EP4284824A2 - Récepteurs antigéniques chimériques ciblant cd20 - Google Patents

Récepteurs antigéniques chimériques ciblant cd20

Info

Publication number
EP4284824A2
EP4284824A2 EP22746521.8A EP22746521A EP4284824A2 EP 4284824 A2 EP4284824 A2 EP 4284824A2 EP 22746521 A EP22746521 A EP 22746521A EP 4284824 A2 EP4284824 A2 EP 4284824A2
Authority
EP
European Patent Office
Prior art keywords
seq
car
copies
amino acid
gdna
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.)
Pending
Application number
EP22746521.8A
Other languages
German (de)
English (en)
Inventor
Yihong Yao
Jiaqi Huang
Xin Yao
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.)
Cellular Biomedicine Group Inc
Original Assignee
Cellular Biomedicine Group Inc
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
Priority claimed from US17/352,915 external-priority patent/US11472858B2/en
Application filed by Cellular Biomedicine Group Inc filed Critical Cellular Biomedicine Group Inc
Publication of EP4284824A2 publication Critical patent/EP4284824A2/fr
Pending legal-status Critical Current

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    • 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
    • A61K39/46—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
    • A61K39/463—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
    • A61K39/464—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
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—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
    • A61K39/46—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
    • A61K39/464424—CD20
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • 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

Definitions

  • Adoptive T cell therapy has shown its powerful efficacy and bright prospect in the clinical treatment of malignant tumors.
  • multiple centers independently using chimeric antigen receptor (CAR)-modified T cells to target recurrent, refractory malignant tumors of CD19-expressed B cell have achieved unprecedented success.
  • CAR chimeric antigen receptor
  • R/R B-ALL refractory acute B-cell lymphoma
  • CD20 is specifically expressed in more than 95% of normal and cancerous B cells. These cells are in the pre-B cell stage and subsequent developmental stages, and CD20 stops expression until the cells differentiated into plasma cells. Therefore, CD20 is an ideal target for immunotherapy of B cell malignancies.
  • Rituximab (MabThera®, Rituxan®) is the first generation of chimeric monoclonal antibody targeting CD20 which was firstly approved by the US FDA and the European EMA for treating indolent lymphoma.
  • Rituximab recognizes and binds to the big loop structure of the extracellular domain of CD20, and it kills tumor cells by ADCC-mediated killing effect.
  • Rituximab alone shows limited activity and short duration of response, but its combination with chemotherapy can significantly enhance the efficacy of chemotherapy.
  • Rituximab is used for the treatment of lymphoma, and half of the patients have a complete response (CR) or a partial response (PR).
  • Ofatumumab (Arzerra ® ) is the first completely humanized CD20 therapeutic antibody.
  • the epitope recognized by Ofatumumab contains parts of the big loop and the small loop of CD20.
  • the tumor killing method of Ofatumumab is mainly through the complement-dependent pathway, followed by ADCC-dependent tumor killing effect.
  • LCDR1, LCDR2 and LCDR3 may have amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, respectively.
  • HCDR1, HCDR2 and HCDR3 may have amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, respectively.
  • LCDR1, LCDR2 and LCDR3 may have amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, respectively.
  • HCDR1, HCDR2 and HCDR3 may have amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, respectively.
  • LCDR1, LCDR2 and LCDR3 may have amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, respectively.
  • V H is located at the N-terminus of V L .
  • V L is located at the N-terminus of V H .
  • V H and V L have amino acid sequences about 80% to about 100% identical to amino acid sequences set forth in (a) SEQ ID NO: 7 and SEQ ID NO: 11, respectively; (b) SEQ ID NO: 9 and SEQ ID NO: 13, respectively; or (c) SEQ ID NO: 33 and SEQ ID NO: 35, respectively.
  • the anti-CD20 antigen-binding region is a single-chain variable fragment (scFv) that specifically binds CD20.
  • the CAR may further comprise one or more of the following: (a) a signal peptide, (b) a hinge region, (c) a transmembrane domain, (d) a co-stimulatory region, and (e) a cytoplasmic signaling domain.
  • the co-stimulatory region comprises a co-stimulatory region of 4-1BB (CD137), CD28, or combinations thereof.
  • the co-stimulatory region comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 23, or SEQ ID NO: 39.
  • the cytoplasmic signaling domain comprises a cytoplasmic signaling domain of CD3 ⁇ .
  • the cytoplasmic signaling domain comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the hinge region comprises a hinge region of CD8, CD28, CD137, IG4, or combinations thereof.
  • the hinge region comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 17, or SEQ ID NO: 19.
  • the transmembrane domain comprises a transmembrane domain of CD8, CD28, or combinations thereof.
  • the transmembrane domain comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 21.
  • the CAR comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 29, or SEQ ID NO: 31. In one embodiment, the CAR comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 5.
  • the present disclosure provides for an immune cell expressing or comprising the CAR.
  • the immune cell may be a T cell or a natural killer (NK) cell.
  • NK natural killer
  • the present disclosure also provides for a nucleic acid encoding the CAR, or a vector comprising the nucleic acid.
  • the present disclosure provides for a pharmaceutical composition comprising the immune cell, the nucleic acid, the vector, or the CAR.
  • the cancer may be a hematologic cancer.
  • the cancer may be a B-cell malignancy.
  • the B-cell malignancy may be acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), B-cell leukemia, or B cell lymphoma.
  • the cancer may be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and/or multiple myeloma (MM).
  • the immune cell may be administered by infusion, injection, transfusion, implantation, and/or transplantation.
  • the immune cell may be administered intravenously, subcutaneously, intradermally, intranodally, intratumorally, intramedullary, intramuscularly, or intraperitoneally.
  • the immune cell may be administered via intravenous infusion.
  • the immune cell may be allogeneic or autologous.
  • the subject may be a human.
  • the present disclosure provides for a method for treating cancer. The method may comprise administering the immune cell to a subject in need thereof.
  • the chimeric antigen receptor (CAR) may generate an area under the curve (AUC) ranging from about 1.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+07 copies/gDNA, from about 2.0e+06 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+07 copies/gDNA, from about 1.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 4.0e+06 copies/gDNA, from about 1.0e+06 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.0e+07 copies/gDNA, from about 5.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.3e+07 copies/gDNA, from about 5.0e+06 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.0e+07 copies/gDNA, from about 5.0e+06 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.3e+07 copies/gDNA, or from about 7.0e+
  • the chimeric antigen receptor (CAR) may generate a maximum plasma concentration (C max ) ranging from about 1.0e+04 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+06 copies/gDNA, from about 1.0e+04 copies/ ⁇ g genomic DNA (copies/gDNA) to about 3.0e+05 copies/gDNA, from about 2.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+06 copies/gDNA, from about 5x10 4 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.3x10 6 copies/gDNA, from about 5x10 5 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.3x10 6 copies/gDNA, or from about 7.5x10 5 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1x10 6 copies/gDNA, in the blood of the subject.
  • C max maximum plasma concentration
  • the CAR may have a T max ranging from about 10 days to about 25 days, from about 10 days to about 20 days, from about 12 days to about 15 days, from about 12 days to about 25 days, from about 14 days to about 20 days, or from about 6 days to about 22 days.
  • the anti-CD20 antigen-binding region includes a heavy chain variable region (V H ) comprising an amino acid sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 33.
  • V H heavy chain variable region
  • the anti-CD20 antigen-binding region includes a light chain variable region (V L ) comprising an amino acid sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 35.
  • V L light chain variable region
  • a heavy chain variable region of the anti-CD20 antigen-binding region can comprise one, two, or three complementarity determining regions (CDRs) that are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the CDRs of a heavy chain variable region of the Ofatumumab antibody (CDR1, CDR2 and CDR3
  • a light chain variable region of the anti-CD20 antigen-binding region can comprise one, two, or three complementarity determining regions (CDRs) that are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the CDRs of a light chain variable region of the Ofatumumab antibody (CDR1, CDR2 and CDR3
  • a heavy chain variable region of the anti-CD20 antigen-binding region includes three CDRs that are identical (e.g., 80% - 100% identical) to the CDRs of a heavy chain variable region of the Ofatumumab antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, respectively), and a light chain variable region of the anti-CD20 antigen-binding region includes three CDRs that are identical (e.g., 80% - 100% identical) to the CDRs of a light chain variable region of the Ofatumumab antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, respectively).
  • a heavy chain variable region of the anti-CD20 antigen-binding region includes three CDRs that are identical (e.g., 80% - 100% identical) to the CDRs of a heavy chain variable region of the Rituximab antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, respectively), and a light chain variable region of the anti-CD20 antigen-binding region includes three CDRs that are identical (e.g., 80% - 100% identical) to the CDRs of a light chain variable region of the Rituximab antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, respectively).
  • a heavy chain variable region of the anti-CD20 antigen-binding region includes three CDRs that are identical (e.g., 80% - 100% identical) to the CDRs of a heavy chain variable region of the Obinutuzumab antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, respectively), and a light chain variable region of the anti-CD20 antigen-binding region includes three CDRs that are identical (e.g., 80% - 100% identical) to the CDRs of a light chain variable region of the Obinutuzumab antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, respectively).
  • the CAR may comprise an amino acid sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO
  • the CAR may generate an area under the curve (AUC) ranging from about 1.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+07 copies/gDNA, from about 2.0e+06 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+07 copies/gDNA, from about 1.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 4.0e+06 copies/gDNA, from about 1.0e+06 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.0e+07 copies/gDNA, from about 0.5e+06 copies/ ⁇ g genomic DNA (copies/gDNA) to about 2e+07 copies/gDNA, from about 5.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.3e+07 copies/gDNA, from about 5.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 2e+07 copies/gDNA, from about 5.0e+05 copies/ ⁇ g genomic DNA (copies/
  • the AUC may be a median AUC.
  • the CAR generates a maximum plasma concentration (C max ) ranging from about 1.0e+04 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+06 copies/gDNA, from about 1.0e+04 copies/ ⁇ g genomic DNA (copies/gDNA) to about 3.0e+05 copies/gDNA, from about 2.0e+05 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.1e+06 copies/gDNA, from about 5x10 4 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.3x10 6 copies/gDNA, from about 5x10 4 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.5x10 6 copies/gDNA, from about 5x10 5 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1.3x10 6 copies/gDNA, from about 7.5x10 5 copies/ ⁇ g genomic DNA (copies/gDNA) to about 1x10 6 copies/gDNA, from about 7x
  • the C max may be a median C max .
  • the CAR has a T max (the time it takes the CAR to reach C max ) ranging from about 10 days to about 25 days, from about 10 days to about 20 days, from about 12 days to about 15 days, from about 12 days to about 25 days, from about 14 days to about 20 days, from about 6 days to about 22 days, from about 3 days to about 20 days, from about 4 days to about 18 days, from about 5 days to about 17 days, from about 6 days to about 16 days, from about 7 days to about 15 days, from about 9 days to about 15 days, from about 10 days to about 15 days, from about 10 days to about 14 days, from about 8 days to about 12 days, from about 6 days to about 14 days, from about 12 days to about 14 days, from about 8 days to about 11 days, from about 8 days to about 15 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about
  • the T max may be a median T max .
  • the CAR has a T last (the time corresponding to the last quantifiable CAR level) ranging from about 10 days to about 200 days, from about 10 days to about 100 days, from about 10 days to about 90 days, from about 50 days to about 80 days, from about 70 days to about 90 days, from about 30 days to about 90 days, from about 30 days to about 80 days, from about 30 days to about 200 days, from about 50 days to about 150 days, from about 50 days to about 100 days, from about 60 days to about 80 days, from about 60 days to about 150 days, from about 80 days to about 150 days, from about 50 days to about 200 days, from about 50 days to about 60 days, from about 50 days to about 80 days, from about 50 days to about 100 days, from about 60 days to about 100 days, from about 80 days to about 100 days, from about 60 days to about 200 days, from about 80 days to about 200 days, from about 50 days to about 140 days, from about 60 days to about 140 days, or from about 80 days,
  • the T last may be a median T last .
  • the disclosure provides new and effective methods and preparations for clinical application of CAR- T in the treatment of CD20-positive leukemia and lymphoma. It is an object of the present disclosure to provide chimeric antigen receptors targeting CD20, a preparation method and application thereof.
  • the present disclosure relates to the construction of chimeric antigen receptors targeting CD20, a preparation method of chimeric antigen receptor engineered T cells targeting CD20, and activity identification thereof.
  • a chimeric antigen receptor (CAR) sequence
  • scFv antigen binding domain
  • the antigen binding domain of the chimeric antigen receptor is as follows: V H -V L wherein V H is an antibody heavy chain variable region; V L is an antibody light chain variable region; and "-" is a linker peptide or a peptide bond.
  • the amino acid sequence of the linker peptide is as shown in SEQ ID NO: 15.
  • the amino acid sequence of V H is as shown in SEQ ID NO: 7, and the amino acid sequence of V L is as shown in SEQ ID NO: 11.
  • the amino acid sequence of V H is as shown in SEQ ID NO: 9, and the amino acid sequence of V L is as shown in SEQ ID NO: 13.
  • the amino acid sequence of V H is as shown in SEQ ID NO: 33
  • the amino acid sequence of V L is shown in SEQ ID NO: 35.
  • the structure of the chimeric antigen receptor is as follows: L-V H -V L -H-TM-CS-CD3 ⁇ wherein, L is an optional leader sequence (i.e., signal peptide); H is a hinge region; TM is a transmembrane domain; CS is a co-stimulatory region or molecule derived from 4-1BB and/or CD28; CD3 ⁇ is a cytoplasmic signaling domain or sequence derived from CD3 ⁇ ; V H , V L , and "-" are as described above, respectively.
  • the sequence of L is as shown in SEQ ID NO: 27.
  • the signal peptide may comprise an amino acid sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in S
  • the sequence of H is as shown in SEQ ID NO: 17 or 19.
  • the hinge region may comprise an amino acid sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ
  • the sequence of TM comprises a transmembrane region derived from CD8a or CD28.
  • the sequence of TM is as shown in SEQ ID NO: 21 or 37.
  • the transmembrane domain may comprise an amino acid sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or or
  • the CS structure is: CD28-4-1BB, wherein CD28 is a co- stimulatory molecule derived from CD28; and 4-1BB is a co-stimulatory molecule derived from 4-1BB.
  • the sequence of the co-stimulatory molecule derived from 4-1BB is as shown in SEQ ID NO: 23.
  • the sequence of the co-stimulatory molecule derived from CD28 is as shown in SEQ ID NO: 39.
  • the co-stimulatory region may comprise an amino acid sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 39.
  • the sequence of CD3 ⁇ is as shown in SEQ ID NO: 25.
  • the cytoplasmic signaling domain may comprise an amino acid sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid
  • sequence of the chimeric antigen receptor is as shown in SEQ ID NOs: 1, 3, 5, 29, or 31.
  • a nucleic acid molecule is provided, encoding the chimeric antigen receptor (CAR) of the first aspect of the disclosure.
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region selected from the group consisting of: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 17 or 19; (b) a polynucleotide having a sequence as shown in SEQ ID NO: 18 or 20; (c) a polynucleotide having a nucleotide sequence with ⁇ 90% (preferably ⁇ 95%) homologous to the sequence of SEQ ID NO: 18 or 20, and encoding the amino acid sequence of SEQ ID NO: 17 or 19; (d) a polynucleotide complementary to the polynucleotide of any of (a) to (c).
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the CD8a transmembrane region selected from the group consisting of: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 21; (b) a polynucleotide having a sequence as shown in SEQ ID NO: 22; (c) a polynucleotide having a nucleotide sequence with ⁇ 90% (preferably ⁇ 95%) homologous to the sequence of SEQ ID NO: 22 and encoding the amino acid sequence of SEQ ID NO: 21; (d) a polynucleotide complementary to the polynucleotide of any of (a) to (c).
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the 4-1BB (CD137) intracellular signal domain selected from the group consisting of: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 23; (b) a polynucleotide having a sequence as shown in SEQ ID NO: 24; (c) a polynucleotide having a nucleotide sequence with ⁇ 90% (preferably ⁇ 95%) homologous to the sequence of SEQ ID NO: 24 and encoding the amino acid sequence of SEQ ID NO: 23; (d) a polynucleotide complementary to the polynucleotide of any of (a) to (c).
  • 4-1BB CD137 intracellular signal domain
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the CD28 intracellular signal domain selected from the group consisting of: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 39; (b) a polynucleotide having a sequence as shown in SEQ ID NO: 40; (c) a polynucleotide having a nucleotide sequence with ⁇ 90% (preferably ⁇ 95%) homologous to the sequence of SEQ ID NO: 40 and encoding the amino acid sequence of SEQ ID NO: 39; (d) a polynucleotide complementary to the polynucleotide of any of (a) to (c).
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the CD3 ⁇ intracellular signal domain selected from the group consisting of: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 25; (b) a polynucleotide having a sequence as shown in SEQ ID NO: 26; (c) a polynucleotide having a nucleotide sequence with ⁇ 90% (preferably ⁇ 95%) homologous to the sequence of SEQ ID NO: 26 and encoding the amino acid sequence of SEQ ID NO: 25; (d) a polynucleotide complementary to the polynucleotide of any of (a) to (c).
  • the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of: (a) a polynucleotide encoding a polypeptide as shown in SEQ ID NOs: 1, 3, 5, 29 or 31; (b) a polynucleotide having the sequence as shown in SEQ ID NOs: 2, 4, 6, 30 or 32; (c) a polynucleotide having a nucleotide sequence with ⁇ 95% (preferably ⁇ 98%) homologous to the sequence of SEQ ID NOs: 2, 4, 6, 30 or 32, and encoding the amino acid sequence of SEQ ID NOs: 1, 3, 5, 29 or 31; (d) a polynucleotide complementary to the polynucleotide of any of (a) to (c).
  • the nucleic acid molecule is isolated.
  • the nucleic acid molecule further comprises a polynucleotide encoding the leader sequence (directing sequence, signal peptide), and the amino acid sequence of the leader sequence is as shown in SEQ ID NO: 27; the polynucleotide encoding the leader sequence (signal peptide) may be as shown in SEQ ID NO: 28.
  • the sequence of the nucleic acid molecule is as shown in SEQ ID NOs: 2, 4, 6, 30 or 32.
  • it provides a vector, comprising the nucleic acid molecule of the second aspect of the invention.
  • the vector is a lentiviral vector.
  • a host cell comprising the vector of the third aspect of the disclosure or having the exogenous nucleic acid molecule of the second aspect of the disclosure integrated into its genome.
  • the cell is an isolated cell, and/or the cell is a genetically engineered cell.
  • the cell is a mammalian cell.
  • the cell is a T cell.
  • it provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor of the first aspect of the disclosure, the nucleic acid molecule of the second aspect of the disclosure, the vector of the third aspect of the disclosure, or the cell of the fourth aspect of the disclosure.
  • a seventh aspect of the disclosure provides a method for treating a disease comprising administering an appropriate amount of the chimeric antigen receptor of the first aspect of the disclosure, the nucleic acid molecule of the second aspect of the disclosure, the vector of the third aspect of the disclosure, the cell of the fourth aspect of the disclosure, or the pharmaceutical composition of the fifth aspect of the disclosure, to a subject in need of treatment.
  • the disease is tumor.
  • it provides a method for preparing a CAR-T cell (CAR-modified T cell) expressing the chimeric antigen receptor of the first aspect of the disclosure.
  • FIG. 1 shows the structure of the chimeric antigen receptor targeting CD20.
  • Each element of the designed CAR structure is shown in the figure, and the listed elements include: a leader sequence, an antigen recognition sequence (Ofatumumaband, Obinutuzumab, Rituximab), a hinge region, a transmembrane region, a co-stimulatory region, and a CD3 ⁇ signaling region.
  • CAR-T20.14, CAR-T20.13 and CAR-T20.16 are CAR structures constructed based on the antibody variable region sequences of Ofatumumab, Obinutuzumab and Rituxmab, respectively.
  • CAR-T20.19 and CAR-20.20 are the mutant form of CAR-T20.14, having L235E-N297Q mutation in IgG4 Hinge-CH2-CH3 linker region.
  • CAR-T20.20 is a third-generation chimeric antigen receptor structure with coding sequences of both CD28 and 4-1BB co-stimulatory signaling molecule.
  • Figures 2A-2B show detection of transfection efficiency of engineered T cell with chimeric antigen receptors targeting CD20.
  • the expression level of the CAR gene-encoded protein on the surface of the T cell membrane in CAR-T20s cells cultured on day 7 ( Figure 2A) and day 11 ( Figure 2B) was identified by the Protein L method.
  • Figures 3A-3B.1*10 5 of NT, CART-20.13, CART-20.14 and CAR-T20.16 cells (cultured on day 6) were co-cultured respectively with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell line in 200 ⁇ l GT-551 medium for 18h in a ratio of 1:1. Then the expression level of CD137 on the surface of T cell membrane (Figure 3A) and the secretion level of IFN ⁇ in the co-culture supernatant ( Figure 3B) were detected.
  • Figure 4 shows detection of apoptosis levels of tumor cells induced by CART-20.1*10 4 of CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines were co-cultured respectively with NT, CART-20.13, CART-20.14 and CAR-T20.16 cells (cultured on day 11) in 200 ⁇ l GT-551 medium for 4h according to the ratio as shown in figure. Then the cell pellet was collected by centrifugation. The cells were washed twice with PBS and stained for 30 min with Annexin V-APC dye in a ratio of 1:50 in 100 ⁇ l of dyeing solution.
  • FIG. 5A-5C show identification of the activation ability in vitro of the third-generation chimeric antigen receptor and the chimeric antigen receptor with mutation in hinge region (which are constructed based on the sequence of Ofatumumaband antibody).
  • the expression level of the CAR gene-encoded protein (Figure 5A) on the surface of the T cell membrane in CAR-T20.14, CAR-T20.19 and CAR-T20.20 cells cultured on day 7 was identified by the Protein L method.1*10 5 of NT, CART-20.14, CART-20.19 and CAR-T20.20 cells (cultured on day 7) were cultured respectively with K562, K562 stable transfected cells of CD19 single positive, CD20 single positive, CD19 and CD20 double positive, and RAJI target cell in 200 ⁇ l GT-551 medium for 18h in a ratio of 1:1.
  • Figure 5B shows the expression level of CD137 on the surface of T cell membrane
  • Figure 5C shows the secretion level of IFN ⁇ in the culture supernatant
  • Figure 6 shows the detection results of the ability of CAR-T20 cells to scavenge CD20- positive cells in vivo. The results indicate that CAR-T20.19 can effectively inhibit the in vivo expansion of CD20-positive tumor cells.
  • Figures 7A-7D show the screening of scFv for anti-CD20-CARs.
  • Figure 7A shows the structures of CAR-T20.1, CAR-T20.9 to CAR-T20.16.
  • Figure 7B shows the secretion levels of IFN ⁇ .
  • Figure 7C shows the structures of CAR-T20.9, CAR-T20.12, CAR-T20.14, CAR-T20.17 to CAR-T20.19 (C-CAR066).
  • Figure 7B shows the secretion levels of IFN ⁇ .
  • Figures 8A-8D show the lead selection for anti-CD20-CARs.
  • Figure 8A shows the structures of CAR-T20.17, CAR-T20.18 and CAR-T20.19 (C-CAR066).
  • Figure 8B shows the secretion levels of IFN ⁇ .
  • CAR-T20.17 CART20-LEU (3rd), which is the third-generation CAR with Leu-16 scFv.
  • CAR-T20.18 CART20-LEU (2nd), which is the second-generation CAR with Leu-16 scFv.
  • Figure 8C shows the cytotoxicity of CAR- T20.17, CAR-T20.18 and CAR-T20.19 (C-CAR066).
  • Figure 8D shows in vivo anti-tumor efficacy of CAR-T20.17, CAR-T20.18 and CAR-T20.19 (C-CAR066).
  • Figure 9A shows the structures of CAR-T20.19 (C-CAR066) and CAR-T20.29.
  • FIG. 9B shows that CAR-T20.19 (C-CAR066) has the optimized V H -V L scFv structure.
  • Figures 10A-10D show that CAR-T20.19 (C-CAR066) had superior in vivo anti-tumor activity.
  • C-CAR011 is anti-CD1941BB CAR with FMC63.
  • Figure 11 shows an example of the CAR (C-CAR066) manufacture process. The process includes the usage of serum free media, and a functionally closed, highly automated system. Stars indicate improved processes.
  • Figure 12 shows CAR066 Phase I clinical study design and flow chart. A Phase I, first in human, open-label study targeting/r B-NHL patients after failing CD19 CAR-T therapy conducted at two sites.
  • Enrollment key eligibility criteria include 18-75 years old; measurable lesion, CD19 CAR-T failure, no active infection, adequate organ function and no CNS lesion.
  • Objectives include the following. Primary objectives include incidence and severity of TEAEs (CTCAE V5.0 and ASTCT). Secondary objectives include ORR, DOR, PFS, OS (Lugano 2014). Exploratory objectives include CAR-T expansion and persistence.
  • Figure 13 shows the CRS safety profile of C-CAR066.
  • Figure 14A shows the C-CAR066 clinical response, including SD, PR, CR, and PD. SD: stable disease; PR: partial response; CR: complete response; PD: progressive disease.
  • Figure 14B shows the Kaplan Meyer estimation of progression-free survival (PFS).
  • Figure 14C shows the tumor burden (% change) in the seven patients.
  • Figure 15A shows a case study where CR was achieved at 4 weeks with bulky disease.
  • Figure 15B shows the PET-CT images of the cancer lesions for one patient, patient No.2.
  • Figures 16A-16F show C-CAR066 PK/PD profiles.
  • Figure 16A shows the changes of C- CAR066 CAR copies in the peripheral blood of the patients after CAR administration over time.
  • Figure 16B shows the changes of CD20+ B cell levels in the peripheral blood of the patients after CAR administration over time.
  • Figure 16C shows C max levels in the blood of the patients after CAR administration.
  • Figure 16D shows AUC levels in the blood of the patients after CAR administration.
  • Figure 16E shows T max levels in the blood of the patients after CAR administration.
  • Figure 16F shows T last levels in the blood of the patients after CAR administration.
  • Low dose 2.0 x 10 6 CAR-T cells/kg
  • mid dose 3.0 x 10 6 CAR-T cells/kg
  • high dose 4.8 x 10 6 CAR-T cells/kg.
  • Figure 17 shows CD19/CD20 expression and PK/PD in C-CAR066 relapsed patients, including CAR-T expansion and B cell depletion in peripheral blood.
  • the present disclosure provides for chimeric antigen receptors (CARs) targeting CD20.
  • the CARs are based on three antibodies: Ofatumumab, Rituximab and Obinutuzumab.
  • the present disclosure also provides for the in vitro activities and tumor cell killing efficacy of these chimeric antigen receptors.
  • the chimeric antigen receptors of the present disclosure target CD20-positive cells and can be used to treat a hematologic cancer including a B-cell malignancy such as acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), B-cell leukemia, or B cell lymphoma.
  • B-cell malignancy such as acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), B-cell leukemia, or B cell lymphoma.
  • the present CARs may be used to treat Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and/or multiple myeloma (MM).
  • Chimeric antigen receptors targeting CD20 and the preparation and application thereof are provided.
  • the extracellular antigen binding domain of the chimeric antigen receptor includes the antibody heavy chain variable region and the antibody light chain variable region.
  • the experimental results show that the chimeric antigen receptor provided by the present disclosure shows significantly high killing ability against tumor cells.
  • a series of chimeric antigen receptors targeting CD20 were constructed in the present disclosure by combining various transmembrane and intracellular components with the amino acid sequences of the variable regions in various anti-CD20 antibodies.
  • the expression of such chimeric antigen receptors in T cells (e.g., primary T cells) was completed.
  • the detection method of receptor expression intensity was established.
  • Chimeric antigen receptors The disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain comprises a target-specific binding element (also known as an antigen binding region or domain).
  • the intracellular domain includes a co-stimulatory (signaling) region and a ⁇ chain moiety.
  • the co-stimulatory signaling region refers to a part of the intracellular domain that includes a co-stimulatory molecule.
  • the co-stimulatory molecule is a cell surface molecule for efficient response of lymphocytes to antigens, rather than an antigen receptor or its ligand.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • linker generally refers to any oligopeptide or polypeptide that plays a role of linking the two components of the CAR.
  • a linker can link the transmembrane domain to the extracellular domain or the cytoplasmic domain in a polypeptide chain.
  • the linker may comprise 0-300 amino acids, 2-100 amino acids, or 3-50 amino acids.
  • the extracellular domain of the CAR provided by the present disclosure comprises an antigen binding domain targeting CD20.
  • antigen recognition can be performed based on antigen binding specificity. When it binds to its cognate antigen, it affects a tumor cell so that the tumor cell fails to grow, is prompted to die, or otherwise is affected so that the tumor burden in a patient is diminished or eliminated.
  • the antigen binding domain may be fused with an intracellular domain from one or more of a co-stimulatory molecule and a ⁇ chain.
  • the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB signaling domain and/or a CD28 signaling domain, and a CD3 ⁇ signaling domain.
  • the CAR targeting CD20 comprises the specific signaling domain (e.g., the transmembrane region of CD8, the intracellular signal domains of CD137 and CD3 ⁇ are in series).
  • the signaling domain of the disclosure significantly increases anti-tumor activity and in vivo persistence of CAR-T cells compared to an otherwise identical CAR targeting CD20.
  • the amino acid sequence of the chimeric antigen receptor (CAR) provided by the present disclosure is as follows.
  • the DNA sequence encoding CAR-T20.13 (SEQ ID NO: 30) may be as follows:
  • the DNA sequence encoding CAR-T20.14 (SEQ ID NO: 2) is as follows:
  • the DNA sequence encoding CAR-T20.16 (SEQ ID NO: 4) is as follows:
  • the amino acid sequence of the chimeric antigen receptor (CAR) provided by the disclosure is as follows.
  • the amino acid sequence of CAR-T20.19 (SEQ ID NO:5)
  • the DNA sequence encoding CAR-T20.19 (SEQ ID NO: 6) is as follows:
  • the amino acid sequence of the chimeric antigen receptor (CAR) provided by the invention is as follows.
  • the amino acid sequence of CAR-T20.20 (SEQ ID NO:31)
  • the coding DNA sequence of CAR-T20.20 (SEQ ID NO: 32) is as follows:
  • the CAR of the disclosure comprises a target-specific binding element referred to as antigen binding region or domain.
  • the antigen binding domain of the present CAR is a specific binding element targeting CD20.
  • the antigen binding domain comprises a heavy chain variable region and a light chain variable region of an anti-CD20 antibody.
  • the amino acid sequence of the heavy chain variable region of the Ofatumumab antibody is as follows: The DNA sequence encoding the heavy chain variable region of the Ofatumumab antibody is as follows: The amino acid sequence of the heavy chain variable region of the Rituximab antibody is as follows: The DNA sequence encoding the heavy chain variable region of the Rituximab antibody is as follows: Further, the amino acid sequence of the heavy chain variable region of the Obinutuzumab antibody used in the present disclosure is as follows: The DNA sequence encoding the heavy chain variable region of the Obinutuzumab antibody is as follows: In another embodiment, the amino acid sequence of the light chain variable region of the Ofatumumaband antibody is as follows: The DNA sequence of Ofatumumaband antibody is as follows: The anti-CD20 CAR comprises an anti-CD20 antigen-binding region which comprises a light chain variable region (V L)
  • V L comprises three complementarity determining regions (CDRs), LCDR1, LCDR2 and LCDR3, and V H comprises three CDRs, HCDR1, HCDR2 and HCDR3.
  • the CDRs of Ofatumumab are as follows.
  • V H comprises three CDRs: CDR-H1 (HCDR1), CDR-H2 (HCDR2) and CDR-H3 (HCDR3);
  • V L comprises three CDRs: CDR-L1 (LCDR1), CDR- L2 (LCDR2) and CDR-L3 (LCDR3).
  • CDR-H1 NDYAMH (SEQ ID NO: 41)
  • CDR-H2 TISWNSGSIGYADSVKG (SEQ ID NO: 42)
  • CDR-H3 DIQYGNYYYGMDV (SEQ ID NO: 43)
  • CDR-L1 RASQSVSSYLA (SEQ ID NO: 44)
  • CDR-L2 DASNRAT (SEQ ID NO: 45)
  • CDR-L3: QQRSNWPIT SEQ ID NO: 46
  • the amino acid sequence of the light chain variable region of the Rituximab antibody is as follows:
  • the DNA sequences encoding the light chain (VL) of single-chain variable region derived from the Rituximab antibody is:
  • the CDRs of Rituximab are as follows.
  • V H comprises three CDRs: CDR-H1 (HCDR1), CDR-H2 (HCDR2) and CDR-H3 (HCDR3);
  • V L comprises three CDRs: CDR-L1 (LCDR1), CDR- L2 (LCDR2) and CDR-L3 (LCDR3).
  • Rituximab Heavy Chain SEQ ID NO: 9
  • Rituximab Light Chain SEQ ID NO:13
  • the amino acid sequence of the light chain variable region of the Obinutuzumab antibody used in the present disclosure is as follows:
  • the DNA sequence encoding the light chain variable region of the Obinutuzumab antibody is as follows:
  • the CDRs of Obinutuzumab are as follows.
  • V H comprises three CDRs: CDR-H1 (HCDR1), CDR-H2 (HCDR2) and CDR-H3 (HCDR3);
  • V L comprises three CDRs: CDR-L1 (LCDR1), CDR-L2 (LCDR2) and CDR-L3 (LCDR3).
  • Obinutuzumab Heavy Chain SEQ ID NO:33
  • transmembrane domains may be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complexes.
  • the hinge region comprises the following amino acid sequence (IgG4 Hinge-CH2-CH3 hinge region): Its coding DNA sequence is as follows: In one embodiment, the hinge region comprises the following amino acid sequence (IgG4 Hinge-CH2-CH3 (L235E, N297Q)): Its coding DNA sequence is as follows: In a preferred embodiment of the invention, the amino acid sequence of the transmembrane region derived from CD8 (CD8TM) is as follows: The coding DNA sequence thereof is as follows: In a preferred embodiment of the invention, the amino acid sequence of the transmembrane region derived from CD28 (CD28TM) is as follows: The DNA sequence encoding the transmembrane region derived from CD28 (CD28TM) is as follows:
  • the intracellular signaling domain of 4-1BB comprises the following amino acid sequence:
  • the coding DNA sequence thereof is as follows:
  • the intracellular signaling domain derived from CD28 comprises the following amino acid sequence:
  • the coding DNA sequence thereof is as follows:
  • the intracellular signaling domain of CD3 ⁇ comprises the following amino acid sequence:
  • the coding DNA sequence thereof is as follows:
  • Vector The present disclosure also provides a nucleic acid, a vector, or a DNA construct encoding the present CAR.
  • the nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically.
  • the present disclosure also provides vectors in which the DNA construct of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • the expression constructs of the present disclosure may also be used for nucleic acid immune and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S, Pat. Nos.5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • the present disclosure provides a gene therapy vector.
  • the nucleic acid can be cloned into any suitable types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors, Further, the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al, (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S, Pat. No. 6,326, 193).
  • a number of virus-based systems have been developed for gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • tk thymidine kinase
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-1 ⁇ Elongation Growth Factor-1 ⁇
  • constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter MoMuLV promoter
  • an avian leukemia virus promoter an Epstein-Barr virus immediate early promoter
  • Rous sarcoma virus promoter as well as human gene promoters such
  • inducible promoters are also contemplated as part of the disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure.
  • Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic- resistance genes, such as neo and the like. Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • Methods of introducing and expressing genes into a cell are known in the art.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat, Nos.5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • a liposome e.g., an artificial membrane vesicle
  • an exemplary delivery vehicle is a liposome.
  • the use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • genome editing technique may be exemplarily employed, for example CRISPR-Cas9, ZFN or TALEN.
  • the vector is a lentiviral vector.
  • the DNA construct further comprises a signal peptide coding sequence.
  • the signal peptide sequence is ligated upstream of the nucleic acid sequence of antigen binding domain.
  • the signal peptide is a human CD8a signal peptide.
  • the amino acid sequence of the signal peptide is as follows.
  • the amino acid sequence of CD8 leader sequence is:
  • the DNA sequence encoding CD8 leader sequence is: As used herein, the terms "CAR-T cell”, “CAR-T”, and “CART”, may be used interchangeably.
  • the administered (e.g., infused) cell is able to kill tumor cells in the recipient.
  • CAR-T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.
  • the CAR mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified T cells induce an immune response specific to the antigen binding moiety in the CAR.
  • an anti-CD20 CAR-T cell elicits an immune response specific against cells expressing CD20.
  • CD20-positive tumors and diseases e.g., caused by excessive B cells (such as autoimmune diseases, for example, lupus erythematosus, etc.).
  • CD20 positive tumors may include CD20 positive non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or solid tumors.
  • hematological (or hematogenous) cancers include leukemias, e.g., acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non- Hodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • acute leukemias such as acute lymphocytic leuk
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer and ovarian cancer.
  • the CAR-modified T cells of the disclosure may also serve as a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. Preferably, the mammal is a human.
  • At least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a CAR to the cells, and/or iii) cryopreservation of the cells.
  • Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit.
  • the mammalian recipient may be a human and the CAR-modified cell can be autologous with respect to the recipient.
  • the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.
  • the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
  • the CAR-modified T cells are used in the treatment of CCL.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids
  • antioxidants e.g., antioxidants
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • an immunologically effective amount When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, or 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al uncomfortable Eng. J. of Med.319: 1676, 1988).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the administration of the compositions or cells may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient subcutaneously, intradermaliy, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • compositions or cells of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the compositions or cells of the present disclosure are administered by i.v. injection.
  • the compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients.
  • compositions or cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents.
  • the compositions or cells of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, or the use of chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded immune cells of the present disclosure.
  • expanded cells are administered before or following surgery.
  • the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the scaling of dosages for human administration can be performed according to art-accepted practices.
  • 1x10 6 to 1x10 10 of the modified T cells of the invention e.g., CAR-T 20 cells
  • the advantages of the certain embodiments of the present disclosure include: (1) As for the chimeric antigen receptor of the present disclosure, the extracellular antigen binding domain thereof is a specific anti-CD20 scFv.
  • the CAR formed by binding the specific anti-CD20 scFv to a specific hinge region and an intracellular domain shows a great capability of killing tumor cells with low cytotoxicity and low side effects.
  • the chimeric antigen receptor provided by the disclosure can achieve stable expression and membrane localization of CAR protein after T cells is infected by lentivirus carrying CAR gene.
  • the CAR-modified T cell of the present disclosure has a longer survival time in vivo and strong anti-tumor efficacy.
  • the optimized CAR with the IgG4 Hinge-CH2-CH3 linker region can avoid the binding of the Fc receptor and the subsequent ADCC effect (antibody- dependent cytotoxicity).
  • administering refers to the physical introduction of a product of the disclosure into a subject using any one of various methods and delivery systems known to those skilled in the art, including, but not limited to, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration, such as by injection or infusion.
  • Example 1 Construction of lentiviral expression vector The full-length DNA synthesis and cloning construction of coding plasmids were conducted. Different anti-CD20 scFv coding sequences were used in each plasmid. The cloning vector was selected as pWPT lentiviral vector. The cloning sites were BamH I and Sal I sites. The specific sequence structure is shown in Figure 1. The amino acid and nucleotide sequences of each element are as described above.
  • CAR-T20.13, CAR-T20.14, CAR-T20.16, CAR-T20.19, CAR-T20.20 with better effects are taken as examples.
  • Example 2 Preparation of CAR-T cell (1) After taking venous blood from healthy subjects, mononuclear cells (PBMCs) were isolated by density gradient centrifugation. (2) On day 0, PBMCs were cultured in GT-T551 cell culture medium containing 2% human albumin, and the final concentration of cells was adjusted to 2x10 6 cells/mL.
  • PBMCs mononuclear cells
  • the cells were seeded in a cell culture flask previously coated with Retronectin (purchased from TAKARA) at a final concentration of 10 ⁇ g/mL and CD3 monoclonal antibody (OKT3) at a final concentration of 5 ⁇ g/mL.
  • Retronectin purchased from TAKARA
  • CD3 monoclonal antibody OKT3
  • Recombinant human interleukin 2 IL-2
  • the cells were cultured in an incubator with a saturated humidity and 5% CO 2 at 37 °C.
  • fresh medium, concentrated and purified CAR20 lentivirus solution, protamine sulfate (12 ⁇ g/ml), and IL-2 (at a final concentration of 1000 U/mL) were added.
  • Example 5 Detection of the CAR-T20s cells induced early apoptosis activity of tumor cells CART-20.13, CART-20.14 and CAR-T20.16 cells (cultured on day 11) from Example 2 were co-cultured respectively with 1x10 4 of CFSE-labeled CD20-negative (MOLT-4) or CD20- positive (RAJI, RAMOS) tumor cell lines in 200 ⁇ l GT-551 medium for 4h. Then the cell pellet was collected by centrifugation. The cells were washed twice with PBS and stained for 30 min with Annexin V-APC dye in a ratio of 1:50 in 100 ⁇ l of dyeing solution.
  • Example 6 Identification of the in vitro activation ability of the third-generation chimeric antigen receptor and the chimeric antigen receptor with mutation in hinge region (1) Under the condition that the transfection rate was basically equal (Fig.5A), the CAR- T20s cells (prepared by the method of Example 2) cultured on the day 7 were cultured respectively with K562, K562 stable transfected cells of CD19 single positive, CD20 single positive, CD19 and CD20 double positive, and RAJI target cell ( each taking 1x10 5 cells) in 200 ⁇ l GT-551 medium for 18h in a ratio of 1:1. Then the up-regulated level of CD137 (Fig.5B) and the secretion level of IFN ⁇ in the culture supernatant (Fig.5C) were detected.
  • Fig.5A the up-regulated level of CD137
  • Fig.5C secretion level of IFN ⁇ in the culture supernatant
  • Example 8 We prepared two anti-CD20 CARs having the same V H (SEQ ID NO: 7) and V L (SEQ ID NO: 11) but in different orders: CAR-T20.19 (SEQ ID No.5) has V H -V L (i.e., V H is located at the N-terminus of V L ), while CAR-T20.29 has V L -V H ( Figure 9A). As shown in Figure 9B, CAR-T20.19 (V H -V L ) demonstrated significantly higher in vitro activities (e.g., inducing interferon- ⁇ or IFN- ⁇ release) against the CD20-positive cells than CAR-T20.29 (V L -V H ).
  • CAR-T20.19 (V H -V L ) showed 190%, 80%, and 38% greater activities compared to CAR-T20.29 (V L -V H ) for the CD20-positive cell lines K562-CD20, A549-CD20, and Raji, respectively.
  • V H and V L directly impacts the function of the CAR T cells.
  • CARs having the V H -V L structure (e.g., CAR-T20.19) demonstrated significantly higher in vitro activities than CARs having the reversed V L - V H structure (e.g., CAR-T20.29).
  • Example 9 Our studies demonstrated that CAR-T20.19 was considerably more cytotoxic towards tumor cells both in vitro and in vivo, compared to CAR-T cells based on another anti-CD20 antibody, Leu16.
  • Figures 8B-8C show CAR-T20.19 induced higher levels of IFN- ⁇ release and greater cell killing in CD20-positive tumor cells, including RAJI and RAMOS, compared to CART20-Leu.
  • NSG mice were xenografted with Raji-Luc cells which are human Burkitt’s lymphoma Raji cells expressing firefly luciferase as a reporter. Different CAR-T cells or negative control were then administered to the mice.
  • CART20-OF(2nd) CAR-T20.19
  • B-cell lymphomas can be stratified into Hodgkin lymphoma ( ⁇ 10% of all cases) and non- Hodgkin lymphoma (NHL; ⁇ 90% of all cases), both of which comprise many subtypes.
  • NHL non- Hodgkin lymphoma
  • the response rates to conventional salvage chemotherapy are approximately 40–50%.
  • CAR-T20.19 also termed “C-CAR066”
  • R/R DLBCL relapsed/refractory DLBCL
  • the patients’ baseline demographics and clinical characteristics prior to the start of our anti-CD20 CAR treatment are shown in Table 1.
  • the patients received lymphodepletion pretreatment, including fludarabine (30 mg/m 2 /d, intravenous, once per day for three days), and cyclophosphamide (300 mg/m 2 /d, intravenous, once per day for three days).
  • lymphodepletion pretreatment including fludarabine (30 mg/m 2 /d, intravenous, once per day for three days), and cyclophosphamide (300 mg/m 2 /d, intravenous, once per day for three days).
  • the patients were administered 2.0 x 10 6 , 3.0 x 10 6 or 4.8 x 10 6 CAR-T cells/kg on day 0 as a single infusion.
  • follow-ups with the patients were carried out from day 1 to month 24 (e.g., day 4, day 7, day 10, week 2, week 3, week 4, etc.) after the infusion.
  • the first clinical response assessment was at week 4 after the CAR-T infusion.
  • the Kaplan Meier progression-free survival (PFS) estimates include a 6-month PFS of 57.1%, with 95% confidence intervals (CIs) of ⁇ 30% - 100%. See Table 2 and Figure 14B.
  • Table 2 The tumor burden in the patients decreased significantly ( Figure 14C).
  • the patients’ adverse reactions (treatment-emergent adverse events, TEAE) were recorded (Table 3 and Table 4). There was only 1 (10.0%) grade ⁇ 3 cytokine release syndrome (CRS). No neurotoxicity was observed in the patients.
  • Cytopenia such as neutropenia and thrombocytopenia, was mostly related to the fludarabine/cyclophosphamide (Cy/Flu) lymphodepletion. The cytopenia was also reversible. These demonstrated that our anti-CD20 CAR had an excellent safety profile.
  • Table 3 1 CRS uniformly graded according to the ASTCT Guidelines. See, Lee, Biol Blood Marrow Transplant, 2019, 25: 625. Table 4 8 out of 10 patients had grade 1-2 CRS, while 1 out of 10 patients had grade 4 CRS. This patient presented with high fever, hypotension and hypoxia on day 6 and resolved on day 10. The patient was treated with tocilizumab and steroids, and with non-invasive ventilation support. The patient was not admitted to the ICU.
  • C-CAR066 has optimal structure and superior anti-tumor activity compared to anti-CD20 CAR-Ts derived from scFvs of Leu16, Rituximab, and Obinutuzumab and anti-CD19 CAR-T.
  • C-CAR066 shows a favorable safety profile and very promising efficacy in patients with r/r NHL following CD19 CAR-T therapy (with a median DOR of 2.1 months) compared to CD20/CD3 bispecific antibody.
  • Example 11 In one case study, a 67-year-old male with double-expressor DLBCL was diagnosed in May 2019. The patient had 4 prior lines of therapy, including anti-CD19 CAR-T treatment.
  • the patient had right and left calve lesions.
  • the patient’s bulky disease was 25.9 *6.3*10.1 cm in the right leg at baseline.
  • the prior anti-CD19 CAR-T treatment had a best response of PR and duration of response of 1.2 months.
  • the C-CAR066 treatment included 3.0x10 6 /kg dosage, grade 2 CRS (onset on day 2, resolved on day 11), no neurotoxicity.
  • CR was achieved by day 27 ( Figure 15A).
  • the scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions and dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of this invention.

Abstract

L'invention concerne des récepteurs antigéniques chimériques ciblant CD20 et des procédés de préparation de ceux-ci. La région de liaison à l'antigène du récepteur d'antigène chimérique peut comprendre une région variable de chaîne lourde représentée dans SEQ ID NO : 7, 9 ou 33 et une région variable de chaîne légère représentée dans SEQ ID NO : 11, 13 ou 35.
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