WO2022011846A1 - Récepteur antigénique chimérique ciblé par cd19- et cd22- et utilisation associée - Google Patents

Récepteur antigénique chimérique ciblé par cd19- et cd22- et utilisation associée Download PDF

Info

Publication number
WO2022011846A1
WO2022011846A1 PCT/CN2020/119213 CN2020119213W WO2022011846A1 WO 2022011846 A1 WO2022011846 A1 WO 2022011846A1 CN 2020119213 W CN2020119213 W CN 2020119213W WO 2022011846 A1 WO2022011846 A1 WO 2022011846A1
Authority
WO
WIPO (PCT)
Prior art keywords
variable region
chain variable
chain antibody
antibody
series
Prior art date
Application number
PCT/CN2020/119213
Other languages
English (en)
Chinese (zh)
Inventor
李鹏
秦乐
汤朝阳
邓殷建
魏志辉
Original Assignee
广东昭泰体内生物医药科技有限公司
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 广东昭泰体内生物医药科技有限公司 filed Critical 广东昭泰体内生物医药科技有限公司
Publication of WO2022011846A1 publication Critical patent/WO2022011846A1/fr

Links

Images

Classifications

    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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

Definitions

  • the present application belongs to the technical field of biomedicine, and relates to CD19 and CD22 dual-target chimeric antigen receptors and applications thereof.
  • a chimeric antigen receptor consists of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signal transduction region.
  • the single chain fragment variable (scFv) of the antibody contained in the CAR molecule has a specific binding effect on the tumor associated antigen (TAA), which is bound to the signaling molecule through the hinge and the transmembrane region. Cytoplasmic domain coupling.
  • CAR-T chimeric antigen receptor T-cell immunotherapy
  • CAR-T cell therapy has been widely used in the treatment of B-cell malignancies.
  • CAR-T cells targeting CD19 are the pioneers of CAR-T therapy for the treatment of B-cell malignancies, providing an effective solution for the treatment of B-cell malignancies.
  • medical diagnosis has shown that tumor cells in some blood tumors do not express CD19 molecules, but express CD22 molecules.
  • the treatment effect of only CAR-T cells targeting CD19 molecules is not effective, and some patients have tumor recurrence after a period of time. Phenomenon.
  • the present application provides CD19 and CD22 dual-target chimeric antigen receptors and applications thereof.
  • the chimeric antigen receptors can simultaneously target CD19 and CD22 molecules and have broad prospects in the treatment of hematological malignancies.
  • the present application provides a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and a signal transduction domain;
  • the antigen-binding domain includes the light chain variable region of anti-CD19 single-chain antibody, the heavy chain variable region of anti-CD19 single-chain antibody, the light chain variable region of anti-CD22 single-chain antibody, and the heavy chain variable region of anti-CD22 single-chain antibody. chain variable region.
  • anti-CD19 and CD22 dual-target chimeric antigen receptors have stronger targeting activity to CD19-positive and/or CD22-positive cells, and have stronger targeting activity to CD19 antigen expression levels.
  • Tumor cells with little or no expression, and tumor cells with little or no expression of CD22 antigen have efficient targeting, which is beneficial to avoid immune escape.
  • the antigen binding domain comprises a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are connected in series in sequence.
  • Heavy chain variable region of CD22 single chain antibody is a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are connected in series in sequence.
  • Heavy chain variable region of CD22 single chain antibody is a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are connected in series in sequence.
  • Heavy chain variable region of CD22 single chain antibody
  • the antigen binding domain comprises a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody in series.
  • Light chain variable region of CD22 single chain antibody is a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody in series.
  • the antigen binding domain comprises a light chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody, which are serially connected in series.
  • Heavy chain variable region of CD22 single chain antibody is preferred.
  • the antigen binding domain comprises a light chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody in series in series Heavy chain variable region of CD19 single chain antibody.
  • the antigen binding domain comprises a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series in series Light chain variable region of CD22 single chain antibody.
  • the antigen binding domain comprises a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • Heavy chain variable region of CD19 single chain antibody is preferred.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • Heavy chain variable region of CD22 single chain antibody is a heavy chain variable region of CD22 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • Light chain variable region of CD22 single chain antibody is a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • the antigen-binding domain comprises a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody, which are serially connected in series.
  • Heavy chain variable region of CD22 single chain antibody is a heavy chain variable region of CD22 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • Light chain variable region of CD19 single chain antibody is a heavy chain variable region of an anti-CD19 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series in series Light chain variable region of CD22 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody in series in series Light chain variable region of CD19 single chain antibody.
  • the antigen binding domain comprises a light chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series.
  • Heavy chain variable region of CD22 single chain antibody is a light chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series.
  • Heavy chain variable region of CD22 single chain antibody is a light chain variable region of an anti-CD22 single-chain antibody.
  • the antigen binding domain comprises a light chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • Heavy chain variable region of CD19 single chain antibody is preferred.
  • the antigen binding domain comprises a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody, which are connected in series in sequence.
  • Heavy chain variable region of CD22 single chain antibody is a light chain variable region of an anti-CD22 single-chain antibody.
  • the antigen binding domain comprises a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • Light chain variable region of CD19 single chain antibody is a light chain variable region of an anti-CD22 single-chain antibody.
  • the antigen binding domain comprises a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series in series Heavy chain variable region of CD19 single chain antibody.
  • the antigen binding domain comprises a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series in series Light chain variable region of CD19 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series.
  • Light chain variable region of CD22 single chain antibody is a heavy chain variable region of an anti-CD22 single-chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody, which are serially connected in series.
  • Heavy chain variable region of CD19 single chain antibody is preferred.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series in series Light chain variable region of CD22 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, and an anti-CD22 single-chain antibody in series in series Light chain variable region of CD19 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series.
  • Heavy chain variable region of CD19 single chain antibody is a heavy chain variable region of CD19 single chain antibody.
  • the antigen binding domain comprises a heavy chain variable region of an anti-CD22 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody, a heavy chain variable region of an anti-CD19 single-chain antibody, and an anti-CD19 single-chain antibody in series in series Light chain variable region of CD19 single chain antibody.
  • the light chain variable region of the anti-CD19 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 1;
  • the heavy chain variable region of the anti-CD19 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 2;
  • the light chain variable region of the anti-CD22 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 3;
  • the heavy chain variable region of the anti-CD22 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 4;
  • the light chain variable region of the anti-CD19 single-chain antibody, the heavy chain variable region of the anti-CD19 single-chain antibody, the light chain variable region of the anti-CD22 single-chain antibody, and the heavy chain of the anti-CD22 single-chain antibody can be The variable regions are linked by linker peptides.
  • the connecting peptide comprises the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7;
  • SEQ ID NO: 5 GSGGGGSGGGGSGGGGS;
  • SEQ ID NO: 6 GGGGSGGGGSGGGGSGGGGS;
  • SEQ ID NO: 7 GTSSGSGKPGSGEGSTKG.
  • the transmembrane domain comprises CD28 and/or CD8 ⁇ .
  • the signal transduction domain comprises any one or a combination of at least two of CD3 ⁇ , 4-1BB, CD28, TLR1, TLR2, CD27, OX40 or DAP10.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide comprises a GM-CSF signal peptide.
  • the GM-CSF signal peptide comprises the amino acid sequence shown in SEQ ID NO: 8;
  • SEQ ID NO: 8 MLLLVTSLLLCELPHPAFLL.
  • the chimeric antigen receptor is composed of GM-CSF signal peptide, light chain variable region of anti-CD19 single chain antibody, first linking peptide, heavy chain variable region of anti-CD19 single chain antibody, second linking peptide , the heavy chain variable region of the anti-CD22 single-chain antibody, the third linking peptide, the light chain variable region of the anti-CD22 single-chain antibody, CD28 and CD3 ⁇ in series.
  • the present application provides an encoding gene encoding the chimeric antigen receptor of the first aspect.
  • the coding genes include the coding sequence of the light chain variable region of anti-CD19 single-chain antibody, the coding sequence of the heavy chain variable region of anti-CD19 single-chain antibody, the coding sequence of the heavy chain variable region of anti-CD22 single-chain antibody, and Light chain variable region coding sequence of anti-CD22 single chain antibody.
  • the coding gene further comprises a linker peptide coding sequence.
  • the coding gene further comprises a GM-CSF signal peptide coding sequence, a CD28 coding sequence and a CD3 ⁇ coding sequence.
  • the light chain variable region coding sequence of the anti-CD19 single-chain antibody comprises the nucleic acid sequence shown in SEQ ID NO: 9;
  • the heavy chain variable region coding sequence of the anti-CD19 single-chain antibody comprises the nucleic acid sequence shown in SEQ ID NO: 10;
  • the heavy chain variable region coding sequence of the anti-CD22 single-chain antibody comprises the nucleic acid sequence shown in SEQ ID NO: 11;
  • the light chain variable region coding sequence of the anti-CD22 single-chain antibody comprises the nucleic acid sequence shown in SEQ ID NO: 12;
  • the connecting peptide coding sequence comprises the nucleic acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15;
  • the GM-CSF signal peptide coding sequence comprises the nucleic acid sequence shown in SEQ ID NO: 16;
  • the present application provides an expression vector, the expression vector comprising the encoding gene described in the second aspect.
  • the expression vector is a viral vector containing the encoding gene described in the second aspect.
  • the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated virus vector containing the encoding gene described in the second aspect.
  • the present application provides a recombinant lentivirus, wherein the recombinant lentivirus is a mammalian cell transfected with the expression vector and the helper plasmid described in the third aspect.
  • the application provides a CAR-T cell expressing the chimeric antigen receptor of the first aspect.
  • T cells expressing anti-CD19 and CD22 dual-target chimeric antigen receptors have high targeting activity and killing efficacy against CD19-positive and/or CD22-positive cells, and can be used against tumors with little or no expression of CD19 antigen.
  • Cells and tumor cells with little or no expression of CD22 antigen have a killing effect, which is beneficial to avoid immune escape and reduce the possibility of disease recurrence.
  • the encoding gene of the second aspect is integrated into the genome of the CAR-T cell.
  • the CAR-T cells comprise the expression vector described in the third aspect and/or the recombinant lentivirus described in the fourth aspect.
  • the present application provides a method for preparing a CAR-T cell according to the fifth aspect, the method comprising the step of introducing the gene encoding the chimeric antigen receptor according to the first aspect into the T cell.
  • the present application provides the chimeric antigen receptor described in the first aspect, the encoding gene described in the second aspect, the expression vector described in the third aspect, the recombinant lentivirus described in the fourth aspect or The application of the CAR-T cell described in the fifth aspect in the preparation of a disease treatment drug.
  • the disease comprises a hematological neoplasm.
  • the disease comprises CD19 positive and/or CD22 positive disease.
  • the anti-CD19 and CD22 dual-target chimeric antigen receptors constructed in this application have stronger targeting activity to CD19-positive and/or CD22-positive cells, and have stronger targeting activity to CD19-positive and/or CD22-positive cells.
  • Tumor cells with little or no antigen expression, and tumor cells with little or no expression of CD22 antigen have efficient targeting, which is beneficial to avoid immune escape phenomenon;
  • T cells expressing anti-CD19 and CD22 dual-target chimeric antigen receptors in the present application have efficient targeting activity and killing effect on CD19-positive and/or CD22-positive cells.
  • Tumor cells and tumor cells with little or no expression of CD22 antigen have a killing effect, which is beneficial to avoid the phenomenon of immune escape and reduce the possibility of disease recurrence.
  • Fig. 1 is the lentiviral vector map containing the encoding gene of CAR targeting CD19 and CD22 dual targets;
  • Figure 2A shows the flow cytometry results of WT and 2228zT2
  • Figure 2B shows the flow cytometry results of 1928zT2, 31A2 and 11H8.22.28.zGFP;
  • Figure 3 shows the killing efficiency of WT, 2228zT2 and 11H8.22.28.zGFP on tumor cells K52-CD22-GL at different E:T ratios;
  • Figure 4 shows the killing efficiency of WT, 31A2, 1928zT2 and 11H8.22.28.zGFP on tumor cells K52-CD19-GL at different E:T ratios.
  • the coding gene for anti-CD19 and CD22 dual-target chimeric antigen receptors was firstly synthesized, and the restriction endonuclease Pme1 restriction site and its protective bases were added to the C-terminal and N-terminal of the coding gene, respectively. Restriction endonuclease Spe1 restriction site and its protective base;
  • the coding gene was double digested with restriction enzymes Pme1 and Spe1, and the digested product containing sticky ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing the same double digested by Pme1 and Spe1).
  • the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent Company) to obtain a lentiviral vector containing the encoding gene of the CAR targeting CD19 and CD22 dual targets.
  • T4 DNA polymerase Invitrogent Company
  • antigen-binding domains of anti-CD19 scFv CAR (antiCD19 scFv-CD28-CD3 ⁇ ) and anti-CD22 scFv CAR (antiCD22 scFv-CD28-CD3 ⁇ ) were constructed simultaneously, and corresponding lentiviral vectors were constructed.
  • 293T cells were used to prepare recombinant lentiviruses.
  • lentivirus packaging was performed:
  • the medium was replaced with DMEM containing 1% fetal bovine serum, and the addition amount was 6mL/100mm culture dish;
  • the plasmid mixture shown in Table 1 was prepared.
  • the pWPXLd-expression plasmid includes a lentiviral vector containing the coding gene of CAR targeting CD19 and CD22 dual targets, and lentivirus containing the coding gene of CAR targeting CD19 single target.
  • Vector, lentiviral vector containing the coding gene of CAR targeting CD22 single target, pWPXLd-eGFP plasmid is an empty vector without CAR coding gene;
  • the medium was replaced with DMEM containing 1% fetal bovine serum, and the addition amount was 7mL/100mm culture dish;
  • the virus supernatant was collected, and the medium was added to the 293T cells at the same time, and the addition amount was 7mL/100mm culture dish;
  • PBMCs Peripheral blood mononuclear cells
  • GE Company Ficoll density gradient centrifugation kit
  • T cells were sorted by MACS Pan-T magnetic beads;
  • the sorted T cells were diluted to a cell concentration of 2.5 ⁇ 10 6 cells/mL with medium (AIM-V medium + 5% FBS + penicillin 100 U/mL + streptomycin 0.1 mg/mL) for use;
  • medium AIM-V medium + 5% FBS + penicillin 100 U/mL + streptomycin 0.1 mg/mL
  • the CD2/CD3/CD28 T cell activation and expansion kit (Miltenyi) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells in a ratio of 1:2, and the final density of T cells was 5 ⁇ 10 6 cells/mL /cm 2 , after mixing, placed in a 37°C, 5% CO 2 incubator for stimulation for 48 hours;
  • the CAR-T cell density was maintained at about 1 ⁇ 10 6 cells/mL, and the medium was changed in half every 2 to 3 days. After two weeks, the number of CAR-T cells expanded 100-fold.
  • the CAR-T cells constructed in this example are 11H8.22.28.zGFP (expressing anti-CD19 and CD22 dual-target CAR), 31A2 (expressing anti-human CD19 single-target CAR), 1928zT2 (expressing anti-mouse CD19 single-target CAR) CAR), 2228zT2 (expressing anti-mouse CD22 single-target CAR), and a WT control group (transfecting blank control eGFP lentivirus).
  • the lentiviral transfection conditions are shown in Table 1.
  • the expression of CAR molecule on T cells can be indicated by eGFP.
  • the flow cytometer ACEA Novocyte is used to detect eGFP on T cells.
  • Figure 2A and Figure 2B are the flow cytometry results of the experimental group and different control groups. It can be seen that the transfection efficiency of lentivirus to WT cells was 0.72%, the transfection efficiency of 2228zT2 was 3.71%, and the transfection efficiency of 1928zT2 was 0.72%. The transfection efficiency was 18.5%, 7.77% for 31A2 and 9.79% for 11H8.22.28.zGFP.
  • WT, 2228zT2 and 11H8.22.28.zGFP prepared in Example 3 were respectively mixed with 1 ⁇ 10 4 tumor cells K52-CD22-GL according to E:T ratio of 4:1, 2:1, 1:1, 1:2, The ratios of 1:4, 1:8, and 1:16 were mixed and added to a 96-well plate. Each group was set up with 3 duplicate wells. After centrifugation at 250g for 5 min, it was placed in a 37°C, 5% CO 2 incubator for co-cultivation for 18 hours;
  • WT, 31A2, 1928zT2 and 11H8.22.28.zGFP prepared in Example 3 were respectively mixed with 1 ⁇ 10 4 tumor cells K52-CD19-GL according to E:T ratio of 8:1, 4:1, 2:1, 1: 1,1: 2,1: 4,1: 8 ratio mixed, added to a 96-well plate, each group of three holes, 250g centrifugation after 5min, placed in 37 °C, 5% cO 2 incubator coculture 18h;
  • luciferase substrate 1 ⁇ was added to the 96-well plate, the cells were resuspended, and RLU (relative light unit) was measured immediately by a multi-plate reader for 1 second.
  • RLU relative light unit
  • the anti-CD19 and CD22 dual-target chimeric antigen receptors constructed in this application have targeting activity to CD19-positive and/or CD22-positive cells, and T cells expressing anti-CD19 and CD22 dual-target chimeric antigen receptors.
  • Cells have a killing effect on tumor cells with little or no expression of CD19 antigen and tumor cells with little or no expression of CD22 antigen, which is beneficial to avoid immune escape and reduce the possibility of disease recurrence.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

Landscapes

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

Abstract

La présente invention concerne un récepteur antigénique chimérique ciblé par CD19 et CD22 et une utilisation associée.. .Le récepteur antigénique chimérique comprend un domaine de liaison à l'antigène, un domaine transmembranaire et un domaine de transduction de signal ; le domaine de liaison à l'antigène comprend une région variable de chaîne légère d'un anticorps à chaîne unique anti-CD19, une région variable de chaîne lourde d'un anticorps à chaîne unique anti-CD19, une région variable de chaîne légère d'un anticorps à chaîne unique anti-CD22, et une région variable de chaîne lourde d'un anticorps à chaîne unique anti-CD22. Le récepteur antigénique chimérique ciblé par CD19 et CD22 de la présente invention présente une activité de ciblage d'une cellule CD19-positive et/ou CD22-positive, et un lymphocyte T exprimant le récepteur antigénique chimérique ciblé par CD19-et CD22 possède un effet destructeur à la fois sur une cellule tumorale ayant une expression faible ou nulle sur un antigène CD19 et sur une cellule tumorale ayant une expression faible ou nulle sur un antigène CD22, ce qui facilite l'évitement de l'évasion immunitaire et réduit la possibilité de récidive de maladie.
PCT/CN2020/119213 2020-07-16 2020-09-30 Récepteur antigénique chimérique ciblé par cd19- et cd22- et utilisation associée WO2022011846A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010687675.3 2020-07-16
CN202010687675.3A CN111808200B (zh) 2020-07-16 2020-07-16 Cd19和cd22双靶点嵌合抗原受体及其应用

Publications (1)

Publication Number Publication Date
WO2022011846A1 true WO2022011846A1 (fr) 2022-01-20

Family

ID=72866374

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/119213 WO2022011846A1 (fr) 2020-07-16 2020-09-30 Récepteur antigénique chimérique ciblé par cd19- et cd22- et utilisation associée

Country Status (2)

Country Link
CN (1) CN111808200B (fr)
WO (1) WO2022011846A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112521515B (zh) * 2020-12-21 2022-02-15 汤朝阳 Cd19和cd10双靶点嵌合抗原受体及其应用
CN113527518A (zh) * 2021-07-19 2021-10-22 广州百暨基因科技有限公司 靶向cd22和cd19的双特异性嵌合抗原受体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108504668A (zh) * 2018-05-23 2018-09-07 上海恒润达生生物科技有限公司 靶向cd19和cd22嵌合抗原受体及其用途
CN109678965A (zh) * 2018-10-12 2019-04-26 中国人民解放军总医院 嵌合抗原受体及其基因和重组表达载体、cd22-cd19双靶向性的t细胞及其应用
CN109734813A (zh) * 2019-01-28 2019-05-10 广东昭泰体内生物医药科技有限公司 一种嵌合抗原受体及其应用
CN110157738A (zh) * 2018-02-13 2019-08-23 亘喜生物科技（上海）有限公司 靶向cd19和cd22的工程化免疫细胞及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109517799B (zh) * 2018-11-30 2022-07-26 北京美康基免生物科技有限公司 一种基于cd19和cd22的双重嵌合抗原受体基因修饰的免疫细胞及其应用
CN111320703A (zh) * 2020-03-11 2020-06-23 北京双赢科创生物科技有限公司 靶向cd22的嵌合抗原受体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110157738A (zh) * 2018-02-13 2019-08-23 亘喜生物科技（上海）有限公司 靶向cd19和cd22的工程化免疫细胞及其应用
CN108504668A (zh) * 2018-05-23 2018-09-07 上海恒润达生生物科技有限公司 靶向cd19和cd22嵌合抗原受体及其用途
CN109678965A (zh) * 2018-10-12 2019-04-26 中国人民解放军总医院 嵌合抗原受体及其基因和重组表达载体、cd22-cd19双靶向性的t细胞及其应用
CN109734813A (zh) * 2019-01-28 2019-05-10 广东昭泰体内生物医药科技有限公司 一种嵌合抗原受体及其应用

Also Published As

Publication number Publication date
CN111808200A (zh) 2020-10-23
CN111808200B (zh) 2022-05-20

Similar Documents

Publication Publication Date Title
WO2018121712A1 (fr) Nouveau récepteur chimère de l'antigène et utilisation correspondante
CN108409840B (zh) 抗cd123单链抗体及其组合的嵌合抗原受体和应用
CN111748044B (zh) Cd19和pd-l1双靶点嵌合抗原受体及其应用
CN110734931A (zh) 一种靶向CD19的人源化scFv嵌合抗原受体T细胞及制备方法、应用
WO2022011846A1 (fr) Récepteur antigénique chimérique ciblé par cd19- et cd22- et utilisation associée
CN112029729B (zh) Cd19和cd22双靶点嵌合抗原受体nk细胞及其应用
CN112195157B (zh) Cd19和cd22双靶点嵌合抗原受体t细胞及其应用
JP2024045111A (ja) ガイダンス及びナビゲーションコントロール蛋白質の生産及び使用方法
CN111848822B (zh) Cd19和cd30双靶点嵌合抗原受体及其应用
CN113248622B (zh) 一种靶向cll1和nkg2d配体的双靶点嵌合抗原受体及其应用
CN112251412B (zh) 一种靶向bcma的嵌合抗原受体t细胞及其应用
CN111848818A (zh) 一种增强型免疫细胞及其应用
CN111875711A (zh) 一种增强型免疫细胞及其应用
JP7386848B2 (ja) 改良されたレンチウイルスベクター
WO2017219916A1 (fr) Molécule, cellules l'exprimant, son procédé de préparation et ses applications
CN110819596A (zh) 具有增强的迁移能力的修饰的细胞
CN113248621B (zh) Cll1和cd33双靶点嵌合抗原受体及其应用
CN111826353A (zh) 调节t细胞功能和反应的方法
CN112608387B (zh) Cd19和cd20双靶点嵌合抗原受体及其应用
CN111875710A (zh) 异质性肿瘤治疗用免疫细胞及其应用
WO2023016576A1 (fr) Récepteur d'antigène chimérique ciblant le bcma à base d'anticorps monocaténaire entièrement humain et murin et son utilisation
WO2023134718A1 (fr) Récepteur antigénique chimérique ciblant gprc5d et son application
RU2800920C2 (ru) Способы экспандирования антигенспецифических car-t-клеток, композиции и применения, связанные с ними
CN112521515B (zh) Cd19和cd10双靶点嵌合抗原受体及其应用
US20240075061A1 (en) Cell therapy activating lymphocyte in tme

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20945432

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20945432

Country of ref document: EP

Kind code of ref document: A1