[go: up one dir, main page]

CN113773402B - Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition - Google Patents

Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition Download PDF

Info

Publication number
CN113773402B
CN113773402B CN202111168331.2A CN202111168331A CN113773402B CN 113773402 B CN113773402 B CN 113773402B CN 202111168331 A CN202111168331 A CN 202111168331A CN 113773402 B CN113773402 B CN 113773402B
Authority
CN
China
Prior art keywords
chimeric antigen
antigen receptor
cells
target
target chimeric
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111168331.2A
Other languages
Chinese (zh)
Other versions
CN113773402A (en
Inventor
曾桂芳
肖非笛
汤赞
胡樾
蔡车国
胡隽源
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Beike Bio Technology Co ltd
Original Assignee
Shenzhen Beike Bio Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Beike Bio Technology Co ltd filed Critical Shenzhen Beike Bio Technology Co ltd
Priority to CN202111168331.2A priority Critical patent/CN113773402B/en
Publication of CN113773402A publication Critical patent/CN113773402A/en
Application granted granted Critical
Publication of CN113773402B publication Critical patent/CN113773402B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A61K39/001112—CD19 or B4
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001117—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR] or CD30
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention discloses a double-target chimeric antigen receptor, a nucleic acid molecule, a carrier, cells and a pharmaceutical composition, and relates to the technical field of tumor immunity. The dual-target chimeric antigen receptor comprises two chimeric antigen receptors with independent targeting and containing an antigen binding domain, a transmembrane domain and a signal transduction domain; the two chimeric antigen receptors are linked by a self-cleaving 2A short peptide. The double-target chimeric antigen receptor provided by the invention has the advantages of simple structure, safety and effectiveness, and the constructed CAR-T cells have a strong killing effect on tumor cells, generate lower cytokine level, and are expected to obviously improve the clinical treatment effect of cancer. Compared with the common single-target chimeric antigen receptor, the double-target chimeric antigen receptor has stronger recognition capability to tumor cells, has high-efficiency targeting effect to the tumor cells of the corresponding targets, and is beneficial to preventing the occurrence of antigen escape phenomenon.

Description

Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition
Technical Field
The invention relates to the technical field of tumor immunity, in particular to a double-target chimeric antigen receptor, a nucleic acid molecule, a carrier, cells and a pharmaceutical composition.
Background
Substantial progress has been made in the areas of gene therapy, cell biology and cancer target recognition over the past few years, making it possible to genetically modify T cells to have tumor antigen specificity and greatly expanding their range of applications by quality control of cellular products. T cells genetically modified with chimeric antigen receptor (chimeric antigen receptor abbreviated CAR) are referred to as CAR-T cells. CAR-T cell therapy is an innovative, revolutionary cancer treatment immunotherapy that relies on the ability of transgenic T cells to continually expand in vivo and ultimately destroy cancer cells.
Most of the existing CAR structures use cd3ζ as a T cell activating domain, and there is a paper indicating that T Cell Receptors (TCRs) mediate antigen-induced signaling through their associated cd3ε, δ, γ and ζ, but the effects of different CD3 chains are different. Due to the selectivity of Lck kinases, a subset of CD3 epsilon ITAMs are mono-phosphorylated and specifically recruit inhibitory Csk kinases to attenuate TCR signaling, suggesting that TCR is a self-constraining signaling mechanism comprising activating and inhibitory motifs. Furthermore, it was found that the incorporation of the CD3 epsilon cytoplasmic domain into the second generation Chimeric Antigen Receptor (CAR) can increase the anti-tumor activity of CAR-T cells. Mechanistically, csk recruitment of CD3 epsilon to ITAM reduced CAR-T cytokine production, while BRS sequence of CD3 epsilon promoted persistence of CAR-T through p85 recruitment. Overall, CD3 epsilon is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next generation CARs. Therefore, replacing the traditional transmembrane and intracellular structure with a simple CD3 epsilon chain as the transmembrane and signaling domain may be a good choice.
On the other hand, tumors can still develop, metastasize and recur under the action of human immune function, which indicates that certain tumors have the ability of evading the immune surveillance of the organism. Thus, it can be considered that: tumor cells can escape from monitoring, recognizing and attacking the immune system of the organism to continue dividing and growing by means of such approaches as modification of self surface antigens, recruitment of inhibitory immune cells and molecules, change of microenvironment around tumor tissues, and the like, which is the immune escape of tumors.
In view of the above, the immune escape of tumor antigens is prevented, a double-targeting chimeric antigen receptor is constructed, the significance of improving the recognition and killing ability of the CAR-T to tumor cells is great, and the targeting and clearing effect of the CAR-T cells to multiple myeloma cells is improved.
Disclosure of Invention
The technical problem to be solved by the invention is the shortages in the background technology, and provides a double-target chimeric antigen receptor, a nucleic acid molecule and a carrier with a double-target chimeric antigen receptor structure, wherein the double-target chimeric antigen receptor structure can respectively target two different antigen targets, and has wide prospects in the aspects of treating malignant tumors and preventing antigen from escaping.
In order to solve the problems, the invention provides the following technical scheme:
in a first aspect, the present invention provides a dual-target chimeric antigen receptor having a dual-target chimeric antigen receptor structure comprising two chimeric antigen receptors having a single targeting comprising an antigen binding domain, a transmembrane domain, and a signaling domain; the two chimeric antigen receptors are linked by a self-cleaving 2A short peptide.
Preferably, the self-cleaving 2A short peptide in the middle of the two chimeric antigen receptors can perform cleavage treatment independently of protease, so that the two chimeric antigen receptors are separated in structure and independently function.
Preferably, the self-cleaving 2A short peptide is P2A or T2A.
Preferably, the antigen binding domain is an scFv structure or a VHH structure directed against any target.
Compared with the common single-target chimeric antigen receptor, the double-target chimeric antigen receptor can effectively avoid the occurrence of antigen escape phenomenon and obviously kill tumor cells.
Preferably, the tumor cells comprise B cell tumors.
Preferably, the target is a tumor cell surface antigen selected from the group consisting of CD19, CD22, BCMA.
In a further technical scheme, at least one chimeric antigen receptor in the double-target chimeric antigen receptor contains a CD3 epsilon chain as a transmembrane domain and a signal transduction domain.
Preferably, the CD3 epsilon chain comprises the amino acid sequence shown in SEQ ID NO. 1;
SEQ ID NO:1：
DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI。
preferably, the chimeric antigen receptor further comprises a signal peptide; the signal peptide is selected from GM-CSF signal peptide, CD8 leader, etc.
In a second aspect, the invention provides nucleic acid molecules encoding said dual-target chimeric antigen receptor.
In a third aspect, the invention provides a vector comprising said nucleic acid molecule.
In the present invention, the carrier may be a linear carrier or a cyclic carrier. The vector may be a non-viral vector such as a plasmid, a viral vector, or a vector using a transposon. The vector can contain regulatory sequences such as promoters, terminators and the like, and marker sequences such as drug resistance genes, reporter genes and the like. In addition, the vectors described above may also contain sequences encoding suicide genes, and the number of CAR-T cells in vivo may be controlled by administering a substance that activates the suicide gene, depending on the course of treatment.
The viral vectors may be retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like. In one embodiment of the invention, lentiviral expression vectors are used.
In a fourth aspect, the invention provides a cell comprising said dual-target chimeric antigen receptor, said nucleic acid molecule, or said vector.
In one embodiment of the invention, the cells are human T cells. The T cells can be derived from body fluid such as blood and bone marrow, or from tissue such as spleen, thymus, lymph, or cancer tissue such as primary tumor, metastatic tumor, cancerous ascites, etc., and can be obtained by separation and purification. Meanwhile, the T cells may be CD4 + T cells, CD8 + T cells, αβ T cells, or γδ T cells. The T cells can be replaced in a suitable manner by NK cells, which are also considered to compriseWithin the scope of the invention.
In a fifth aspect, the invention provides a pharmaceutical composition comprising said dual-target chimeric antigen receptor, said nucleic acid molecule, said vector, said cell, and a pharmaceutically acceptable carrier.
The pharmaceutical composition provided by the invention can contain any pharmaceutically acceptable additive besides the components, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and a composition, a stabilizer, a surfactant, a preservative, an isotonic agent and the like thereof.
Likewise, the pharmaceutical compositions of the present invention may be used in combination with other suitable anticancer agents.
Furthermore, the invention provides application of the double-target chimeric antigen receptor in preparation of anti-B cell tumor drugs.
Furthermore, the invention provides application of the nucleic acid molecule in preparing anti-B cell tumor medicines.
Furthermore, the invention provides application of the vector in preparation of anti-B cell tumor drugs.
Furthermore, the invention provides an application of the cell in preparing an anti-B cell tumor drug.
Compared with the prior art, the invention has the following technical effects:
(1) Compared with the common single-target chimeric antigen receptor, the double-target chimeric antigen receptor provided by the invention has stronger recognition capability to tumor cells, has high-efficiency targeting effect to the tumor cells of the corresponding targets, and is beneficial to preventing the occurrence of antigen escape phenomenon;
(2) The double-target chimeric antigen receptor has the advantages of simple structure, safety and effectiveness, and the constructed CAR-T cells have strong killing effect on tumor cells, generate lower cytokine level, and are expected to obviously improve the clinical treatment effect of cancer.
Drawings
FIG. 1 is a schematic structural diagram of a dual-target chimeric antigen receptor structure provided by the invention;
FIG. 2 is a block diagram of a dual-target chimeric antigen receptor 19-BCMA-CAR according to an embodiment of the present invention;
FIG. 3 is a block diagram of a dual-target chimeric antigen receptor 19-22-CAR according to another embodiment of the present invention;
FIG. 4 is a diagram of the 19-CAR structure of the present invention provided as a comparison;
FIG. 5 is a diagram of the BCMA-CAR structure of the present invention provided by way of comparison;
FIG. 6 is a diagram of a 22-CAR structure of the present invention provided by way of comparison;
FIG. 7 shows the in vitro killing results of the dual-target chimeric antigen receptor 19-22-CAR-T provided by an embodiment of the present invention;
FIG. 8 is an in vitro killing result of the dual-target chimeric antigen receptor 19-BCMA-CAR-T provided by an embodiment of the invention;
FIG. 9 shows cytokine levels after killing of the dual-target chimeric antigen receptor 19-22-CAR-T provided by an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments. It will be apparent that the embodiments described below are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Specific techniques or conditions are not described in detail in the examples and are not to be construed as being limiting in accordance with the general techniques or conditions in the art or are in accordance with the product specifications. The reagents or equipment used are conventional products used daily in the field without the manufacturer's attention.
The embodiment of the invention provides a double-target chimeric antigen receptor, which comprises two chimeric antigen receptors with independent targeting and containing an antigen binding domain, a transmembrane domain and a signal transduction domain; the two chimeric antigen receptors are connected by a self-cleaving 2A short peptide; the antigen binding domain is an scFv structure or a VHH structure aiming at any target point; and at least one of said chimeric antigen receptors comprises a CD3 epsilon chain as a transmembrane domain and a signaling domain. The specific structure diagram is shown in figure 1.
EXAMPLE 1 construction of CAR molecule vector
The embodiment constructs a double-target chimeric antigen receptor 19-BCMA-CAR of anti-CD 19 and anti-BCMA and a double-target chimeric antigen receptor 19-22-CAR of anti-CD 19 and anti-CD 22, and the structure schematic diagram is shown in figures 2-3;
the embodiment simultaneously constructs an anti-CD 19 single-target chimeric antigen receptor 19-CAR, an anti-BCMA single-target chimeric antigen receptor BCMA-CAR and an anti-CD 22 single-target chimeric antigen receptor 22-CAR as a control group, and the structure schematic diagrams are shown in figures 4-6;
the sequencing company is entrusted to synthesize corresponding 5 CAR sequences, a lentiviral vector pLTR-CMV-MCS is inserted, and plasmids and bacterial solutions are sent back after transformation and sequencing are completed.
EXAMPLE 2 plasmid Mass amplification
Adding the corresponding bacterial liquid into a 2 xTY culture medium, placing a culture system shown in table 1 into a shaking table, and culturing at 37 ℃ at 225r/min for 18h;
TABLE 1
Component (A) The amount (/ L)
Tryptone 16.0g
Yeast extract 10.0g
Sodium chloride 5.0g
Extracting plasmid with endotoxin-free plasmid extraction kit after culturing, sterilizing with 0.22 μm filter, packaging, and storing at-20deg.C.
EXAMPLE 3 lentiviral packaging
This example uses the plasmid constructed in example 1 for lentiviral packaging using a three plasmid system, the steps are as follows:
helper plasmids pCMV-dR8.91 and pCMV-VSV-G are mixed with lentiviral vectors in proportion and added to serum-free DMEM of 1/20 volume of 293T culture system;
adding PEI 3 times of the total mass of the plasmid into serum-free DMEM with the same volume as that of the previous step, then dripping PEI solution into the plasmid solution, uniformly mixing and standing for 20-30min;
adding the above mixture into a cell culture flask paved with 293T cells, gently mixing, and heating at 37deg.C with 5% CO 2 Culturing in a cell incubator for 4-6h; after the completion, changing a fresh culture medium, continuing to culture, and adding 10mM sodium butyrate solution; the lentiviral culture supernatants were collected twice after 48h and 72h for concentration, purification and detection.
Example 4T cell activation and lentiviral infection
Separating Peripheral Blood Mononuclear Cells (PBMC) from whole blood by adopting a density gradient centrifugation method, and separating T cells by utilizing MACS CD3 magnetic beads;
the sorted T cells were diluted to a cell concentration of 2X 10 with medium (AIM-V medium+10% FBS) 6 individual/mL for use;
activating T cells by adopting MACS TransAct CD3/CD 28T cell activating and amplifying kit, adding magnetic beads with culture volume of 1/100, and obtaining final density of T cells of 2×10 6 After mixing, the mixture was placed at 37℃and 5% CO 2 Culturing and stimulating for 72 hours in an incubator;
after 72h of T cell activation, the beads were removed, centrifuged at 300g for 5min, the supernatant was removed, T cells were resuspended in fresh medium, recombinant lentivirus expressing CAR (moi=5) was added separately, and 8 μg was addedPer mL of polybrene and 300IU/mL of IL-2, at 37℃in 5% CO 2 Culturing in an incubator;
after 24h, 300g is centrifuged for 5min, the supernatant is removed, and the infection is repeated once; re-suspending the T cells by using a fresh culture medium containing 300IU/mL IL-2 to obtain CAR-T cells;
after 48h, the T cells were resuspended in fresh medium containing 300IU/mL IL-2 to maintain the CAR-T cell density at 1X 10 6 About one half of the liquid is changed every 2 to 3 days, and the liquid can be used for killing experiments and other detection after the liquid is sufficiently changed.
Example 5 in vitro detection of the killing function of 19-22-CAR-T cells on target cells
The constructed 3M-CD19-Luc cells, 3M-CD22-Luc cells and 3M-CD19-CD22-Luc cells were plated in 96-well plates 10000 cells/well one day in advance;
the following day, T cells, 19-CAR-T, 22-CAR-T and 19-22-CAR-T prepared in example 4 were mixed with tumor cells of the corresponding targets in the ratios of E: T of 10:1, 5:1, 2.5:1, and added to 96-well plates, 6 duplicate wells were set in each group, and placed at 37℃and 5% CO 2 Co-culturing in an incubator for 18-24 hours;
after 18-24h, the supernatant was aspirated for later use, 100. Mu.L/well was added with D-PBS, 50. Mu.L/well of lysate was added to lyse the cells, 50. Mu.L/well was taken out and mixed with Luciferase substrate (1X) uniformly in a new full black 96-well plate, the luminescence intensity was measured by a multifunctional microplate reader after 30min, the killing efficiency was quantitatively evaluated by Luciferase (Luciferase) of the remaining tumor cells after killing, and the killing effect of T cells, 19-CAR-T, 22-CAR-T, and 19-22-CAR-T on the corresponding target tumor cells was compared in vitro, and the killing ratio was calculated as follows:
100% × (target cell group reading-effector cell group reading)/target cell group reading
The results are shown in figure 7, where all CAR-T groups were significantly more efficient in vitro than T cells, and 19-22-CAR-T groups were significantly killed.
Example 6 in vitro detection of the killing function of 19-BCMA-CAR-T cells on target cells
Spreading the constructed 3M-CD19-Luc cells, 3M-BCMA-Luc cells and 3M-CD19-BCMA-Luc cells into 96-well plates 10000 cells/well one day in advance;
the following day, T cells, 19-CAR-T, BCMA-CAR-T and 19-BCMA-CAR-T prepared in example 4 were mixed with tumor cells of the respective targets in the ratios of E: T10:1, 5:1, 2.5:1, respectively, added to 96-well plates, each set was provided with 6 duplicate wells, and placed at 37 ℃, 5% co 2 Co-culturing in an incubator for 18-24 hours;
sucking out the supernatant for standby after 18-24h, adding D-PBS into 100 mu L/hole, adding 50 mu L/hole lysate to lyse cells, taking out 50 mu L/hole and Luciferase substrate (1X) to mix uniformly in a new full black 96-well plate, measuring the luminous intensity by a multifunctional microplate reader after 30min, quantitatively evaluating the killing efficiency by utilizing Luciferase (Luciferase) of the remained tumor cells after killing, and comparing the killing effect of T cells, 19-CAR-T, BCMA-CAR-T and 19-BCMA-CAR-T on the corresponding target tumor cells in vitro, wherein the killing proportion is calculated by the following formula:
100% × (target cell group reading-effector cell group reading)/target cell group reading
The results are shown in figure 8, where all CAR-T groups were significantly more efficient in vitro than T cells, and 19-BCMA-CAR-T groups were significantly killed.
EXAMPLE 7 killer cytokine level detection
Detecting the concentration of TNF-a, IFN-y and IL-2 by ELISA detection kit from the culture supernatant after killing in example 5;
the results are shown in figure 9, with the T cell group as a reference, the levels of cytokines were significantly reduced after killing of the 19-22-CAR-T group compared to the 19-CAR-T and 22-CAR-T groups, especially compared to the 22-CAR-T group.
In conclusion, the novel double-target chimeric antigen receptor structure constructed by the invention is feasible and effective, has a strong killing effect on tumor cells of corresponding targets, simultaneously generates lower cytokine level, and is expected to obviously improve the clinical treatment effect of cancer.
In the foregoing embodiments, the descriptions of the embodiments are focused on, and for those portions of one embodiment that are not described in detail, reference may be made to the related descriptions of other embodiments.
While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.
Sequence listing
<110> Northc Biotechnology Co., ltd in Shenzhen City
<120> a double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition
<130> 1
<141> 2021-09-29
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 185
<212> PRT
<213> Artificial Sequence
<400> 1
Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val
1 5 10 15
Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly
20 25 30
Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu
35 40 45
Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu
50 55 60
Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly
65 70 75 80
Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val
85 90 95
Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile Val
100 105 110
Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr Tyr
115 120 125
Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala
130 135 140
Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro
145 150 155 160
Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu
165 170 175
Tyr Ser Gly Leu Asn Gln Arg Arg Ile
180 185

Claims (7)

1. A dual-target chimeric antigen receptor, comprising two chimeric antigen receptors having an antigen binding domain, a transmembrane domain, and a signaling domain that are individually targeted; the two chimeric antigen receptors are connected by a self-cleaving 2A short peptide; the antigen binding domain is an scFv structure against CD19, CD22 or BCMA; namely, the structure of the double-target chimeric antigen receptor is CD19scFv-TM-ICOS-CD3 zeta-2A-BCMAScFv-CD 3 epsilon or CD19scFv-TM-ICOS-CD3 zeta-2A-CD 22scFv-CD3 epsilon;
the protein sequence of the CD3 epsilon chain is as follows:
DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI。
2. the dual-target chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor further comprises a signal peptide; the signal peptide is selected from GM-CSF signal peptide and CD8 leader.
3. The dual-target chimeric antigen receptor of claim 1, wherein the self-cleaving 2A short peptide is P2A or T2A.
4. A nucleic acid molecule encoding the dual-target chimeric antigen receptor of any one of claims 1-3.
5. A vector comprising the nucleic acid molecule of claim 4.
6. A cell comprising the dual-target chimeric antigen receptor of any one of claims 1-3, the nucleic acid molecule of claim 4, or the vector of claim 5; the cells are T cells or NK cells.
7. A pharmaceutical composition comprising the dual-target chimeric antigen receptor of any one of claims 1-3, the nucleic acid molecule of claim 4, the vector of claim 5 or the cell of claim 6, and a pharmaceutically acceptable carrier.
CN202111168331.2A 2021-09-29 2021-09-29 Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition Active CN113773402B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111168331.2A CN113773402B (en) 2021-09-29 2021-09-29 Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111168331.2A CN113773402B (en) 2021-09-29 2021-09-29 Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition

Publications (2)

Publication Number Publication Date
CN113773402A CN113773402A (en) 2021-12-10
CN113773402B true CN113773402B (en) 2024-03-19

Family

ID=78854707

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111168331.2A Active CN113773402B (en) 2021-09-29 2021-09-29 Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition

Country Status (1)

Country Link
CN (1) CN113773402B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114560949B (en) * 2022-03-07 2023-09-26 中国人民解放军空军军医大学 A chimeric antigen receptor and D-CAR-T cell that enhances the anti-tumor ability of CAR-T cells and their applications
CN114573712B (en) * 2022-03-07 2023-05-12 中国人民解放军空军军医大学 Chimeric antigen receptor, CAR-T cell and application thereof
CN114560948B (en) * 2022-03-07 2023-05-23 中国人民解放军空军军医大学 A kind of chimeric antigen receptor, CAR-T cell and application thereof
CN118843693A (en) * 2022-04-03 2024-10-25 上海先博生物科技有限公司 Chimeric antigen receptor targeting BCMA and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111848820A (en) * 2020-07-31 2020-10-30 广东昭泰体内生物医药科技有限公司 CD19 and BCMA double-target chimeric antigen receptor and application thereof
CN112195157A (en) * 2020-10-12 2021-01-08 广东昭泰体内生物医药科技有限公司 CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof
CN112979820A (en) * 2019-12-17 2021-06-18 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) CD 123-targeted chimeric antigen receptor and double-target chimeric antigen receptor containing CD 123-targeted chimeric antigen receptor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112979820A (en) * 2019-12-17 2021-06-18 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) CD 123-targeted chimeric antigen receptor and double-target chimeric antigen receptor containing CD 123-targeted chimeric antigen receptor
CN111848820A (en) * 2020-07-31 2020-10-30 广东昭泰体内生物医药科技有限公司 CD19 and BCMA double-target chimeric antigen receptor and application thereof
CN112195157A (en) * 2020-10-12 2021-01-08 广东昭泰体内生物医药科技有限公司 CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof

Also Published As

Publication number Publication date
CN113773402A (en) 2021-12-10

Similar Documents

Publication Publication Date Title
CN113773402B (en) Double-target chimeric antigen receptor, nucleic acid molecule, vector, cell and pharmaceutical composition
CN111094345B (en) Anti-B cell maturation antigen chimeric antigen receptor with human domain
EP3599251A1 (en) Construction of chimeric antigen receptor targeting cd20 antigen and activity identification of engineered t cells thereof
CN111511911B (en) Immunocompetent cells expressing cell surface molecules, IL-7 and CCL19 that specifically recognize human mesothelin
CN110627909B (en) Chimeric antigen receptor for specifically activating NK cells and application thereof
CN108409840A (en) The Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and application
CN113583139A (en) Chimeric receptor and application thereof
CN113416260B (en) Claudin18.2-targeted specific chimeric antigen receptor cell and preparation method and application thereof
CN115838439A (en) Preparation method and application of chimeric transition receptor gene modified NK cell
CN107624119A (en) A kind of molecule, the cell for expressing it and its production and use
CN110423767A (en) Express Chimeric antigen receptor CAR gene and the application of solubility PD-1
AU2020242520B2 (en) Method for the expansion and differentiation of T lymphocytes and NK cells in adoptive transfer therapies
CN110054698B (en) Construction and application of novel CD19-CAR vector of anti-CD 19 antibody
CN110903401A (en) Second-generation chimeric antigen receptor targeting CD19, and expression vector and application thereof
CN108392492B (en) Application of LDLR overexpression in NK cell adoptive therapy
CN117247466B (en) Chimeric antigen receptor against glypican 3 and its use
CN111499766B (en) Immune effector cell aiming at chronic lymphocytic leukemia, preparation method and application thereof
CN113528452A (en) Immune cells co-expressing IL-21 and hrCD16 chimeric receptors and their applications
CN111956795A (en) Application of chimeric antigen receptor combined anti-tumor drug taking CD99 as target
CN113980133B (en) Antibody and application thereof in anti-tumor
CN113956359B (en) Antibody and application thereof in anti-tumor
CN109280087A (en) A dual-targeting switch receptor, encoding gene, recombinant expression vector, anti-tumor NK cell and preparation method and application thereof
CN109096404A (en) A kind of chimeric costimulation switch acceptor, encoding gene, recombinant expression carrier, antitumor NK cell and preparation method thereof, application
CN116254230A (en) Method for preparing and amplifying universal humanized anti-CD 19CAR-NK cells and uses thereof
CN115925974A (en) Preparation method of universal IPS (in-plane switching) -derived CAR-NK (CAR-NK cell) cell for solid tumor

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant