WO2023246908A1 - Préparation et utilisation d'une cellule immunitaire à récepteur antigénique chimérique ciblant csf1r - Google Patents

Préparation et utilisation d'une cellule immunitaire à récepteur antigénique chimérique ciblant csf1r Download PDF

Info

Publication number
WO2023246908A1
WO2023246908A1 PCT/CN2023/101865 CN2023101865W WO2023246908A1 WO 2023246908 A1 WO2023246908 A1 WO 2023246908A1 CN 2023101865 W CN2023101865 W CN 2023101865W WO 2023246908 A1 WO2023246908 A1 WO 2023246908A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
amino acid
car
sequence
chimeric antigen
Prior art date
Application number
PCT/CN2023/101865
Other languages
English (en)
Chinese (zh)
Inventor
赵旭东
孙彬
马海燕
Original Assignee
四川大学华西医院
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 CN202210707586.XA external-priority patent/CN115806626B/zh
Priority claimed from CN202210707541.2A external-priority patent/CN115819614B/zh
Application filed by 四川大学华西医院 filed Critical 四川大学华西医院
Publication of WO2023246908A1 publication Critical patent/WO2023246908A1/fr

Links

Classifications

    • 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
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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

Definitions

  • the invention belongs to the field of immune cell therapy, and specifically relates to the preparation and application of chimeric antigen receptor immune cells targeting CSF1R.
  • Cancer is the second largest disease threatening human health. In 2018, there were 18.1 million new cancer patients and 9.5 million cancer deaths worldwide. It is estimated that by 2040, there will be 29.5 million new cancer cases and 16.4 million deaths every year. Although traditional cancer treatments such as radiotherapy, chemotherapy, and surgical resection can delay the survival of cancer patients, the patient's declining quality of life and easy recurrence still restrict traditional cancer treatments.
  • Chimeric Antigen Receptor-T cell (CART) T cells refer to T cells that have been genetically modified to recognize specific target antigens in an MHC-unrestricted manner and to continuously activate and expand.
  • the structure of CAR includes a tumor-associated antigen binding region, extracellular hinge region, transmembrane region and intracellular signaling region.
  • CART therapy has shown strong killing ability in hematological malignancies.
  • the application of CART therapy in solid tumors is limited by tumor heterogeneity, lack of tumor-specific antigens, and tumor immunosuppressive microenvironment.
  • Colony-stimulating factor 1 receptor belongs to the platelet-derived growth factor family. In addition to being expressed in myeloid cells such as macrophages, Langerhans cells, and osteoclasts, CSF1R is also overexpressed in various tumors such as breast cancer, gastric cancer, and colorectal cancer. Inhibiting or knocking down CSFIR can increase the apoptosis of T-cell lymphoma cells. At the same time, inhibiting CSF1R activity can inhibit tumor growth in mouse transplant tumor models. In addition, studies have demonstrated that activation of the CSF1R paracrine pathway in osteosarcoma can promote tumor invasion, and autocrine activation of CSF1R in breast cancer is associated with tumor metastasis and growth and implies poor prognosis.
  • CSF1R also exists widely in the tumor microenvironment (Tumor Microenvironment, TME).
  • TME Tumor Microenvironment
  • the CSF1R signaling pathway activates a variety of proteins by regulating tyrosine phosphorylation, promoting the differentiation of myeloid cells, the orientation of monocytes, and the survival, proliferation, and chemotaxis of macrophages.
  • CSF1R regulates the function and survival of tumor-associated macrophages (TAMs), which play a crucial role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression, and therapy.
  • TAMs tumor-associated macrophages
  • CSF1R expression can also be detected on tumor-associated dendritic cells, tumor-associated neutrophils, and myeloid-derived suppressor cells.
  • CSF1 is the ligand of CSF1R.
  • CSF1R is an important target for tumor treatment.
  • Targeting CSF1R may inhibit tumor growth, invasion, metastasis and drug resistance, thereby prolonging the survival rate of patients.
  • the purpose of the present invention is to provide a chimeric antigen receptor immune cell targeting the CS1 receptor (CSF1R) and its preparation and application methods.
  • a chimeric antigen receptor (CAR)
  • the CAR contains an extracellular binding domain
  • the extracellular binding domain includes:
  • the extracellular binding domain can specifically bind to the CSFl receptor.
  • the binding is ligand-receptor binding.
  • the CSFl receptor includes CSF1R located on the cell membrane.
  • the CSF1R is derived from humans or non-human mammals.
  • the non-human mammals include: rodents (such as rats, mice), primates (such as monkeys); preferably primates.
  • the extracellular binding domain of the CAR in addition to the first extracellular domain targeting CSF1R, also includes a second extracellular domain targeting additional targets.
  • the additional target is a tumor-specific target.
  • the extracellular binding domain has an amino acid sequence derived from CSFl.
  • the extracellular binding domain includes CSFl protein or a fragment thereof.
  • the extracellular binding domain includes wild-type or mutant CSFl protein domain.
  • the extracellular binding domain has the amino acid sequence shown in SEQ ID NO: 1, preferably the amino acid sequence at positions 33 to 554 of the sequence shown in SEQ ID NO: 1, and more Preferably, it has the amino acid sequence of positions 33 to 496 of the sequence shown in SEQ ID NO:1.
  • amino acid sequence of the extracellular binding domain is selected from the following group:
  • amino acid sequence of the extracellular binding domain is shown in positions 33 to 496 of SEQ ID NO: 1.
  • the extracellular binding domain has an amino acid sequence derived from IL34.
  • the extracellular binding domain includes IL34 protein or a fragment thereof.
  • the extracellular binding domain includes wild-type or mutant IL34 protein domain.
  • the extracellular binding domain has the amino acid sequence shown in SEQ ID NO: 10, preferably the amino acid sequence at positions 21 to 242 of the sequence shown in SEQ ID NO: 10.
  • amino acid sequence of the extracellular binding domain is selected from the following group:
  • amino acid residues are replaced, deleted, changed or inserted, or at its N terminus or C 1 to 30 amino acid residues are added to the end, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence is the same as SEQ ID NO :
  • the sequence shown at positions 21 to 242 of the sequence shown in 10 has ⁇ 85% (preferably ⁇ 90%, more preferably ⁇ 95%, such as ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%) Sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).
  • the structure of the CAR is as shown in Formula I below: L-EB-H-TM-C-CD3 ⁇ -RP (I)
  • Each "-" is independently a connecting peptide or peptide bond
  • L is none or signal peptide sequence
  • EB is an extracellular binding domain that specifically binds to CSF1R;
  • H is the null or hinge region
  • TM is the transmembrane domain
  • C is no or co-stimulatory signaling molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • RP is a null or reporter protein.
  • the reporter protein RP is a fluorescent protein (such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein).
  • the reporter protein RP is mKate2 red fluorescent protein.
  • the red fluorescent reporter protein RP (mKate2) also includes a self-cleavage recognition site located at its N-terminus, preferably a T2A sequence.
  • the amino acid sequence of the mKate2 red fluorescent protein is shown in SEQ ID NO: 2.
  • the L is a signal peptide selected from the following group of proteins: CD8, CD28, GM-CSF, CD4, CD137, CD7 or a combination thereof.
  • the L is a signal peptide derived from CD8.
  • amino acid sequence of L is shown in SEQ ID NO: 3.
  • the H is the hinge region of a protein selected from the following group: CD8, CD28, CD137, or a combination thereof.
  • the H is a hinge region derived from CD8.
  • amino acid sequence of H is shown in SEQ ID NO: 4.
  • the TM is the transmembrane region of a protein selected from the following group: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.
  • the TM is a transmembrane region derived from CD28.
  • amino acid sequence of the TM is shown in SEQ ID NO: 5.
  • the C is a costimulatory signal molecule selected from the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS(CD278), NKG2D, GITR, TLR2, or combinations thereof.
  • the C is a costimulatory signal molecule derived from 4-1BB.
  • amino acid sequence of C is shown in SEQ ID NO: 6.
  • amino acid sequence of the cytoplasmic signaling sequence derived from CD3 ⁇ is shown in SEQ ID NO: 7.
  • amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO: 8.
  • amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO: 11.
  • nucleic acid molecule encoding a chimeric antigen receptor as described in the first aspect of the invention is provided.
  • the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 9.
  • the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 12.
  • a vector is provided, said vector containing the nucleic acid molecule according to the second aspect of the present invention.
  • the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
  • the vector is a lentiviral vector.
  • the vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.
  • the vector is a pTomo lentiviral vector.
  • the vector also includes a promoter selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.
  • the vector includes the nucleotide sequence shown in SEQ ID NO:9 or SEQ ID NO:12.
  • a host cell in the fourth aspect of the present invention, contains the vector as described in the third aspect of the present invention or the exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated into the chromosome. Or express the CAR as described in the first aspect of the invention.
  • an engineered immune cell contains the vector as described in the third aspect of the present invention or the exogenous DNA as described in the second aspect of the present invention is integrated into the chromosome. Nucleic acid molecules or expressions of a CAR as described in the first aspect of the invention.
  • the engineered immune cells are selected from the following group: T cells, NK cells, NKT cells, macrophages, or combinations thereof.
  • the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).
  • the engineered immune cells are CAR-T cells.
  • a method for preparing engineered immune cells as described in the fifth aspect of the present invention comprising the following steps: converting the nucleic acid molecule as described in the second aspect of the present invention or the nucleic acid molecule as described in the second aspect of the present invention.
  • the vectors described in the three aspects are transduced into immune cells, thereby obtaining the engineered immune cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • a pharmaceutical composition which pharmaceutical composition contains the CAR as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, and the third aspect of the present invention.
  • the preparation is a liquid preparation.
  • the dosage form of the preparation is an injection.
  • the concentration of the engineered immune cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml ml.
  • a CAR as described in the first aspect of the present invention a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, or a vector as described in the third aspect of the present invention.
  • the host cells described in the fourth aspect, and/or the use of the engineered immune cells described in the fifth aspect of the present invention are used to prepare drugs or preparations for preventing and/or treating diseases with high expression of CSFl receptors.
  • the diseases associated with high CSF1R expression include but are not limited to tumors, aging, obesity, cardiovascular disease, diabetes, neurodegenerative diseases, infectious diseases, etc.
  • the diseases associated with high CSF1R expression include: tumors, aging, cardiovascular disease, obesity, etc.
  • the disease is a malignant tumor with high expression of CSF1R.
  • the high expression of CSF1R means that the ratio of CSF1R expression (F1) to expression under normal physiological conditions (F0) (i.e., F1/F0) ⁇ 1.5, preferably ⁇ 2, more preferably ⁇ 2.5.
  • the tumors include solid tumors and hematological tumors.
  • the solid tumor is selected from the following group: pancreatic cancer, osteosarcoma, breast cancer, gastric cancer, colorectal cancer, hepatobiliary cancer, bladder cancer, non-small cell lung cancer, ovarian cancer, and esophageal cancer. Cytoma, lung cancer, prostate cancer, nasopharyngeal cancer, or combinations thereof.
  • the blood tumor is selected from the following group: T-cell lymphoma, acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL) ), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • the tumor is pancreatic cancer.
  • the ninth aspect of the present invention there is provided a use of the engineered immune cells as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention, for preventing and/or treating cancer. or tumors.
  • the tumor is pancreatic cancer.
  • a method for treating diseases comprising administering to a subject in need of treatment an effective amount of engineered immune cells as described in the fifth aspect of the present invention, or as described in the seventh aspect of the present invention.
  • pharmaceutical compositions comprising administering to a subject in need of treatment an effective amount of engineered immune cells as described in the fifth aspect of the present invention, or as described in the seventh aspect of the present invention.
  • the disease is a disease with high expression of CSFl receptor.
  • the disease is cancer or tumor, preferably pancreatic cancer.
  • the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from the subject (autologous cells).
  • the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).
  • the method can be used in combination with other treatment methods.
  • the other treatment methods include chemotherapy, radiotherapy, targeted therapy and other methods.
  • FIG. 1 shows a schematic diagram of CSF1-CAR vector construction.
  • A is a schematic diagram of the CSF1 sequence
  • 1-32AA in CSF1 is the signal peptide
  • the 33-554AA sequence is the mature protein.
  • B is a schematic structural diagram of the CD19-CAR control vector and CSF1-CAR vector.
  • the signal peptide, hinge region, and transmembrane region are all derived from human CD8 molecules
  • 4-1BB is derived from human CD137
  • CD3 ⁇ is derived from human CD3
  • mKate2 is a fluorescent marker.
  • C is the enzyme digestion identification of pTomo-CSF1-CAR vector HindIII and PstI.
  • Figure 2 shows the CAR infection efficiency assay.
  • A is the fluorescence expression of T cells after CD19-CAR and CSF1-CAR infection for 72 hours
  • BF is the bright field
  • mKate2 is the fluorescence expression of CAR.
  • B is flow cytometric detection of fluorescence expression.
  • FIG. 3 shows immunofluorescence detection of CSF1R expression in different pancreatic cancer cell lines.
  • Figure 4 shows the gradient killing results of CSF1-CAR on different pancreatic cancer cell lines.
  • Figure 5 shows the IFN ⁇ release results after CSF1-CAR kills ASPC1 pancreatic cancer cell line.
  • FIG. 6 shows overexpression of CSF1R in the pancreatic cancer cell line PANC1.
  • A is a schematic diagram of the overexpression vector structure.
  • B is immunofluorescence detection of CSF1R expression in PANC1 cells.
  • C is flow cytometric detection of CSF1R expression.
  • Figure 7 shows the killing effect and IFN ⁇ release of CSF1-CAR on PANC1 overexpressing CSF1R.
  • Figure 8 shows that knocking down CSF1R expression in ASPC1 cells reduces the killing effect of CSF1-CAR.
  • A is the cell phenotype of ASPC1 cells 96 hours after knocking down CSF1R.
  • B is qPCR detection of CSF1RmRNA level.
  • C is the detection of CSF1-CAR killing of ASPC1-shCSF1R.
  • D is the detection of IL34-CAR killing of ASPC1-shCSF1R.
  • Figure 9 shows the inhibitory effects of CSF1-CAR and IL34-CAR on transplanted tumors in ASPC1 nude mice.
  • A shows the in vivo imaging of ASPC1 nude mouse transplanted tumors at different time periods after CART reinfusion.
  • B is a statistical graph of fluorescence intensity of transplanted tumors.
  • Figure 10 shows a schematic diagram of IL34-CAR vector construction.
  • A is a schematic diagram of the IL34 sequence, 1-20AA in IL34 is the signal peptide, and the 21-242AA sequence is the mature polypeptide.
  • B is a schematic structural diagram of the control plasmids CD19-CAR and IL34-CAR.
  • the signal peptide, hinge region, and transmembrane region are all derived from human CD8 molecules
  • 4-1BB is derived from human CD137
  • CD3 ⁇ is derived from human CD3
  • mKate2 is a fluorescent marker.
  • C is the enzyme digestion identification of pTomo-IL34-CAR vector HindIII and PstI.
  • Figure 11 shows the CAR transfection efficiency assay.
  • A is the fluorescence expression of T cells after CD19-CAR and IL34-CAR infection for 72 hours, where BF (upper row) is the bright field, and mKate2 (lower row) is the fluorescence expression of CAR.
  • B is flow cytometric detection of fluorescence expression.
  • Figure 12 shows the gradient killing results of IL34-CAR on different pancreatic cancer cell lines.
  • Figure 13 shows the IFN ⁇ release results after IL34-CAR kills ASPC1 pancreatic cancer cell line.
  • Figure 14 shows the killing effect and IFN ⁇ release of IL34-CAR on PANC1 overexpressing CSF1R.
  • Figure 15 shows the cytotoxic effect of IL34-CAR on CSF1R + /Syndecan-1 + MCF7 cells.
  • Figure 16 shows the results of ligand screening suitable for constructing chimeric antigen receptors.
  • the present invention has constructed a total of two CAR immune cells targeting CSF1R, whose extracellular binding domains are partial fragments of full-length CSF1 (i.e., amino acid sequence 33 to 496) and partial fragments of full-length IL34 (i.e., amino acid sequences 21 to 242). amino acid sequence).
  • CAR-T cells of the present invention have high specificity and excellent cell killing power
  • in vivo experiments also indicate that they have in vivo inhibitory capabilities. On this basis, the present invention was completed.
  • the term “contains” or “includes” can mean open, semi-closed, and closed. In other words, the term also includes “consisting essentially of” or “consisting of.”
  • Transduction refers to the process of delivering exogenous polynucleotides into host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to convert exogenous polynucleotides into host cells.
  • the polynucleotide is introduced into a host cell (eg, E. coli).
  • Gene expression or “expression” refers to the process of gene transcription, translation, and post-translational modification to produce the gene's RNA or protein product.
  • Polynucleotide refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. As used herein, the term polynucleotide refers to interchangeable single- and double-stranded molecules. Unless otherwise stated, the polynucleotides in any embodiment described herein include double-stranded forms and the two complementary single strands known or predicted to constitute the double-stranded form.
  • potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine, and proline; aspartic acid, glutamic acid Acid; asparagine, glutamine; serine, threonine, lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine .
  • the present invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.
  • Colony-stimulating factor receptor CSF1R
  • CSF1R Colony-stimulating factor receptor
  • T-cell lymphoma inhibiting or knocking down CSFIR can increase tumor cell apoptosis.
  • inhibiting CSF1R activity can inhibit tumor growth.
  • CSF1R widely exists in the tumor microenvironment (Tumor Microenvironment, TME).
  • TME Tumor Microenvironment
  • the CSF1R signaling pathway activates a variety of proteins by regulating tyrosine phosphorylation, promoting the differentiation of myeloid cells, the orientation of monocytes, and the survival, proliferation, and chemotaxis of macrophages.
  • CSF1R regulates the function and survival of tumor-associated macrophages (TAMs), which play a crucial role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression, and therapy.
  • TAMs tumor-associated macrophages
  • CSF1R expression can also be detected in tumor-associated dendritic cells, tumor-associated neutrophils, and myeloid-derived suppressor cells.
  • CSF1R has two ligands: colony-stimulating factor-1 (CSF-1) and IL34.
  • CSF-1 colony-stimulating factor-1
  • IL34 colony-stimulating factor-1
  • the binding of CSF1 to CSF1R is mainly through salt bonds, while the binding of IL34 to CSF1R requires Hydrophobic amino acids and hydrophobic interactions bond, and CSF1 binds to one CSF1R and IL34 binds to two CSF1Rs.
  • CSF1 is the ligand of CSF1R. It mainly exists in the circulation system in the form of proteoglycans and is secreted by a variety of cells of mesenchymal and epithelial origin. Various diseases, including infection, cancer, and chronic inflammatory diseases, can cause increased expression of CSF1 in the blood, and CSF1 binds to CSF1R to activate downstream signaling pathways.
  • Interleukin-34 is a cytokine discovered in 2008. It is found in spleen, thymus, heart, brain, lung, liver, kidney, testis, prostate, ovary, small intestine, colon and other tissues. Express. IL34 is a secreted homodimeric glycoprotein consisting of 242 amino acids in the human body with a molecular weight of 39kD. It is highly conserved between humans and chimpanzees (99.6% similarity) and 72% similarity between humans and mice.
  • PTP- ⁇ is highly expressed in a variety of tumors (including but not limited to lung cancer, uterine cancer, hepatocellular carcinoma, renal cancer, prostate cancer, glioma and astrocytoma). Activation of PTP- ⁇ increases the phosphorylation of multiple signaling pathways and promotes tumor metastasis.
  • IL34 binding to CSF1R can cause stronger ERK1/2 and AKT phosphorylation in a short period of time, thereby affecting cell morphology and phenotype.
  • internal survival signaling cascades are triggered to prevent cell death and defend against future insults.
  • accumulating evidence has revealed the importance of the IL-34/CSF1R axis in cancer chemoresistance.
  • IL-34 derived from cancer cells activates ERK1/2 and AKT downstream of CSF1R in an autocrine manner, thereby providing a critical survival signal for CSF1R-expressing cancer cells.
  • IL34 has three receptors: CSF1R, tyrosine phosphatase zeta receptor (the receptor-type protein-tyrosine phosphatase zeta, PTP- ⁇ ), and Syndecan-1.
  • IL34 receptor PTP- ⁇ is highly expressed in a variety of tumors (including but not limited to lung cancer, uterine cancer, hepatocellular carcinoma, renal cancer, prostate cancer, glioma and astrocytoma). Blocking PTP- ⁇ can inhibit glioblastoma. Blastoma tumors grow and extend survival in mice. Currently, there are multiple antibodies targeting PTP- ⁇ (7E4B11-SAP, SCB4380) that have shown very effective anti-tumor effects in vitro and in transplanted tumor models.
  • Syndecan-1 Another representative IL34 receptor is Syndecan-1.
  • Syndecan-1 is highly expressed in various tumors such as myeloma, melanoma, liver cancer, lung cancer, and pancreatic cancer.
  • the migration of bone marrow cells depends on the interaction between IL34 and Syndecan-1.
  • the binding of PTP- ⁇ and Syndecan-1 to IL34 depends on chondroitin sulfate. Both are transmembrane proteoglycans, with glycosaminoglycans outside the cell and PDZ domains at the intracellular C-terminus.
  • IL34 has the strongest binding ability to CSF1R, followed by PTP- ⁇ and Syndecan-1.
  • the Kd of IL34 and CSF1R is 10 -12 M
  • the Kd of IL34 and PTP- ⁇ is 10 -7 M
  • Kd with Syndecan-1 is approximately 10 -8 M. Therefore, the IL34-CAR of the present invention also has the potential to recognize PTP- ⁇ and Syndecan-1.
  • IL34-CART also has a killing effect on CSF1R + /Syndecan-1 + MCF7 cells.
  • the main way to construct CAR-T targeting specific tumor antigens is to design CAR based on related antibodies.
  • the antibody affinity is too low, the ability to target and bind to tumor cells is poor, and if the antibody affinity is too high, excessive immune responses may occur, which may lead to patient tolerance. Poor sex.
  • the present invention selects a receptor/ligand that naturally binds to the target molecule, and takes advantage of the conservative characteristics of the binding evolved by the two under natural conditions to design a CAR sequence whose affinity is more suitable and can better overcome artificially designed antibodies. Affinity inappropriateness issues.
  • the research of the present invention proves that CAR-T cells constructed using the natural ligand of CSF1R as the extracellular recognition domain can express well in vivo and produce tumor suppressive effects.
  • the present invention integrates the CSF1 fragment or IL34 fragment targeting CSF1R into a CAR vector for the first time through genetic engineering, and modifies related immune cells, thereby achieving specific killing of CSF1R-positive cells and can be used for the treatment of related diseases.
  • Chimeric Antigen Receptor consists of an extracellular antigen recognition region, a transmembrane region, and an intracellular costimulatory signal region.
  • the design of CAR has gone through the following process: the first generation CAR only has one intracellular signaling component, CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause short-term T cell proliferation and less cytokine secretion. , but cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical efficacy.
  • the second-generation CAR introduces a co-stimulatory molecule, such as CD28, 4-1BB, OX40, and ICOS, based on the original structure. Compared with the first-generation CAR, its function is greatly improved, further enhancing the persistence of CAR-T cells and its ability to target tumor cells. of lethality. On the basis of second-generation CAR, some new immune costimulatory molecules such as CD27 and CD134 are connected in series to develop into third- and fourth-generation CAR.
  • the extracellular segment of CAR can recognize a specific antigen and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines, thereby eliminating target cells.
  • the patient's autologous cells or allogeneic donors
  • CAR immune cells are isolated, activated and genetically modified to produce CAR immune cells, and then injected into the same patient. In this way, the probability of developing graft-versus-host disease is extremely low, and the antigen is recognized by immune cells in a non-MHC-restricted manner.
  • CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. Such a high response rate has not been achieved by any previous treatment method, triggering an upsurge in clinical research around the world.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements.
  • the extracellular domain may be an antibody ScFv based on the specific binding of an antigen-antibody, or may be a natural sequence or a derivative thereof based on the specific binding of a ligand-receptor.
  • the extracellular domain of the chimeric antigen receptor is a CSFl protein or a fragment thereof that can specifically bind to the CSF1R target site. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence from positions 33 to 496 of the sequence shown in SEQ ID NO:1.
  • the extracellular domain of the chimeric antigen receptor is an IL34 protein or a fragment thereof that can specifically bind to the CSF1R target. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence at positions 21 to 242 of the sequence shown in SEQ ID NO: 10.
  • the intracellular domain includes costimulatory signaling regions and zeta chain portions.
  • a costimulatory signaling domain refers to the portion of the intracellular domain that includes costimulatory molecules.
  • Costimulatory molecules are cell surface molecules that are required for effective lymphocyte response to antigen, rather than antigen receptors or their ligands.
  • Linkers can be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that serves to connect a transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the CAR of the present invention When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to fail to grow, be driven to death, or otherwise affected, and cause the patient's tumor burden to shrink or be eliminated.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain.
  • the antigen binding domain is fused to the intracellular domain of a combination of the CD28 signaling domain and the CD3 ⁇ signaling domain.
  • the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which means that compared with the amino acid sequence of positions 33 to 496 of SEQ ID NO: 1, there are at most 10, preferably At most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide; or compared with the amino acid sequence at positions 21 to 242 of SEQ ID NO: 10, At most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • the number of added, deleted, modified and/or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, and more preferably 1-33%. More preferably, it is 5-30%, more preferably, it is 10-25%, and even more preferably, it is 15-20%.
  • the number of added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, Optimally 1.
  • the CAR can be designed to include the transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, using one of the domains naturally associated with the CAR linked transmembrane domain. In some examples, transmembrane domains may be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thus minimizing interaction with the receptor complex. Interactions with other members.
  • the intracellular domain in the CAR of the present invention includes the 4-1BB costimulatory domain and the signaling domain of CD3 ⁇ .
  • the CAR is a CAR that can specifically target CSF1R.
  • a chimeric antigen receptor immune cell which contains the chimeric antigen receptor of the present invention that specifically targets CSF1R.
  • the chimeric antigen receptor immune cells of the present invention can be CAR-T cells, CAR-NK cells, or CAR-macrophages.
  • the chimeric antigen receptor immune cells of the present invention are CAR-T cells.
  • CAR-T cell As used herein, the terms "CAR-T cell”, “CAR-T” and “CAR-T cell of the present invention” all refer to the CAR-T cell described in the fifth aspect of the present invention.
  • CAR-T cells have the following advantages over other T cell-based treatments: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, targeting a certain Once the CAR gene construction of tumor markers is completed, it can be widely used; (3) CAR can use both tumor protein markers and glycolipid non-protein markers, expanding the target range of tumor markers; ( 4) Using the patient’s autologous cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • CAR-NK cells As used herein, the terms “CAR-NK cells”, “CAR-NK” and “CAR-NK cells of the present invention” all refer to the CAR-NK cells described in the fifth aspect of the present invention.
  • the CAR-NK cells of the present invention can be used for tumors with high expression of CSF1R.
  • Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen-specific pathways.
  • Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells of the body; (2) they release very A small amount of cytokines thus reduces the risk of cytokine storm; (3) It is easy to amplify in vitro and develop into "off-the-shelf" products. Otherwise, it is similar to CAR-T cell therapy.
  • Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating it from the cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.
  • the invention also provides vectors comprising the nucleic acid molecules of the invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia virus, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector.
  • This vector is suitable for replication and integration into eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.
  • the expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated by reference in their entirety.
  • the present invention provides gene therapy vectors.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
  • the expression vector can be provided to the 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 other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • a suitable vector will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected genes can be inserted into the vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the 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.
  • lentiviral vectors are used.
  • promoter elements can modulate the frequency with which transcription is initiated.
  • these are located in a region of 30-110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the promoter element The spacing between them can be increased by 50 bp before activity begins to decrease.
  • individual elements appear to act cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the invention.
  • an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification of populations of cells that are transfected or infected by the viral vector. Identify and select expressing cells.
  • the selectable marker can be carried on a separate stretch of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to evaluate 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 clearly indicated by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at appropriate times after the DNA has been introduced into the recipient cell.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82).
  • the reporter gene is a gene encoding mKate2 red fluorescent protein.
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.
  • the construct with a minimum of 5 flanking regions that shows the highest level of reporter gene expression is identified as the promoter.
  • Such promoter regions can be ligated to a reporter gene and used to evaluate the ability of an agent to regulate promoter-driven transcription.
  • the vector can be readily introduced into the host cell by any method known in the art, e.g. Mammal, bacterial, yeast or insect cells.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells including 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). A preferred method of introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenovirus and adeno-associated virus, among others. See, for example, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Exemplary colloidal systems useful as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).
  • an exemplary delivery vehicle is liposomes.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids can be encapsulated into the aqueous interior of the liposomes, dispersed within the lipid bilayer of the liposomes, attached via linker molecules associated with both the liposomes and the oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexed with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in solution.
  • Lipids are fatty substances, which may be naturally occurring or synthetic lipids.
  • lipids include lipid droplets that occur naturally in the cytoplasm as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the invention provides a method containing the chimeric antigen receptor CAR according to the first aspect of the invention, the nucleic acid molecule according to the second aspect of the invention, the vector according to the third aspect of the invention, or the fourth aspect of the invention.
  • Host cells or engineered immune cells according to the fifth aspect of the present invention and pharmaceutically acceptable carriers, diluents or excipients.
  • the formulation is a liquid formulation.
  • the preparation is injection Injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • the invention includes therapeutic applications of cells (eg, T cells) transduced with lentiviral vectors (LV) encoding expression cassettes of the invention.
  • the transduced T cells can target the tumor cell marker CSF1R, synergistically activate T cells, and induce immune cell immune responses, thus significantly improving their killing efficiency against tumor cells.
  • the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal, comprising the steps of administering to the mammal a CAR-cell of the present invention.
  • the present invention includes a type of cell therapy in which a patient's autologous T cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • This method has a very low probability of suffering from graft-versus-host disease, and the antigen is recognized by T cells in an MHC-free manner.
  • one CAR-T can treat all cancers that express this antigen.
  • CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • CAR-T cells of the invention can undergo robust in vivo T cell expansion for an extended amount of time.
  • CAR-mediated immune responses can be part of an adoptive immunotherapy step, in which CAR-modified T cells induce an immune response specific for the antigen-binding domain in the CAR.
  • CSF1R CAR-T cells elicit a cell-specific immune response against CSF1R.
  • Treatable cancers include tumors that are not vascularized or substantially unvascularized, as well as tumors that are vascularized.
  • Cancer includes non-solid tumors (such as hematological tumors, such as leukemias and lymphomas) and solid tumors.
  • Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas.
  • Solid tumors in the present invention include, but are not limited to, pancreatic cancer, osteosarcoma, breast cancer, gastric cancer, colorectal cancer, hepatobiliary cancer, bladder cancer, non-small cell lung cancer, ovarian cancer and esophageal cancer, glioblastoma, lung cancer, and prostate cancer. , nasopharyngeal cancer, etc.
  • the therapeutic application of the present invention is for the treatment of pancreatic cancer.
  • Hematologic cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia leukemias and myeloblastoid, promyelocytic, myelomonocytic, monocytic and erythroleukemias), chronic leukemias such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia , heavy chain diseases, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasi
  • the CAR-modified T cells of the present invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is human.
  • cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic, or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the invention provides methods of treating tumors comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
  • the CAR-modified T cells of the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • a pharmaceutical composition of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelates Adjuvants such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • the compositions of the present invention are preferably formulated for intravenous administration.
  • compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease - although appropriate dosages may be determined by clinical trials.
  • compositions of the invention to be administered can be determined by the physician, It takes into account the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease. It may generally be stated that pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight (including all integers within those ranges). value) application. T cell compositions can also be administered multiple times at these dosages.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). best for a specific patient Optimal dosages and treatment regimens can be readily determined by those skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into the tumor, lymph node or site of infection.
  • cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., before , simultaneously or subsequently) administered to a patient, such forms of treatment include, but are not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as for ARA-C) or natalizumab treatment in patients with MS or elfalizumab treatment in patients with psoriasis or other treatments in patients with specific tumors.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as for ARA-C) or natalizumab treatment in patients with MS or elfalizumab treatment in patients with psoriasis or other treatments in patients with specific tumors.
  • the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cellular compositions of the invention are administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, use of a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatments administered to a patient will vary depending on the precise nature of the condition being treated and the recipient of the treatment. Dosage proportions for human administration may be implemented in accordance with art-accepted practice. Generally, 1 ⁇ 10 6 to 1 ⁇ 10 10 CAR-T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, by intravenous infusion.
  • CSF1R is lowly expressed on the cell membrane of normal cells, but highly expressed on the cell membrane of tumor tissue and macrophages, so the CAR of the present invention specifically kills tumor cells and macrophages that highly express CSF1R on their membranes. , but has no killing effect on other cells or tissues that do not express or have low expression of CSF1R.
  • the present invention utilizes the binding mode of ligand and receptor instead of the binding mode of single chain variable region (ScFv) and antigen.
  • the conservation of receptor-ligand interactions determines that safety tests in animals, especially primates, can better reflect their safety in humans.
  • Table 2 shows the cell lines used in the examples.
  • CSF1-CAR vector construction artificially synthesized based on gene sequence information of CSF1 (NM_000757.6), human CD8 signal peptide, human CD8 ⁇ hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3 ⁇ intracellular region method or PCR method to obtain the corresponding nucleotide sequence.
  • the CD8 signal peptide and CSF1 extracellular region were synthesized, and the nucleotide sequence of the CAR molecule was digested with AgeI (Thermo) and NheI (Thermo), and the CD8 transmembrane region, The 4-1BB costimulatory domain and CD3 ⁇ signaling region were inserted into the lentiviral vector pTomo. Transform competent E. coli (Stbl3).
  • IL34-CAR vector construction The same method was used to construct the IL34-CAR vector based on the nucleotide sequence of IL34 (NM_001172772.2).
  • the recombinant plasmid was sequenced, and the sequencing results were compared to confirm whether the plasmid was correct.
  • the sequencing primers were universal sequencing primers. Both sequencing and enzyme digestion identification results showed that the CAR coding sequence was correctly inserted into the predetermined position of the plasmid ( Figure 1C and Figure 10C).
  • All plasmids were extracted using QIAGEN's endotoxin-free large extraction kit, and the purified plasmids were transfected into HEK-293T cells using Beyotime lipo6000 for lentivirus packaging.
  • HEK-293T cells were cultured in 15 cm culture dishes for virus packaging.
  • 2ml OPTIMEM-dissolved plasmid mixture core plasmid 20 ⁇ g, pCMV ⁇ R8.9 10 ⁇ g, PMD2.G 4 ⁇ g
  • 2ml OPTIMEM and 68 ⁇ l of lipo 6000 After standing at room temperature for 5 minutes, add the plasmid complex to the liposome complex and let stand at room temperature for 20 minutes.
  • the above mixture was added dropwise to HEK-293T cells, incubated at 37°C for 6 hours and then the medium was removed. Re-add pre-warmed complete medium.
  • Example 4 Detection of positive rate of infected CART cells by flow cytometry
  • CAR-T cells and NTD cells 72 hours after virus infection were collected by centrifugation respectively. After washing once with PBS, the supernatant was discarded and the cells were resuspended in PBS containing 2% FBS. The positive rate was detected by flow cytometry.
  • Figure 2B and Figure 11B show that flow cytometry was used to detect, indicating that the positive expression rate of CAR or mKate2CAR-T was approximately 50%.
  • (1) Cell immunofluorescence Spread the target cells on a disc in a 24-well plate, fix the cells with 4% paraformaldehyde (PFA) for 20 minutes after 24 hours, wash three times with PBST, 5 minutes each time; use 10% goat serum Block for 1 hour at room temperature and incubate overnight with an antibody that specifically recognizes CSF1R at four degrees. The next day, wash three times with PBST for five minutes each time. Incubate with CY5-labeled secondary antibody that specifically recognizes the primary antibody for 1 hour at room temperature. After washing three times with PBS, the nuclei were stained with DAPI. Confocal microscopy imaging.
  • PFA paraformaldehyde
  • PANC1, BXPC3, ASPC1, and MCF-7 cells After the virus infected PANC1, BXPC3, ASPC1, and MCF-7 cells, they were screened with Puromycin (1ug/ml) for 2 weeks, and PANC1, BXPC3, ASPC1, and MCF-7-luciferase cells were successfully obtained.
  • the target cells used include: target cells BXPC3 and ASPC1 that highly express CSF1R; target cells PANC1 that do not express or have low expression of CSF1R; and CSF1R + /Syndecan-1 + MCF7 cells.
  • MCF7 and PANC1-luciferase cells were digested and counted and then the cell density was adjusted to 2 ⁇ 10 4 /ml. Inoculate 100 ⁇ l of luciferase cells into a 96-well plate. Adjust the cell density of CAR-T and control cells to 1 ⁇ 10 5 /ml and inoculate them into a black 96-well plate according to E:T ratio of 5:1. Inoculate 100 ⁇ l in each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.
  • BXPC3 and ASPC1-luciferase cells were digested and counted and then the cell density was adjusted to 2 ⁇ 10 4 /ml. Inoculate 100 ⁇ l BXPC3 and ASPC1-luciferase cells in a 96-well plate. Adjust the cell density of CAR-T and control cells to 8 ⁇ 10 4 /ml. According to E:T, it is 0.5:1, 1:1, 2:1, 4:1 was inoculated into a black 96-well plate, and 100 ⁇ l was inoculated into each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.
  • the cell supernatant was collected and frozen at -80°C to detect IFN ⁇ release (see Example 8).
  • Cell killing was detected using the promega fluorescence detection kit.
  • the cells were treated with 20 ⁇ l of 1 ⁇ PLB lysis buffer for 20 minutes. After adding 100 ⁇ l of substrate to each well, the cells were immediately detected using a BioTek microplate reader.
  • Cytotoxic Killing Cells (1-target cell fluorescence value when containing effector cells/target cell fluorescence value when there are no effector cells) ⁇ 100%
  • the killing results of IL34-CAR on different pancreatic cancer cell lines are shown in Figure 12 and Figure 15.
  • the results show that the killing effect of IL34-CART cells on tumor cells with high expression of CSF1R gradually increases with the increase of the effect-to-target ratio (E:T). It has basically no killing effect on tumor cells that basically do not express CSF1R. It has no killing effect on CSF1R + / Syndecan-1 + MCF7 cells also had a significant killing effect.
  • the release of cytokines when the CAR-T cells of the present invention are co-incubated with target cells is detected.
  • the cell supernatant incubated in the cell killing experiment was used for detection.
  • the method is as follows: take the cell supernatant of the CAR-T cells of the present invention co-incubated with target cells in Example 7 and detect IFN ⁇ according to IFN gamma Human ELISA Kit (life technology).
  • Primers were designed based on the CDS region sequence of CSF1R, and the CDS sequence of CSF1R was amplified using 293T cell cDNA as a template and digested and ligated to construct the pTomo-CMV-CSF1R-T2A-luciferase-IRES-puro vector. Lentivirus was packaged as described in Example 2, and PANC1 cells were infected and selected with puromycin (1 ⁇ g/ml) for 2 weeks to obtain PANC1-CSF1R-luc cells.
  • PANC1-luc cells and PANC1-CSF1R-luc cells were digested and counted, and then the cell density was adjusted to 2 ⁇ 10 4 /ml. Inoculate 100 ⁇ l of luciferase cells into a 96-well plate, adjust the cell density of CAR-T/NTD cells to 1 ⁇ 10 5 /ml, and inoculate into a black 96-well plate according to E:T ratio of 5:1, with 100 ⁇ l in each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.
  • FIG. 7 The killing results of CSF1-CAR-T cells after overexpressing CSF1R in the pancreatic cancer cell line PANC1 are shown in Figure 7.
  • Figure 6 shows the detection of overexpression of CSF1R in PANC1 cells.
  • the results show that PANC1 overexpressing CSF1R was successfully constructed.
  • Figures 7 and 14 respectively show the killing effect and IFN ⁇ release of CSF1-CAR-T and IL34-CAR-T on PANC1 after overexpression of CSF1R.
  • the results showed that compared with the control group PANC1-con, the killing rate and IFN ⁇ release of CSF1-CAR-T cells and IL34-CAR-T cells on PANC1-CSF1R cells overexpressing CSF1R were significantly increased. This result shows that the killing effect of CSF1-CAR-T cells and IL34-CAR-T on CSF1R-overexpressing tumor cells is significantly enhanced.
  • Example 10 Effect of specific knockdown of CSF1R on the killing effect of CAR-T cells
  • shRNA sequence library targeting CSF1R provided by Sigma, select shRNAs that have been verified in the CDS region, and BLAST each shRNA selected on NCBI to ensure the specificity of the target.
  • the shRNAs are constructed into the pLKO.1 vector and passed Enzyme digestion identification and sequencing ensure the correctness of the knockdown vector.
  • shRNA virus packaging One day before transfection, 1 million HEK-293T cells per dish were inoculated into a 6cm dish for culture. Before transfection, replace the 6cm dish with 5ml of fresh culture medium (containing serum, without antibiotics); take two clean sterile centrifuge tubes and add 250 ⁇ l to each. Medium, then add 5 ⁇ g shRNA plasmid, 2.5 ⁇ g pCMV ⁇ R8.9, 1 ⁇ g PMD2.G plasmid to one tube, and gently pipet and mix with a gun; add 17 ⁇ l Lipo6000 TM transfection reagent to the other tube, and gently pipet and mix with a gun. .
  • shRNA virus infection One day before infection, 500,000 ASPC1 cells per well were seeded into a six-well plate for culture. Before infection, replace each well of the six-well plate with 1 ml of fresh culture medium (containing serum, without antibiotics), then add 1 ml of virus supernatant and 2 ⁇ l of polybrane (10 mg/ml) to prepare ASPC1-shCOO2 and ASPC1-shCSF1R cells; 24 hours later The complete culture medium was replaced, and shRNA knockdown efficiency was measured 96 hours later, and CAR-T cell killing test was performed.
  • CSF1-CAR-T test Inoculate 100 ⁇ l luciferase cells in a 96-well plate, adjust the cell density of CAR-T/NT cells to 1 ⁇ 10 5 , and inoculate them into a black 96-well plate according to E:T ratio of 4:1. Inoculate 100 ⁇ l per well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours before detecting the killing effect. As mentioned before, the killing of ASPC1 and ASPC1-shCSF1R by CSF1-CAR-T cells was detected by changes in fluorescence values.
  • IL34-CAR-T test Inoculate 100 ⁇ l luciferase cells into a 96-well plate, adjust the cell density of CAR-T cells to 8 ⁇ 10 4 /ml, and follow the E:T ratio of 4:1, 2:1, and 1:1 , 0.5:1 was inoculated into a black 96-well plate, and 100 ⁇ l was inoculated into each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours to detect the killing effect. The killing of ASPC1 and ASPC1-shCSF1R by IL34-CAR-T cells is detected by changes in fluorescence value.
  • Figure 8-A is a phenotypic diagram of knockdown CSF1R cells in ASPC1 cells.
  • Figure 8-B shows qPCR detection of CSF1RmRNA levels.
  • Figure 8-C shows the killing effect of CSF1-CAR-T on ASPC1 after silencing CSF1R.
  • Figure 8-D shows the killing effect of IL34-CAR-T on ASPC1 after silencing CSF1R.
  • Example 11 Inhibitory effect of CAR-T on ASPC1-luc nude mouse transplanted tumors
  • ASPCl-luc cells were constructed as described in Example 6. After digestion and counting of the ASPC1-luc cell line, 30% matrigel was added to adjust the cell density to 5 ⁇ 10 ⁇ 6/ml.
  • Six-week-old female NCG mice were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. Each mouse was subcutaneously inoculated with 100 ⁇ l of cell suspension, and CART cells were reinfused 7 days later. CART cells were prepared as described in Example 3.
  • mice were anesthetized with 0.025% Avertin (300 ⁇ l/20g), intraperitoneally injected with 200 ⁇ l D-luciferin plus salt (15mg/ml), and 10 minutes later, small animal in vivo imaging was performed, and according to the fluorescence Value size groups NTD, CD19-CAR, CSF1/IL34-CAR. Each mouse was infused with 1 ⁇ 10 ⁇ 7/200 ⁇ l CART cells through the tail vein. Thereafter, nude mice were imaged every 7 days.
  • chimeric antigen receptors were constructed using the methods of Examples 1-3, in which Sigmarl, Gmbp1, VAP, WDL, GIR, Vaspin, GRP78, TF-ED, SAC, Cop35 and PEP42 targeted GRP78, CSFl and IL34 targets Towards CSF1R.

Landscapes

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

Abstract

L'invention concerne la préparation et l'utilisation d'une cellule immunitaire à récepteur antigénique chimérique ciblant CSF1R. Plus précisément, l'invention concerne un récepteur antigénique chimérique (CAR) modifié sur la base d'un ligand CSF1R naturel, le CAR comprenant un domaine de liaison extracellulaire pouvant cibler spécifiquement un récepteur CSF1. La cellule immunitaire à CAR possède une spécificité élevée et une capacité de destruction hautement efficace, et des tests in vivo montrent qu'elle présente une excellente capacité de suppression de tumeur.
PCT/CN2023/101865 2022-06-21 2023-06-21 Préparation et utilisation d'une cellule immunitaire à récepteur antigénique chimérique ciblant csf1r WO2023246908A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202210707586.XA CN115806626B (zh) 2022-06-21 2022-06-21 一种基于csf1的嵌合抗原受体免疫细胞制备及其应用
CN202210707541.2A CN115819614B (zh) 2022-06-21 2022-06-21 一种基于il34的嵌合抗原受体免疫细胞制备及其应用
CN202210707541.2 2022-06-21
CN202210707586.X 2022-06-21

Publications (1)

Publication Number Publication Date
WO2023246908A1 true WO2023246908A1 (fr) 2023-12-28

Family

ID=89379198

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/101865 WO2023246908A1 (fr) 2022-06-21 2023-06-21 Préparation et utilisation d'une cellule immunitaire à récepteur antigénique chimérique ciblant csf1r

Country Status (1)

Country Link
WO (1) WO2023246908A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105121466A (zh) * 2013-04-12 2015-12-02 莫佛塞斯公司 靶向m-csf的抗体
CN105142659A (zh) * 2013-02-28 2015-12-09 爱丁堡大学董事会 Csf1治疗剂
CN106084030A (zh) * 2016-04-25 2016-11-09 北京大学 一种提高白细胞介素il‑34热稳定性的方法
CN108276495A (zh) * 2018-01-24 2018-07-13 博生吉医药科技(苏州)有限公司 靶向csf1r嵌合抗原受体修饰的nk92mi细胞和t细胞及其制法和应用
CN113508129A (zh) * 2019-01-15 2021-10-15 法国国家健康和医学研究院 突变的白介素-34(il-34)多肽及其在治疗中的用途
CN115806626A (zh) * 2022-06-21 2023-03-17 四川大学华西医院 一种基于csf1的嵌合抗原受体免疫细胞制备及其应用
CN115819614A (zh) * 2022-06-21 2023-03-21 四川大学华西医院 一种基于il34的嵌合抗原受体免疫细胞制备及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105142659A (zh) * 2013-02-28 2015-12-09 爱丁堡大学董事会 Csf1治疗剂
CN105121466A (zh) * 2013-04-12 2015-12-02 莫佛塞斯公司 靶向m-csf的抗体
CN106084030A (zh) * 2016-04-25 2016-11-09 北京大学 一种提高白细胞介素il‑34热稳定性的方法
CN108276495A (zh) * 2018-01-24 2018-07-13 博生吉医药科技(苏州)有限公司 靶向csf1r嵌合抗原受体修饰的nk92mi细胞和t细胞及其制法和应用
CN113508129A (zh) * 2019-01-15 2021-10-15 法国国家健康和医学研究院 突变的白介素-34(il-34)多肽及其在治疗中的用途
CN115806626A (zh) * 2022-06-21 2023-03-17 四川大学华西医院 一种基于csf1的嵌合抗原受体免疫细胞制备及其应用
CN115819614A (zh) * 2022-06-21 2023-03-21 四川大学华西医院 一种基于il34的嵌合抗原受体免疫细胞制备及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, PING ET AL.: "Effects of CSF1R-targeted chimeric antigen receptor-modified NK92MI & T cells on tumor-associated macrophages", IMMUNOTHERAPY, vol. 10, no. 11, 31 August 2018 (2018-08-31), pages 935 - 949, XP055981097, DOI: 10.2217/imt-2018-0012 *

Similar Documents

Publication Publication Date Title
CN108018299B (zh) 靶向bcma的嵌合抗原受体及其用途
CN109306016B (zh) 共表达细胞因子il-7的nkg2d-car-t细胞及其用途
CN109320615B (zh) 靶向新型bcma的嵌合抗原受体及其用途
WO2020135870A1 (fr) Cellule nk-92mi modifiée par un récepteur d'antigène chimère cd7 et son utilisation
CN109503721B (zh) 靶向cd19的嵌合抗原受体及其用途
CN111378625A (zh) 一种cxcl13趋化型car－t细胞的制备和应用
WO2021136040A1 (fr) Préparation et applications de cellule t de récepteur antigénique chimérique coexprimant une molécule immunomodulatrice
CN109777783B (zh) 表达cxcr2的car-nk92细胞、制备方法及其应用
CN113416260B (zh) 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
WO2021239020A1 (fr) Procédé d'immunothérapie pour combiner un récepteur d'antigène chimère et un interféron de type i et application de celui-ci
CN110923255A (zh) 靶向bcma和cd19嵌合抗原受体及其用途
CN110054698B (zh) 抗cd19抗体的新型cd19-car载体的构建及应用
WO2023217192A1 (fr) Préparation d'une cellule immunitaire de récepteur antigénique chimérique construite sur la base d'une protéine précurseur de msln et son utilisation
CN108707619B (zh) 靶向ror1的嵌合抗原受体及其用途
WO2020034081A1 (fr) Récepteur d'antigène chimère visant bcma et ses utilisations
CN115819614B (zh) 一种基于il34的嵌合抗原受体免疫细胞制备及其应用
CN115806626B (zh) 一种基于csf1的嵌合抗原受体免疫细胞制备及其应用
CN111378624A (zh) 一种靶向性抗肿瘤t细胞及其制备方法和应用
WO2022262764A1 (fr) Préparation et application d'une cellule immunitaire de récepteur antigénique chimérique à base de lox1
CN109897114B (zh) 具有自杀基因开关的靶向cd47的工程化免疫细胞
WO2023246908A1 (fr) Préparation et utilisation d'une cellule immunitaire à récepteur antigénique chimérique ciblant csf1r
WO2022151959A1 (fr) Cellule car t ciblant b7-h3 et son utilisation dans le traitement de la leucémie aiguë myéloïde
WO2023083195A1 (fr) Préparation pour cellule immunitaire de récepteur antigénique chimérique construite sur la base de gas6 et utilisation d'une cellule immunitaire de récepteur antigénique chimérique
WO2022262765A1 (fr) Préparation et utilisation d'une cellule immunitaire de récepteur antigénique chimérique construite sur la base de granzyme b
CN114907485A (zh) 一种以内源性蛋白质分子替代单结构域抗体的嵌合抗原受体

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: 23826553

Country of ref document: EP

Kind code of ref document: A1