CN115819614A

CN115819614A - Preparation and application of chimeric antigen receptor immune cells based on IL34

Info

Publication number: CN115819614A
Application number: CN202210707541.2A
Authority: CN
Inventors: 赵旭东; 孙彬; 马海燕
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2023-03-21
Anticipated expiration: 2042-06-21
Also published as: CN115819614B

Abstract

The invention provides preparation and application of a chimeric antigen receptor immune cell constructed based on IL34. In particular, the invention provides a Chimeric Antigen Receptor (CAR) engineered based on IL34, said CAR comprising an extracellular binding domain capable of specifically targeting an IL34 receptor (in particular CSF 1R). The CAR immune cell has high specificity and high killing capacity, and has excellent tumor inhibition capacity through in vivo tests.

Description

Preparation and application of chimeric antigen receptor immune cells based on IL34

Technical Field

The invention belongs to the field of immune cell therapy, and particularly relates to preparation and application of a chimeric antigen receptor immune cell constructed based on IL34.

Background

The tumor is the second disease threatening the health of human beings, 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 survival quality, easy relapse and the like still restrict the traditional tumor treatment means.

Biological immunotherapy has become a fourth tumor treatment means after surgery, radiotherapy and chemotherapy, and will become a necessary means for future tumor treatment. Chimeric Antigen antibody Receptor-Tcell (CART) T cells are T cells that are genetically modified to recognize a specific Antigen of interest in an MHC-unrestricted manner and to continuously activate expanded cells. The structure of the CAR includes a tumor associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. At present, CART therapy shows strong killing capacity in hematologic malignancies, but solid tumors have limitations on the application of CART therapy in solid tumors due to tumor heterogeneity, lack of tumor-specific antigens, tumor immunosuppressive microenvironment and the like.

Interleukin-34 (Interleukin-34, IL-34) is a cytokine found in 2008 and is expressed in tissues such as spleen, thymus, heart, brain, lung, liver, kidney, testis, prostate, ovary, small intestine, colon, etc. IL34 is a secreted homodimeric glycoprotein consisting of 242 amino acids in the human body and having a molecular weight of 39kD. Highly conserved between human and chimpanzee (99.6% similarity), 72% similarity between human and mouse. It has now been found that there are three receptors for IL 34: colony-stimulating factor receptor (CSF1R), tyrosine phosphatase zeta receptor (PTP-zeta), syncan-1.

CSF1R is a receptor tyrosine kinase and, when bound to a ligand, induces phosphorylation of proteins on the cytoplasmic interior of CSF1R and formation of dimers which phosphorylate a range of other proteins (e.g., ERK1/2 or AKT). IL-34 from cancer cells activates ERK1/2 and AKT downstream of CSF1R in an autocrine manner, thereby providing a key survival signal for cancer cells expressing CSF1R. CSF1R is over-expressed in various solid tumors such as breast cancer, gastric cancer, colorectal cancer and the like, and is related to metastasis and prognosis of tumors, for example, the activation of CSF1R paracrine pathway in osteosarcoma can promote tumor invasion, and the autocrine activation of CSF1R in breast cancer is related to tumor metastasis and growth, and suggests poor prognosis. In addition, inhibition or knockdown of CSFIR causes increased T cell lymphoma cell apoptosis, while inhibition of CSF1R activity inhibits tumor growth in a mouse transplant tumor model. Currently, a plurality of small molecule inhibitors (PLX 3397, ARRY-382, PLX7486, BLZ945 and JNJ 40346527) targeting CSF1R and antibodies are applied to the clinical stage I/II. And when the small-molecule inhibitor or the antibody is combined with a CTLA-4/PDL1 blocker, the growth of pancreatic cancer and colorectal cancer can be obviously inhibited, and the survival period of mice is prolonged.

Meanwhile, CSF1R is also widely present in Tumor Microenvironment (TME). The CSF1R signaling pathway activates a variety of proteins, promoting differentiation of myeloid cells, targeting of monocytes, and survival, proliferation and chemotaxis of macrophages. In TME, IL34, in combination with CSF1R, causes ERK1/2 and AKT phosphorylation, which in turn affects macrophage morphology and phenotype, regulates the function and survival of Tumor Associated Macrophages (TAMs), which play a crucial role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression and tumor resistance.

The IL34 receptor PTP-zeta is highly expressed in various tumors (including but not limited to lung cancer, uterine cancer, hepatocellular carcinoma, renal cancer, prostatic cancer, glioma and astrocytoma), and the blockage of PTP-zeta can inhibit the growth of glioblastoma and prolong the survival of mice. At present, a plurality of antibodies (7E 4B11-SAP, SCB 4380) targeting PTP-zeta show very effective anti-tumor effects in vitro and in a transplantation tumor model.

IL34 receptor Syndecano-1 is highly expressed in myeloma, melanoma, liver cancer, lung cancer, pancreatic cancer and other tumors, and the migration of bone marrow cells depends on the interaction between IL34 and Syndecano-1. The binding of IL34 and Syndecano-1 can regulate the binding of IL34 and CSF1R, and a small amount of Syndecano-1 retains IL34 on the surface of a cell membrane through a chondroitin sulfate chain so as to reduce the binding of IL34 and CSF1R. In contrast, overexpression of Syndecano-1 promotes the binding of IL34 to CSF1R. Syndecano-1-targeted antibodies and CAR-T therapy show good efficacy in the treatment of multiple myeloma patients.

However, no chimeric antigen receptor immune cells based on IL-34 to target these receptors are currently available in the art.

Therefore, there is an urgent need in the art to develop a novel chimeric antigen receptor immune cell targeting IL-34 receptor and a method for treating the same.

Disclosure of Invention

The invention aims to provide a chimeric antigen receptor immune cell taking an IL34 receptor (especially CSF 1R) as a target and a preparation and application method 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 IL-34 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 the IL34 receptor.

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

In another preferred embodiment, the IL34 receptor comprises: CSF1R, PTP-zeta, syndecanon-1, or a combination thereof.

In another preferred embodiment, the IL34 receptor is a cell membrane-localized IL34 receptor.

In another preferred embodiment, the IL34 receptor 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, in addition to a first extracellular domain directed to the IL34 receptor, a second extracellular domain directed to an additional target.

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 IL34.

In another preferred embodiment, the extracellular binding domain comprises an IL34 protein or a 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 the amino acid sequence from position 21 to 242 of the sequence shown in SEQ ID NO. 1.

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

(i) 1, as shown in 21 st to 242 nd positions of the sequence shown in SEQ ID NO; and

(ii) 1, or 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are added at the N-terminus or C-terminus thereof, thereby obtaining an amino acid sequence; and the obtained amino acid sequence has a sequence identity of more than or equal to 85 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent, such as more than or equal to 96 percent, more than or equal to 97 percent, more than or equal to 98 percent or more than or equal to 99 percent) with the sequence shown in the 21 st to the 242 nd positions 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 as shown in SEQ ID NO 1 at positions 21 to 242.

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

L-EB-H-TM-C-CD3ζ-RP (I)

in the formula (I), the compound is shown in the specification,

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

l is a null or signal peptide sequence;

EB is an extracellular binding domain that specifically binds to the IL34 receptor;

h is a none or hinge region;

TM is a transmembrane domain;

c is a no or co-stimulatory signal molecule;

CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ;

RP is a null or reporter protein.

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 example, the reporter protein RP is mKate2 red fluorescent protein.

In another preferred example, the red fluorescent reporter protein RP (mKate 2) further comprises a self-cleavage recognition site at its N-terminus, preferably a T2A sequence. 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, said L is a signal peptide derived from CD 8.

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, said H is a CD 8-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 domain.

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

In another preferred embodiment, C is a costimulatory signal molecule for 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 a combination thereof.

In another preferred embodiment, C is a co-stimulatory signaling molecule from 4-1 BB.

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 ζ is as set forth in SEQ ID NO 7.

In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor CAR is shown as 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 lentivirus vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV and the like.

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

In another preferred embodiment, the vector further comprises one or more selected from the group consisting of: promoter, transcription enhancing element WPRE, long terminal repeat LTR, etc.

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 having integrated therein 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 having integrated therein 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: transducing 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 the engineered immune cell.

In another preferred embodiment, the method further comprises the step of performing functional and effective detection 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 said engineered immune cells in said formulation is 1 × 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 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 use in the preparation of a medicament or formulation for the prevention and/or treatment of a disease in which an IL34 receptor is highly expressed.

In another preferred embodiment, the IL34 receptor comprises CSF1R, PTP-zeta, syndecano-1, or a combination thereof.

In another preferred embodiment, the diseases related to the high expression of IL34 receptor include, but are not limited to, tumors, aging, obesity, cardiovascular diseases, diabetes, neurodegenerative diseases, infectious diseases, inflammatory diseases, and the like.

In another preferred embodiment, the diseases related to the high expression of IL34 receptor comprise: tumors, infections, inflammatory diseases, etc.

In another preferred embodiment, the disease is a malignancy in which the IL34 receptor is highly expressed.

In another preferred embodiment, the IL34 receptor high expression means that the ratio of the IL34 receptor expression level (F1) to the expression level (F0) under normal physiological conditions (i.e., F1/F0) is not less than 1.5, preferably not less than 2, more preferably not less than 2.5.

In another preferred embodiment, the tumor includes a solid tumor and a hematologic tumor.

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

In another preferred embodiment, the hematological tumor is selected from the group consisting of: t cell lymphoma, acute Myeloid Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

In another preferred embodiment, the tumor is pancreatic cancer.

In a ninth aspect of the invention, there is provided a use of the engineered immune cell according to the fifth aspect of the invention, or the pharmaceutical composition according to the seventh aspect of the invention, for the prevention and/or treatment of cancer or tumor.

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 thereof 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 the IL34 receptor is highly expressed.

In another preferred embodiment, the IL34 receptor comprises CSF1R, PTP-zeta, syndecanon-1, or a combination thereof.

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

In another preferred embodiment, the engineered immune cell or the CAR immune cell comprised in the pharmaceutical composition is a cell derived from the subject (autologous cell).

In another preferred embodiment, the engineered immune cell or the CAR immune cell comprised in the pharmaceutical composition is a cell derived from a healthy individual (allogeneic cell).

In another preferred embodiment, the method may 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 to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 shows a schematic diagram of the IL34-CAR vector construction. Wherein, A is a schematic diagram of an IL34 sequence, 1-20AA in the IL34 is a signal peptide, and 21-242AA is a mature polypeptide. B is a structural schematic diagram of control group plasmids CD19-CAR and IL34-CAR, wherein the signal peptide, the hinge region and the transmembrane region are all derived from a human CD8 molecule, 4-1BB is derived from human CD137, CD3 zeta is derived from human CD3, and mKate2 is a fluorescent marker for detecting CAR expression. C is the restriction enzyme identification of pTomo-IL34-CAR vector HindIII and PstI.

Figure 2 shows CAR transfection efficiency assays. Wherein, A is cell fluorescence expression of CD19-CAR and IL34-CAR infected T cells 72 hours later, BF (upper row) is bright field, and mKate2 (lower row) is CAR fluorescence expression. B is flow detection fluorescence expression.

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

FIG. 4 shows the results of gradient killing of IL34-CAR on different pancreatic cancer cell lines.

FIG. 5 shows the results of IFN γ release following killing of IL34-CAR on the ASPC1 pancreatic cancer cell line.

Fig. 6 shows the overexpression of CSF1R in the pancreatic cancer cell line PANC 1. Wherein A is a structural schematic diagram of an overexpression vector. B is immunofluorescence detection of CSF1R overexpression in PANC1 cells. C is flow detection of CSF1R expression.

FIG. 7 shows the killing effect of IL34-CAR on CSF1R overexpressing PANC1 and IFN γ release.

Figure 8 shows that knockdown of CSF1R expression in ASPC1 cells reduces IL34-CAR killing. Wherein A is the cell phenotype of ASPC1 cells after CSF1R knockdown. B is qPCR for CSF1RmRNA level. C is the killing detection of the ASPC1-shCSF1R by the IL34-CAR.

FIG. 9 shows IL34-CAR vs CSF1R ⁺ /Syndecan-1 ⁺ MCF7 cells of (1)

FIG. 10 shows the inhibitory effect of IL34-CAR on ASPC1 nude mouse transplantable tumors. Wherein, A is the living body imaging of ASPC1 nude mouse transplantation tumor in different time periods of CART reinfusion. B is a fluorescence intensity statistical chart of the transplanted tumor.

Detailed Description

The inventor of the invention has extensively and deeply studied, and has developed a chimeric antigen receptor immune cell constructed based on IL34 for the first time through a large amount of screening. The invention uses partial fragments (namely 21 to 242 amino acid sequences) of the full-length IL34 as the extracellular binding domain of the CAR, and obtains CAR-T cells targeting IL34 receptors (especially CSF 1R). In vitro experiments suggest that the CAR-T cells have high specificity and excellent cell killing ability, and in vivo experiments also suggest that the CAR-T cells have in vivo inhibitory ability. The present invention has been completed based on this finding.

Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined 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 the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies 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, since 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, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (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 terms "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of or" consisting of 82303030A ".

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

"Gene expression" or "expression" refers to the process of transcription, translation and post-translational modification of a gene to produce the RNA or protein product of the gene.

"Polynucleotide" refers to a polymeric form of nucleotides of any length, including Deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to interchangeable single-and double-stranded molecules. Unless otherwise indicated, a polynucleotide in any of the embodiments described herein includes a double-stranded form and two complementary single strands that are known or predictable to make up 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. In addition, the invention also provides non-conservative amino acid substitutions that allow for amino acid substitutions from different groups.

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

All definitions, as defined and used herein, should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.

All references, patents, and patent applications cited herein are hereby incorporated by reference with respect to the subject matter to 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 disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the 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 determined.

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

Colony stimulating factor receptor (CSF 1R)

The IL 34-based CARs provided herein can specifically bind to the IL34 receptor. A representative IL34 receptor is CSF1R. CSF1R is a receptor tyrosine kinase and, when bound to a ligand, induces phosphorylation of proteins on the cytoplasmic interior of CSF1R and formation of dimers which phosphorylate a range of other proteins (e.g., ERK1/2 or AKT). CSF1R is overexpressed in various tumors such as breast cancer, gastric cancer, colorectal cancer and the like. In T cell lymphomas, increased apoptosis of tumor cells was observed by inhibition or knockdown of CSFIR, while inhibition of CSF1R activity inhibited tumor growth in a mouse transplanted tumor model. It has also been shown 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, suggesting poor prognosis.

CSF1R has two ligands: colony stimulating factor-1 (Colony-stimulating factor-1, CSF-1) and IL34.CSF-1 exists mainly in the circulatory system in the form of proteoglycans, secreted by a variety of cells of mesenchymal and epithelial origin. Various diseases including infection, cancer and chronic inflammatory diseases cause an increase in CSF1 expression in blood. Binding of CSF1 to CSF1R is mainly through salt bond, whereas binding of IL34 to CSF1R requires hydrophobic amino acids and hydrophobic interaction bond, and CSF1 binds to 1 CSF1R while IL34 binds to two CSF1 rs, therefore IL34 has stronger affinity to CSF1R. In macrophages, binding of IL34 to CSF1R results in more intense ERK1/2 and AKT phosphorylation in a shorter time than CSF1, which in turn affects cell morphology and phenotype. When cancer cells are stressed (e.g., in chemotherapy), an internal survival signaling cascade is triggered to prevent cell death and defend against future insults. In this regard, increasing evidence has revealed the importance of the IL-34/CSF1R axis in cancer chemoresistance. IL-34 from cancer cells activates ERK1/2 and AKT downstream of CSF1R in an autocrine manner, thereby providing a key survival signal for cancer cells expressing CSF1R.

Meanwhile, CSF1R is also widely present in Tumor Microenvironment (TME). The CSF1R signaling pathway activates a variety of proteins by regulating tyrosine phosphorylation, promoting differentiation of myeloid cells, monocyte targeting, and macrophage survival, proliferation, and chemotaxis. In TME, CSF1R regulates the function and survival of Tumor-Associated Macrophages (TAMs), which play a crucial role in Tumor growth, invasion, metastasis, angiogenesis, immunosuppression and Tumor resistance.

During cancer progression, IL-34 is a powerful tool to reprogram macrophages into tumor-promoting macrophages in the primary tumor. In solid tumors, TAMs are the most abundant immunosuppressive cells in TME, the number of which correlates with poor prognosis. TAMs have a M2 polarized phenotype, enabling them to modulate immune responses, promote angiogenesis and promote tumor growth, invasion and metastasis. Early studies of IL-34 biology showed that IL-34-derived macrophages exhibit most of the phenotypic and functional properties of TAM (low T cell co-stimulatory properties, inhibition of activated effector T cell responses). Bone marrow-derived macrophages appear as M2-like macrophages, exhibit most of the phenotypic and functional characteristics associated with tumors, show elevated levels of Arg-1, tie-2 and TNF- α, and contribute to angiogenesis and teratoma development. In addition, IL 34-derived macrophages support the switching of memory T cells to Th17 cells through constitutive expression of membrane IL-1 α. In addition to TAMs, CSF1R expression can also be detected in tumor-associated dendritic cells, tumor-associated neutrophils, and myeloid-derived suppressor cells. Thus, the effect of CSF-1 on these myeloid cells in the tumor microenvironment is equally important as IL-34.

PTP-ζ

Another representative IL34 receptor is PTP-zeta. PTP- ζ is highly expressed in a variety of tumors, including but not limited to lung, uterine, hepatocellular, renal, prostate, glioma, and astrocytoma tumors. Activation of PTP-zeta increases phosphorylation of multiple signaling pathways and promotes tumor metastasis.

Syndecan-1

Another representative IL34 receptor is Syndecano-1. Syndecano-1 is highly expressed in myeloma, melanoma, liver cancer, lung cancer, pancreatic cancer and other tumors, and the migration of bone marrow cells depends on the interaction between IL34 and Syndecano-1. In addition to IL34, syndecano-1 binds to various growth factors such as Epidermal Growth Factor (EGF), hepatocyte Growth Factor (HGF), vascular Endothelial Growth Factor (VEGF), WNT factor, etc., via chondroitin sulfate. The binding of IL34 and Syncan-1 can regulate the binding of IL34 and CSF1R, and the binding of IL34 and CSF1R is reduced by retaining IL34 on the surface of a cell membrane through a chondroitin sulfate chain in a small amount of Syncan-1. In contrast, overexpression of Syndecano-1 promotes the binding of IL34 to CSF1R.

No cell line satisfying CSF1R negative, PTP-zeta and Syndecano-1 positive at the same time is found, so that the binding effect of the CAR-T cell of the invention on the two receptors cannot be verified at present. However, the mechanism of the binding of PTP-zeta and Syndecano-1 to IL34 is dependent on chondroitin sulfate. Both belong to transmembrane proteoglycans, with glycosaminoglycans outside the cell and PDZ domains at the intracellular C-terminus. In addition, IL34 binds most strongly to CSF1R, followed by PTP-zeta and Syndecano-1. Specifically, IL34 and CSF1R have a Kd of 10 ^-12 M, kd to PTP-zeta is 10 ^-7 M, kd of Syndecano-1 is approximately10 ^-8 And M. Thus, it is expected that the IL34-CAR of the invention may also recognize PTP- ζ and Syndecano-1. And, the experiment confirmed: IL34-CART vs CSF1R ⁺ /Syndecan-1 ⁺ The MCF7 cells also have a killing effect.

IL34

The invention designs a chimeric antigen receptor with an extracellular binding domain based on IL34, and the CAR-T cell can simultaneously aim at a plurality of tumor treatment targets, and can effectively inhibit the growth, invasion, metastasis and drug resistance of tumors, thereby prolonging the survival rate of patients.

On the one hand, the current major way to construct CAR-T targeting specific tumor antigens is to design CAR based on related antibodies, however, too low affinity of the antibody targets the ability to bind tumor cells poorly, too high affinity of the antibody is prone to excessive immune response, 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 advantage of conservative combination of the receptor/ligand and the target molecule, and the CAR sequence has proper affinity and can better overcome the problem of inappropriate artificial antibody affinity. The present study demonstrates that CAR-T cells constructed using the natural ligand of the IL34 receptor as the extracellular recognition domain are well expressed in vivo and produce tumor suppressive effects.

Based on the method, the IL34 fragment is integrated into the CAR vector by a genetic engineering mode for the first time, and related immune cells are modified, so that the specific killing of the IL34 receptor positive cells is realized, and the method can be used for treating related diseases.

Chimeric Antigen Receptors (CAR) of the invention

A Chimeric immune antigen receptor (CAR) consists of an extracellular antigen recognition region, a transmembrane region, and an intracellular costimulatory signal region.

The design of the CAR goes through the following process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and, because of the single intracellular activation domain, were only capable of inducing 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 costimulatory molecule such as CD28, 4-1BB, OX40, ICOS based on the original structure, and compared with the first generation CAR, the function is greatly improved, and the persistence of CAR-T cells and the killing ability to tumor cells are further enhanced. On the basis of the second generation CAR, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and the development of the second generation CAR and the fourth generation CAR is realized.

The extracellular domain of the CAR recognizes a specific antigen and subsequently transduces this signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity and secretion of cytokines, which in turn clear the target cell. Autologous cells from the patient (or a heterologous donor) are first isolated, activated and genetically engineered to produce immune cells for CAR production, and then injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by immune cells in a non-MHC restricted manner.

CAR-immune cell therapy has achieved very high clinical response rates in the treatment of hematological malignancies, which rates were previously unattainable by any therapeutic approach, and have triggered a hot surge of clinical research in the world.

Specifically, the Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain.

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

In the present invention, the extracellular domain of the chimeric antigen receptor is an IL34 protein or fragment thereof that specifically binds to the IL34 receptor target of the CAR of the invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence from position 21 to 242 of the sequence shown in SEQ ID NO. 1.

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

A linker may 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 an ectodomain or a 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 its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the CD28 signaling domain, and CD3 zeta signaling domain combination.

In the present invention, the extracellular binding domain of the CAR of the invention also includes sequence-based conservative variants, which refer to polypeptides formed by substituting up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having similar or similar properties, as compared to the amino acid sequence at positions 21 to 242 of SEQ ID No. 1.

In the present invention, the number of amino acids to be 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 number of amino acids in the original amino acid sequence.

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

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

The intracellular domains in the CAR of the invention include the 4-1BB co-stimulatory domain and the signaling domain of CD3 ζ.

In one embodiment of the invention, the CAR is a CAR that can specifically target CSF1R.

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 specific targeting to an IL34 receptor (especially CSF 1R).

The chimeric antigen receptor immune cells of the invention may be CAR-T cells, and may also be CAR-NK cells, CAR-macrophages. Preferably, the chimeric antigen receptor immune cell of the invention is a CAR-T cell.

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

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

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to a CAR-NK cell according to the fifth aspect of the invention. The CAR-NK cells of the invention can be used for tumors with high IL34 receptor expression.

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. By engineering (genetically modifying) NK cells it is possible to obtain new functions, including the ability to specifically recognize tumor antigens and having an enhanced anti-tumor cytotoxic effect.

CAR-NK cells also have the following advantages compared to CAR-T cells, for example: (1) Directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) Is easy to be amplified in vitro and can be developed into ready-made products. Otherwise, similar to CAR-T cell therapy.

Carrier

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from 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, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, an 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 into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. 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 can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, molecular cloning. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise 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 gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral 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 that promoter function is maintained 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 apart, before activity begins to decrease. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.

An 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 level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). 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 cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous 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 present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells 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 separate 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 a host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, the reporter gene is the following: 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 determined at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al, 2000FEBS letters 479. In one embodiment of the invention, the reporter gene is a gene encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Methods for introducing 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 by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a 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, e.g., sambrook et al (2001, molecular cloning. A preferred method for 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 for 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. Pat. nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells 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. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (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 in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker 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 as a suspension in the lipid, contained in or complexed with a 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 bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain 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.

Preparation

The invention provides a pharmaceutical composition 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 an engineered immune cell according to the 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 CAR-T cells are present in the formulation at a concentration of 1X 10 ³ -1×10 ⁸ Individual cells/ml, more preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; 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 of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target a marker IL34 receptor of tumor cells, and synergistically activate the T cells to cause immune cell immune response, so that the killing efficiency of the 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 to the mammal the CAR-cells of the invention.

In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or allogeneic donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained 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. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells of the IL34 receptor elicit a cell-specific immune response against the IL34 receptor.

Although the data disclosed herein specifically disclose lentiviral vectors comprising an IL34 protein or fragment thereof, a hinge and transmembrane region, 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 substantially vascularized, as well as vascularized tumors. Cancers include non-solid tumors (such as hematological tumors, e.g., leukemias and lymphomas) and solid tumors. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

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

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

The CAR-modified T cells of the invention may also be used as a type of vaccine 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 cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified 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 using 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, a pharmaceutical composition 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 compositions 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 an "effective amount", "immunologically effective amount", "anti-tumor 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, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It is possible to generally point out: pharmaceutical compositions comprising T cells described herein can be in the range of 10 ⁴ To 10 ⁹ Dosage of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Doses of individual cells per kg body weight (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 infusion 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 those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out 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, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is 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 conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment 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 efavirenz therapy for psoriasis patients or other therapy for specific tumor patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with 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, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

The dosage 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 proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ⁶ 1 to 10 ¹⁰ The CAR-T cells of the invention are administered to a patient, for example, by intravenous infusion.

The main advantages of the present invention include:

1) And (3) specific target spots: the IL34 receptor (especially CSF 1R) related by the invention is not expressed on the cell membrane of normal cells basically, but is expressed on the cell membrane and macrophages of tumor tissues, so that the CAR specifically kills the tumor cells and macrophages which express the IL34 receptor on the membrane, and has no killing effect on normal cells or tissues.

2) 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. Conservation of receptor-ligand interactions determines that safety tests in animals, particularly primates, are more responsive to safety in humans.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

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

TABLE 1 summary of sequences to which the invention relates

Table 2 shows the cell lines used in the examples.

TABLE 2 cell lines

Cell lines	Type (B)
		PANC1	Pancreatic cancer cell
BXPC3	Pancreatic cancer cell
		ASPC1	Pancreatic cancer cell
MCF-7	Breast cancer cell

Example 1: preparation of IL34-CAR vectors

Based on the nucleotide sequence (NM-001172772.2) of IL34, human CD8 signal peptide, human CD8 alpha hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3 zeta intracellular region gene sequence information, the corresponding nucleotide sequence was obtained by artificial synthesis method or PCR method. The CD8 signal peptide and IL34 extracellular domain were synthesized and the nucleotide sequence of the CAR molecule was cleaved by AgeI (Thermo) and NheI (Thermo) and inserted into the lentiviral vector pTomo into which the CD8 transmembrane region, 4-1BB co-stimulatory domain, CD3 zeta signaling region had been inserted by T4 DNA ligase (NEB) ligation. Competent E.coli (Stbl 3) was transformed.

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

All plasmids were extracted using a QIAGEN endotoxin-free macroextraction kit, and purified plasmids were packaged with lentiviruses transfected into HEK-293T cells using Bilongtian lipo 6000.

Example 2: virus package

HEK-293T cells were cultured in 15cm dishes for virus packaging. 2ml OPTIMEM solubilized plasmid mixture (20. Mu.g core plasmid, 8.9. Mu.g pCMV. DELTA.R, 4. Mu.g PMD2. G) was prepared for transfection with HEK-293T cells at about 80% -90% confluency; in another centrifuge tube 2ml OPTIMEM and 68. Mu.l lipo6000 were added. Standing at room temperature for 5min, adding the plasmid complex into the liposome complex, and standing at room temperature for 20min. The mixture was added dropwise to HEK-293T cells, incubated at 37 ℃ for 6 hours and the medium was removed. Add again the pre-warmed complete medium. After collecting the virus supernatants for 48 hours and 72 hours, they were centrifuged at 3000rpm at 4 ℃ for 20 minutes. After filtration through a 0.45 μm filter, the virus was concentrated by centrifugation at 25000rpm at 4 ℃ for 2.5 hours. After the concentrated virus was dissolved overnight in 30. Mu.l of a virus dissolving solution, the virus titer was measured by QPCR. The results show that the virus titer meets the requirements.

Example 3: CAR-T cell preparation

Mononuclear cells were isolated from Human peripheral blood using Ficoll separation, and purified CD3+ T cells were obtained from RosettSeep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3/CD28 magnetic beads (Life technology) and virus infected after 48 hours of incubation with IL2 (PeproTech) at RPMI1640+10% FBS +1% PS + 200U/ml. Lentiboost in the presence of lentivirus according to MOI =100 infection of T cells to prepare CAR-T cells. The medium was changed one day after infection.

Example 4: positive rate of detecting infected CART cells by flow cytometry

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

As a result: the results of transfection efficiency are shown in FIG. 2.

As shown in FIG. 2A, after the CAR-T2A-mKate2 fusion protein expressed by the CAR-T cell is cut, the formed mKate2 protein shows red fluorescence in cells.

FIG. 2B shows that flow cytometry was used to demonstrate a positive expression rate of CAR or mKate2CAR-T of about 50%.

Example 5: detection of CSF1R expression in target cells

(1) Cellular immunofluorescence: target cells were plated on discs in 24-well plates, 24 hours later cells were fixed with 4% Paraformaldehyde (PFA) for 20 minutes, and PBST was washed three times for 5 minutes each; blocking with 10% goat serum for 1 hour at room temperature and incubating overnight at four degrees with an antibody that specifically recognizes CSF1R. The following day, three washes with PBST for five minutes each. The secondary antibody specifically recognizing the primary antibody, labelled with CY5, was incubated for 1 hour at room temperature. After three PBS washes, DAPI stained nuclei. Confocal microscopy imaging.

(2) Flow cytometry: 100 ten thousand cells were collected, washed with 4% PFA room temperature fixed cells 15min, centrifuged with 1 XPBS; cells were permeabilized with 100% methanol on ice for 15min, washed centrifugally with 1 × PBS; 100 μ l of diluted primary antibody (1; 1PBS centrifugal washing. The supernatant was discarded. And repeating the operation. The cells were resuspended in 100. Mu.l of a fluorescent-coupled secondary antibody (Cy 5-anti-rabbitt), incubated for 30 minutes at room temperature in the dark and washed by centrifugation in 1 XPBS. The supernatant was discarded. And repeating the operation. Resuspend cells with 300. Mu.l of 1 XPBS and flow cytometric analyzer.

(3) qPCR: collecting 6-well plate cells, removing culture medium, adding 1ml Trizol to crack cells, standing at room temperature for 5min, adding 200 μ l/1ml Trizol chloroform, reversing and mixing uniformly for 6-8 times, and standing at room temperature for 5min;12000g, centrifuging for 15 minutes at 4 ℃, and sucking supernatant liquid into another centrifuge tube; adding isopropanol with equal volume, mixing by inversion, and standing at room temperature for 10min;12000g, centrifuging for 10 minutes at 4 ℃, and discarding the supernatant; adding 1ml 70% ethanol (with RNase free H) ₂ O preparation), washing, 7500g, centrifuging for 5min at room temperature; removing supernatant, standing at room temperature for 10min, drying RNA, and adding 30 μ l RNase free water to dissolve RNA; nandrop 2000 measures the concentration of RNA and detects the integrity of the RNA and the accuracy of the quantification by electrophoresis on a 1% agarose gel. cDNA was synthesized and assayed for mRNA levels according to the RevertAID First Strand cDNA Synthesis Kit (Thermo Scientific) instructions.

As a result: the results of the CSF1R expression assay for each cell line are shown in FIG. 3. The expression of CSF1R of target cells is detected by immunofluorescence, and the results show that BXPC3 and ASPC1 highly express CSF1R and PANC1 lowly express CSF1R.

Example 6: construction of target cells carrying luciferase

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

After the virus is infected with the PANC1, BXPC3, ASPC1 and MCF-7 cells, the cells are screened by Puromycin (1 ug/ml) for 2 weeks, and the PANC1, BXPC3, ASPC1 and MCF7-luciferase cells are successfully obtained.

Example 7: CAR-T cell killing

In this example, the killing ability of CAR-T cells of the invention to different target cells was examined. Target cells employed included: target cells highly expressing CSF 1R: BXPC3, ASPC1; target cells that do not express or under-express CSF 1R: PANC1; CSF1R ⁺ /Syndecan-1 ⁺ The MCF7 cell of (1).

The cell density is adjusted to 2 x 10 after MCF7 and PANC1-luciferase cell digestion and counting ⁴ And (4) the concentration is/ml. Mu.l of luciferase cells were seeded in 96-well plates and CAR-T and control cells were adjusted to a cell density of 1X 10 ⁵ Perml, according to E: T of 5, were inoculated into black 96-well plates in 100. Mu.l/well. The target cells and T cells were mixed well and incubated in an incubator for 24 hours.

Adjusting the cell density to 2 x 10 after digesting and counting BXPC3 and ASPC1-luciferase cells ⁴ And/ml. 100 μ l 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, according to E: T0.5, 1, 2, 1, 4. The target cells and T cells were mixed well and incubated in an incubator for 24 hours.

Cell supernatants were collected and frozen at-80 ℃ for IFN γ release (see example 8). Cell killing was detected using a promega fluorescence detection kit, cells were first treated with 20 μ l of 1 × PLB lysate for 20 minutes, and 100 μ l of substrate was added to each well and immediately detected using a BioTek plate reader.

Cytotoxic killer cell% = (1-fluorescence of target cell with effector cell/fluorescence of target cell without effector cell) × 100%

As a result: the results of gradient killing of IL34-CAR on different pancreatic cancer cell lines are shown in fig. 4 and fig. 9. The result shows that the killing effect of IL34-CART cells on CSF1R high-expression tumor cells is gradually enhanced along with the increase of the effective target ratio (E: T), the killing effect on CSF 1R-hardly expressed tumor cells is basically not enhanced, and the killing effect on CSF1R is basically not enhanced ⁺ /Syndecan-1 ⁺ The MCF7 cells also had significant killing effect.

Example 8: IFN gamma cytokine release

In this example, cytokine release was examined in the case of CAR-T cells of the invention incubated with target cells. The cell supernatants were co-incubated in a cell killing experiment for detection.

The method comprises the following steps: the supernatant of the cells of example 7 in which the CAR-T cells of the present invention were co-incubated with target cells such as PANC1 and ASPC1 was subjected to IFN gamma Human ELISA Kit (life technology) for detecting IFN γ.

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

Mu.l of incorporation buffer, 50. Mu.l of assay sample, and 50. Mu.l of IFN γ biotin conjugated solution were added to each well, mixed and allowed to stand at room temperature for 90 minutes.

Then the operation is carried out according to the following steps in sequence:

(1) The wells were washed 4 times with 1 × Wash Buffer, each for 1 min.

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

(3) The wells were washed 4 times with 1 × Wash Buffer, each for 1 min.

(4) Mu.l of Stabilized chromogen was added and left to stand at room temperature for 30 minutes in the dark.

(5) Add 100. Mu.l of Stop solution to each well and mix well.

(6) The absorbance was measured at 450 nm.

As a result: the results are shown in FIG. 5. Cytokine IFN gamma after IL34-CAR-T kills ASPC1 is obviously increased. Figure 7 right panel control results show that IL34-CAR has no IFN γ release on CSF1R negative PANC1 cells. The results indicate that the killing effect of IL34-CAR-T cells on tumor cells is accompanied by IFN γ release, suggesting that the killing effect is related to IFN γ release.

Example 9: effect on killing of IL34-CAR-T cells after overexpression of IL34 receptor

Designing a specific primer according to the CDS region sequence of the CSF1R, amplifying the CSF1R by using 293T cell cDNA as a template, and performing enzyme digestion and connection to construct a pTomo-CMV-CSF1R-T2A-luciferase-IRES-puro vector. Lentiviral packaging As described in example 2, PANC1-CSF1R-luc cells were obtained by screening with puromycin (1. Mu.g/ml) for 2 weeks following viral infection of PANC1 cells.

Digesting and counting PANC1-luc cells and PANC1-CSF1R-luc cells, and adjusting cell density to 2 × 10 ⁴ And/ml. Mu.l of luciferase cells were seeded in 96-well plates and CAR-T cells were adjusted to a cell density of 1X 10 ⁵ Perml, according to E: T of 5, were inoculated into black 96-well plates in 100. Mu.l/well. The target cells and T cells were mixed well and incubated in an incubator for 24 hours.

As a result: the results of killing of the pancreatic cancer cell line PANC1 by IL34-CAR-T cells after overexpression of CSF1R are shown in fig. 6 and fig. 7. FIG. 6 shows the detection of overexpression of CSF1R by PANC1 cells. FIG. 7 shows the killing effect of IL34-CAR-T on PANC1 after over-expression of CSF1R and IFN γ release. The results show that both the killing rate and the IFN γ release of IL34-CAR-T cells on CSF1R overexpressing PANC1-CSF1R cells are significantly increased compared to the control PANC 1-con. The result shows that the killing effect of the IL34-CAR-T cell on CSF1R over-expression tumor cells is obviously enhanced.

Example 10: effect of specific CSF1R knockdown on killing effect of IL34-CAR-T cells

The validated shRNA of the CDS region was selected according to the CSF1R shRNA sequence library provided by Sigma, and the specificity of the target was ensured for each shRNA selected by BLAST at NCBI. The shRNA is connected into a pLKO.1 vector, and the correctness of the vector is confirmed by enzyme digestion identification and sequencing.

Packaging shRNA virus: the 6cm dishes were seeded with 100 ten thousand HEK-293T cells per dish for culture the day before transfection. Before transfection, 6cm dishes were placedChanging to 5ml fresh culture medium (containing serum and no antibiotics); two clean sterile centrifuge tubes were added to 250. Mu.l each

Adding 5 mu g shRNA plasmid, 2.5 mu g pCMV delta R8.9 and 1 mu g PMD2.G into one tube of the Medium, and gently blowing and uniformly mixing by using a gun; another tube was charged with 17. Mu.l Lipo6000 ^TM The transfection reagent was gently blown and mixed well with a gun. After standing at room temperature for 5 minutes, the DNA-containing culture solution was gently added by a gun to the solution containing Lipo6000 ^TM And (3) slightly inverting the centrifuge tube or slightly blowing and beating the mixture by using a gun in the culture solution of the transfection reagent, standing the mixture for 20 minutes at room temperature, adding the mixture into a 6cm dish, mixing the mixture uniformly, collecting the supernatant after 48 hours and 72 hours respectively, centrifuging the mixture for 20 minutes at 4 ℃ at 3000r/min, and filtering the mixture by using a 0.45-micrometer filter membrane to obtain the virus-containing supernatant.

shRNA virus infection: 50 ten thousand of ASPC1 cells per well were seeded into a six-well plate for culture the day before infection. Before infection, the six well plates were replaced with 1ml of fresh medium (containing serum without antibiotics) per well, and 1ml of viral supernatant and 2. Mu.l of polybrane (10 mg/ml) were added; and after 24h, replacing the complete culture medium, detecting shRNA knockdown efficiency after 96h, and performing IL34-CAR-T cell killing detection.

Infecting ASPC1 cells with pLKO.1-shCOO2, pLKO.1-shCSF1R-1#, and pLKO.1-shCSF1R-2# lentivirus to prepare ASPC1-shCOO2, ASPC1-shCSF1R #1, and ASPC1-shCSF1R #2-luciferase cells, digesting and counting after 48 hours, and adjusting cell density to 2 × 10 ⁴ And/ml. Mu.l of luciferase cells were seeded in 96-well plates and CAR-T cells were adjusted to a cell density of 8X 10 ⁴ Per ml, according to E: T4, 1, 0.5. And (3) uniformly mixing the target cells and the T cells, placing the mixture into an incubator, incubating for 24 hours, detecting the killing effect, and detecting the killing of the IL34-CAR-T cells on ASPC1 and ASPC1-shCSF1R through the change of fluorescence values. .

As a result: the results are shown in FIG. 8. FIG. 8-A is a cytophenotype of knocked-down CSF1R in ASPC1 cells. FIG. 8-B shows qPCR detection of CSF1RmRNA levels. FIG. 8-C shows the killing effect of IL34-CAR-T on ASPC1 after silencing CSF1R. The results showed that the killing rate and the apparent decrease of IL34-CAR-T cells on CSF 1R-knocked-down ASPC1-shCSF1R #1 and ASPC1-shCSF1R #1 cells compared to the control ASPC1-shCOO2 cells. This result indicates that knocking down CSF1R expression reduces the killing effect of IL 34-CAR-T.

Example 11: inhibitory effect of IL34-CAR-T on ASPC1-luc nude mouse transplantation tumor

ASPC1-luc cells were as described in example 6. Addition of 30% after digestion and counting of ASPC1-luc cell line to adjust cell density to 5X 10 ⁶ And/ml. Female NCG mice of 6 weeks of age were purchased from tokyo kuro pharmacon biotech gmbh, and 100 μ l of cell suspension was subcutaneously inoculated to each mouse, and CART cells were reinfused 7 days later, and prepared as described in example 7. Nude mice were imaged 1 day before CART reinfusion: 0.025% Avertin (300. Mu.l/20 g) anesthetized mice were intraperitoneally injected with 200. Mu.l of D-fluorescein plus salt (15 mg/ml), 10 minutes later were followed by live imaging of the small animals, and NTD, CD19-CAR, IL34-CAR were grouped according to the fluorescence value size. 1X 10 of the tail vein of each mouse is returned ⁷ Per 200. Mu.l CART cells. Nude mice were imaged every 7 days thereafter.

As a result: the results are shown in FIG. 10. The result shows that the IL34-CAR-T has obvious inhibition effect on ASPC1 nude mouse transplantation tumor.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Sichuan university Hospital in western China

<120> preparation and application of chimeric antigen receptor immune cell based on IL34

<130> P2022-0323

<160> 9

<170> PatentIn version 3.5

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Met Pro Arg Gly Phe Thr Trp Leu Arg Tyr Leu Gly Ile Phe Leu Gly

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

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Glu Glu Cys Thr Val Thr Gly Phe Leu Arg Asp Lys Leu Gln Tyr Arg

35 40 45

Ser Arg Leu Gln Tyr Met Lys His Tyr Phe Pro Ile Asn Tyr Lys Ile

50 55 60

Ser Val Pro Tyr Glu Gly Val Phe Arg Ile Ala Asn Val Thr Arg Leu

65 70 75 80

Gln Arg Ala Gln Val Ser Glu Arg Glu Leu Arg Tyr Leu Trp Val Leu

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

100 105 110

His Pro Ser Trp Lys Tyr Leu Gln Glu Val Glu Thr Leu Leu Leu Asn

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

130 135 140

Val Leu Ser Leu Leu Asn Ala Pro Gly Pro Asn Leu Lys Leu Val Arg

145 150 155 160

Pro Lys Ala Leu Leu Asp Asn Cys Phe Arg Val Met Glu Leu Leu Tyr

165 170 175

Cys Ser Cys Cys Lys Gln Ser Ser Val Leu Asn Trp Gln Asp Cys Glu

180 185 190

Val Pro Ser Pro Gln Ser Cys Ser Pro Glu Pro Ser Leu Gln Tyr Ala

195 200 205

Ala Thr Gln Leu Tyr Pro Pro Pro Pro Trp Ser Pro Ser Ser Pro Pro

210 215 220

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

225 230 235 240

Leu Pro

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

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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

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

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His Ala Ala Arg Pro

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

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

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

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

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

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Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

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

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

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

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

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

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

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

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

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His Ala Ala Arg Pro Asn Glu Pro Leu Glu Met Trp Pro Leu Thr Gln

20 25 30

Asn Glu Glu Cys Thr Val Thr Gly Phe Leu Arg Asp Lys Leu Gln Tyr

35 40 45

Arg Ser Arg Leu Gln Tyr Met Lys His Tyr Phe Pro Ile Asn Tyr Lys

50 55 60

Ile Ser Val Pro Tyr Glu Gly Val Phe Arg Ile Ala Asn Val Thr Arg

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Leu Gln Arg Ala Gln Val Ser Glu Arg Glu Leu Arg Tyr Leu Trp Val

85 90 95

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

100 105 110

Gly His Pro Ser Trp Lys Tyr Leu Gln Glu Val Glu Thr Leu Leu Leu

115 120 125

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

130 135 140

Ser Val Leu Ser Leu Leu Asn Ala Pro Gly Pro Asn Leu Lys Leu Val

145 150 155 160

Arg Pro Lys Ala Leu Leu Asp Asn Cys Phe Arg Val Met Glu Leu Leu

165 170 175

Tyr Cys Ser Cys Cys Lys Gln Ser Ser Val Leu Asn Trp Gln Asp Cys

180 185 190

Glu Val Pro Ser Pro Gln Ser Cys Ser Pro Glu Pro Ser Leu Gln Tyr

195 200 205

Ala Ala Thr Gln Leu Tyr Pro Pro Pro Pro Trp Ser Pro Ser Ser Pro

210 215 220

Pro His Ser Thr Gly Ser Val Arg Pro Val Arg Ala Gln Gly Glu Gly

225 230 235 240

Leu Leu Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

245 250 255

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

260 265 270

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

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

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

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

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

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Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

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

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

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

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

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

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

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Pro Arg

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<210> 9

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<213> Artificial Sequence (Artificial Sequence)

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atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccgaatgagc ctttggagat gtggcccttg acgcagaatg aggagtgcac tgtcacgggt 120

tttctgcggg acaagctgca gtacaggagc cgacttcagt acatgaaaca ctacttcccc 180

atcaactaca agatcagtgt gccttacgag ggggtgttca gaatcgccaa cgtcaccagg 240

ctgcagaggg cccaggtgag cgagcgggag ctgcggtatc tgtgggtctt ggtgagcctc 300

agtgccactg agtcggtgca ggacgtgctg ctcgagggcc acccatcctg gaagtacctg 360

caggaggtgg agacgctgct gctgaatgtc cagcagggcc tcacggatgt ggaggtcagc 420

cccaaggtgg aatccgtgtt gtccctcttg aatgccccag ggccaaacct gaagctggtg 480

cggcccaaag ccctgctgga caactgcttc cgggtcatgg agctgctgta ctgctcctgc 540

tgtaaacaaa gctccgtcct aaactggcag gactgtgagg tgccaagtcc tcagtcttgc 600

agcccagagc cctcattgca gtatgcggcc acccagctgt accctccgcc cccgtggtcc 660

cccagctccc cgcctcactc cacgggctcg gtgaggccgg tcagggcaca gggcgagggc 720

ctcttgccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgctagc 780

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 840

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 900

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 960

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1020

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1080

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1200

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1320

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1380

caggccctgc cccctcgc 1398

Claims

1. A Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular binding domain comprising the structure of IL34 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 an IL34 receptor.

2. The chimeric antigen receptor according to claim 1, wherein the extracellular binding domain has an amino acid sequence as shown in SEQ ID No. 1, or an amino acid sequence from position 21 to 242 of the sequence as shown in SEQ ID No. 1.

3. The chimeric antigen receptor according to claim 1, having the structure of formula I:

L-EB-H-TM-C-CD3ζ-RP (I)

in the formula (I), the compound is shown in the specification,

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

l is a null or signal peptide sequence;

h is a none or hinge region;

TM is a transmembrane domain;

c is a no or co-stimulatory signal molecule;

CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ;

RP is a null or reporter protein.

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

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the vector or chromosome of claim 5 integrated with an exogenous nucleic acid molecule of claim 4 or expressing the chimeric antigen receptor of claim 1.

7. An engineered immune cell comprising the vector or chromosome of claim 5 integrated with an exogenous nucleic acid molecule of claim 4 or expressing the chimeric antigen receptor of claim 1.

8. A method of making the engineered immune cell of claim 7, comprising the steps of: transferring the nucleic acid molecule of claim 4 or the vector of claim 5 into an immune cell, thereby obtaining the engineered immune cell.

9. A pharmaceutical composition comprising the CAR of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, and/or the engineered immune cell of claim 7, and a pharmaceutically acceptable carrier, diluent or excipient.

10. Use of the CAR of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, or the host cell of claim 6, and/or the engineered immune cell of claim 7, for the preparation of a medicament or formulation for the prevention and/or treatment of a disease in which the IL34 receptor is highly expressed.