CN115806626B - Preparation and application of chimeric antigen receptor immune cells based on CSF1 - Google Patents

Abstract

The invention provides a chimeric antigen receptor immune cell preparation constructed based on CSF1 and application thereof. In particular, the invention provides a Chimeric Antigen Receptor (CAR) based on CSF1 engineering, the CAR comprising an extracellular binding domain capable of specifically targeting the CSF1 receptor. The CAR immune cells have high specificity and high killing capacity, and have excellent tumor inhibition capacity through in vivo tests.

Description

Preparation and application of chimeric antigen receptor immune cells based on CSF1

Technical Field

The invention belongs to the field of immune cell treatment, and particularly relates to a preparation method and application of chimeric antigen receptor immune cells constructed based on CSF 1.

Background

Tumor is the second largest disease threatening human health, with 18,100,000 new tumor patients worldwide in 2018, tumor death cases 9,500,000. It is estimated that tumors increase 29,500,000 cases annually by 2040 years, with death cases 16,400,000. Although the traditional tumor treatment means such as radiotherapy, chemotherapy, surgical excision and the like can delay the survival time of tumor patients, the characteristics of the patients such as reduced life quality, easy recurrence and the like still restrict the traditional tumor treatment means.

Biological immunotherapy has become a fourth tumor treatment means following surgery, radiotherapy and chemotherapy, and will become a necessary means for future tumor treatment. Chimeric antigen antibody receptor (Chimeric Antigen Receptor-Tcell, CART) T cells refer to T cells that, after genetic modification, recognize a specific antigen of interest in an MHC non-limiting manner and continue to activate expansion. The CAR structure includes a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. At present, the CART therapy shows strong killing capability in hematological malignant tumors, but the application of the CART therapy in the solid tumors is limited by the existence of tumor heterogeneity, lack of tumor specific antigens, tumor immunosuppression microenvironment and the like of the solid tumors.

Colony stimulating factor receptors (Colony-stimulating factor receptor, CSF1R) belong to the platelet-derived growth factor family. In addition to being expressed in myeloid cells such as macrophages, langerhans cells, osteoclasts, CSF1R is also overexpressed in various tumors such as breast cancer, gastric cancer, colorectal cancer, and the like. Inhibition or knockdown of CSFIR can observe increased apoptosis of T cell lymphoma cells, while inhibition of CSF1R activity can inhibit tumor growth in a mouse engraftment tumor model. Furthermore, studies have demonstrated that activation of the CSF1R paracrine pathway in osteosarcoma can promote tumor invasion, and that autocrine activation of CSF1R in breast cancer is associated with tumor metastasis and growth and implies a poor prognosis.

CSF1R is also widely found in tumor microenvironments (Tumor Microenvironment, TME). The CSF1R signaling pathway promotes differentiation of myeloid cells, orientation of monocytes, and survival, proliferation and chemotaxis of macrophages by modulating tyrosine phosphorylation, activating a variety of proteins. In TME, CSF1R regulates the function and survival of tumor-associated macrophages (Tumor Associated Macrophages, TAM), which play a vital role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression and therapy. In addition to TAM, CSF1R expression can also be detected in tumor-associated dendritic cells, tumor-associated neutrophils, and myeloid-derived suppressor cells. CSF1 is a ligand for CSF 1R.

Thus, CSF1R is an important target for tumor treatment, and targeting CSF1R may be capable of inhibiting tumor growth, invasion, metastasis, and resistance, thereby prolonging survival of patients.

Thus, there is a great need in the art to develop chimeric antigen receptor immune cells targeting CSF1R and methods of treatment thereof.

Disclosure of Invention

The invention aims at providing chimeric antigen receptor immune cells targeting a CSF1 receptor and preparation and application methods thereof.

In a first aspect of the invention there is provided a Chimeric Antigen Receptor (CAR), said CAR comprising an extracellular binding domain, and said extracellular binding domain comprising the structure of CSF1 or a fragment thereof based on the amino acid sequence shown in SEQ ID NO. 1,

and, the extracellular binding domain is capable of specifically binding to CSF1 receptor.

In another preferred embodiment, the binding is ligand receptor binding.

In another preferred embodiment, the CSF1 receptor comprises CSF1R.

In another preferred embodiment, the CSF1 receptor comprises CSF1R located on a cell membrane.

In another preferred embodiment, the CSF1R is derived from a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises: rodents (e.g., rats, mice), primates (e.g., monkeys); preferably a primate.

In another preferred embodiment, the extracellular binding domain of the CAR comprises a second extracellular domain for an additional target in addition to the first extracellular domain for CSF1R.

In another preferred embodiment, the additional target is a tumor specific target.

In another preferred embodiment, the extracellular binding domain has an amino acid sequence derived from CSF 1.

In another preferred embodiment, the extracellular binding domain comprises a CSF1 protein or fragment thereof.

In another preferred embodiment, the extracellular binding domain comprises a wild-type and a mutant domain.

In another preferred embodiment, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO. 1, preferably has the amino acid sequence at positions 33 to 554 of the sequence shown in SEQ ID NO. 1, more preferably has the amino acid sequence at positions 33 to 496 of the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the extracellular binding domain is selected from the group consisting of:

(i) A sequence shown in positions 33 to 496 of the sequence shown in SEQ ID NO. 1; and

(ii) An amino acid sequence obtained by performing substitution, deletion, alteration or insertion of one or more amino acid residues, or adding 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to the N-terminus or C-terminus thereof based on the sequence shown at positions 33 to 496 of the sequence shown in SEQ ID NO. 1; and the amino acid sequence obtained has a sequence identity of ≡85% (preferably ≡90%, more preferably ≡95%, for example ≡96%,. Gtoreq.97%,. Gtoreq.98% or ≡99%) with the sequence shown at positions 33 to 496 of the sequence shown in SEQ ID NO. 1; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

In another preferred embodiment, the amino acid sequence of the extracellular binding domain is shown at positions 33 to 496 of SEQ ID NO. 1.

In another preferred embodiment, the CAR has the structure shown in formula I below:

L-EB-H-TM-C-CD3ζ-RP (I)

in the method, in the process of the invention,

each "-" is independently a connecting peptide or peptide bond;

l is an absent or signal peptide sequence;

EB is an extracellular binding domain that specifically binds to CSF1R;

h is a no or hinge region;

TM is a transmembrane domain;

c is an absent or co-stimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

RP is absent or reporter.

In another preferred embodiment, the reporter protein RP is a fluorescent protein (e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein).

In another preferred embodiment, the reporter protein RP is mKate2 red fluorescent protein.

In another preferred embodiment, the red fluorescent reporter protein RP (mKate 2) further comprises a self-cleaving recognition site, preferably a T2A sequence, at its N-terminus. In another preferred example, the amino acid sequence of the mKate2 red fluorescent protein is shown as SEQ ID NO. 2.

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, CD7 or a combination thereof.

In another preferred embodiment, L is a CD8 derived signal peptide.

In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO. 3.

In another preferred embodiment, said H is a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.

In another preferred embodiment, the H is a CD8 derived hinge region.

In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO. 4.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, the TM is a CD 28-derived transmembrane region.

In another preferred embodiment, the amino acid sequence of said TM is shown in SEQ ID NO. 5.

In another preferred embodiment, said C is a costimulatory signaling molecule of a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), NKG2D, GITR, TLR2, or combinations thereof.

In another preferred embodiment, said C is a costimulatory signaling molecule of 4-1BB origin.

In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO. 6.

In another preferred embodiment, the amino acid sequence of the cytoplasmic signaling sequence derived from CD3 zeta is shown in SEQ ID NO. 7.

In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO. 8.

In a second aspect of the invention there is provided a nucleic acid molecule encoding a chimeric antigen receptor according to the first aspect of the invention.

In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO. 9.

In a third aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.

In another preferred embodiment, the vector is a lentiviral vector.

In another preferred embodiment, the carrier is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM, pHCMV, pLBS.CAG, pHR, pLV, etc.

In another preferred embodiment, the vector is a pTomo lentiviral vector.

In another preferred embodiment, the carrier further comprises a member selected from the group consisting of: promoters, transcription enhancing elements WPRE, long terminal repeat LTR, and the like.

In another preferred embodiment, the vector comprises the nucleotide sequence shown as SEQ ID NO. 9.

In a fourth aspect of the invention there is provided a host cell comprising a vector or chromosome according to the third aspect of the invention incorporating an exogenous nucleic acid molecule according to the second aspect of the invention or expressing a CAR according to the first aspect of the invention.

In a fifth aspect of the invention there is provided an engineered immune cell comprising a vector or chromosome according to the third aspect of the invention incorporating an exogenous nucleic acid molecule according to the second aspect of the invention or expressing a CAR according to the first aspect of the invention.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of: t cells, NK cells, NKT cells, macrophages, or a combination thereof.

In another preferred embodiment, the engineered immune cell is a chimeric antigen receptor T cell (CAR-T cell) or a chimeric antigen receptor NK cell (CAR-NK cell).

In another preferred embodiment, the engineered immune cell is a CAR-T cell.

In a sixth aspect of the invention there is provided a method of preparing an engineered immune cell according to the fifth aspect of the invention comprising the steps of: transduction of a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention into an immune cell, thereby obtaining said engineered immune cell.

In another preferred embodiment, the method further comprises the step of performing functional and validity assays on the obtained engineered immune cells.

In a seventh aspect of the invention there is provided a pharmaceutical composition comprising a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and/or an engineered immune cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the formulation is a liquid formulation.

In another preferred embodiment, the formulation is in the form of an injection.

In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1X 10 ³ -1×10 ⁸ Individual cells/ml, preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

In an eighth aspect of the invention there is provided the use of a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, and/or an engineered immune cell according to the fifth aspect of the invention, for the preparation of a medicament or formulation for the prevention and/or treatment of a disease in which a CSF1 receptor is highly expressed.

In another preferred embodiment, the CSF1 receptor includes, but is not limited to, CSF1R.

In another preferred embodiment, the CSF1R high expression associated disease includes, but is not limited to, tumor, aging, obesity, cardiovascular disease, diabetes, neurodegenerative disease, infectious disease, and the like.

In another preferred embodiment, the CSF1R high expression associated disease comprises: tumors, aging, cardiovascular diseases, obesity, etc.

In another preferred embodiment, the disease is a malignant tumor in which CSF1R is highly expressed.

In another preferred embodiment, the CSF1R high expression means that the ratio of the CSF1R expression level (F1) to the expression level (F0) under normal physiological conditions (i.e., F1/F0) is 1.5 or more, preferably 2 or more, more preferably 2.5 or more.

In another preferred embodiment, the tumor comprises a solid tumor or a hematological tumor.

In another preferred embodiment, the solid tumor is selected from the group consisting of: pancreatic cancer, osteosarcoma, breast cancer, gastric cancer, colorectal cancer, hepatobiliary cancer, bladder cancer, non-small cell lung cancer, ovarian and esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal cancer, or combinations thereof.

In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: t cell lymphoma, acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse Large B Cell Lymphoma (DLBCL), or combinations thereof.

In another preferred embodiment, the tumor is pancreatic cancer.

In a ninth aspect of the invention there is provided the use of an engineered immune cell as described in the fifth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention, for the prevention and/or treatment of cancer or tumour.

In another preferred embodiment, the tumor is pancreatic cancer.

In a tenth aspect of the invention there is provided a method of treating a disease comprising administering to a subject in need of treatment an effective amount of an engineered immune cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.

In another preferred embodiment, the disease is a disease in which CSF1 receptor is highly expressed.

In another preferred embodiment, the disease is cancer or a tumor, preferably pancreatic cancer.

In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells).

In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).

In another preferred embodiment, the methods described can be used in combination with other therapeutic methods.

In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, and the like.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a schematic of CSF1-CAR vector construction.

Wherein A is a schematic diagram of CSF1 sequence, 1-32AA in CSF1 is signal peptide, and 33-554AA sequence is mature protein. B is a schematic structural diagram of a CD19-CAR control vector and a CSF1-CAR vector, wherein the signal peptide, the hinge region and the transmembrane region are all derived from a human CD8 molecule, 4-1BB is derived from a human CD137, CD3 zeta is derived from a human CD3, and mKate2 is a fluorescent label and used for detecting CAR expression. C is the HindIII and PstI digestion identification of pTomo-CSF1-CAR vector.

Figure 2 shows CAR infection efficiency detection. Wherein A is CD19-CAR, and CSF1-CAR is expressed by cell fluorescence after T cells are infected for 72 hours, BF is bright field, and mKate2 is expressed by CAR fluorescence. B is the flow detection fluorescence expression.

FIG. 3 shows immunofluorescence detection of CSF1R expression from different pancreatic cancer cell lines.

Figure 4 shows the gradient killing results of CSF1-CAR against different pancreatic cancer cell lines.

Figure 5 shows ifnγ release results after killing ASPC1 pancreatic cancer cell lines by CSF 1-CAR.

Fig. 6 shows overexpression of CSF1R in pancreatic cancer cell line PANC 1.

Wherein A is a schematic structural diagram of the over-expression vector. B is immunofluorescence detection of CSF1R expression in PANC1 cells. C is a flow assay for CSF1R expression.

Figure 7 shows the killing effect of CSF1-CAR on PANC1 overexpressing CSF1R and ifnγ release.

Figure 8 shows that knockdown CSF1R expression in ASPC1 cells reduces CSF1-CAR killing.

Wherein A is the phenotype of ASPC1 cells 96 hours after knocking down CSF1R. B is qPCR to detect CSF1RmRNA levels. C is the detection of killing of ASPC1-shCSF1R by CSF 1-CAR.

FIG. 9 shows the inhibition of ASPC1 nude mice engraftment by CSF 1-CAR.

Wherein A is the live imaging of ASPC1 nude mice transplanted tumor in different time periods of CART reinfusion. B is a fluorescence intensity statistical graph of the transplanted tumor.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result of extensive screening, developed for the first time a chimeric antigen receptor immune cell constructed based on CSF 1. The invention uses part of fragments (namely 33 to 496 amino acid sequences) in the full-length CSF1 as the extracellular binding domain of the CAR, and obtains the CAR-T cell targeting CSF 1R. In vitro experiments indicate that the CAR-T cells have high specificity and excellent cell killing power, and in vivo experiments also indicate that the CAR-T cells have in vivo inhibition capability. The present invention has been completed on the basis of this finding.

Terminology

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …" or "consisting of …".

"transduction," "transfection," "transformation," or the terms used herein refer to the process of transferring an exogenous polynucleotide into a host cell, and transcription and translation to produce a polypeptide product, including the use of plasmid molecules to introduce the exogenous polynucleotide into the host cell (e.g., E.coli).

"Gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce an RNA or protein product of the gene.

"Polynucleotide" refers to polymeric forms of nucleotides of any length, including Deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to single-and double-stranded molecules that are interchangeable. Unless otherwise indicated, polynucleotides in any of the embodiments described herein include a double stranded form and two complementary single strands that are known or predicted to constitute the double stranded form.

Conservative amino acid substitutions are known in the art. In some embodiments, the 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; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine. Furthermore, the invention provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.

The meaning of all parameters, dimensions, materials and configurations described herein will be readily understood by those skilled in the art. The actual parameters, dimensions, materials, and/or configurations may depend upon the specific application for which the invention is used. It will be appreciated by those skilled in the art that the examples or claims are given by way of example only and that the scope of the invention which can be covered by the embodiments of the invention is not limited to the specifically described and claimed scope within the scope of the equivalents or claims.

All definitions and uses herein should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.

All references, patents and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, and in some cases may contain the entire document.

It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these embodiments.

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.

The term "administering" refers to physically introducing a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion.

CSF1 and CSF1R

CSF1R is widely found in tumor microenvironments (Tumor Microenvironment, TME). The CSF1R signaling pathway promotes differentiation of myeloid cells, orientation of monocytes, and survival, proliferation and chemotaxis of macrophages by modulating tyrosine phosphorylation, activating a variety of proteins. In TME, CSF1R regulates the function and survival of tumor-associated macrophages (Tumor Associated Macrophages, TAM), which play a vital role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression and therapy. In addition to TAM, CSF1R expression can also be detected in tumor-associated dendritic cells, tumor-associated neutrophils and myeloid-derived suppressor cells.

CSF1 is a ligand for CSF1R, which exists in the circulatory system mainly in the form of proteoglycans, and is secreted by a variety of cells of mesenchymal and epithelial origin. A variety of diseases, including infection, cancer and chronic inflammatory diseases, cause increased expression of CSF1 in the blood, and CSF1 binds to CSF1R to activate downstream signaling pathways.

The current major CAR-T building approach to target specific tumor antigens is to design CARs based on related antibodies, however, the ability of antibodies to target tumor cells with too low an affinity is poor, excessive immune responses are likely to occur with too high an affinity, and patient tolerance is poor.

Therefore, the invention selects the receptor/ligand naturally combined with the target molecule, designs the CAR sequence by utilizing the characteristic of the combination conservation of the receptor/ligand and the ligand evolved under natural conditions, and has more suitable affinity, thereby better overcoming the problem of unsuitable affinity of the artificially designed antibody. The studies of the present invention demonstrate that CAR-T cells constructed using the natural ligand of CSF1R as an extracellular recognition domain are well expressed in vivo and produce tumor-inhibiting effects.

Based on the above, the invention integrates the CSF1 fragment into the CAR carrier for the first time in a genetic engineering way and modifies the related immune cells, thereby realizing the specific killing of the CSF1R positive cells and being applicable to the treatment of related diseases.

Chimeric Antigen Receptor (CAR) of the invention

Chimeric immune antigen receptor (Chimeric Antigen Receptor, CAR) consists of extracellular antigen recognition region, transmembrane region and intracellular co-stimulatory signaling region.

The design of the CAR goes through the following process: the first generation of CARs had only one intracellular signaling component, cd3ζ or fcγri molecule, which, due to the presence of only one activation domain within the cell, only caused transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve 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, and has greatly improved function compared with the first generation CAR, and further enhances the persistence of CAR-T cells and the killing ability to tumor cells. Some new immune co-stimulatory molecules such as CD27, CD134 are concatenated on the basis of the second generation CARs, developing into third and fourth generation CARs.

The extracellular segment of the CAR recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity, and secretion of cytokines, thereby clearing the target cell. Patient autologous cells (or heterologous donors) are first isolated, CAR-producing immune cells are activated and genetically engineered, and then injected into the same patient. This way the probability of graft versus host disease is very 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 hematological malignancy therapy, which is not achieved by any conventional therapeutic means, and has triggered a hot tide of clinical research worldwide.

In particular, the Chimeric Antigen Receptor (CAR) of the invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.

The extracellular domain includes a target-specific binding member. The extracellular domain may be ScFv of an antibody based on specific binding of an antigen-antibody, or may be a native sequence or a derivative thereof based on specific binding of a ligand-receptor.

In the present invention, the extracellular domain of the chimeric antigen receptor is a CSF1 protein or fragment thereof that specifically binds to a CSF1R target of a CAR of the invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence at positions 33 to 496 of the sequence shown as SEQ ID NO. 1.

The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

The linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to the extracellular domain 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 CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the CD28 signaling domain, and the cd3ζ signaling domain.

In the present invention, the extracellular binding domain of the CAR of the invention also includes sequence-based conservative variants, meaning that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence at positions 33 to 496 of SEQ ID NO. 1 to form a polypeptide.

In the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids of the original amino acid sequence.

In the present invention, the number of the added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, most preferably 1.

For hinge and transmembrane regions (transmembrane domains), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

The intracellular domains in the CARs of the invention include a 4-1BB costimulatory domain and a signaling domain of cd3ζ.

In one embodiment of the invention, the CAR is a CAR that can specifically target CSF 1R.

Chimeric antigen receptor immune cells (CAR-immune cells)

In the present invention, there is provided a chimeric antigen receptor immune cell comprising the chimeric antigen receptor of the present invention having a specific targeting CSF 1R.

The chimeric antigen receptor immune cells of the invention can be CAR-T cells, also can be CAR-NK cells and CAR-macrophages. Preferably, the chimeric antigen receptor immune cells of the invention are CAR-T cells.

As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to CAR-T cells according to the fifth aspect of the invention.

CAR-T cells have the following advantages over other T cell-based therapies: (1) the course of action of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor markers, CAR gene construction for a certain tumor marker can be widely utilized once completed; (3) The CAR can utilize not only tumor protein markers but also glycolipid non-protein markers, so that the target range of the tumor markers is enlarged; (4) The use of autologous patient cells reduces the risk of rejection; (5) The CAR-T cells have an immunological memory function and can survive in vivo for a long time.

As used herein, the terms "CAR-NK cells", "CAR-NK cells of the invention" all refer to CAR-NK cells of the fifth aspect of the invention. The CAR-NK cells of the invention can be used for tumors with high expression of CSF 1R.

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion by non-antigen specific pathways. New functions may be obtained by engineered (genetically modified) NK cells, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

CAR-NK cells also have advantages over CAR-T cells, such as: (1) The perforin and the granzyme are released to directly kill tumor cells, and the perforin and granzyme have no killing effect on normal cells of the organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storms; (3) Is easy to expand and develop into a ready-made product in vitro. In addition, similar to CAR-T cell therapy.

Carrier body

Nucleic acid sequences encoding a 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 vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may 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 for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used in 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. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid may be cloned into many types of vectors. For example, the nucleic acid may 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-generating vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are 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 may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act cooperatively or independently to initiate transcription.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may 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.

The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. Typically, the reporter gene is the following gene: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al 2000FEBS Letters479:79-82). In one embodiment of the invention, the reporter gene is a gene encoding a mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, constructs with a minimum of 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to regulate promoter-driven transcription.

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell 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 for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). A preferred method of introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery tool is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery means is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated into the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

Formulations

The invention provides an engineered immune cell comprising a chimeric antigen receptor CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, or a fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said CAR-T cells in said formulation is 1 x 10 ³ -1×10 ⁸ Individual cells/ml, more preferably 1X 10 ⁴ -1×10 ⁷ Each thinCells/ml.

In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications with cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding the expression cassettes of the invention. The transduced T cells can target a tumor cell marker CSF1R, and synergistically activate the T cells to cause immune cell immune response, so that the killing efficiency of the transduced T cells on the tumor cells is remarkably improved.

Accordingly, 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 the CAR-cells of the invention to a mammal.

In one embodiment, the invention includes a class of cell therapies in which autologous T cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This way the probability of graft versus host disease is very low and the antigen is recognized by T cells in a non-MHC restricted manner. Furthermore, a CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to persistent tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-modified T cells induce an immune response specific for an antigen binding domain in the CAR. For example, CAR-T cells of CSF1R elicit a cell-specific immune response against CSF 1R.

Although the data disclosed herein specifically disclose lentiviral vectors comprising CSF1 protein or a fragment thereof, hinge and transmembrane regions, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of variations to each of the construct components.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Cancers include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) and solid tumors. Types of cancers treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemia or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Solid tumors in the present invention include, but are not limited to, pancreatic cancer, osteosarcoma, breast cancer, gastric cancer, colorectal cancer, liver and gall cancer, bladder cancer, non-small cell lung cancer, ovarian and esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal cancer, etc., and preferably the therapeutic application of the present invention is for the treatment of pancreatic cancer.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematogenic) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous (myelogenous) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, hodgkin's disease, non-hodgkin's lymphomas (indolent and high grade forms), multiple myelomas, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

The CAR-modified T cells of the invention can also be used as a vaccine type for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) Expanding the cells, ii) introducing nucleic acid encoding the CAR into the cells, and/or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with vectors expressing the CARs disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.

In addition to the use of cell-based vaccines for ex vivo immunization, 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 a method of treating a tumor 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 a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the invention may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to "effective amount", "immunologically effective amount", "antineoplastic effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be administered at 10 ⁴ To 10 ⁹ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Individual cells/kg body weight doses (including all integer values within those ranges) are administered. T cell compositions may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intradesmally, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, 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.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapies for specific tumor patients. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.

The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, 1X 10 will be administered per treatment or per course of treatment ⁶ Up to 1X 10 ¹⁰ The CAR-T cells of the invention are administered to a patient by, for example, intravenous infusion.

The main advantages of the invention include:

1) Target specificity: CSF1R is low-expressed on the cell membrane of normal cells, but is high-expressed on tumor tissue cell membranes and macrophages, so that the CAR specifically kills tumor cells and macrophages on the membrane that highly express CSF1R, but has no killing effect on other cells or tissues that do not express or low-express CSF 1R.

(b) The present invention utilizes the mode of ligand binding to the receptor, rather than the mode of single chain variable region (ScFv) binding to the antigen. The conservation of receptor-ligand interactions determines that safety assays in animals, particularly primates, are more responsive to their safety in humans.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Reagents, plasmids, and cells in the examples of this application are commercially available unless otherwise indicated. Table 1 summarizes the sequences of the present invention.

Table 1 sequence summary of the sequences to which the present invention relates

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Table 2 shows the cell lines used in the examples.

TABLE 2 cell lines

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Example 1: preparation of CSF1-CAR vectors

Based on gene sequence information of CSF1 (NM-000757.6), human CD8 signal peptide, human CD8 alpha hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3 zeta intracellular region, the corresponding nucleotide sequence is obtained by artificial synthesis method or PCR method. The CD8 signal peptide and the extracellular domain of CSF1 are synthesized or, alternatively, the nucleotide sequence of the CAR molecule is double digested by AgeI (Thermo) and NheI (Thermo), and inserted into the lentiviral vector pTomo into which the CD8 transmembrane region, 4-1BB costimulatory domain, CD3 zeta signaling region have been inserted via a T4 DNA ligase (NEB) ligation. Competent E.coli (Stbl 3) was transformed.

Results: sequencing the recombinant plasmid, and comparing the sequencing results to confirm whether the plasmid is correct, wherein the sequencing primer is a universal sequencing primer. Both sequencing and restriction characterization showed that the coding sequence of the CAR was correctly inserted into the predetermined position of the plasmid (fig. 1C).

All plasmids were extracted with QIAGEN endotoxinfree megapump kit and purified plasmids were lentivirally packaged with Biyundian lipo6000 transfected HEK-293T cells.

Example 2: virus package

HEK-293T cells were cultured in 15cm dishes for virus packaging. 2ml of OPTIMEM-dissolved plasmid mixture (core plasmid 20. Mu.g, pCMV. DELTA.R 8.9. Mu.g, PMD2.G 4. Mu.g) was prepared after transfection at about 80% -90% confluence of HEK-293T cells; in another centrifuge tube 2ml OPTIMEM and 68. Mu.l lipo 6000. After standing at room temperature for 5min, the plasmid complex was added to the liposome complex, and standing at room temperature for 20min. The mixture was added dropwise to HEK-293T cells and the medium was removed after incubation at 37℃for 6 hours. The preheated complete medium was re-added. After collecting the virus supernatant for 48 hours and 72 hours, it was centrifuged at 3000rpm at 4℃for 20 minutes. After filtration through a 0.45 μm filter, the virus was concentrated by centrifugation at 25000rpm for 2.5 hours at 4 ℃. After the concentrated virus was solubilized with 30. Mu.l of the virus lysate overnight, the virus titer was detected by QPCR. The results show that the virus titer meets the requirements.

Example 3: CAR-T cell preparation

Monocytes were isolated from human peripheral blood using Ficoll isolation and purified cd3+ T cells were obtained from RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3/CD28 magnetic beads (Life technology) and virus infection was performed after 48 hours incubation with RPMI1640+10% FBS+1% PS+200U/ml IL2 (PeproTech). Lentiviruses infected T cells in the presence of leptaboost to prepare CAR-T cells at moi=100. The medium was changed one day after infection.

Example 4: detection of positive Rate of infected CART cells by flow cytometry

CAR-T cells and NTD cells (control) after 72 hours of virus infection were collected separately by centrifugation, the supernatant was washed once with PBS, the cells were resuspended in PBS containing 2% fbs, and the positive rate was detected by flow.

Results: the results of transfection efficiency are shown in FIG. 2.

As shown in fig. 2A, CAR-T2A-mKate2 fusion proteins expressed by CAR-T cells, after cleavage, formed mKate2 proteins exhibited red fluorescence in cells.

Fig. 2B shows that detection using flow cytometry indicated a positive expression rate of CAR or mKate2CAR-T of about 50%.

Example 5: detecting the expression of CSF1R from each target cell

(1) Cellular immunofluorescence: pancreatic cancer cells were plated on discs of 24-well plates, after 24 hours, the cells were fixed with 4% Paraformaldehyde (PFA) for 20 minutes, and PBST washed three times for 5 minutes each; blocking with 10% goat serum for 1 hour at room temperature was performed, and incubation with antibodies specifically recognizing CSF1R was performed four times overnight. The next day, wash with PBST three times, five minutes each. Secondary antibodies specifically recognizing primary antibodies labeled with CY5 were incubated for 1 hour at room temperature. After three washes with PBS, DAPI stained nuclei. Confocal microscopy imaging.

(2) Flow cytometry: 100 ten thousand cells were collected, cells were fixed with 4% PFA at room temperature for 15min, and washed by centrifugation with 1 XPBS; cells were permeabilized with 100% methanol on ice for 15min, washed by centrifugation with 1 XPBS; 100 μl of diluted primary antibody (1:300) resuspended cells and incubated for 1 hour at room temperature; centrifuge washing with 1 XPBS. The supernatant was discarded. The operation is repeated. Cells were resuspended in 100. Mu.l of diluted fluorescent material conjugated secondary antibody (Cy 5 anti-rubbit), incubated at room temperature for 30 min in the absence of light, and washed by centrifugation in 1 XPBS. The supernatant was discarded. The operation is repeated. Cells were resuspended with 300 μl of 1XPBS and flow cytometer.

(3) qPCR: collecting 6-well plate cells, removing culture medium, adding 1ml Trizol to lyse cells, standing at room temperature for 5min, adding 200 μl/1ml chloroform of Trizol, mixing for 6-8 times, and standing at room temperature for 5min; centrifuging 12000g at 4 ℃ for 15 minutes, and sucking the supernatant into another centrifuge tube; adding etcMixing the isopropyl alcohol in volume, and standing at room temperature for 10min;12000g, centrifuging at 4 ℃ for 10 minutes, and discarding the supernatant; 1ml of 70% ethanol (with RNase free H ₂ O) washing, 7500g, centrifuging at room temperature for 5min; removing the supernatant, standing at room temperature for 10min, drying RNA, and adding 30 μl RNase free water to dissolve RNA; nandrop 2000 measured the concentration of RNA and 1% agarose gel electrophoresis was used to detect the integrity of RNA and the accuracy of quantification. cDNA was synthesized and mRNA levels were measured according to the instructions of RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).

Results: the results of the CSF1R expression detection of each cell line are shown in FIG. 3, and the results of the CSF1R expression detection of target cells by immunofluorescence show that BXPC3 and ASPC1 highly express the CSF1R and PANC1 lowly express the CSF1R.

Example 6: construction of target cells carrying luciferases

The pTomo-CMV-Luciferase-IRES-Puro lentivirus packaging procedure was the same as in example 2.

PANC1, BXPC3, ASPC1 cells were infected with the virus and then screened with Puromycin (1 ug/ml) for 2 weeks to successfully obtain PANC1, BXPC3, ASPC1-luciferase cells.

Example 7: CAR-T cell killing

In this example, the killing ability of CAR-T cells of the invention against different target cells was tested. The target cells used include: target cells highly expressing CSF 1R: BXPC3, ASPC1; target cells that do not express or underexpress CSF 1R: PANC1.

Cell density was adjusted to 2X 10 after PANC1-luciferase cell digestion and counting ⁴ /ml. 100 mu lluciferase cells were seeded in 96-well plates and CAR-T and control cells were adjusted to a cell density of 1X 10 ⁵ Per ml, 100 μl per well was plated in black 96-well plates at 5:1 E:T. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours.

The cell density was adjusted to 2X 10 after the BXPC3, ASPC1-luciferase cell digestion and counting ⁴ /ml. Mu.l of BXPC3, ASPC1-luciferase cells were seeded in 96-well plates and CAR-T and control cells were adjusted to a cell density of 8X 10 ⁴ Per ml, 0.5:1, 1:1, 2:1, 4:1 according to E:T to black 96 wellsIn the plate, 100 μl was inoculated per well. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours.

Cell supernatants were collected and frozen at-80℃for detection of IFNγ release (see example 8). Cell killing was detected with the promega fluorescence detection kit, cells were first treated with 20 μl of 1×plb lysate for 20 min, and immediately after addition of 100 μl of substrate per well, detected with a BioTek microplate reader.

Cytotoxic killer cell% = (1-target cell fluorescence value at effector cell-containing/target cell fluorescence value at effector cell-null) ×100%

Results: the results of gradient killing of CSF 1-CARs on different pancreatic cancer cell lines are shown in figure 5. The result shows that the killing effect of CSF1-CART cells on CSF1R high expression tumor cells is basically gradually enhanced along with the increase of the effective target ratio (E: T). While having substantially no killing effect on cell lines negative or underexpressing CSF 1R.

Example 8: IFNgamma cytokine release

In this example, cytokine release was detected in the case of co-incubation of CAR-T cells of the invention with target cells. Cell supernatants co-incubated in cell killing experiments were used for detection.

The method comprises the following steps: ifnγ was detected according to IFN gamma Human ELISA Kit (life technology) from cell supernatants of example 7 in which CAR-T cells of the invention were incubated with ASPC1 target cells (E: T ratio 4:1).

The standard was dissolved with Standard Dilution Buffer and diluted in a gradient to 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0 pg/ml.

50 mu l Incubation buffer, 50 mu l of detection sample and 50 mu l of IFN gamma biotin conjugated solution are added into each hole, and the mixture is stirred uniformly and then kept stand at room temperature for 90 minutes.

Then sequentially operating according to the following steps:

(1) The wells were washed 4 times with 1 XWash Buffer, each for 1 minute.

(2) Mu.l of 1 Xstrepitavidin-HRP solution was added to each well, and the mixture was allowed to stand at room temperature for 45 minutes.

(3) The wells were washed 4 times with 1 XWash Buffer, each for 1 minute.

(4) 100 mu l Stabilized chromogen was added thereto, and the mixture was allowed to stand at room temperature for 30 minutes.

(5) Mu.l of Stop solution was added to each well and mixed well.

(6) Absorbance was measured at 450 nm.

Results: the results are shown in FIG. 5. The cytokine increases significantly after CSF1-CAR killing ASPC1 suggesting that this killing is associated with ifnγ release.

Example 9: effect on CSF1-CAR-T cell killing after overexpression of CSF1R

Primers were designed according to CDS region sequences of CSF1R, CDS sequences of CSF1R were amplified using 293T cell cDNA as a template and digested and ligated to construct pTomo-CMV-CSF1R-T2A-luciferase-IRES-puro vector. Lentiviral packaging was as described in example 2 and PANC1-CSF1R-luc cells were obtained 2 weeks after infection of PANC1 cells with puromycin (1 ug/ml) screening.

The cell density was adjusted to 2X 10 after the PANC1-luc cells and PANC1-CSF1R-luc cells were digested and counted ⁴ /ml. Mu.l of luciferase cells were seeded in 96-well plates and CAR-T/NTD cells were adjusted to a cell density of 1X 10 ⁵ Per ml, 100 μl per well was plated in black 96-well plates at 5:1 E:T. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours.

Results: the killing results of CSF1-CAR-T cells on pancreatic cancer cell line PANC1 after overexpression of CSF1R are shown in fig. 6 and 7. FIG. 6 shows the success of PANC1 construction by over-expressing CSF1R as measured by PANC1 cells over-expressing CSF 1R. FIG. 7 shows the killing effect of CSF1-CAR-T on PANC1 after overexpression of CSF1R and IFN gamma release. The results show a significant increase in both the killing rate and ifnγ release of CSF1-CAR-T cells on PANC1-CSF1R cells over CSF 1R-expressing compared to the control group PANC 1-con. The results indicate that the killing effect of CSF1-CAR-T cells on CSF1R over-expressed tumor cells is obviously enhanced.

Example 10: effect on CSF1-CAR-T cell killing after specific knockdown of CSF1R

Selecting shRNA verified by CDS region according to shRNA sequence library of targeting CSF1R provided by Sigma company, BLAST selecting each shRNA on NCBI to ensure target specificity, constructing shRNA into pLKO.1 vector, and ensuring correct knocking-down vector through enzyme digestion identification and sequencing. .

shRNA virus packaging: the day before transfection, 100 ten thousand HEK-293T cells per dish were seeded into 6cm dishes for culture. Before transfection, 6cm dishes were replaced with 5ml fresh medium (serum-containing, antibiotic-free); two clean sterile centrifuge tubes were taken and 250 μl each was addedMedium, then 5. Mu.g shRNA plasmid DNA, 2.5. Mu.g pCMV DeltaR 8.9, 1. Mu.g PMD2.G plasmid were added to one of the tubes and gently mixed with a gun; another tube was filled with 17. Mu.l Lipo6000 ^TM The transfection reagent was gently beaten and mixed with a gun. After standing at room temperature for 5 minutes, the culture solution containing DNA was gently added with a gun to the culture solution containing Lipo6000 ^TM In the culture solution of the transfection reagent, a centrifuge tube is gently inverted or a gun is used for gently blowing and mixing, after standing for 20 minutes at room temperature, a 6cm dish is added for mixing, the supernatant is collected after 48 hours and 72 hours respectively, and is centrifuged for 20 minutes at the temperature of 3000r/min and filtered by a 0.45 mu m filter membrane to obtain the supernatant containing the virus.

shRNA virus infection: the day before infection, 50 ten thousand ASPC1 cells per well were seeded into six well plates for culture. Before infection, 1ml of fresh culture solution (containing serum and no antibiotics) is changed into each of six holes, and then 1ml of virus supernatant and 2 μl of polybrane (10 mg/ml) are added to prepare ASPC1-shCOO2 and ASPC1-shCSF1R cells; and replacing the complete culture medium after 24 hours, detecting the shRNA knocking-down efficiency after 96 hours, and performing CSF1-CAR-T cell killing detection.

Preparation of ASPC1-shCOO2, ASPC1-shCSF1r#1, ASPC1-shCSF1r#2-luciferase cells by infection of ASPC1 cells with plko.1-shCSF1R-1#, plko.1-shCSF1R-2# lentivirus, and adjustment of cell density to 2×10 after 48 hours of digestion and counting ⁴ /ml. Mu.l of luciferase cells were seeded in 96-well plates and the CAR-T/NT cells were adjusted to a cell density of 1X 10 ⁵ The wells were plated in black 96-well plates at 4:1 for E:T, with 100 μl each. And uniformly mixing the target cells and the T cells, incubating in an incubator for 24 hours, and detecting the killing effect. As described previously, by fluorescenceThe change in the light value detects the killing of ASPC1 and ASPC1-shCSF1R by CSF1-CAR-T cells.

The results are shown in FIG. 8. FIG. 8-A is a phenotype of knockdown CSF1R cells in ASPC1 cells. FIG. 8-B shows qPCR detection of CSF1RmRNA levels. FIG. 8-C is the post-silencing CSF1R killing effect of CSF1-CAR-T on ASPC 1. The results show that the killing rate of CSF1-CAR-T cells against ASPC1-shCSF1r#1 and ASPC1-shCSF1r#2 cells knocked down CSF1R is significantly reduced compared to control ASPC1-shCOO2 cells, and that the killing rate against cells is further reduced with increasing degree of knockdown. This result shows that knockdown of CSF1R on cell membranes significantly reduces the killing effect of CSF1-CAR-T, suggesting that CSF1-CAR-T of the invention is highly specific for CSF 1R.

Example 11: inhibition of ASPC1-luc nude mice transplantation tumor by CSF1-CAR-T

ASPC1-luc cells were constructed as described in example 6. ASPC1-luc cell line digestion counts were followed by addition of 30% matrigel to adjust cell density to 5X 10≡6/ml. Female NCG mice of 6 weeks old were purchased from south kyo collection pharmaceutical biotechnology, inc, and 100 μl of cell suspension was inoculated subcutaneously into each of the mice, and CART cells were returned after 7 days and prepared as described in example 3. Nude mice were imaged 1 day prior to CART reinfusion: 200 mu l D-fluorescein plus salt (15 mg/ml) was intraperitoneally injected after 0.025% Avertin (300. Mu.l/20 g) anesthetized mice, and after 10 minutes, live-small animals were imaged and NTD, CD19-CAR, CSF1-CAR were grouped according to fluorescence value size. Each mouse tail vein was reinfused with 1X 10≡7/200. Mu.l CART cells. The nude mice were imaged every 7 days thereafter.

The results are shown in FIG. 9. The result shows that CSF1-CAR-T has a remarkable inhibiting effect on ASPC1 nude mice transplantation tumor.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Huaxi Hospital at university of Sichuan

<120> preparation of chimeric antigen receptor immune cell based on CSF1 and application thereof

<130> P2022-0322

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 554

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 1

Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu

1 5 10 15

Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr

20 25 30

Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu

35 40 45

Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln

50 55 60

Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys

65 70 75 80

Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr

85 90 95

Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu

100 105 110

Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu

115 120 125

Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln

130 135 140

Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu

145 150 155 160

Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala

165 170 175

Glu Cys Ser Ser Gln Asp Val Val Thr Lys Pro Asp Cys Asn Cys Leu

180 185 190

Tyr Pro Lys Ala Ile Pro Ser Ser Asp Pro Ala Ser Val Ser Pro His

195 200 205

Gln Pro Leu Ala Pro Ser Met Ala Pro Val Ala Gly Leu Thr Trp Glu

210 215 220

Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu Pro Gly Glu Gln Pro

225 230 235 240

Leu His Thr Val Asp Pro Gly Ser Ala Lys Gln Arg Pro Pro Arg Ser

245 250 255

Thr Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro Val Val Lys Asp Ser

260 265 270

Thr Ile Gly Gly Ser Pro Gln Pro Arg Pro Ser Val Gly Ala Phe Asn

275 280 285

Pro Gly Met Glu Asp Ile Leu Asp Ser Ala Met Gly Thr Asn Trp Val

290 295 300

Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu Ile Pro Val Pro Gln Gly

305 310 315 320

Thr Glu Leu Ser Pro Ser Arg Pro Gly Gly Gly Ser Met Gln Thr Glu

325 330 335

Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser Ser Pro Leu Pro Ala

340 345 350

Ser Ala Lys Gly Gln Gln Pro Ala Asp Val Thr Gly Thr Ala Leu Pro

355 360 365

Arg Val Gly Pro Val Arg Pro Thr Gly Gln Asp Trp Asn His Thr Pro

370 375 380

Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg Asp Pro Pro Glu Pro

385 390 395 400

Gly Ser Pro Arg Ile Ser Ser Leu Arg Pro Gln Gly Leu Ser Asn Pro

405 410 415

Ser Thr Leu Ser Ala Gln Pro Gln Leu Ser Arg Ser His Ser Ser Gly

420 425 430

Ser Val Leu Pro Leu Gly Glu Leu Glu Gly Arg Arg Ser Thr Arg Asp

435 440 445

Arg Arg Ser Pro Ala Glu Pro Glu Gly Gly Pro Ala Ser Glu Gly Ala

450 455 460

Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly

465 470 475 480

His Glu Arg Gln Ser Glu Gly Ser Phe Ser Pro Gln Leu Gln Glu Ser

485 490 495

Val Phe His Leu Leu Val Pro Ser Val Ile Leu Val Leu Leu Ala Val

500 505 510

Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro

515 520 525

Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr

530 535 540

Gln Asp Asp Arg Gln Val Glu Leu Pro Val

545 550

<210> 2

<211> 233

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Ser Glu Leu Ile Lys Glu Asn Met His Met Lys Leu Tyr Met Glu

1 5 10 15

Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly

20 25 30

Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Ala Val Glu Gly

35 40 45

Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr

50 55 60

Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe

65 70 75 80

Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr

85 90 95

Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp

100 105 110

Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser

115 120 125

Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr

130 135 140

Glu Thr Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ala Asp Met

145 150 155 160

Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr

165 170 175

Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val

180 185 190

Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu

195 200 205

Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu

210 215 220

Pro Ser Lys Leu Gly His Lys Leu Asn

225 230

<210> 3

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 4

<211> 45

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 5

<211> 24

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 6

<211> 43

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

35 40

<210> 7

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln

1 5 10 15

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

20 25 30

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

35 40 45

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

50 55 60

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

65 70 75 80

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

85 90 95

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 8

<211> 708

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile

20 25 30

Gly Ser Gly His Leu Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met

35 40 45

Glu Thr Ser Cys Gln Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu

50 55 60

Lys Asp Pro Val Cys Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Asp

65 70 75 80

Ile Met Glu Asp Thr Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile

85 90 95

Ala Ile Val Gln Leu Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe

100 105 110

Thr Lys Asp Tyr Glu Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr

115 120 125

Glu Thr Pro Leu Gln Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu

130 135 140

Thr Lys Asn Leu Leu Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys

145 150 155 160

Asn Asn Ser Phe Ala Glu Cys Ser Ser Gln Asp Val Val Thr Lys Pro

165 170 175

Asp Cys Asn Cys Leu Tyr Pro Lys Ala Ile Pro Ser Ser Asp Pro Ala

180 185 190

Ser Val Ser Pro His Gln Pro Leu Ala Pro Ser Met Ala Pro Val Ala

195 200 205

Gly Leu Thr Trp Glu Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu

210 215 220

Pro Gly Glu Gln Pro Leu His Thr Val Asp Pro Gly Ser Ala Lys Gln

225 230 235 240

Arg Pro Pro Arg Ser Thr Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro

245 250 255

Val Val Lys Asp Ser Thr Ile Gly Gly Ser Pro Gln Pro Arg Pro Ser

260 265 270

Val Gly Ala Phe Asn Pro Gly Met Glu Asp Ile Leu Asp Ser Ala Met

275 280 285

Gly Thr Asn Trp Val Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu Ile

290 295 300

Pro Val Pro Gln Gly Thr Glu Leu Ser Pro Ser Arg Pro Gly Gly Gly

305 310 315 320

Ser Met Gln Thr Glu Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser

325 330 335

Ser Pro Leu Pro Ala Ser Ala Lys Gly Gln Gln Pro Ala Asp Val Thr

340 345 350

Gly Thr Ala Leu Pro Arg Val Gly Pro Val Arg Pro Thr Gly Gln Asp

355 360 365

Trp Asn His Thr Pro Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg

370 375 380

Asp Pro Pro Glu Pro Gly Ser Pro Arg Ile Ser Ser Leu Arg Pro Gln

385 390 395 400

Gly Leu Ser Asn Pro Ser Thr Leu Ser Ala Gln Pro Gln Leu Ser Arg

405 410 415

Ser His Ser Ser Gly Ser Val Leu Pro Leu Gly Glu Leu Glu Gly Arg

420 425 430

Arg Ser Thr Arg Asp Arg Arg Ser Pro Ala Glu Pro Glu Gly Gly Pro

435 440 445

Ala Ser Glu Gly Ala Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro

450 455 460

Leu Thr Asp Thr Gly His Glu Arg Gln Ser Glu Gly Ser Phe Ser Pro

465 470 475 480

Gln Leu Gln Glu Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

485 490 495

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

500 505 510

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

515 520 525

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

530 535 540

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

545 550 555 560

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

565 570 575

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

580 585 590

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

595 600 605

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

610 615 620

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

625 630 635 640

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

645 650 655

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

660 665 670

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

675 680 685

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

690 695 700

Leu Pro Pro Arg

705

<210> 9

<211> 2124

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccggaggagg tgtcggagta ctgtagccac atgattggga gtggacacct gcagtctctg 120

cagcggctga ttgacagtca gatggagacc tcgtgccaaa ttacatttga gtttgtagac 180

caggaacagt tgaaagatcc agtgtgctac cttaagaagg catttctcct ggtacaagac 240

ataatggagg acaccatgcg cttcagagat aacaccccca atgccatcgc cattgtgcag 300

ctgcaggaac tctctttgag gctgaagagc tgcttcacca aggattatga agagcatgac 360

aaggcctgcg tccgaacttt ctatgagaca cctctccagt tgctggagaa ggtcaagaat 420

gtctttaatg aaacaaagaa tctccttgac aaggactgga atattttcag caagaactgc 480

aacaacagct ttgctgaatg ctccagccaa gatgtggtga ccaagcctga ttgcaactgc 540

ctgtacccca aagccatccc tagcagtgac ccggcctctg tctcccctca tcagcccctc 600

gccccctcca tggcccctgt ggctggcttg acctgggagg actctgaggg aactgagggc 660

agctccctct tgcctggtga gcagcccctg cacacagtgg atccaggcag tgccaagcag 720

cggccaccca ggagcacctg ccagagcttt gagccgccag agaccccagt tgtcaaggac 780

agcaccatcg gtggctcacc acagcctcgc ccctctgtcg gggccttcaa ccccgggatg 840

gaggatattc ttgactctgc aatgggcact aattgggtcc cagaagaagc ctctggagag 900

gccagtgaga ttcccgtacc ccaagggaca gagctttccc cctccaggcc aggagggggc 960

agcatgcaga cagagcccgc cagacccagc aacttcctct cagcatcttc tccactccct 1020

gcatcagcaa agggccaaca gccggcagat gtaactggta ccgccttgcc cagggtgggc 1080

cccgtgaggc ccactggcca ggactggaat cacacccccc agaagacaga ccatccatct 1140

gccctgctca gagacccccc ggagccaggc tctcccagga tctcatcact gcgcccccag 1200

ggcctcagca acccctccac cctctctgct cagccacagc tttccagaag ccactcctcg 1260

ggcagcgtgc tgccccttgg ggagctggag ggcaggagga gcaccaggga tcggaggagc 1320

cccgcagagc cagaaggagg accagcaagt gaaggggcag ccaggcccct gccccgtttt 1380

aactccgttc ctttgactga cacaggccat gagaggcagt ccgagggatc cttcagcccg 1440

cagctccagg agtctaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 1500

gctagccagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 1560

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 1620

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1680

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1740

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1800

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 1860

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1920

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1980

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 2040

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 2100

cacatgcagg ccctgccccc tcgc 2124

Claims (14)

1. A Chimeric Antigen Receptor (CAR), characterized in that the chimeric antigen receptor has the structure according to formula I:

L-EB-H-TM-C-CD3ζ-RP(I)

in the method, in the process of the invention,

each "-" is independently a connecting peptide or peptide bond;

l is an absent or signal peptide sequence, said signal peptide being a signal peptide of a protein selected from the group consisting of: CD8, CD28, CD4;

EB is an extracellular binding domain, the amino acid sequence of which is shown in positions 33 to 496 of SEQ ID NO. 1;

h is a hinge region which is a hinge region of a protein selected from the group consisting of: CD8, CD28;

TM is a transmembrane domain which is a transmembrane region of a protein selected from the group consisting of: CD28, CD8;

c is a costimulatory signaling molecule, which is a costimulatory signaling molecule of a protein selected from the group consisting of: CD28, 4-1BB, or a combination thereof;

CD3 zeta is cytoplasmic signaling sequence from CD3 zeta and its amino acid sequence is shown in SEQ ID NO. 7;

RP is a null or reporter protein, wherein the reporter protein is a fluorescent protein.

2. The chimeric antigen receptor according to claim 1,

l is a CD8 derived signal peptide;

h is a CD8 derived hinge region;

TM is a CD 8-derived transmembrane region; and is also provided with

C is a 4-1 BB-derived costimulatory signaling molecule.

3. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of L is shown in SEQ ID No. 3; the amino acid sequence of H is shown as SEQ ID NO. 4; the amino acid sequence of the TM is shown as SEQ ID NO. 5; and the amino acid sequence of C is shown as SEQ ID NO. 6.

4. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID No. 8.

5. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence of said nucleic acid molecule is set forth in SEQ ID NO. 9.

7. A vector comprising the nucleic acid molecule of claim 5.

8. A host cell comprising the vector or chromosome of claim 7 integrated with an exogenous nucleic acid molecule of claim 5 or expressing the chimeric antigen receptor of claim 1.

9. An engineered immune cell comprising the vector or chromosome of claim 7 integrated with an exogenous nucleic acid molecule of claim 5 or expressing the chimeric antigen receptor of claim 1.

10. The engineered immune cell of claim 9, wherein the engineered immune cell is a chimeric antigen receptor T cell or a chimeric antigen receptor NK cell.

11. A method of preparing the engineered immune cell of claim 9, comprising the steps of: transduction of the nucleic acid molecule according to claim 5 or the vector according to claim 7 into an immune cell, thereby obtaining the engineered immune cell.

12. A pharmaceutical composition comprising the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 5, the vector of claim 7, the host cell of claim 8, and/or the engineered immune cell of claim 9, and a pharmaceutically acceptable carrier, diluent or excipient.

13. Use of a chimeric antigen receptor according to claim 1, a nucleic acid molecule according to claim 5, a vector according to claim 7, or a host cell according to claim 8, and/or an engineered immune cell according to claim 9, for the preparation of a medicament or formulation for the treatment of a disease in which CSF1 receptor is highly expressed, said disease being selected from the group consisting of: pancreatic cancer, osteosarcoma, breast cancer, gastric cancer, colorectal cancer, hepatobiliary cancer, bladder cancer, ovarian cancer, esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal cancer, or a combination thereof.

14. The use according to claim 13, wherein the disease is non-small cell lung cancer.