WO2021073626A1 - Récepteur antigénique chimérique et lymphocytes t exprimant des récepteurs antigéniques chimériques en leur sein - Google Patents

Récepteur antigénique chimérique et lymphocytes t exprimant des récepteurs antigéniques chimériques en leur sein Download PDF

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WO2021073626A1
WO2021073626A1 PCT/CN2020/121674 CN2020121674W WO2021073626A1 WO 2021073626 A1 WO2021073626 A1 WO 2021073626A1 CN 2020121674 W CN2020121674 W CN 2020121674W WO 2021073626 A1 WO2021073626 A1 WO 2021073626A1
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seq
chimeric antigen
antigen receptor
amino acid
acid sequence
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Chinese (zh)
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芦志华
杜宝华
刘永峰
王方圆
戴卫国
朱滨
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北京门罗生物科技有限公司
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Publication of WO2021073626A1 publication Critical patent/WO2021073626A1/fr

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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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present invention relates to the field of immunotherapy, and relates to chimeric antigen receptors and T cells expressing the chimeric antigen receptors.
  • T cells or T lymphocytes are the armed forces of our immune system, which constantly look for foreign antigens and distinguish abnormal (cancer or infected cells) from normal cells.
  • Genetic modification of T cells with CAR is a common method for designing tumor-specific T cells.
  • CAR-T cells targeting tumor-associated antigens can be infused into patients (called adoptive T cell therapy), which represents an effective immunotherapy method.
  • adoptive T cell therapy represents an effective immunotherapy method.
  • the advantage of CAR-T technology is that reprogrammed engineered T cells can proliferate and persist in the patient's body, acting like living drugs.
  • CAR-T therapy for tumor immunotherapy CAR-T, full name Chimeric Antigen Receptor T-Cell Immunotherapy, namely chimeric antigen receptor T-cell immunotherapy; the principle is that T cells modified by chimeric antigen receptors can specifically Recognizing tumor-associated antigens enables effector T cells to have higher targeting, killing activity and durability than conventionally used immune cells, and can overcome the tumor local immunosuppressive microenvironment and break the host's immune tolerance state.
  • Chimeric antigen receptor (CAR) is the core component of CAR-T, which gives T cells the ability to recognize tumor antigens in an HLA-independent manner. This makes CAR-modified T cells more capable of recognizing than natural T cell surface receptor TCR. Broad goals.
  • CAR Chimeric Antigen Receptor
  • the extracellular domain is composed of a single-chain variable fragment (scFv) of a monoclonal antibody that is responsible for recognizing and binding antigens and a hinge region (Hinge) that functions as a connection.
  • the intracellular domain consists of a costimulatory domain (Costimulatory Domain) and a signal transduction domain (Signaling Domain).
  • One of the objectives of the present invention is to provide the application of CD3 in preparing a chimeric antigen receptor targeting CD3.
  • the second objective of the present invention is to provide the application of an antibody against CD3 or an antigen-binding fragment thereof in preparing a chimeric antigen receptor targeting CD3.
  • the third objective of the present invention is to provide a chimeric antigen receptor targeting CD3 or a CAR-T containing the same.
  • the fourth object of the present invention is to provide the application of the aforementioned CD3-targeting chimeric antigen receptor or CAR-T cell in immunotherapy.
  • the fifth object of the present invention is to provide the application of the aforementioned CD3-targeting chimeric antigen receptor or CAR-T cell in anti-transplant rejection.
  • the present invention provides the use of CD3 in preparing a chimeric antigen receptor targeting CD3.
  • the present invention provides the use of an antibody against CD3 or an antigen-binding fragment thereof in the preparation of a chimeric antigen receptor targeting CD3.
  • Antigen-binding fragments can be Fab fragments (Fab), F(ab') 2 fragments, double-chain antibodies, tri-chain antibodies, quadru-chain antibodies, single-chain variable region fragments (scFv), or disulfide stabilized variable region fragments (dsFv).
  • the antigen-binding fragment is a scFv.
  • the scFv is a truncated Fab fragment that contains the V domain of the antibody heavy chain connected to the variable (V) domain of the antibody light chain via a synthetic peptide, which can be generated using conventional recombinant DNA technology.
  • the present invention provides a chimeric antigen receptor targeting CD3, the chimeric antigen receptor comprising a CD3 binding domain.
  • the chimeric antigen receptor includes from N-terminal to C-terminal: a CD3 binding domain, a hinge region, a transmembrane domain, and a signal transduction domain.
  • the chimeric antigen receptor includes from N-terminus to C-terminus: a CD3 binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signal transduction domain.
  • the chimeric antigen receptor includes from N-terminus to C-terminus: CD3 binding domain, hinge region, transmembrane domain, costimulatory domain, and signal transduction domain.
  • the CD3 binding domain can comprise any antigen binding portion of a CD3 antibody.
  • the CD3 binding domain can be a Fab fragment (Fab), F(ab') 2 fragment, double-chain antibody, tri-chain antibody, four-chain antibody, single-chain variable region fragment (scFv), or disulfide stabilized Variable region fragment (dsFv).
  • the CD3 binding domain is scFv.
  • the scFv is a truncated Fab fragment that contains the V domain of the antibody heavy chain connected to the variable (V) domain of the antibody light chain via a synthetic peptide, which can be generated using conventional recombinant DNA technology.
  • the CD3 binding domain used in the CAR of the present invention is not limited to these exemplary types of antibody fragments.
  • the CD3 binding domain may comprise a light chain variable region and/or a heavy chain variable region.
  • the heavy chain variable region includes one or more of complementarity determining region (CDR) 1, CDR2, and CDR3.
  • the CD3 binding domain comprises a human heavy chain CDR1, a human heavy chain CDR2, and a human heavy chain CDR3.
  • the heavy chain CDR1 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 50 Amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 50 Amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 50 Amino acid sequence of
  • the heavy chain CDR2 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 51 Amino acid sequence of
  • the heavy chain CDR3 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 41, SEQ ID NO: 52 The amino acid sequence.
  • the light chain variable region comprises complementarity determining region (CDR) 1, CDR2, and CDR3.
  • the CD3 binding domain comprises a human light chain CDR1, a human light chain CDR2, and a human light chain CDR3.
  • the light chain CDR1 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 53 Amino acid sequence of
  • the light chain CDR2 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 54 Amino acid sequence of
  • the light chain CDR3 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 55 The amino acid sequence.
  • the CD3 binding domain comprises a heavy chain variable region and a light chain variable region.
  • the CD3 binding domain comprises a human heavy chain variable region and a human light chain variable region.
  • the heavy chain variable region of the CD3 binding domain has an amino acid sequence shown in any of SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 56 An amino acid sequence with at least 95% sequence identity.
  • the light chain variable region of the CD3 binding domain has an amino acid sequence shown in any of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 57 An amino acid sequence with at least 95% sequence identity.
  • variable region of the light chain and the variable region of the heavy chain may be connected by a linker.
  • the linker can comprise any suitable amino acid sequence.
  • the linker may include SEQ ID NO: 9 or its homologous sequence.
  • the homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • the CD3 binding domain includes a CD3 binding domain that has a combination with any one of SEQ ID NO: 10, SEQ ID NO: 25, SEQ ID NO: 36, SEQ ID NO: 47, SEQ ID NO: 58
  • the amino acid sequence shown is an scFv with an amino acid sequence of at least 95% sequence identity.
  • the CD3 binding domain may also include a leader sequence, or called a signal peptide sequence.
  • the leader sequence can be located at the amino terminus of the variable region of the light chain or the variable region of the heavy chain.
  • the leader sequence is located at the amino terminus of the variable region of the heavy chain.
  • the leader sequence can include any suitable leader sequence.
  • the CD3 binding domain may include a leader sequence that contains SEQ ID NO: 11 or a homologous sequence thereof.
  • the homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • the leader sequence can promote the expression of the CAR on the cell surface, the presence of the leader sequence in the expressed CAR may not be necessary for the CAR to function.
  • all or part of the leader sequence can be excised from the CAR.
  • the CD3 binding domain containing the signal peptide sequence or the leader sequence has the same characteristics as SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 38, SEQ ID NO: 49, SEQ ID NO :
  • the amino acid sequence shown in any one of 60 has an amino acid sequence with at least 95% sequence identity.
  • the function of the hinge region is to promote the binding of the antigen receptor to the antigen; the transmembrane domain is used to immobilize the CAR.
  • the hinge region is a human hinge region
  • the transmembrane domain is a human transmembrane domain.
  • the hinge region and the transmembrane domain may comprise the hinge region and the transmembrane domain of any one or more of the following molecules: CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.
  • the selected hinge region and transmembrane domain may comprise the amino acid sequence of SEQ ID NO: 12 or its homologous sequence.
  • the homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • the signaling domain can also be called the T cell activation domain, which provides the first signal for T cell activation.
  • the most commonly used signaling domain is the CD3 ⁇ intracellular domain.
  • the intracellular domain of CD3 ⁇ may include the amino acid sequence of SEQ ID NO: 13 or a homologous sequence thereof.
  • the homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • the costimulatory domain provides the second signal of T cell activation, and includes the intracellular domain of costimulatory factors, including CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2C, B7-H3 .
  • the costimulatory domain may include an amino acid sequence containing SEQ ID NO: 14 or a homologous sequence thereof.
  • the homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
  • the chimeric antigen receptor of the present invention includes SEQ ID NO: 15, SEQ ID NO: 26, SEQ ID NO: 37, SEQ ID NO: 48, SEQ ID NO: 59 The amino acid sequence shown.
  • the CAR of the embodiments of the present invention may comprise synthetic amino acids instead of one or more naturally occurring amino acids.
  • synthetic amino acids include, for example, aminocyclohexanoic acid, norleucine, ⁇ -amino n-decanoic acid, homoserine, S-acetamidomethyl-cysteine, Trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine Acid, ⁇ -phenylserine, ⁇ -hydroxyphenylalanine, phenylglycine, ⁇ -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1, 2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benz
  • the CAR of the embodiments of the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted into an acid addition salt and/or optionally Ground dimerization or multimerization.
  • the CAR of the embodiment of the present invention can be obtained by methods known in the art.
  • the CAR can be prepared by any suitable method for preparing polypeptides or proteins. Suitable methods for de novo synthesis of polypeptides and proteins are known in the art. In addition, you can use, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (No. 4
  • the CAR described herein can be synthesized commercially by a company.
  • the CAR of the present invention may be synthetic and/or recombinant.
  • the present invention provides a nucleic acid molecule encoding the aforementioned chimeric antigen receptor or its component parts.
  • the nucleic acid molecule of the present invention may include one or more of the leader sequence, CD3 binding domain, hinge region and transmembrane domain, signal transduction domain, costimulatory domain, and chimeric antigen receptor as described herein. Nucleotide sequence.
  • Nucleic acid as used herein includes “polynucleotide”, “oligonucleotide” and “nucleic acid molecule”, and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or natural Obtained from a source (for example, isolated and/or purified), it may contain natural, non-natural or altered nucleotides, and it may contain natural, unnatural or altered internucleotide linkages, Such as phosphoramidate bond or phosphorothioate bond, which replaces the phosphodiester existing between the nucleotides of the unmodified oligonucleotide.
  • the nucleic acid does not contain any insertions, deletions, inversions, and/or substitutions. However, in some cases it may be suitable for the nucleic acid to contain one or more insertions, deletions, inversions, and/or substitutions.
  • the nucleic acid of the embodiment of the present invention may be a recombinant.
  • the term "recombinant” as used herein refers to (i) molecules constructed outside living cells by linking natural or synthetic nucleic acid segments with nucleic acid molecules that can replicate in living cells, or (ii) from the above (i) ) The molecules produced by the replication of those molecules described in. For the purposes herein, replication can be in vitro replication or in vivo replication.
  • the nucleic acid may consist essentially of one or more designated nucleotide sequences as described herein, so that other components (such as other nucleotides) do not substantially alter the biological activity of the encoded CAR.
  • the recombinant nucleic acid may be a nucleic acid having a non-naturally occurring sequence or a sequence prepared by artificial combination of two originally separated segments of the sequence. This artificial combination is usually accomplished by chemical synthesis, or more usually by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques, such as those described in Green et al. above.
  • the nucleic acid can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green et al. above.
  • nucleotides or different modified nucleotides designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed during hybridization can be used.
  • Pyridine substituted nucleotides to chemically synthesize nucleic acids.
  • modified nucleotides that can be used to produce nucleic acids include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine Pyrimidine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, ⁇ -D galactosyl Q Nucleoside (beta-D-galactosylqueosine), creatinine, N 6-isopentene adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl Adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5 -Methoxyamin
  • the nucleic acid may comprise any isolated or purified nucleotide sequence encoding any CAR described herein.
  • the nucleotide sequence may comprise any sequence of degenerate nucleotide sequences or a combination of degenerate sequences.
  • Embodiments of the present invention also provide an isolated or purified nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that is identical to any nucleic acid described herein under stringent conditions. Sequence of nucleotide sequences that hybridize to.
  • Nucleotide sequences that hybridize under stringent conditions can hybridize under highly stringent conditions.
  • “Highly stringent conditions” means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization.
  • Highly stringent conditions include combining polynucleotides containing exact complementary sequences or polynucleotides containing only some scattered mismatches with random sequences that happen to have small regions (e.g. 3-10 bases) of matching nucleotide sequences. Distinguish the conditions. Such small complementary regions are easier to melt than full-length complements of 14-17 bases or more, and highly stringent hybridization makes them easier to distinguish.
  • Relatively highly stringent conditions will include, for example, low-salt and/or high-temperature conditions, such as those provided by about 0.02-0.1M NaCl or equivalent at a temperature of about 50-70°C.
  • Such highly stringent conditions tolerate very few (if any) mismatches between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any CAR of the invention described herein. It is generally believed that more stringent conditions can be caused by adding an increased amount of formamide.
  • the nucleic acid comprises a codon-optimized nucleotide sequence encoding a CAR.
  • codon optimization of the nucleotide sequence increases the translation efficiency of mRNA transcripts. Codon optimization of a nucleotide sequence can involve replacing the natural codon with another codon that encodes the same amino acid, but can be translated from a tRNA that is more readily available in the cell, thereby improving translation efficiency. The optimization of the nucleotide sequence can also reduce the secondary mRNA structure that interferes with translation, thereby improving translation efficiency.
  • the nucleic acid encoding the CAR may comprise the codon-optimized nucleotide sequence of any one of SEQ ID NO: 16-25.
  • the present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 95% or more, such as about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of any nucleic acid described herein Nucleotide sequence.
  • the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecule.
  • the nucleic acid of the present invention can be incorporated into a recombinant expression vector.
  • embodiments of the present invention provide a recombinant expression vector comprising any nucleic acid of the present invention.
  • the term "recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct, when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and is sufficient to make When the mRNA, protein, polypeptide or peptide is expressed in the host cell when the carrier is contacted with the cell, it allows the cell to express the mRNA, protein, polypeptide or peptide.
  • the carrier of the present invention as a whole is not naturally occurring.
  • the recombinant expression vector of the present invention may contain any type of nucleotides, including but not limited to DNA and RNA, it may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and it may contain natural , Unnatural or altered nucleotides.
  • Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.
  • the recombinant expression vector of the present invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell.
  • Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.
  • the vector can be selected from: pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX Series (Clontech, Palo Alto, CA).
  • Phage vectors such as ⁇ GT10, ⁇ GT11, ⁇ ZapII (Stratagene), ⁇ EMBL4 and ⁇ NM1149 can also be used.
  • plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).
  • the recombinant expression vector may be a viral vector, such as a retroviral vector.
  • the vector is a gamma retroviral vector, a lentiviral vector or a transposon.
  • the circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells.
  • the replication system can be derived from, for example, ColEl, 2 ⁇ plasmid, lambda, SV40, bovine papilloma virus and the like.
  • Recombinant expression vectors can contain regulatory sequences, such as transcription and translation start and stop codons, depending on the situation and considering whether the vector is DNA-based or RNA-based, which is important for the type of host cell into which the vector is to be introduced (such as bacteria, fungi) , Plant or animal) is specific.
  • Recombinant expression vectors may contain restriction sites to facilitate cloning.
  • the recombinant expression vector may contain one or more marker genes that allow selection of transformed or transfected host cells.
  • Marker genes include resistance to antimicrobial agents (for example, resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic hosts to provide prototrophs, and the like.
  • Suitable marker genes for the expression vector of the present invention include, for example, neomycin/G418 resistance gene, hygromycin resistance gene, histidine resistance gene, tetracycline resistance gene, and ampicillin resistance gene.
  • the recombinant expression vector may comprise a natural or non-natural promoter operably linked to the following sequence: a nucleotide sequence encoding the CAR of the present invention or a nucleotide sequence that is complementary or hybridizing to the nucleotide sequence encoding the CAR of the present invention sequence.
  • a promoter such as strong, weak, inducible, tissue-specific and development-specific, is within the abilities of those of ordinary skill in the art.
  • the combination of nucleotide sequence and promoter is also within the abilities of those of ordinary skill in the art.
  • the promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, or a promoter present in the long terminal repeat of murine stem cell virus.
  • CMV cytomegalovirus
  • the recombinant expression vector of the present invention can be designed for transient expression, stable expression, or both.
  • the recombinant expression vector can be prepared for constitutive expression or inducible expression.
  • the recombinant expression vector can be prepared to contain a suicide gene.
  • suicide gene refers to a gene that causes the death of cells expressing the suicide gene.
  • a suicide gene may be a gene that imparts sensitivity to an agent (e.g., a drug) to the cell in which the gene is expressed, and causes cell death when the cell comes into contact with the agent or is exposed to the agent.
  • agent e.g., a drug
  • Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.
  • the present invention provides a host cell comprising the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, or the aforementioned recombinant expression vector.
  • a host cell comprising any of the recombinant expression vectors described herein.
  • the term "host cell” as used herein refers to any type of cell that can contain the recombinant expression vector of the present invention.
  • the host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacteria or protozoa.
  • the host cell may be a cultured cell or a primary cell, that is, a cell directly isolated from an organism such as a human.
  • the host cell may be an adherent cell or a suspension cell, that is, a cell that grows in suspension.
  • Suitable host cells are known in the art, and include, for example, DH5 ⁇ E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like.
  • the host cell may be a prokaryotic cell, such as a DH5 ⁇ cell.
  • the host cell may be a mammalian cell.
  • the host cell may be a human cell.
  • the host cell can be any type of cell, can be derived from any type of tissue, and can be at any stage of development, the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).
  • PBL peripheral blood lymphocyte
  • PBMC peripheral blood mononuclear cell
  • the host cell can be a B cell, a natural killer (NK) cell or a T cell.
  • the T cell may be any T cell, such as a cultured T cell, such as a primary T cell; or a T cell derived from a cultured T cell line, such as Jurkat, SupT1, etc.; or a T cell obtained from a mammal T cells. If obtained from a mammal, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified.
  • the T cell may be a human T cell.
  • the T cell may be a T cell isolated from a human.
  • T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cells) Toxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc.
  • CD4+/CD8+ double positive T cells CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cells) Toxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc.
  • T cells can be autologous or allogeneic.
  • Autologous means that the cells to be used in the treatment method or use (ie, to be transduced with a nucleic acid or vector) are derived or obtained from the subject to be subjected to the treatment method. Therefore, autologous cells are obtained from the subject, transduced with nucleic acid or vector, and returned to the same subject.
  • Allogeneic means that the cells to be used in the treatment method or use (ie, to be transduced with a nucleic acid or vector) are derived or obtained from a different subject than the subject to be treated. Therefore, allogeneic cells are obtained from the first subject, transduced with the nucleic acid or vector, and administered to the second subject.
  • the host cell of the present invention may contain more than one nucleic acid or vector.
  • the cells of the present invention may contain 2, 3, 4, or 5 or more nucleic acids or vectors, each of which express a different chimeric antigen receptor molecule. Therefore, the cells of the present invention may contain different chimeric antigen receptor molecules that are capable of binding CD3, for example at the same or different positions of CD3.
  • the cell of the present invention may comprise a chimeric antigen receptor molecule comprising an scFv that binds to CD3 and a chimeric antigen receptor molecule that comprises a ligand that binds to CD3.
  • the cell of the present invention may contain at least one other receptor (especially exogenous) (for example, multiple receptors), which may be combined with the CAR The method is used to bind the target.
  • the binding of CAR and at least one other receptor to the target cell may be required to stimulate the immune response against the target cell (for example, each CAR/receptor may only provide immune cells Part of the signal of stimulation, which alone may not be sufficient for immune cell stimulation, but together allows immune cell stimulation).
  • the cells of the present invention are T cells
  • both CAR binding to CD3 and at least one other receptor binding to its ligand on CD3 expressing cells may be necessary to stimulate T cells.
  • At least one other receptor can be another CAR molecule.
  • Additional receptors can be used in combination with the CAR of the present invention, where the two receptors bind to different targets and induce different effects to treat diseases. Therefore, the effects of the two receptors can be completely independent of each other, but together can present an effective treatment for the disease.
  • the present invention provides a method for preparing the aforementioned host cell, the method comprising introducing the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector into the host cell, and The host cell is cultured under conditions suitable for the cell to express the nucleic acid molecule or vector.
  • the present invention provides a cell population, which includes the aforementioned host cell.
  • the cell population may be a heterogeneous population including host cells containing any of the recombinant expression vectors in addition to at least one other cell, and the other cells are, for example, host cells (such as T cells) that do not contain any recombinant expression vectors or in addition to Cells other than T cells, such as B cells, macrophages, neutrophils, red blood cells, liver cells, endothelial cells, epithelial cells, muscle cells, brain cells, etc.
  • the cell population may be a substantially homogeneous population, wherein the population mainly comprises host cells containing a recombinant expression vector (for example, consisting essentially of host cells containing a recombinant expression vector).
  • the population may also be a clonal cell population, in which all cells of the population are clones of a single host cell containing a recombinant expression vector, so that all cells of the population contain the recombinant expression vector.
  • the cell population is a clonal population comprising host cells containing the recombinant expression vector described herein.
  • the number of cells in the population can be rapidly expanded.
  • the expansion of the number of CAR-expressing cells can be accomplished by any of a variety of methods known in the art as described below, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012/0244133 ; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990).
  • CARs CARs, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) are collectively referred to as "CAR materials" hereinafter.
  • the present invention provides a pharmaceutical composition comprising the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, or the aforementioned The cell population described.
  • the pharmaceutical composition includes a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used for the particular CAR material of the invention under consideration.
  • the method of preparing the applicable composition is known or obvious to those skilled in the art, and is described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012).
  • the pharmaceutically acceptable carrier is a carrier that has no harmful side effects or toxicity under the conditions of use.
  • Suitable formulations may include any of those formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration. More than one route can be used to administer the CAR material of the present invention, and in some cases, a particular route can provide a more direct and effective response than another route.
  • the CAR material of the present invention is administered by injection, for example intravenously.
  • the pharmaceutically acceptable carrier for the injected cells may include any isotonic carrier, such as, for example, physiological saline (containing about 0.90% w /v NaCl water, containing about 300mOsm/L NaCl water, or about 9.0g NaCl per liter of water, NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), containing about 5% dextrose water or lactated Ringer's solution.
  • the pharmaceutically acceptable carrier is supplemented with human serum albumin.
  • the pharmaceutical composition of the present invention may also include other pharmaceutically active agents or drugs administered in combination with the CAR material of the present invention, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, Cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutic agents such as asparaginase, busulfan, carboplatin, Cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the one or more additional therapeutic agents can be co-administered to the mammal.
  • “Co-administration” means to administer one or more additional therapeutic agents and the CAR material of the present invention sufficiently close in time so that the CAR material of the present invention can enhance the effect of the one or more additional therapeutic agents, vice versa.
  • the CAR material of the present invention can be administered first, followed by one or more additional therapeutic agents, and vice versa.
  • the CAR material of the present invention and one or more additional therapeutic agents can be administered simultaneously.
  • Additional therapeutic agents that can enhance the function of CAR-expressing cells can include, for example, one or more cytokines or one or more antibodies (e.g., antibodies that inhibit PD-1 function).
  • the CAR materials and pharmaceutical compositions of the present invention can be used in methods of treating or preventing conditions in mammals.
  • the CAR material of the present invention has biological activities, such as the ability to recognize CD3, so that when expressed by cells, CAR can mediate an immune response against CD3-expressing cells.
  • the embodiments of the present invention provide a method of treating or preventing a condition in a mammal, which comprises administering to the mammal an amount of the CAR, nucleic acid, recombinant expression of the present invention in an amount effective to treat or prevent the condition in the mammal Any of the vector, host cell, cell population and/or pharmaceutical composition.
  • the condition can be any condition characterized by the expression or overexpression of CD3.
  • the condition is cancer.
  • the cell may be a cell allogeneic to a mammal or an autologous cell.
  • autologous cells are removed from the mammal, stored (and optionally modified), and returned to the same mammal.
  • the mammal receives cells from a genetically similar but different donor.
  • the cell is mammalian autologous.
  • the cells administered to the mammal have undergone gene editing.
  • the mammal mentioned herein can be any mammal.
  • the term "mammal” as used herein refers to any mammal, including but not limited to mammals of the Rodent order, such as mice and hamsters; and mammals of the Lagomorph order, such as rabbits.
  • Mammals can be from the order Carnivora, including the cat family (cats) and canine family (dogs).
  • Mammals can be from the order Artiodactyla, including Bovidae (bovine) and Suidae (pigs); or from the order Perissodactyla, including Equididae (horse).
  • Mammals can be from the order of the Primates, Ceboids, or Simoids (monkeys), or from the order of the Apes (humans and apes).
  • the mammal is a human.
  • treatment and “prevention” as used herein and the words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are different degrees of treatment or prevention that one of ordinary skill in the art considers to have potential benefits or therapeutic effects.
  • the methods of the present invention can provide treatment or prevention of conditions in mammals at any level in any amount.
  • the treatment or prevention provided by the method of the present invention may include the treatment or prevention of one or more conditions or symptoms of the disease (for example, cancer) being treated or prevented.
  • prevention can encompass delaying the onset of a disease, such as cancer or its symptoms or conditions. Alternatively or additionally, “prevention” can encompass delaying the recurrence of a disease, such as cancer or its symptoms or conditions.
  • the present invention provides a kit comprising the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, and the aforementioned cell Group, or the aforementioned pharmaceutical composition.
  • kits of the kit can be packaged in an aqueous medium or lyophilized form.
  • the container device of the kit usually includes at least one vial, test tube, flask, bottle, syringe or other container device, in which the components can be placed, preferably appropriately divided. If there are multiple components in the kit, the kit will usually also contain a second, third or other additional container, where the additional components can be placed in the container separately. However, various combinations of components may be included in the vial.
  • the kits of the present invention will generally also include a device for containing commercially available sealed restriction components. Such containers may include injection- or blow-molded plastic containers in which the required vials are held.
  • the liquid solution is an aqueous solution
  • the sterile aqueous solution is particularly useful.
  • the container device itself may be a syringe, pipette, and/or other similar device from which the formulation may be applied to the infected area of the body, injected into the animal, and/or even applied to other parts of the kit Components and/or mixing with them.
  • the components of the kit can be provided as dry powder.
  • the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent can also be provided in another container device.
  • the kit may also include a second container device for containing a sterile pharmaceutically acceptable buffer and/or other diluent.
  • the present invention provides an immunotherapy method comprising administering the aforementioned host cell, the aforementioned cell population or the aforementioned drug to a person in need combination.
  • the applicable diseases of the method include autoimmune diseases and cancer.
  • the cancer includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin's lymphoma, neuroblastoma, Ewing's sarcoma, multiple myeloma, myelodysplastic syndrome, BPDCN, glioma, Or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.
  • the present invention provides a method for resisting transplant rejection, the method comprising administering the aforementioned host cell, the aforementioned cell population or the aforementioned Pharmaceutical composition.
  • transplant rejection reaction includes graft versus host reaction and host versus graft reaction.
  • the present invention provides the use of the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector in the preparation of the aforementioned host cell or the aforementioned cell population.
  • the present invention provides the use of the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector in the preparation of CAR or CAR-T.
  • the present invention provides the use of the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned host cell in the preparation of the aforementioned pharmaceutical composition.
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned drug combination
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, and the aforementioned pharmaceutical composition
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, and the aforementioned pharmaceutical composition
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned drug combination The application of drugs in immunotherapy.
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned drug combination
  • the application of drugs in anti-graft rejection The application of drugs in anti-graft rejection.
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned Application of the pharmaceutical composition in the preparation of drugs for immunotherapy.
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, or the aforementioned cell population, or the aforementioned The application of the pharmaceutical composition in the preparation of a medicament for resisting transplantation rejection.
  • the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, or the aforementioned cell population, or the aforementioned Application of the pharmaceutical composition in the preparation of anti-cancer drugs.
  • the cancer of the present invention can be any cancer, including but not limited to acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin's lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, bone marrow Dysplasia syndrome, BPDCN, glioma, or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.
  • the cancer is lymphoma.
  • the cancer is T cell lymphoma (such as, for example, anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma-non-specific (PTCL-NOS), angioimmunoblastic T cell Lymphoma (AITL) and other T-cell lymphomas).
  • ACL anaplastic large cell lymphoma
  • PTCL-NOS peripheral T cell lymphoma-non-specific
  • AITL angioimmunoblastic T cell Lymphoma
  • the cancer is characterized by the expression or overexpression of CD3.
  • Figure 1 shows a schematic diagram of the LV-CD3CAR plasmid constructed by the present invention
  • Figure 2 shows the result of detecting the transduction rate of lentivirus LV-CD3CAR-291 by flow cytometry
  • Figure 3 shows the result of detecting the transduction rate of lentivirus LV-CD3CAR-cel by flow cytometry
  • Figure 4 shows the result graph of detecting the transduction rate of lentivirus LV-CD3CAR-OKT31 by flow cytometry
  • Figure 5 shows the result of using flow cytometry to detect the TCR knockout effect in LV-CD3CAR-291-T;
  • Figure 6 shows the result of using flow cytometry to detect the TCR knockout effect in LV-CD3CAR-cel-T;
  • Figure 7 shows the result of using flow cytometry to detect the TCR knockout effect in LV-CD3CAR-OKT31-T;
  • Figure 8 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-291-T cells on Jurkat-GFP cells
  • Figure 9 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-cel-T cells on Jurkat-GFP cells;
  • Figure 10 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-OKT31-T cells on Jurkat-GFP cells;
  • Figure 11 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-sp34-T cells on Jurkat-GFP cells;
  • Figure 12 shows the result of using flow cytometry to detect the killing effect of LV-CD3CAR-UCHT1-T cells on Jurkat-GFP cells;
  • Figure 13 shows a result diagram of using animal models to study the effect of LV-CD3CAR-cel-T cells constructed in the present invention on tumors;
  • Figure 14 shows a statistical graph of fluorescence intensity in mice
  • Figure 15 shows a statistical chart of the survival time of mice
  • Figure 16 shows a statistical diagram of the clearance effect of LV-CD3CAR-T on CD3 positive cells.
  • the schematic diagram of the LV-CD3CAR plasmid is shown in Figure 1 (intracellular co- The stimulation domain is 4-1BB, EGFR D III-D VI can be used as a marker for CAR expression detection and a suicide gene for CAR-T cells, increasing the safety of the product).
  • Restriction site XbaI, EcoRI. Transform, plate, and sequence to confirm that the plasmid is constructed successfully. Large-scale extraction of plasmids to obtain endotoxin-free expression plasmids for packaging lentivirus.
  • PEI transfection method for T75 culture flask.
  • Virus packaging will be performed in the afternoon of day6. Observe the cell status before transfection, and proceed to transfection when the confluence is about 90%. The culture medium in the bottle was discarded, replaced with 15ml fresh DMEM medium (without antibiotics), and cultured for 30 minutes.
  • Solution A Take LV-CD3CAR expression plasmid 17.7 ⁇ g, helper plasmid pRSV-REV 8.8 ⁇ g, helper plasmid pMDLg/pRRE 8.8 ⁇ g and helper plasmid pMD2.G 4.4 ⁇ g, the transfection ratio is 4:2:2:1, total The amount is 40 ⁇ g, after mixing, dilute to 0.75ml with serum-free DMEM, and let stand at room temperature for 5min after mixing.
  • Solution B preparation Take 630 ⁇ l DMEM, and then add 120 ⁇ l PEI working solution (1mg/ml, stored at 4°C), mix well, and let stand for 5min at room temperature.
  • T cell complete medium preparation OpTmizer TM CTS TM T-cell Expansion SFM + 5% CTS Immune Cell SR + 1% L-glutamine + 10ng/ml IL-7/15.
  • the starting cell number is 3M+Human T-Activator CD3/CD28 Dynabeads 75ul.
  • the initial cell concentration is 1M/ml. Cultivate in a 37°C incubator. Activate for 48 hours.
  • the CRISPR/cas9 system was used to design sgRNA and knock out TCR by electrotransformation.
  • Cas9 protein and sgRNA were purchased from ThermFisher Company.
  • TCR sgRNA sequence is as follows:
  • cagggttctggatatctgt (SEQ ID NO: 67)
  • LV-CD3CAR-291-T knock out LV-CD3CAR-291 transfected T cells, CAR-T cells
  • TCR-positive cells are less than 1%
  • LV-CD3CAR-cel-T knock out T cells transfected with LV-CD3CAR-cel, CAR-T cells
  • LV-CD3CAR-OKT31-T knock out the T cells transfected with LV-CD3CAR-OKT31, CAR-T cells
  • cells have a small amount of CD3/ ⁇ TCR/ ⁇ , and the TCR-positive cells are less than 1%.
  • PanT represents untreated T cells
  • PanT TCRKO represents TCR knock-out T cells
  • LV-CD3CAR-291-T, LV-CD3CAR-cel-T, LV-CD3CAR-OKT31-T represent CAR-T cells.
  • the transduction rate was measured by flow cytometry. The results are shown in Figures 5-7. After 3 days of lentiviral transduction, the CAR expression rate is not less than 50%.
  • PanT represents untreated T cells
  • PanT TCRKO represents TCR knocked out T cells
  • LV-CD3CAR- 291-T, LV-CD3CAR-cel-T, LV-CD3CAR-OKT31-T represent CAR-T cells.
  • the Jurkat-GFP cell line is co-cultured with the CAR-T cells prepared in Example 1, and the E/T (Jurkat-GFP: CAR-T) ratio is 8:1, 4:1, 2:1, and 1:1 respectively. , 0.5:1, 0:1.
  • Jurkat-GFP group 0.5M per well, three multiple wells;
  • PanT TCRKO (T cell knockout TCR) group 0.5M per well, three duplicate wells;
  • PanT TCRKO T cell knockout TCR + Jurkat-GFP group: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1;
  • LV-CD3CAR-T(CAR-T)+Jurkat-GFP group 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1;
  • NPG mice aged 5-8 weeks, all female, were injected with 1 ⁇ 10 6 Jurkat-Fluc cells through the tail vein. One week later, the biofluorescence test confirmed that the NPG mouse tumor model was successfully constructed.
  • mice were divided into tumor model group (negative control group), LV-CD3CAR-cel-T group, LV-TCRCAR-T group, a total of three groups, each with three mice.
  • the results of in vivo effectiveness are shown in Figure 13.
  • the LV-CD3CAR-T group significantly inhibited tumor growth, and the biofluorescence intensity was significantly lower than that of the tumor group.
  • the survival time of mice in the LV-CD3CAR-T group was significantly prolonged. The mice in the experimental group were still alive during the observation period.
  • CD3-targeted CAR-T constructed in the present invention can effectively kill CD3 positive cells, and can be used to treat T cell-derived lymphocytic leukemia and T cell-derived lymphoma.

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Abstract

La présente invention concerne un récepteur antigénique chimérique et des lymphocytes T exprimant le récepteur antigénique chimérique en leur sein, et plus particulièrement un récepteur antigénique chimérique ciblant CD3, le récepteur antigénique chimérique comprenant un domaine de liaison à CD3 ; une région charnière et un domaine transmembranaire ; un domaine de co-stimulation ; et un domaine de transduction de signal. La présente invention concerne en outre des molécules d'acide nucléique codant pour le récepteur antigénique chimérique et un vecteur d'expression, des cellules hôtes, une composition pharmaceutique et un kit les comprenant. Le récepteur antigénique chimérique selon la présente invention peut être utilisé en immunothérapie et en réaction de rejet de greffe, et a une grande valeur d'application commerciale.
PCT/CN2020/121674 2019-10-17 2020-10-16 Récepteur antigénique chimérique et lymphocytes t exprimant des récepteurs antigéniques chimériques en leur sein WO2021073626A1 (fr)

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CN102958942A (zh) * 2009-12-29 2013-03-06 新兴产品开发西雅图有限公司 异二聚体结合蛋白及其应用
CN107249602A (zh) * 2015-02-27 2017-10-13 美商生物细胞基因治疗有限公司 靶向血液恶性肿瘤之嵌合抗原受体（car）、其组合物及使用方法

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CA3054064A1 (fr) * 2017-03-03 2018-09-07 F1 Oncology, Inc. Procedes et compositions pour la transduction et l'expansion de lymphocytes et la regulation de l'activite de ces derniers
CN109777782A (zh) * 2019-02-15 2019-05-21 北京门罗生物科技有限公司 一种通用型car-t细胞及其制备方法和用途

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Publication number Priority date Publication date Assignee Title
CN102958942A (zh) * 2009-12-29 2013-03-06 新兴产品开发西雅图有限公司 异二聚体结合蛋白及其应用
CN107249602A (zh) * 2015-02-27 2017-10-13 美商生物细胞基因治疗有限公司 靶向血液恶性肿瘤之嵌合抗原受体（car）、其组合物及使用方法

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