[go: up one dir, main page]

WO2024094004A1 - Anticorps entièrement humain ciblant cd123 et son utilisation - Google Patents

Anticorps entièrement humain ciblant cd123 et son utilisation Download PDF

Info

Publication number
WO2024094004A1
WO2024094004A1 PCT/CN2023/128518 CN2023128518W WO2024094004A1 WO 2024094004 A1 WO2024094004 A1 WO 2024094004A1 CN 2023128518 W CN2023128518 W CN 2023128518W WO 2024094004 A1 WO2024094004 A1 WO 2024094004A1
Authority
WO
WIPO (PCT)
Prior art keywords
car
cell
antibody
seq
chimeric antigen
Prior art date
Application number
PCT/CN2023/128518
Other languages
English (en)
Chinese (zh)
Inventor
赵文旭
么瑞娜
张长江
高诗静
黄宇康
陈运凡
徐艳敏
刘童灿
赵永春
陈军
黄霞
洪娟
代德鹏
秦蕾
沈俊杰
Original Assignee
重庆精准生物技术有限公司
重庆精准生物产业技术研究院有限公司
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
Priority claimed from CN202211370708.7A external-priority patent/CN118027193A/zh
Priority claimed from CN202211370723.1A external-priority patent/CN118027194A/zh
Application filed by 重庆精准生物技术有限公司, 重庆精准生物产业技术研究院有限公司 filed Critical 重庆精准生物技术有限公司
Publication of WO2024094004A1 publication Critical patent/WO2024094004A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention belongs to the technical field of cell engineering, and in particular relates to CD123 antibodies and applications thereof.
  • Adoptive cell therapy refers to the separation of immune-competent cells from cancer patients, their expansion and functional identification in vitro, and then their return to the patient, thereby directly killing the tumor or stimulating the body's immune response to kill tumor cells.
  • CAR Chimeric antigen receptor
  • T cell technology is a rapidly developing cellular immunotherapy technology.
  • CAR is an artificially synthesized fusion receptor, which structurally includes an extracellular antigen binding region, a transmembrane region, an intracellular signal transduction region and a co-stimulatory signal region.
  • the extracellular region is a monoclonal antibody sequence (single chain variable fragment, scFv) that recognizes tumor-associated antigens.
  • the transmembrane region connects the extracellular region and the intracellular region. Commonly used transmembrane region molecules are selected from the transmembrane regions and variants of genes such as CD3, CD4, CD8 and CD28.
  • the intracellular region has an immune receptor tyrosine-based activation motif (ITAM), the most commonly used of which are the T cell receptor TCR/CD3 ⁇ chain and the immunoglobulin FC receptor FC ⁇ RI ⁇ , which are mainly responsible for signal transduction function; people use molecular cloning methods to recombinate the above elements in vitro to form recombinant plasmids, and then transduce the recombinant plasmids into T cells through viral vectors, electroporation and other means, followed by in vitro culture and amplification, and finally back into the patient's body to treat tumors.
  • This genetically modified and transformed T cell is called CAR-T cell.
  • CAR-T cells recognize and bind to tumor antigens through the scFv segment, and then activate T cells through the CD3 ⁇ chain and co-stimulatory signal region.
  • the modified CAR-T cells recognize and kill tumor cells with two major characteristics: 1 Targetedness; 2 Overcoming MHC restriction means no longer relying on MHC, thus overcoming the problem of tumor cells escaping immune function by downregulating MHC expression.
  • NK cells In addition to T cells, NK cells also play an important role in tumor treatment.
  • the CAR structure can also form the corresponding CAR-
  • NK cells and CAR-NK cells are not restricted by HLA matching and can achieve allogeneic adoptive immune therapy.
  • NK cells have a wide range of sources, including hematopoietic stem cells, peripheral blood, umbilical cord blood, induced pluripotent stem cells, etc. Therefore, CAR-NK can also be used as one of the important methods of immunotherapy.
  • AML Acute myeloid leukemia
  • anthracyclines and cytarabine chemotherapy have been used to treat mild patients
  • hematopoietic stem cell transfer has been used to treat medium- and high-risk patients.
  • elderly patients ⁇ 60 years old
  • patients with poor clinical manifestations have a high mortality rate, and the 5-year overall survival rate is about 10%.
  • Due to the heterogeneity of acute myeloid leukemia the current treatment of AML patients is still a challenge. In recent years, researchers have found that CD33,
  • Immune targeted therapy targeting CD123 and CLEC12A is effective in treating AML.
  • CD123 is a subunit of the heterodimeric interleukin-3 receptor (IL-3R), which is a member of the ⁇ co-receptor family. This family of membrane receptors plays an important role in regulating the growth, proliferation, survival and differentiation of hematopoietic cells, as well as immune and inflammatory responses. CD123 is widely expressed in various hematological malignancies. Currently, many monoclonal antibody drugs also use CD123 as a target antigen, so the development of a CD123 antibody for use in adoptive immune cell therapy has also become a research hotspot.
  • IL-3R heterodimeric interleukin-3 receptor
  • the present invention provides a CD123 antibody and a chimeric antigen receptor targeting CD123.
  • the CD123 antibody is an anti-CD123 scFv
  • the anti-CD123 scFv comprises a heavy chain variable region and a light chain variable region
  • the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3
  • the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;
  • the L-CDR1, L-CDR2 and L-CDR3 are respectively the following sequences: QSVSSN (SEQ ID NO.1), GAS (SEQ ID NO.2) and QQRSNWPPALT (SEQ ID NO.3);
  • H-CDR1, H-CDR2 and H-CDR3 are the following sequences respectively: GYSFTSYW (SEQ ID NO.4), IYPGDSDT (SEQ ID NO.5) and ARIRFDKEQLFYYYYGMDV (SEQ ID NO.6).
  • the anti-CD123 scFv comprises the amino acid sequence shown in SEQ ID NO.10 or its functional variant.
  • the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the anti-CD123 scFv as described above.
  • the light chain variable region includes, in addition to the above-mentioned high mutation regions of L-CDR1, L-CDR2 and L-CDR3, a low mutation region (also called skeleton region or framework region).
  • amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a functional variant thereof; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.8 or a functional variant thereof.
  • the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', bispecific antibody and multispecific antibody.
  • the antibody or antigen-binding fragment thereof is ScFv.
  • the arrangement of the ScFv is: VL-Linker-VH or VH-Linker-VL.
  • amino acid sequence of the Linker is shown in SEQ ID NO.9.
  • the chimeric antigen receptor comprises an extracellular domain, a hinge region, a transmembrane region and an intracellular signaling domain.
  • the hinge region is derived from CD4, CD28, CD3, CD40, 4-1BB, OX40, CD84, CD166, CD8 ⁇ , CD8 ⁇ , ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, CD7, IgG1, IgG4 or the CH2/CH3 domain in immunoglobulin or other protein molecules with equivalent functions.
  • the amino acid sequence of the hinge derived from CD8 ⁇ is shown in SEQ ID NO.11 or its functional variant, and the sequences of the hinge regions of the above-mentioned other proteins that can be used as hinges can be used. The sequences that can be queried in NCBI.
  • the nucleotide sequence of the IgG4 hinge is as follows: gcaccacctcgggccagcgccctgcctgca ccacccaccggctccgccctgccagaccctcagacagcatctgccctgccagatcctccagcagcaagcgccctgccc (SEQ ID NO. 23);
  • nucleotide sequence of CD7 hinge is as follows: gcaccacctcgggccagcgccctgcctgcaccaccacccaccggctccgccctgccagaccctcagacagcatctgccctgccagatcctccagcagcaagcgccctgccc (SEQ ID NO.24).
  • the transmembrane region is derived from CD2, CD3D, CD3E, CD3G, CD3 ⁇ , CD4, CD7, CD8 ⁇ , CD8 ⁇ , CD16, CD27, CD28, CD28H, CD40, CD80, CD84, CD86, CD134, CD137, CD166, CD278, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERI ⁇ , IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, , NKp46, NKG2C, NKG2D, CS1, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or the transmembrane region of T cell receptor polypeptide SIRP ⁇ 1 or NKp44, or other protein molecules with equivalent functions; wherein
  • the intracellular signaling domain is derived from CD28, OX40, CD27, DAP10, CD3 ⁇ , Fc ⁇ RI, CD2, CD16, TCR ⁇ , FcR ⁇ , CD30, CD40, OX40, ICOS, LFA-1, IL-2 receptor, Fc ⁇ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), PD1L, B7-H3, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, LFA-1 (CD11a/CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), CD137, 2B4, CD3 ⁇ , DAP10 functional domains, ICAM-1, CD7, CD83, CD86 and CD127 other equivalent functional protein molecules.
  • the amino acid sequence of the functional domain of CD137 is shown in SEQ ID NO.15 or its functional variant
  • the amino acid sequence of the functional domain of 2B4 is shown in SEQ ID NO.16 or its functional variant
  • the amino acid sequence of CD3 ⁇ is shown in SEQ ID NO.17 or its functional variant
  • the amino acid sequence of the functional domain of DAP10 is shown in SEQ ID NO.18 or its functional variant.
  • Its functional variants and other intracellular functional domains of proteins that can serve as intracellular signaling domains can adopt sequences that can be searched in public databases such as NCBI, ENA, and DDBJ.
  • the nucleotide sequence of CD28 also includes the following sequence: aggagcaagcggagcagaggcggccacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccctacgcccctcccagggacttcgccgcctaccggagc (SEQ ID NO.25).
  • the chimeric antigen receptor also includes a signal peptide, which is derived from the signal peptide functional domain of CD8 ⁇ or GM-CSF or GM-CSFR, and the amino acid sequences of the signal peptides derived from CD8 ⁇ and GM-CSFR are respectively shown in SEQ ID NO.19 and SEQ ID NO.20 or their functional variants.
  • the chimeric antigen receptor comprises one or more components of a natural killer cell receptor (NKR), thereby forming a NKR-CAR.
  • the NKR component can be a transmembrane domain, hinge domain, or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIR), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; natural cytotoxicity receptors (NCR), for example, NKp30, NKp44, NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, for example, CD48, CD1239, 2B4, CD84, NTB-A, CRA, BLAME and CD2F-10
  • the chimeric antigen receptor can be a multi-target CAR-T, preferably a dual-target CAR-T, comprising the antibody of the present invention and the recognition of CD123 antigen and the antibody of the present invention without competing for binding antibodies or other tumor surface molecules and/or immune checkpoint molecules that recognize non-CD123 antigens.
  • the dual-target CAR structure can be CAR1-connector peptide-CAR2, or ScFv1-connector peptide-ScFv2-hinge region (hinge)-transmembrane region (TM)-intracellular signaling region (including costimulatory domains and/or intracellular activation domains), or VL1-VL2-VH2-VH1-hinge region (hinge)-transmembrane region (TM)-intracellular signaling region (including costimulatory domains and/or intracellular activation domains) of light and heavy chain cross structures, or any other CAR structure that can play a variety of antigen functions.
  • the chimeric antigen receptor can reverse tumor suppressor molecules, cytokines, Fusion protein combination/combination of T cell activation molecules, etc.
  • the molecule that reverses the tumor suppression signal can be PD1
  • the fusion protein structure can be PD1 extracellular segment-hinge-transmembrane-intracellular signal region, or it can be a fusion protein/polypeptide form of any structure such as PD1 extracellular segment and membrane region-intracellular signal region.
  • the fusion protein has various forms. The structures of different fusion proteins will not affect the function of the chimeric antigen receptor of the present invention to recognize CD123.
  • the tumor suppression molecules that reverse can also be CTLA-4, PD-L1, LAG-3, TIM-3, BTLA, CD47, etc.
  • cytokines can be membrane-bound IL-15, membrane-bound IL12, membrane-bound IL21, etc.
  • T cell activation molecules can be CD80, CD86, etc.
  • the chimeric antigen receptor contains secretable cytokines, antibodies or other polypeptides.
  • the chimeric antigen receptor comprises a recognizable marker molecule, which may be a truncated receptor such as truncated EGFR (EGFRt), truncated HER2 (HER2t), truncated CD34 (CD34t) and truncated CD19 (CD19t).
  • a truncated receptor such as truncated EGFR (EGFRt), truncated HER2 (HER2t), truncated CD34 (CD34t) and truncated CD19 (CD19t).
  • the chimeric antigen receptor can be combined with immune checkpoint inhibitors, which include but are not limited to: CTLA-4 inhibitors, PD-1/PD-L1 inhibitors, LAG-3 (Lymphocyte Activation Gene-3) inhibitors, TIM-3 (T cell immunoglobulin-3), TIGIT (T cell immunoglobulin and ITIM domain protein), and BTLA inhibitors.
  • the inhibitors are small molecule drugs, and in some embodiments, they can be antibodies, active peptides, etc.
  • the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', bispecific antibody and multispecific antibody, and can be directly or indirectly coupled/bound to a cytotoxic agent to form an immunoconjugate or antibody ADC drug.
  • Antibody-drug conjugates are a class of targeted biological agents that connect cytotoxic drugs to monoclonal antibodies through covalent compound linking molecules.
  • the cytotoxic agent can be a microtubule inhibitor and/or a DNA damaging agent, such as ⁇ -amanitin (selective RNA polymerase II inhibitor), DM1, PBD, MMAE (monomethyl auristatin E), N-acetyl-gamma-calicheamicin, mitomycin C, anthracyclines, methotrexate, camptothecin derivatives, SN-38 and other toxin molecules.
  • ⁇ -amanitin selective RNA polymerase II inhibitor
  • DM1 selective RNA polymerase II inhibitor
  • PBD selective RNA polymerase II inhibitor
  • MMAE monomethyl auristatin E
  • N-acetyl-gamma-calicheamicin mitomycin C
  • anthracyclines methotrexate
  • camptothecin derivatives camptothecin derivatives
  • SN-38 and other toxin molecules.
  • the antibody of the present invention may be a multispecific antibody, preferably a bispecific antibody.
  • Bispecific monoclonal antibody BsAb
  • Another targeting molecule of the bispecific antibody may be a surface marker of an immune cell, and the surface marker molecule of the immune cell may be a T cell surface marker, which forms a bifunctional antibody (BispecificT cell engager, BiTE) with the antibody of the present invention in which T cells participate.
  • the T cell surface marker molecule is CD3.
  • the bispecific antibody comprises an antibody against CD123 described in the present invention, and further comprises an antibody against an immune checkpoint, and the surface molecule targeted by the immune checkpoint antibody may be: PD1, PDL1, CTLA4, CD47, TIGHT, TIM3, BTLA, LMTK3, etc.
  • the bispecific antibody comprises the antibody against CD123 described in the present invention, and further comprises an antibody against a tumor surface molecule, wherein the tumor surface molecule includes but is not limited to: CD19, CD20, CD123, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1 or TAG-72.
  • the tumor surface molecule includes but is not limited to: CD19, CD20, CD123, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH
  • the antibody or its antigen-binding fragment is selected from ScFv, Fab, Fab', bispecific antibody and multispecific antibody, and can be combined with immune checkpoint inhibitors, including but not limited to: CTLA-4 inhibitors, PD-1/PD-L1 inhibitors, LAG-3 (Lymphocyte Activation Gene-3) inhibitors, TIM-3 (T cell immunoglobulin-3), TIGIT (T cell immunoglobulinand ITIM domain protein), BTLA inhibitors.
  • the inhibitor is a small molecule drug, and in some embodiments, it can be an antibody, active peptide, etc.
  • the ScFv, Fab, Fab', bispecific antibodies and multispecific antibodies of the present invention can be combined with chemical drugs, such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cyclophosphamide, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228.
  • chemical drugs such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cyclophosphamide, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228.
  • scFv refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.; the linker does not need to affect the function or properties of the antibody itself, and any of the previously disclosed linkers can be used.
  • the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof as described above, or comprising a nucleotide sequence encoding the chimeric antigen receptor as described above.
  • the nucleic acid molecule encodes the chimeric antigen receptor as described in any one of the above, and the nucleotide sequence encoding the light chain variable region of the 1h13 antibody is as shown in SEQ ID NO.21 or its functional variant; wherein the nucleotide sequence encoding the heavy chain variable region of the 1h13 antibody is as shown in SEQ ID NO.22 or its functional variant.
  • the present invention also provides an expression vector, which comprises the nucleic acid molecule described above.
  • the expression vector is selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, a DNA vector, an RNA vector, and a plasmid.
  • the lentiviral vector is selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).
  • HAV-1 human immunodeficiency virus 1
  • HMV-2 human immunodeficiency virus 2
  • VMV visna-maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anemia virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • SIV simian immunodeficiency virus
  • the vector comprises a left (5') retroviral LTR, a Psi ( ⁇ ) packaging signal, a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor encompassed herein, and a right (3') retroviral LTR.
  • the chimeric antigen receptor comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).
  • HPRE hepatitis B virus post-transcriptional regulatory element
  • WPRE woodchuck post-transcriptional regulatory element
  • oPRE optimized woodchuck post-transcriptional regulatory element
  • the promoter of the 5'LTR is replaced by a heterologous promoter.
  • heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter or a simian virus 40 (SV40) promoter.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • SV40 simian virus 40
  • the 5'LTR or 3'LTR is a lentiviral LTR.
  • the 3'LTR is a self-inactivating (SIN) LTR.
  • the present invention also provides an engineered cell, wherein the cell is transduced with the above-mentioned nucleic acid molecule or the above-mentioned expression vector, or is obtained by transfecting an immune cell with the above-mentioned recombinant plasmid, or the immune engineered cell comprises any of the above-mentioned chimeric antigen receptors.
  • the cell is a T cell, a T cell precursor, a macrophage, a NKT cell, a ⁇ T cell or a NK cell.
  • the present invention also provides a cell product, which comprises the engineered cells as described above.
  • the chimeric antigen receptor as described in any one of the above or the nucleic acid molecule as described above or Use of the above-mentioned expression vector, the above-mentioned engineered cell, or the above-mentioned cell product in the preparation of anti-tumor drugs.
  • the present invention also provides the use of any of the above-mentioned antigens or antigen-binding fragments, any of the above-mentioned nucleic acid molecules, any of the above-mentioned recombinant plasmids, any of the above-mentioned chimeric antigen receptors, and any of the above-mentioned immune-engineered cells in the preparation of anti-tumor/autoimmune inflammatory drugs.
  • the tumor/autoimmune inflammation is B-cell lymphoma, diffuse large B-cell lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), acute lymphocytic leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, systemic lupus erythematosus.
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • AML acute myeloid leukemia
  • AML acute lymphocytic leukemia
  • non-Hodgkin's lymphoma non-Hodgkin's lymphoma
  • chronic lymphocytic leukemia systemic lupus erythematosus.
  • the anti-tumor drug is a drug for tumors expressing CD123.
  • drugs against CD123-expressing tumors include drugs against acute lymphoid leukemia, drugs against chronic lymphocytic leukemia, drugs against chronic myeloid leukemia, drugs against non-Hodgkin's lymphoma, drugs against Hodgkin's lymphoma and drugs against acute myeloid leukemia.
  • the CD123scFv in the present invention is applicable to all CAR structures, not just limited to those structures listed in the following examples.
  • the term "functional variant” generally refers to an amino acid sequence having substantially the same function (e.g., having the properties of the chimeric antigen receptor) and having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity therewith.
  • the variant of the amino acid sequence has substantially the same function therewith.
  • the CD123 sequence in the present invention can be used together with all other currently disclosed target sequences to construct a dual-target chimeric antigen receptor, such as a dual-target chimeric antigen receptor together with all currently disclosed scFvs such as CD19, CLL-1, CD33, etc.
  • the cell preparations of the present invention also include other active agents that can enhance the expression activity of CAR.
  • the agent that enhances the activity of CAR-expressing cells can be an agent that blocks inhibitory molecules.
  • Inhibitory molecules such as PD1 can reduce the ability of CAR-expressing cells to initiate immune effector responses in some embodiments.
  • Inhibitory molecules include PD1, PD -L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFR ⁇ ) and TGFR ⁇ .
  • the extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as PD1CAR.
  • the activating agent that enhances the activity of CAR-expressing cells can also be an activating agent of a cytokine receptor chemokine receptor.
  • the cytokine receptor may be derived from a type I cytokine receptor, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15 or IL-21.
  • additional therapeutic agents can be used in combination with the compositions described herein.
  • additional therapeutic agents include PD-1 inhibitors, such as nivolumab, pembrolizumab, pidilizumab, and atezolizumab.
  • additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib, ofatumumab, rituximab, bevacizumab, trastuzumab, imatinib, cetuximab, panitumumab, catumaxomab, ibritumomab, tositumomab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib ), sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazo
  • the cell product can also be used in combination with some therapeutic means, and the therapeutic means can be surgery, chemotherapy, and radiation.
  • the invention also provides a pharmaceutical composition, which includes the antigen or antigen-binding fragment described in any one of the above, the nucleic acid molecule described in any one of the above, the recombinant plasmid described in any one of the above, the chimeric antigen receptor described in any one of the above, the immune engineered cell described in any one of the above, and the cell product described in any one of the above.
  • the present invention also provides a use, which is the use of any one of the above antibodies or antigen-binding fragments thereof in the preparation of a detection reagent/detection kit.
  • the antibody or antigen-binding fragment thereof in the present invention can be linked to a detectable label.
  • the detectable label in the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means.
  • Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for
  • retroviruses provide a convenient platform for gene delivery systems.
  • the gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the cells of a subject in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • a lentiviral vector is used. body.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-4, Cold Spring Harbor Press, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • the method for preparing genetically modified immune cells includes at least one of the following steps: 1 taking a blood sample, 2 enrichment, 3 sorting, 4 activation, 5 transfection, and 6 re-enrichment.
  • the CAR-T preparation method used is a rapid preparation method, and the steps are as follows: 1. Peripheral blood nuclear cell separation: the collected whole blood or single-collected nucleated cells can be frozen; 2. Peripheral blood nuclear cell activation 0-72h: the separated or resuscitated mononuclear cells are sorted by immunomagnetic beads to obtain T lymphocytes or NK cells, which can be frozen; specifically, the sorted T lymphocytes or NK cells or the resuscitated T lymphocytes or NK cells are contacted with antibodies (which may contain magnetic beads) or small molecules of the complex and/or antibodies (which may contain magnetic beads) to co-stimulatory molecules on the surface of stimulating cells; 3. Transfection 0-72h: T lymphocytes or NK cells are contacted with lentiviral vectors; 4. Enrichment 14-72h: The finally obtained CAR-T/CAR-NK cells are collected.
  • Density gradient centrifugation can be used in any step of the above-mentioned rapid preparation, and the solution used can be a solution with a continuous or discontinuous density difference, specifically, polysucrose-diatrizoate with a specific gravity of 1.077 ⁇ 0.001, and the resuspension solution and the washing solution in the centrifugation step can be a buffer solution for maintaining the osmotic pressure of the cells, such as containing Na ions (such as physiological saline or physiological saline containing 0.5wt% to 2wt% HSA) or K ions (such as KCL solution or KCL aqueous solution containing 0.5wt% to 2wt% HSA) or containing protein components (such as different concentrations of human albumin, different concentrations of human autologous plasma).
  • Na ions such as physiological saline or physiological saline containing 0.5wt% to 2wt% HSA
  • K ions such as KCL solution or KCL aqueous solution
  • variable region of the single-chain antibody provided by the present invention is derived from a natural fully human antibody, and the sequence is completely derived from the human antibody gene library. Compared with mouse antibodies, chimeric antibodies, and humanized antibodies, its immunogenicity is greatly reduced, avoiding the immunogenicity of mouse antibodies and ensuring safety to the greatest extent in clinical applications.
  • the single-chain antibody provided by the present invention can specifically recognize the human CD123 antigen and can be applied to immunotherapy of blood or solid tumors targeting CD123 targets.
  • CD123 chimeric antigen receptor provided by the present invention binds to CD123 on tumor cells, it shows obvious anti-tumor activity, wherein the CD123-scFv is a fully humanized antibody with high affinity for human CD123 antigen.
  • the single-chain antibody provided by the present invention has good affinity performance and can bind to CD123 positive cells in flow cytometry detection.
  • Figure 1 is a flow cytometry staining image of 293T cells with fully human ScFv FK-22-C08;
  • Figure 2 is a flow cytometry staining image of 293T-CD123 cells with fully human ScFv FK-22-C08;
  • FIG3 shows the binding kinetics of fully human ScFvFK-22-C08CD123 to CD123 antigen
  • FIG. 4 shows the in vitro killing efficiency of CAR-1-CAR-3
  • FIG. 5 shows the in vitro killing efficiency of CAR-1, CAR-2, and CAR-4;
  • FIG. 6 shows the secretion of CAR-1, CAR-2, and CAR-4 factors
  • Figure 7 shows the in vitro killing efficiency of CAR-1, CAR-2, CAR-5, and CAR-6;
  • FIG. 8 shows the secretion of CAR-1, CAR-2, CAR-5, and CAR-6 factors
  • Figure 9 shows the in vitro killing efficiency of CAR-2, CAR-6, CAR-7, and CAR-8;
  • FIG. 10 shows the secretion of CAR-2, CAR-6, CAR-7, and CAR-8 factors
  • FIG. 11 shows the in vitro killing efficiency of CAR-1 and CAR-2 modified T cells
  • FIG. 12 shows the secretion of CAR-1 and CAR-2 factors
  • Figure 13 is the in vivo efficacy evaluation of NK cells modified with CAR-1 and CAR-2;
  • FIG. 14 shows the in vitro killing efficiency of CAR-1, CAR-8 and CAR-9;
  • FIG. 15 shows the factor secretion of CAR-1, CAR-8 and CAR-9.
  • phage samples centrifuge the overnight cultured phage monoclonal recombinant bacterial solution at 4°C and 8000 rpm for 10 min, and take the supernatant as the test sample;
  • Antibody incubation Wash the plate 3 times with PBS in a plate washer, add 100 ⁇ L of the phage monoclonal recombinant solution to be tested to each well, and incubate at 37°C for 1 hour;
  • Color development Wash the plate 6 times with a plate washer, add 100 ⁇ L of TMB color development solution to each well, and place at room temperature away from light for 25 minutes for color development;
  • Termination Add 50 ⁇ L 2M H2SO4 to each well to terminate the reaction;
  • Detection Place the test plate in a microplate reader to detect the OD450 absorbance. Phage clones that are 2.5 times higher than the negative control are positive clones.
  • Ficoll separation solution is used for PBMC separation, and the Ficoll separation solution is slowly added to normal human blood so that the Ficoll separation solution and normal human blood maintain a clear separation interface.
  • a 50mL centrifuge tube containing the blood and the separation solution is centrifuged at about 15°C for 20min. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid, wherein at the junction of the upper and middle layers, there is a narrow band of white cloud layer mainly composed of PBMC, i.e., the PBMC cell layer. Carefully suck off the upper plasma mixture with a sterile Pasteur pipette, and then suck PBMC with a new sterile Pasteur pipette to obtain separated PBMC.
  • the CD123 protein with the His-Fc tag is co-incubated with proteinG magnetic beads to prepare CD123-proteinG coupled magnetic beads, which are then drawn into the prepared fully human single-chain antibody library phage panning. After 3-4 rounds of co-incubation, washing and elution, specific monoclonal antibodies against the antigen can be enriched.
  • Blocking Dissolve 5% skim milk powder in PBS, filter through a 0.45 ⁇ m filter membrane and use as blocking solution. Resuspend the phage and CD123-proteinG-coupled magnetic beads with appropriate amount of blocking solution, and mix by rolling at 160rpm for 1h at room temperature.
  • washing Place the magnetic bead-phage mixture on a magnetic rack, discard the supernatant, and add an appropriate volume of 0.1% Tween-80 PBST washing solution to wash the magnetic beads.
  • the number of washes was determined as follows: the first round of panning consisted of 5 washes with PBST and 5 washes with PBS, the second round of panning consisted of 6 washes with PBST and 6 washes with PBS, and the third round of panning consisted of 3 washes with 5% FBS and 1 wash with PBS;
  • the phages were inoculated into TGI bacterial solution with an OD600 of about 0.5 for infection, and then centrifuged after standing at 37°C for 30 minutes. 200 ⁇ L of culture medium was reserved to resuspend the precipitate, which was then spread on a 2YTAG plate and inverted for overnight culture.
  • Plate washing Wash the plaques from the plates incubated overnight with the culture medium, which will be used as the seed solution for the next round of library packaging.
  • the specific process is the same as that for the preparation of the single-chain antibody library.
  • the monoclonal plaques finally released from the library were picked for ELISA detection and screening, and the phage clones binding to the CD123 antigen were obtained for downstream research.
  • the following scFv was obtained by panning, numbered: FK-22-C08; it has the specific binding ability to the human CD123 antigen.
  • 100ng of pComb3xss plasmid of positive phage clone was mixed with 100 ⁇ L competent Rosetta gami (DE3) bacteria and placed in an ice bath, heat shock for 90s, and then spread on an LB plate containing ampicillin resistance after ice bath, and placed in a 37°C constant temperature incubator for overnight culture; pick the formed monoclonal plaque and place it in LB medium, and after shaking culture, transfer the bacterial solution to 200mL LB medium, culture at 37°C 250rpm, and add IPTG when the bacterial solution OD reaches 0.5-1.0 and adjust the final concentration to 1mM to induce expression. Collect the bacteria by centrifugation and add PBS to resuspend the precipitate, ultrasonically break it for 2min, centrifuge the lysate, discard the precipitate, and collect the supernatant for protein purification.
  • DE3 Rosetta gami
  • the supernatant of bacterial lysis was filtered through 0.22 ⁇ m, diluted with an equal volume of PBS, and then enriched with GE Ni Sep harose excel purification column. After 5 column volumes of PBS were washed, impurities were removed by washing with 5 column volumes of imidazole-containing PBS solution. The protein was eluted with imidazole-containing PBS solution, and the washing solution was collected and concentrated with a 3KDa ultrafiltration tube. The sample was loaded on a GE molecular exclusion chromatography column, washed with PBS and the ultraviolet absorption peak was collected. After SDS-PAGE to identify the purification effect of scFv, flow staining was performed. Sex detection.
  • the monoclonal plaques finally released from the library were picked for chemiluminescence detection screening, and then re-tested with ELISA detection to obtain phage clones that bind to the CD123 antigen for use in downstream research.
  • Antibodies were screened and numbered h13 and h20 respectively.
  • 293T and 293T-CD123 cells were each divided into 1.5mL Eppendorf tubes, with 1 ⁇ 106 cells in each tube as target cells. All cells were centrifuged at 400g for 5min, and the supernatant was discarded. The cells were resuspended with 50 ⁇ L 15 ⁇ g/mL FK-22-C08 ScFv solution and incubated at 4°C in the dark. After 30min, 1mL PBS was added to resuspend the cells, washed at 400g for 5min, and the supernatant was discarded.
  • the ProA biosensor was subjected to baseline 1 for 60s in the analysis buffer, loading for 120s in a 30ug/mL CD12 3-hFc solution for ligand protein immobilization, transferred to baseline 2 in the analysis buffer for 120s, transferred to a gradient diluted analyte (CD123 ScFv) solution for association for 120s, and finally dissociation for 300s in the analysis buffer.
  • the change in the response value R is used to determine whether specific binding exists. Generally speaking, specific binding exists when the maximum response value Rmax after subtracting the reference is greater than 0.05nm and there is concentration dependence and dissociation.
  • the Data Analysis software that comes with the Fortebio Octet K2 instrument can provide the kinetic parameters represented by the binding-dissociation curve, such as the binding constant (Kon), the dissociation constant (Kdis), and the equilibrium dissociation constant (KD).
  • the unit of Kon is 1/Ms, which is used to express the rate of antigen-antibody binding. The higher the Kon, the faster the antibody-antigen binding to form a complex.
  • the unit of Kdis is 1/s, which is used to express the rate of antigen-antibody dissociation. The higher the Kdis, the faster the antibody-antigen binding to form a complex.
  • KD is the ratio of Kdis to Kon, which is used to comprehensively describe the difficulty of antigen-antibody binding.
  • the binding kinetics of the CD123-specific ScFv obtained by panning and the CD123 antigen are shown in Figure 3.
  • the scFv sequence was obtained by PCR amplification, and then the sequence was connected to a lentiviral vector containing different hinges, different transmembrane regions, and different co-stimulatory signals by restriction endonuclease digestion, thereby obtaining CAR plasmids targeting CD123 with different structures.
  • the structures are as follows (8h in the following structure indicates that the hinge region is derived from the hinge region of human CD8 ⁇ , 8TM indicates that the transmembrane region is derived from the transmembrane region of human CD8 ⁇ , BB indicates that the intracellular signaling domain is derived from human CD137, z indicates that the CD3 ⁇ chain, and 134 indicates that the intracellular Signaling domain, 2B4leader indicates the signal peptide domain derived from 2B4, 2B4 indicates the intracellular signaling domain of the 2B4 molecule, 2B4h indicates the hinge region is the extracellular hinge region derived from 2B4, 2B4TM indicates the transmembrane region is the transmembrane region derived from 2B4, NKp44TM indicates the transmembrane region is the transmembrane region derived from NKp44, SIRP ⁇ 1TM indicates the transmembrane region is the transmembrane region derived from SIRP ⁇ 1TM, GMR indicates the signal peptide of GM-CSFR, and 2
  • CAR-1 CD8 ⁇ -CD123 scFv(1h7)-8h-8TM-BBz
  • CAR-2 CD8 ⁇ -CD123 scFv(h13)-8h-8TM-BBz
  • CAR-3 CD8 ⁇ -CD123 scFv(h20)-8h-8TM-BBz
  • CAR-4 CD8 ⁇ -CD123 scFv(1h7)-8h-8TM-2B4z
  • CAR-6 GMR-CD123 scFv(h13)-8h-8TM-2B4z
  • CAR-7 GMR-CD123 scFv(h13)-8h-SIRP ⁇ 1TM-2B4z
  • CAR-8 GMR-CD123 scFv(h13)-8h-NKp44TM-2B4z
  • CAR-9 GMR-CD123 scFv(h13)-8h-NKp44TM-2B4-DAP10
  • CAR-1 to CAR-9 are transfected into NK cells, and CAR-1 and CAR-2 are transfected into T cells.
  • the calcium phosphate method is used to package the lentivirus, specifically: 293T cells are cultured with DMEM culture medium containing 10% FBS (w/v) to an optimal state, the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5mL centrifuge tube in a certain proportion, CaCl2 and 2 ⁇ H BS are added, and the mixture is allowed to stand at room temperature and then added to the treated 293T cell culture medium. After 3-5 hours, the medium is replaced again with 10mL DMEM culture medium containing 10% FBS. After 48 hours or 72 hours, the cell supernatant is collected, the virus is purified, and the titer is measured.
  • the packaging cell line for NK cells was prepared as follows: the CAR-1 to CAR-9 target plasmids were mixed with three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G in a ratio of 3:1:1:1, and the lentiviral packaging cell line was transfected by calcium transfection. After 6 hours of transfection, fresh 5% FBS-DMEM culture medium was replaced, and the crude virus extract was harvested 48 hours after transfection.
  • the prepared lentivirus titer was determined by using CHO cells. After 1e5/well of CHO cells were infected with the test virus for 48 hours, the total CAR expression was detected with Protein-L, and the positive rate was calculated.
  • the titer of the above CAR structure virus is shown in Table 3.
  • Lymphocytes were separated by gradient centrifugation. After centrifugation, the second white lymphocyte layer was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells.
  • the obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with the activated PBMC at a certain multiplicity of infection (MOI).
  • MOI multiplicity of infection
  • the positivity rate of CAR-T was detected on the 12th day after virus infection.
  • the detection method was flow cytometry and the antibody was Protein-L-PE. Protein-L could recognize the light chain of antibody.
  • the light chain of the scFv sequence in the CAR antigen recognition region could be recognized by Protein-L. Therefore, Protein-L could be used to detect the positivity rate and expression intensity of CAR.
  • NK cells For NK cells, after obtaining PBMC, CD56 antibody-labeled Microbeads need to be used for magnetic sorting to obtain CD56-positive NK cells.
  • NK cells can be transduced with lentivirus 24 hours after activation. Three days after transduction, the positive rate of CAR-NK needs to be detected using CD123-6 ⁇ His (ACRO-Biosystems) or monoclonal antibodies targeting the corresponding CD123-scFv.
  • CD123-6 ⁇ His ACRO-Biosystems
  • monoclonal antibodies targeting the corresponding CD123-scFv The results of the CAR-T/NK positive rate test using CD3-PE/Cy7 and CD56-BV510 (BioLegend) labeled NK cells are shown in Table 4. As shown in the table below, all constructed structures can be expressed well.
  • Example 4 CAR-1-CAR-3 modified NK cell killing experiment in vitro and cytokine secretion experiment
  • CD123+KG1a-Luc-GFP and MV-4-11-Luc-GFP cells were used as positive target cells.
  • CAR-NK cells were plated on target cells at a ratio of 1:1, and killing was detected by luciferase after 24 hours.
  • Luciferase principle During the test, the target cells are lysed with lysis buffer, and the luciferase in them will decompose the substrate to emit fluorescence.
  • Example 5 In vitro killing experiment and cytokine secretion experiment of NK cells modified by CAR-1, CAR-2 and CAR-4
  • CD123+KG1a-Luc-GFP and MV-4-11-Luc-GFP cells were used as positive target cells, and CAR-NK cells were plated on the target cells at a ratio of 1:1. After 24 hours, killing was detected by luciferase.
  • Luciferase principle During the test, the target cells are lysed with lysis buffer, and the luciferase in them will decompose the substrate to emit fluorescence.
  • the cell supernatant was collected 24 hours after killing to detect the IFN- ⁇ secretion ability of CAR-NK cells after being stimulated by target cells.
  • the collected supernatant was tested for IFN- ⁇ secretion using ELISA (enzyme-linked immunosorbent assay). The results are shown in Figure 6 and Table 7.
  • CAR-2 has a stronger IFN- ⁇ secretion ability, and the CAR structure composed of CD123 (1h7)-scFv
  • the modified CAR-NK and CAR-2 have higher factor secretion levels, therefore, CAR-2 constructed with CD123(h13)-scFv is preferred.
  • Example 6 In vitro killing experiment of NK cells and cytokine secretion experiment decorated with CAR-1, CAR-2, CAR-5 and CAR-6
  • CD123+KG1a-Luc-GFP and MV-4-11-Luc-GFP cells were used as positive target cells, and CAR-NK cells were plated on the target cells at a ratio of 1:1. After 24 hours, the killing was detected by luciferase.
  • the cell supernatant was collected 24 hours after killing, and the IFN- ⁇ secretion ability of CAR-NK cells after stimulation by target cells was detected.
  • the collected supernatant was used to detect the secretion of IFN- ⁇ by ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • Example 7 In vitro killing experiment and cytokine secretion experiment of NK cells modified by CAR-2, CAR-6, CAR-7, and CAR-8
  • the cell supernatant was collected 24 hours after killing, and the IFN- ⁇ secretion ability of CAR-NK cells after being stimulated by target cells was detected.
  • the collected supernatant was used to detect the secretion of IFN- ⁇ by ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • Example 8 CAR-1 and CAR-2 modified T cells in vitro killing experiment and cytokine secretion experiment
  • CAR-1 and CAR-2 modified CAR-T cells have obvious and effective killing against KG1a-Luc-GFP and MOLM-13-Luc-GFP, but the killing level for negative cells Raji-Luc-GFP is extremely low.
  • the cell supernatant was collected 24 hours after killing to detect the IFN- ⁇ secretion ability of CAR-T cells after stimulation by target cells.
  • the collected supernatant was used to detect the secretion of IFN- ⁇ by ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • CD123+ cells MV-4-11-Luc-GFP were used as positive target cells, and NCG mice were pre-tumored by tail vein injection at a dose of 1e6/mouse, and the pre-tumor time was 8 days.
  • CAR-1 and CAR-2 modified NK cells were injected into NCG mice with pre-tumored tumors at a dose of 6e6/mouse through the tail vein, and NK cells without CAR modification were injected at the same dose and method as the control group.
  • the fluorescent substrate of luciferase was injected intraperitoneally once a day, and the tumor load was detected by in vivo imaging using a small animal in vivo imaging instrument.
  • CAR-2 modified NK cells have the best in vivo efficacy advantage over CAR-1 modified NK cells and unmodified NK.
  • Example 10 In vitro killing experiment and cytokine secretion experiment of NK cells modified by CAR-1, CAR-8 and CAR-9
  • the cell supernatant was collected 24 hours after killing, and the IFN- ⁇ secretion ability of CAR-NK cells after being stimulated by target cells was detected.
  • the collected supernatant was used to detect the secretion of IFN- ⁇ by ELISA (enzyme-linked immunosorbent assay). The results are shown in Figure 15 and Table 15. After antigen stimulation, both CAR-8 and CAR-9 modified NK cells have a strong IFN- ⁇ secretion ability.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Hospice & Palliative Care (AREA)
  • Developmental Biology & Embryology (AREA)
  • Mycology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)

Abstract

L'invention appartient au domaine technique de l'ingénierie immunitaire cellulaire, et concerne en particulier un fragment de liaison à l'antigène entièrement humain et une variable de fragment à chaîne unique ciblant CD123, et son utilisation. La séquence variable de fragment à chaîne unique fournie par l'invention est complètement dérivée d'une bibliothèque de gènes d'anticorps humains. Par comparaison avec un anticorps murin, un anticorps chimérique et un anticorps humanisé, l'immunogénicité de la variable de fragment à chaîne unique est fortement réduite, et la sécurité peut être assurée au maximum dans une application clinique.
PCT/CN2023/128518 2022-11-03 2023-10-31 Anticorps entièrement humain ciblant cd123 et son utilisation WO2024094004A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202211370723.1 2022-11-03
CN202211370708.7 2022-11-03
CN202211370708.7A CN118027193A (zh) 2022-11-03 2022-11-03 靶向cd123的全人源抗体及其应用
CN202211370723.1A CN118027194A (zh) 2022-11-03 2022-11-03 靶向cd123的全人源抗体及其应用

Publications (1)

Publication Number Publication Date
WO2024094004A1 true WO2024094004A1 (fr) 2024-05-10

Family

ID=90929809

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/128518 WO2024094004A1 (fr) 2022-11-03 2023-10-31 Anticorps entièrement humain ciblant cd123 et son utilisation

Country Status (1)

Country Link
WO (1) WO2024094004A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271582A1 (en) * 2013-03-15 2014-09-18 City Of Hope Cd123-specific chimeric antigen receptor redirected t cells and methods of their use
WO2015193406A1 (fr) * 2014-06-17 2015-12-23 Cellectis Récepteur d'antigène chimère multichaînes spécifique de cd123
CN106103490A (zh) * 2014-03-19 2016-11-09 塞勒克提斯公司 用于癌症免疫疗法的cd123特异性嵌合抗原受体
CN107108744A (zh) * 2014-08-19 2017-08-29 诺华股份有限公司 抗cd123嵌合抗原受体(car)用于癌症治疗

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271582A1 (en) * 2013-03-15 2014-09-18 City Of Hope Cd123-specific chimeric antigen receptor redirected t cells and methods of their use
WO2014144622A2 (fr) * 2013-03-15 2014-09-18 Stephen Forman Lymphocytes t redirigés par des récepteurs d'antigènes chimériques spécifiques de cd123 et leurs procédés d'utilisation
CN106103490A (zh) * 2014-03-19 2016-11-09 塞勒克提斯公司 用于癌症免疫疗法的cd123特异性嵌合抗原受体
WO2015193406A1 (fr) * 2014-06-17 2015-12-23 Cellectis Récepteur d'antigène chimère multichaînes spécifique de cd123
CN107108744A (zh) * 2014-08-19 2017-08-29 诺华股份有限公司 抗cd123嵌合抗原受体(car)用于癌症治疗

Similar Documents

Publication Publication Date Title
AU2019203823B2 (en) CS1-specific chimeric antigen receptor engineered immune effector cells
US20230355673A1 (en) Chimeric antigen receptors targeting tim-1
RU2748281C2 (ru) Полностью человеческие антитела к мезотелину и иммунные эффекторные клетки, нацеленные на мезотелин
AU2019299439B2 (en) Chimeric receptors in combination with trans metabolism molecules enhancing glucose import and therapeutic uses thereof
CN107903324B (zh) 一种结合人cd19和cd3的双特异性抗体
CN109824778B (zh) 抗cd19全人抗体以及靶向cd19的免疫效应细胞
US10093746B2 (en) Glypican-3 antibody and uses thereof
JP7222600B2 (ja) 腫瘍細胞に特異的なキメラ抗原レセプター
US20220017625A1 (en) Tumor-specific anti-egfr antibody and application thereof
CN112119157A (zh) 前列腺特异性膜抗原car及其使用方法
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
JP6960947B2 (ja) 生体外での効率的な定向増幅用のキメラ抗原受容体及びその適用
WO2023046110A1 (fr) Cellules immunitaires artificielles co-exprimant ccr2b, leur preparation et leur application
CN111733186A (zh) 靶向cd19的人源化嵌合抗原受体制备及其应用
CN113717288B (zh) 逆转肿瘤微环境的融合蛋白及其应用
WO2018017649A1 (fr) Utilisation du récepteur des lymphocytes t couplé aux anticorps (actr) avec de multiples anticorps anticancéreux dans le traitement du cancer
WO2023072307A1 (fr) Fragment de liaison à l'antigène ciblant cd70, anticorps à chaîne unique, récepteur antigénique chimérique et utilisation associée
US11672827B2 (en) Pharmaceutical chimeric receptor composition and method thereof
WO2024094004A1 (fr) Anticorps entièrement humain ciblant cd123 et son utilisation
US20230144447A1 (en) Cd22-targeted chimeric antigen receptor, preparation method therefor and application thereof
CN107880131A (zh) 一种抗IL‑13Rα2和CD3双特异抗体及其应用
WO2024153124A1 (fr) Lymphocyte t primaire modifié et son utilisation
CN118027194A (zh) 靶向cd123的全人源抗体及其应用
WO2023016576A1 (fr) Récepteur d'antigène chimérique ciblant le bcma à base d'anticorps monocaténaire entièrement humain et murin et son utilisation
CN119708226A (zh) 靶向cd5全人源单链抗体的筛选及应用

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: 23884933

Country of ref document: EP

Kind code of ref document: A1