5'-VL-first connection Body-VH-3' or 5'-VH-first linker-VL-3'; Preferably, the first linker is a linker composed of glycine (Gly) and serine (Ser);
    更优选地，所述第一连接体的氨基酸序列为一个或串联的多个GGGGS序列；More preferably, the amino acid sequence of the first linker is one or multiple GGGGS sequences in series;
    进一步优选地，所述多个为三个；Further preferably, the plurality is three;
    进一步优选地，所述第一连接体的氨基酸序列如SEQ ID No.34所示。Further preferably, the amino acid sequence of the first linker is shown in SEQ ID No. 34.
  6. 根据权利要求2所述的嵌合抗原受体，其特征在于，所述第二结合结构域为特异性结合所述蛋白质分子的scFv，所述scFv包括所述重链可变区(VH)的氨基酸序列和所述轻链可变区(VL)的氨基酸序列；The chimeric antigen receptor according to claim 2, wherein the second binding domain is an scFv that specifically binds to the protein molecule, and the scFv includes the heavy chain variable region (VH). Amino acid sequence and the amino acid sequence of the light chain variable region (VL);
    优选地，所述scFv的氨基酸序列为经过人源化的序列；Preferably, the amino acid sequence of the scFv is a humanized sequence;
    更优选地，所述蛋白质分子为BCMA蛋白质分子；More preferably, the protein molecule is a BCMA protein molecule;
    进一步优选地，所述scFv的所述重链可变区(VH)的氨基酸序列如SEQ ID No.15所示，所述scFv的所述轻链可变区(VL)的氨基酸序列如SEQ ID No.16所示，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。Further preferably, the amino acid sequence of the heavy chain variable region (VH) of the scFv is as shown in SEQ ID No. 15, and the amino acid sequence of the light chain variable region (VL) of the scFv is as SEQ ID No. No. 16, or a sequence that is 85% to 99% identical to the amino acid sequence and has the same biological function.
  7. 根据权利要求6所述的嵌合抗原受体，其特征在于，所述重链可变区(VH)和所述轻链可变区(VL)的连接顺序为5’-VL-第一连接体-VH-3’或5’-VH-第一连接体-VL-3’；优选地，所述第一连接体为由甘氨酸(Gly)和丝氨酸(Ser)构成的连接体；The chimeric antigen receptor according to claim 6, wherein the connection sequence of the heavy chain variable region (VH) and the light chain variable region (VL) is 5'-VL-first connection Body-VH-3' or 5'-VH-first linker-VL-3'; Preferably, the first linker is a linker composed of glycine (Gly) and serine (Ser);
    更优选地，所述第一连接体的氨基酸序列为一个或串联的多个GGGGS序列；More preferably, the amino acid sequence of the first linker is one or multiple GGGGS sequences in series;
    进一步优选地，所述多个为三个；Further preferably, the plurality is three;
    进一步优选地，所述第一连接体的氨基酸序列如SEQ ID No.34所示。Further preferably, the amino acid sequence of the first linker is shown in SEQ ID No. 34.
  8. 根据权利要求2、5或7所述的嵌合抗原受体，其特征在于，所述第一结合结构域和所述第二结合结构域之间通过第二连接体连接；The chimeric antigen receptor according to claim 2, 5 or 7, characterized in that the first binding domain and the second binding domain are connected through a second linker;
    优选地，所述第二连接体的氨基酸序列为一个或串联的多个EAAAK序列；Preferably, the amino acid sequence of the second linker is one or multiple EAAAK sequences in series;
    更优选地，所述多个为五个；More preferably, the plurality is five;
    进一步优选地，所述第二连接体的氨基酸序列如SEQ ID No.35所示；Further preferably, the amino acid sequence of the second linker is shown in SEQ ID No. 35;
    进一步优选地，所述胞外区的结合结构域包括：Further preferably, the binding domain of the extracellular region includes:
    i)5’-第二结合结构域的VL-第一连接体-第二结合结构域的VH-第二连接体-CD38 VL-第一连接体-CD38 VH-3’；或i) 5’-VL of the second binding domain-first linker-VH of the second binding domain-second linker-CD38 VL-first linker-CD38 VH-3’; or
    ii)5’-CD38 VL-第一连接体-CD38 VH-第二连接体-第二结合结构域的VL- 第一连接体-第二结合结构域的VH-3’；ii) 5’-CD38 VL-first linker-CD38 VH-second linker-VL of the second binding domain-first linker-VH-3’ of the second binding domain;
    进一步优选地，所述胞外区的结合结构域包括：Further preferably, the binding domain of the extracellular region includes:
    i)5’-BCMA VL-第一连接体-BCMA VH-第二连接体-CD38 VL-第一连接体-CD38 VH-3’；或i) 5’-BCMA VL-first linker-BCMA VH-second linker-CD38 VL-first linker-CD38 VH-3’; or
    ii)5’-CD38 VL-第一连接体-CD38 VH-第二连接体-BCMA VL-第一连接体-BCMA VH-3’；ii) 5’-CD38 VL-first linker-CD38 VH-second linker-BCMA VL-first linker-BCMA VH-3’;
    进一步优选地，所述胞外区的结合结构域包括如SEQ ID No.17或SEQ ID No.18所示的氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。Further preferably, the binding domain of the extracellular region includes the amino acid sequence shown in SEQ ID No. 17 or SEQ ID No. 18, or has 85% to 99% identity with the amino acid sequence and is identical to it. Sequences of biological functions.
  9. 根据权利要求2、4或6所述的嵌合抗原受体，其特征在于，所述胞外区的结合结构域包括：The chimeric antigen receptor according to claim 2, 4 or 6, wherein the binding domain of the extracellular region includes:
    i)5’-第二结合结构域的VL-第三连接体-CD38 VH-第四连接体-CD38 VL-第三连接体-第二结合结构域的VH-3’；或i) 5’-VL of the second binding domain-third linker-CD38 VH-fourth linker-CD38 VL-third linker-VH-3’ of the second binding domain; or
    ii)5’-CD38 VL-第三连接体-第二结合结构域的VH-第四连接体-第二结合结构域的VL-第三连接体-CD38 VH-3’；ii) 5’-CD38 VL-third linker-VH of the second binding domain-fourth linker-VL of the second binding domain-third linker-CD38 VH-3’;
    优选地，所述胞外区的结合结构域包括：Preferably, the binding domain of the extracellular region includes:
    i)5’-BCMA VL-第三连接体-CD38 VH-第四连接体-CD38 VL-第三连接体-BCMA VH-3’；或i) 5’-BCMA VL-third linker-CD38 VH-fourth linker-CD38 VL-third linker-BCMA VH-3’; or
    ii)5’-CD38 VL-第三连接体-BCMA VH-第四连接体-BCMA VL-第三连接体-CD38 VH-3’；ii) 5’-CD38 VL-third linker-BCMA VH-fourth linker-BCMA VL-third linker-CD38 VH-3’;
    更优选地，所述第三连接体的氨基酸序列如SEQ ID No.36所示，所述第四连接体的氨基酸序列如SEQ ID No.37所示；More preferably, the amino acid sequence of the third linker is shown in SEQ ID No. 36, and the amino acid sequence of the fourth linker is shown in SEQ ID No. 37;
    进一步优选地，所述胞外区的结合结构域包括如SEQ ID No.19或SEQ ID No.20所示的氨基酸序列，或与所述氨基酸序列具有85％～99％同一性且与其具有相同生物学功能的序列。Further preferably, the binding domain of the extracellular region includes the amino acid sequence shown in SEQ ID No. 19 or SEQ ID No. 20, or has 85% to 99% identity with the amino acid sequence and is identical to it. Sequences of biological functions.
  10. 根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述胞外区还包含构建在所述嵌合抗原受体的氨基末端的信号肽或与所述信号肽具有90％以上同一性且与其具有相同生物学功能的序列；The chimeric antigen receptor according to claim 1 or 2, characterized in that the extracellular region also contains a signal peptide constructed at the amino terminus of the chimeric antigen receptor or has a 90% similarity with the signal peptide. Sequences that have the same identity as above and have the same biological function;
    优选地，所述信号肽为CD8α中的信号肽序列或GM-CSF；Preferably, the signal peptide is the signal peptide sequence in CD8α or GM-CSF;
    更优选地，所述信号肽为如SEQ ID NO.21所示的信号肽。More preferably, the signal peptide is the signal peptide shown in SEQ ID NO. 21.
  11. 根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述胞外区的结合结构域通过铰链区与所述跨膜区连接；The chimeric antigen receptor according to claim 1 or 2, characterized in that the binding domain of the extracellular region is connected to the transmembrane region through a hinge region;
    所述铰链区优选为CD8α中的铰链区序列；The hinge region is preferably a hinge region sequence in CD8α;
    所述跨膜区为选自以下蛋白质的跨膜结构域或与所述蛋白质具有90％以上同一性且与其具有相同生物学功能的序列：T细胞受体的α、β或ζ链、CD2、CD3ε、CD4、CD7、CD8α、CD8β、CD11a、CD11b、CD11c、CD11d、CD18、CD19、CD27、CD28、CD29、CD30、CD40、CD48、CD49a、CD49d、CD49f、CD66a、CD66b、CD66c、CD66d、CD66e、CD69、CD79A、CD79B、CD84、CD96、CD100、CD103、CD134、CD137、CD150、CD158A、CD158B1、CD158B2、CD158C、CD158D、CD158F1、CD158F2、CD158K、CD160、CD162、CD226、CD229、CD244、CD247、CD258、CD268、CD270、CD272、CD276、CD279、CD314、CD319、CD335、CD336、CD337、CD352、CD353、CD355、CD357、LFA-1、NKG2C、DAP-10、ICAM-1、NKp80、IL-2R beta、IL-2Rgamma、IL-7R alpha、LFA-1、SLAMF9、LAT、GADS、SLP-76、PAG1/CBP、CD83配体、Fc gamma受体、整联蛋白、激活性NK细胞受体或Toll配体受体，或其组合；The transmembrane region is a transmembrane domain selected from the following proteins or a sequence that has more than 90% identity with the protein and has the same biological function: α, β or ζ chain of T cell receptor, CD2, CD3ε, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD28, CD29, CD30, CD40, CD48, CD49a, CD49d, CD49f, CD66a, CD66b, CD66c, CD66d, CD66e, CD69, CD79A, CD79B, CD84, CD96, CD100, CD103, CD134, CD137, CD150, CD158A, CD158B1, CD158B2, CD158C, CD158D, CD158F1, CD158F2, CD158K, CD160, CD162, CD226, CD229, CD244, CD247, CD258, CD268, CD270, CD272, CD276, CD279, CD314, CD319, CD335, CD336, CD337, CD352, CD353, CD355, CD357, LFA-1, NKG2C, DAP-10, ICAM-1, NKp80, IL-2R beta, IL -2Rgamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS, SLP-76, PAG1/CBP, CD83 ligand, Fc gamma receptor, integrin, activating NK cell receptor or Toll ligand receptor body, or combination thereof;
    优选地，所述跨膜区为CD8α中的跨膜区序列。Preferably, the transmembrane region is a transmembrane region sequence in CD8α.
  12. 根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述胞内区还包含共刺激因子；The chimeric antigen receptor according to claim 1 or 2, wherein the intracellular region further contains a costimulatory factor;
    优选地，所述共刺激因子为通过选自以下蛋白质或与所述蛋白质具有90％以上同一性且与其具有相同生物学功能的序列获得的功能性信号结构域的一种或几种：整联蛋白、BTLA、Toll配体受体、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CDS、ICAM-1、LFA-1、4-1BB、B7-H3、CD278、GITR、BAFFR、LIGHT、HVEM、KIRDS2、SLAMF7、NKp80、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49α、IA4、CD49D、ITGA6、VLA6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11α、ITGAM、CD11b、ITGAX、CD11c、CD29、ITGB1、ITGB2、CD18、ITGB7、NKG2D、NKG2C、TNFR2、CD226、CD84、CD96、CEACAM1、CRTAM、CD229、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、CD162、LTBR、LAT、GADS或SLP-76；Preferably, the costimulatory factor is one or more functional signaling domains obtained by being selected from the following proteins or sequences having more than 90% identity with the protein and having the same biological function with it: integrin Protein, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, 4-1BB, B7-H3, CD278, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49α, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11α, ITGAM, CD11b, ITGAX, CD11c, CD29, ITGB1, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, CD226, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, CD162, LTBR, LAT, GADS or SLP-76;
    更优选地，所述共刺激因子为CD28或4-1BB，或与其具有90％以上同一性且与其具有相同生物学功能的序列。More preferably, the co-stimulatory factor is CD28 or 4-1BB, or a sequence with more than 90% identity and the same biological function therewith.
  13. 根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述胞内信号转导区为选自以下蛋白质或与所述蛋白质具有90％以上同一性且与其具有相同生物学功能的序列：4-1BB、B7-H3、BAFFR、BLAME、BTLA、CD100、CD103、 CD160、CD18、CD19、CD19a、CD2、CD247、CD27、CD276、CD28、CD29、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8alpha、CD8beta、CD96、CDS、CEACAM1、CRTAM、DAP-10、DNAM1、Fc gamma受体、GADS、GITR、HVEM、IA4、ICAM-1、ICAM-1、Ig alpha、IL2R beta、IL2R gamma、IL7R alpha、整联蛋白、ITGA4、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB2、ITGB7、KIRDS2、LAT、LFA-1、LFA-1、LIGHT、LIGHT、LTBR、Ly9、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80、OX-40、PAG/Cbp、PD-1、PSGL1、SELPLG、SLAMF4、SLAMF6、SLAMF7、SLP-76、TNFR2、Toll配体受体、TRANCE/RANKL、VLA1或VLA-6，或其组合；The chimeric antigen receptor according to claim 1 or 2, characterized in that the intracellular signal transduction region is selected from the following proteins or has more than 90% identity with the protein and has the same biological function Sequences: 4-1BB, B7-H3, BAFFR, BLAME, BTLA, CD100, CD103, CD160, CD18, CD19, CD19a, CD2, CD247, CD27, CD276, CD28, CD29, CD3ζ, CD30, CD4, CD40, CD49a , CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96, CDS, CEACAM1, CRTAM, DAP-10, DNAM1, Fc gamma receptor, GADS, GITR, HVEM, IA4, ICAM-1, ICAM-1, Ig alpha, IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT, LTBR, Ly9, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, OX-40, PAG/Cbp, PD-1, PSGL1, SELPLG, SLAMF4, SLAMF6, SLAMF7, SLP-76, TNFR2, Toll ligand receptor body, TRANCE/RANKL, VLA1 or VLA-6, or a combination thereof;
    优选地，所述胞内信号转导区为CD3ζ。Preferably, the intracellular signal transduction region is CD3ζ.
  14. 根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.22-25所示，或与其具有90％以上同一性且与其具有相同生物学功能的序列。The chimeric antigen receptor according to claim 1 or 2, characterized in that the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO. 22-25, or has more than 90% identity with it and is identical with it. Sequences with the same biological function.
  15. 一种分离的核酸分子，其特征在于，所述核酸分子编码如权利要求1所述的嵌合抗原受体的氨基酸序列；An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes the amino acid sequence of the chimeric antigen receptor as claimed in claim 1;
    优选地，所述核酸分子的序列如SEQ ID NO.30-33所示，或与其具有90％以上同一性且与其具有相同生物学功能的序列。Preferably, the sequence of the nucleic acid molecule is as shown in SEQ ID NO. 30-33, or a sequence that has more than 90% identity with it and has the same biological function.
  16. 一种载体，其特征在于，所述载体包含如权利要求15所述的核酸分子的序列；A vector, characterized in that the vector contains the sequence of the nucleic acid molecule as claimed in claim 15;
    优选地，所述载体为质粒载体、慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体；Preferably, the vector is a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector or a retroviral vector;
    更优选地，所述载体中包含EF1α启动子序列。More preferably, the vector contains an EF1α promoter sequence.
  17. 一种分离的细胞，其特征在于，所述细胞中包含如权利要求1所述的嵌合抗原受体、如权利要求15所述的核酸分子或如权利要求16所述的载体；An isolated cell, characterized in that the cell contains the chimeric antigen receptor as claimed in claim 1, the nucleic acid molecule as claimed in claim 15, or the vector as claimed in claim 16;
    优选地，所述细胞为i)免疫应答细胞，优选为T细胞、NK细胞、NKT细胞或CTL细胞，更优选为T细胞；或ii)诱导多能干细胞(iPSC)。Preferably, the cells are i) immune response cells, preferably T cells, NK cells, NKT cells or CTL cells, more preferably T cells; or ii) induced pluripotent stem cells (iPSC).
  18. 根据权利要求17所述的分离的细胞，其特征在于，所述细胞为自体或异体来源的T细胞；The isolated cell according to claim 17, wherein the cell is a T cell of autologous or allogeneic origin;
    优选地，所述细胞为自体来源的T细胞。Preferably, the cells are T cells of autologous origin.
  19. 根据权利要求18所述的分离的细胞，其特征在于，所述细胞为异体来源的T细胞，所述T细胞为通用型嵌合抗原受体T细胞；The isolated cell according to claim 18, wherein the cell is a T cell of allogeneic origin, and the T cell is a universal chimeric antigen receptor T cell;
    优选地，所述T细胞缺失编码TCR、HLA、CD52、PD-1或CD7的基因。Preferably, the T cells lack genes encoding TCR, HLA, CD52, PD-1 or CD7.
  20. 如权利要求1所述的嵌合抗原受体、如权利要求15所述的分离的核酸分子、如权利要求16所述的载体或如权利要求17、18或19所述的分离的细胞在制备***的药物中的用途；The chimeric antigen receptor as claimed in claim 1, the isolated nucleic acid molecule as claimed in claim 15, the vector as claimed in claim 16 or the isolated cell as claimed in claim 17, 18 or 19 is prepared in Use in drugs to treat tumors;
    优选地，所述肿瘤为血液肿瘤；Preferably, the tumor is a hematological tumor;
    更优选地，所述血液肿瘤为CD38 +和/或BCMA +的血液肿瘤； More preferably, the hematological tumor is a CD38 + and/or BCMA + hematological tumor;
    进一步优选地，所述血液肿瘤为经治疗后复发，或对单独靶向BCMA或CD38的免疫治疗无效的CD38 +和/或BCMA +的血液肿瘤； Further preferably, the hematological tumor is a CD38 + and/or BCMA + hematological tumor that relapses after treatment, or is ineffective in immunotherapy targeting BCMA or CD38 alone;
    进一步优选地，所述血液肿瘤为多发性骨髓瘤。Further preferably, the hematological tumor is multiple myeloma.
  21. 一种药物组合物，其特征在于，所述药物组合物包括如权利要求1所述的嵌合抗原受体、如权利要求15所述的分离的核酸分子、如权利要求16所述的载体或如权利要求17、18或19所述的分离的细胞。A pharmaceutical composition, characterized in that the pharmaceutical composition includes the chimeric antigen receptor as claimed in claim 1, the isolated nucleic acid molecule as claimed in claim 15, the carrier as claimed in claim 16, or The isolated cell of claim 17, 18 or 19.
PCT/CN2022/144325 2022-09-09 2022-12-30 Multi-target chimeric antigen receptor comprising cd38 targeting WO2024051057A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112538114A (en) * 2019-09-20 2021-03-23 上海普铭生物科技有限公司 Anti-human CD38 antibody and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112538114A (en) * 2019-09-20 2021-03-23 上海普铭生物科技有限公司 Anti-human CD38 antibody and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MEI HENG, LI CHENGGONG, JIANG HUIWEN, ZHAO XINYING, HUANG ZHIPING, JIN DAN, GUO TAO, KOU HAIMING, LIU LIN, TANG LU, YIN PING, WANG: "A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma", JOURNAL OF HEMATOLOGY & ONCOLOGY, BIOMED CENTRAL LTD, LONDON UK, vol. 14, no. 1, 1 December 2021 (2021-12-01), London UK , XP093148037, ISSN: 1756-8722, DOI: 10.1186/s13045-021-01170-7 *

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