CN115819614B

CN115819614B - Preparation and application of chimeric antigen receptor immune cells based on IL34

Info

Publication number: CN115819614B
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: 2024-02-23
Anticipated expiration: 2042-06-21
Also published as: CN115819614A

Abstract

The invention provides a preparation method and application of chimeric antigen receptor immune cells constructed based on IL 34. In particular, the invention provides a Chimeric Antigen Receptor (CAR) based on IL34 engineering, said CAR comprising an extracellular binding domain capable of specifically targeting IL34 receptor (especially CSF 1R). The CAR immune cells have high specificity and high killing capacity, and have excellent tumor inhibition capacity through in vivo tests.

Description

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

Technical Field

The invention belongs to the field of immune cell therapy, and particularly relates to preparation and application of an IL 34-constructed chimeric antigen receptor immune cell.

Background

The tumor is the second biggest disease threatening human health, and 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 reduced life quality, easy recurrence and the like of the patients still restrict the traditional tumor treatment means.

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

Interlukin-34 (IL-34) is a cytokine found in 2008 and expressed in spleen, thymus, heart, brain, lung, liver, kidney, testis, prostate, ovary, small intestine, colon and other tissues. IL34 is a secreted homodimeric glycoprotein consisting of 242 amino acids with a molecular weight of 39kD in humans. Highly conserved (99.6% similarity) between human and chimpanzee, 72% similarity between human and mouse. IL34 is now found to have three receptors: colony stimulating factor receptor (Coloney-stimulating factor receptor, CSF1R), tyrosine phosphatase zeta receptor (the receptor-type protein-tyrosine phosphatase zeta, PTP-zeta), syndecan-1.

CSF1R is a receptor tyrosine kinase and, upon binding to a ligand, induces phosphorylation of proteins inside the cytoplasm of CSF1R and forms dimers that phosphorylate a range of other proteins (e.g., ERK1/2 or AKT). IL-34 derived from cancer cells activates ERK1/2 and AKT downstream of CSF1R in an autocrine manner, thereby providing a critical survival signal for CSF 1R-expressing cancer cells. CSF1R is overexpressed in various solid tumors such as breast cancer, gastric cancer, colorectal cancer, and the like, and is associated with metastasis and prognosis of the tumor, e.g., activation of CSF1R paracrine pathway in osteosarcoma can promote tumor invasion, autocrine activation of CSF1R in breast cancer is associated with metastasis and growth of the tumor, and implies poor prognosis. In addition, inhibiting or knocking down CSFIR causes an increase in T cell lymphoma cell apoptosis, while inhibiting CSF1R activity in a mouse engraftment tumor model inhibits tumor growth. There are a number of small molecule inhibitors (PLX 3397, ARRY-382, PLX7486, BLZ945, JNJ 40346527) and antibodies targeting CSF1R currently in clinical phase I/II. And when the small molecule inhibitor or antibody is combined with the CTLA-4/PDL1 blocker, the growth of pancreatic cancer and colorectal cancer can be obviously inhibited, and the survival time of mice can be prolonged.

Meanwhile, CSF1R is also widely present in tumor microenvironments (tumor microenvironment, TME). The CSF1R signaling pathway activates a variety of proteins that promote myeloid cell differentiation, monocyte targeting, and macrophage survival, proliferation and chemotaxis. In TME, IL34, upon binding to CSF1R, causes ERK1/2 and AKT phosphorylation, thereby affecting megaphagy morphology and phenotype, modulating the function and survival of tumor-associated macrophages (tumor associated macrophages, TAM), which play a vital role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression and tumor resistance.

IL34 receptor PTP- ζ is highly expressed in a variety of tumors including, but not limited to, lung cancer, uterine cancer, hepatocellular carcinoma, renal cancer, prostate cancer, glioma, and astrocytoma, and blocking PTP- ζ can inhibit glioblastoma growth and prolong survival of mice. A number of antibodies targeting PTP- ζ (7E 4B11-SAP, SCB 4380) have been shown to be very potent in anti-tumor effects both in vitro and in a model of transplanted tumor.

IL34 receptor Syndecan-1 is highly expressed in various tumors such as myeloma, melanoma, liver cancer, lung cancer, pancreatic cancer and the like, and migration of bone marrow cells depends on the interaction of IL34 and Syndecan-1. Binding of IL34 to Syndecan-1 regulates IL34 binding to CSF1R, and small amounts of Syndecan-1 retain IL34 on the cell membrane surface via chondroitin sulfate chains, thereby reducing its binding to CSF 1R. In contrast, overexpression of Syndecan-1 promotes binding of IL34 to CSF 1R. Antibodies targeting Syndecan-1 and CAR-T treatment show good efficacy in the treatment of patients with multiple myeloma.

However, there is currently no chimeric antigen receptor immune cell constructed based on IL-34 that targets these receptors.

Thus, there is a strong need in the art to develop novel chimeric antigen receptor immune cells targeting the IL-34 receptor and methods of treatment thereof.

Disclosure of Invention

The invention aims to provide chimeric antigen receptor immune cells targeting IL34 receptor (especially CSF 1R) and preparation and application methods thereof.

In a first aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR), said CAR comprising an extracellular binding domain, and said extracellular binding domain comprising the structure of 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 the IL34 receptor.

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

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

In another preferred embodiment, the IL34 receptor is an IL34 receptor located on the cell membrane.

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 a second extracellular domain directed against an additional target in addition to the first extracellular domain directed against the IL34 receptor.

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 IL 34.

In another preferred embodiment, the extracellular binding domain comprises an IL34 protein or fragment thereof.

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

In another preferred embodiment, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO. 1, preferably the amino acid sequence at positions 21 to 242 of the sequence shown in SEQ ID NO. 1.

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

(i) A sequence shown in positions 21 to 242 of the sequence shown in SEQ ID NO. 1; and

(ii) An amino acid sequence obtained by performing substitution, deletion, alteration or insertion of one or more amino acid residues, or adding 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to the N-terminus or C-terminus thereof based on the sequence shown at positions 21 to 242 of the sequence shown in SEQ ID NO. 1; and the amino acid sequence obtained has a sequence identity of ≡85% (preferably ≡90%, more preferably ≡95%, for example ≡96%,. Gtoreq.97%,. Gtoreq.98% or ≡99%) with the sequence shown in positions 21 to 242 of the sequence shown in SEQ ID NO. 1; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

In another preferred embodiment, the amino acid sequence of the extracellular binding domain is shown at positions 21 to 242 of SEQ ID NO. 1.

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

L-EB-H-TM-C-CD3ζ-RP (I)

in the method, in the process of the invention,

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

l is an absent or signal peptide sequence;

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

h is a no or hinge region;

TM is a transmembrane domain;

c is an absent or co-stimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

RP is absent or reporter.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In an eighth aspect of the invention there is provided the use of a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, and/or an engineered immune cell according to the fifth aspect of the invention, for the preparation of a medicament or formulation for the prevention and/or treatment of a disease in which IL34 receptor is highly expressed.

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

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

In another preferred embodiment, the IL34 receptor high expression related diseases include: tumors, infections, inflammatory diseases, and the like.

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

In another preferred embodiment, the high expression of IL34 receptor means that the ratio of the expression level (F1) of IL34 receptor 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 comprises a solid tumor or a hematological 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 carcinoma, melanoma, or a combination thereof.

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

In another preferred embodiment, the tumor is pancreatic cancer.

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

In another preferred embodiment, the tumor is pancreatic cancer.

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

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

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

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

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

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

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

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

Drawings

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

Figure 2 shows CAR transfection efficiency detection. Wherein A is the fluorescent expression of cells 72 hours after T cells are infected with CD19-CAR and IL34-CAR, wherein BF (upper row) is bright field and mKate2 (lower row) is the fluorescent expression of CAR. B is the flow detection fluorescence expression.

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

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

Figure 5 shows ifnγ release results after IL34-CAR killing ASPC1 pancreatic cancer cell line.

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

Figure 7 shows the killing effect of IL34-CAR on PANC1 overexpressing CSF1R and ifnγ release.

Figure 8 shows that knockdown CSF1R expression in ASPC1 cells reduces IL34-CAR killing. Wherein A is the phenotype of the cells after knocking down CSF1R by ASPC1 cells. B is qPCR to detect CSF1RmRNA levels. C is the detection of IL34-CAR killing of ASPC1-shCSF 1R.

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

Figure 10 shows the inhibition of ASPC1 nude mice engraftment by IL 34-CAR. Wherein A is the live imaging of ASPC1 nude mice transplanted tumor in different time periods of CART reinfusion. B is a fluorescence intensity statistical graph of the transplanted tumor.

Detailed Description

Through extensive and intensive studies, the present inventors have developed, for the first time, a chimeric antigen receptor immune cell constructed based on IL34 through a large number of screens. The present invention uses a partial fragment of full-length IL34 (i.e., amino acid sequence from position 21 to 242) as the extracellular binding domain of the CAR, resulting in a CAR-T cell targeting IL34 receptor (in particular CSF 1R). In vitro experiments indicate that the CAR-T cells have high specificity and excellent cell killing power, and in vivo experiments also indicate that the CAR-T cells have in vivo inhibition capability. The present invention has been completed on the basis of this finding.

Terminology

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

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

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

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

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

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

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

Conservative amino acid substitutions are known in the art. In some embodiments, the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine. Furthermore, the invention provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.

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

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

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

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

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

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

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

Colony stimulating factor receptor (CSF 1R)

The IL 34-based CARs provided by the invention can specifically bind to IL34 receptors. A representative IL34 receptor is CSF1R. CSF1R is a receptor tyrosine kinase and, upon binding to a ligand, induces phosphorylation of proteins inside the cytoplasm of CSF1R and forms dimers that 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 tumor apoptosis is observed by inhibition or knockdown of CSFIR, while in mouse engraftment tumor models, inhibition of CSF1R activity inhibits tumor growth. Meanwhile, studies have demonstrated that activation of the CSF1R paracrine pathway in osteosarcoma can promote tumor invasion, and that autocrine activation of CSF1R in breast cancer is associated with tumor metastasis and growth, and implies a poor prognosis.

CSF1R has two ligands: colony stimulating factor-1 (color-stimulating factor-1, CSF-1) and IL34.CSF-1 exists in the circulatory system primarily in the form of proteoglycans, secreted by a variety of cells of mesenchymal and epithelial origin. A variety of diseases including infection, cancer and chronic inflammatory diseases cause increased CSF1 expression in the blood. Binding of CSF1 to CSF1R is primarily through salt bonds, whereas binding of IL34 to CSF1R requires hydrophobic amino acids and hydrophobic interaction bonds, and binding of CSF1 to 1 CSF1R and binding of IL34 to two CSF1 rs, therefore IL34 has a greater affinity for CSF1R. In macrophages, IL34, when bound to CSF1R, causes more intense phosphorylation of ERK1/2 and AKT in a short period of time than CSF1, thereby affecting 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 protect against future invasion. In this regard, increasing evidence reveals the importance of the IL-34/CSF1R axis in cancer chemoresistance. IL-34 derived from cancer cells activates ERK1/2 and AKT downstream of CSF1R in an autocrine manner, thereby providing a critical survival signal for CSF 1R-expressing cancer cells.

Meanwhile, CSF1R is also widely present in tumor microenvironments (Tumor Microenvironment, TME). The CSF1R signaling pathway promotes differentiation of myeloid cells, orientation of monocytes, and survival, proliferation and chemotaxis of macrophages by modulating tyrosine phosphorylation, activating a variety of proteins. In TME, CSF1R regulates the function and survival of tumor-associated macrophages (Tumor Associated Macrophages, TAM), which play a vital role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression and tumor resistance.

During cancer progression, IL-34 is a powerful tool for reprogramming macrophages into tumor-promoting macrophages in primary tumors. In solid tumors, TAMs are the most abundant immunosuppressive cells in TMEs, the number of which is associated with poor prognosis. TAMs have a phenotype of M2 polarization, enabling them to modulate immune responses, promote angiogenesis and promote tumor growth, invasion and metastasis. Early studies on IL-34 biology showed that IL-34-derived macrophages exhibit the phenotypic and functional properties of most TAMs (low T cell costimulatory properties, inhibiting activated effector T cell responses). Bone marrow-derived macrophages appear as M2-like macrophages, exhibit most of the phenotype 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 conversion of memory T cells into Th17 cells through constitutive expression of membrane IL-1α. In addition to TAM, CSF1R expression can also be detected in tumor-associated dendritic cells, tumor-associated neutrophils, and myeloid-derived suppressor cells. Thus, the effects of CSF-1 and IL-34 on these myeloid cells in the tumor microenvironment are also important.

PTP-ζ

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

Syndecan-1

Another representative IL34 receptor is Syndecan-1.Syndecan-1 is highly expressed in various tumors such as myeloma, melanoma, liver cancer, lung cancer, pancreatic cancer and the like, and migration of bone marrow cells depends on the interaction of IL34 and Syndecan-1. In addition to IL34, syndecan-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. IL34 binding to Syndecan-1 regulates IL34 binding to CSF1R, and IL34 is retained on the cell membrane surface by chondroitin sulfate chains in small amounts in Syndecan-1, thereby reducing its binding to CSF 1R. In contrast, overexpression of Syndecan-1 promotes binding of IL34 to CSF 1R.

At present, a cell line which is positive for CSF1R, PTP- ζ and Syndecan-1 simultaneously is not found, so that the binding of the CAR-T cells according to the invention to these two receptors cannot be verified. However, both PTP- ζ and Syndecan-1 binding to IL34 are mechanistically dependent on chondroitin sulfate. Both belong to transmembrane proteoglycans, both extracellular glycosaminoglycans and intracellular C-terminal PDZ domains. In addition, IL34 binds to CSF1R most strongly, followed by PTP- ζ and Syndecan-1. Specifically, IL34 has a Kd of 10 with CSF1R ^-12 M, kd with PTP- ζ is 10 ^-7 M, kd with Syndecan-1 is about 10 ^-8 M. Thus, it is contemplated that IL 34-CARs of the invention may also recognize PTP- ζ and Syndecan-1. And, experiments confirm that: IL34-CART vs CSF1R ⁺ /Syndecan-1 ⁺ Also has killing effect on MCF7 cells.

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

Therefore, the invention selects the receptor/ligand naturally combined with the target molecule, designs the CAR sequence by utilizing the characteristic advantage of the combination conservation of the receptor/ligand and the ligand which are developed by natural selection, and has more proper affinity, thereby better overcoming the problem of unsuitable affinity of the artificially designed antibody. The studies of the present invention demonstrate that CAR-T cells constructed using natural ligands of the IL34 receptor as extracellular recognition domains are well expressed in vivo and produce tumor-inhibiting effects.

Based on the above, the invention integrates the IL34 fragment into the CAR carrier in a genetic engineering way for the first time, and modifies the related immune cells, thereby realizing the specific killing of the positive cells of the IL34 receptor, and being applicable to the treatment of related diseases.

Chimeric Antigen Receptor (CAR) of the invention

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

The design of the CAR goes through the following process: the first generation of CARs had only one intracellular signaling component, cd3ζ or fcγri molecule, which, due to the presence of only one activation domain within the cell, only caused transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CAR introduces a co-stimulatory molecule such as CD28, 4-1BB, OX40 and ICOS based on the original structure, and has greatly improved function compared with the first generation CAR, and further enhances the persistence of CAR-T cells and the killing ability to tumor cells. Some new immune co-stimulatory molecules such as CD27, CD134 are concatenated on the basis of the second generation CARs, developing into third and fourth generation CARs.

The extracellular segment of the CAR recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity, and secretion of cytokines, thereby clearing the target cell. Patient autologous cells (or heterologous donors) are first isolated, CAR-producing immune cells are activated and genetically engineered, and then injected into the same patient. This way the probability of graft versus host disease is very low and the antigen is recognized by immune cells in a non-MHC restricted manner.

CAR-immune cell therapy has achieved a very high clinical response rate in hematological malignancy therapy, which is not achieved by any conventional therapeutic means, and has triggered a hot tide of clinical research worldwide.

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

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

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

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

The linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the CD28 signaling domain, and the cd3ζ signaling domain.

In the present invention, the extracellular binding domain of the CAR of the invention also includes sequence-based conservative variants, meaning that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence at positions 21 to 242 of SEQ ID NO. 1 to form a polypeptide.

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

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

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

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

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

Chimeric antigen receptor immune cells (CAR-immune cells)

In the present invention, there is provided a chimeric antigen receptor immune cell comprising a chimeric antigen receptor of the present invention having a specific targeting of the IL34 receptor (particularly CSF 1R).

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

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

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

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

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

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

Carrier body

Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The invention also provides vectors comprising the nucleic acid molecules of the invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

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

The expression constructs of the invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid may be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

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

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

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

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

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

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). A preferred method of introducing the polynucleotide into a host cell is calcium phosphate transfection.

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

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

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

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

Formulations

The present 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 carrierIs a carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said CAR-T cells in said formulation is 1 x 10 ³ -1×10 ⁸ Individual cells/ml, more preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

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

Therapeutic applications

The invention includes therapeutic applications with cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding the expression cassettes of the invention. The transduced T cells can target a tumor cell marker IL34 receptor, and synergistically activate the T cells to cause immune cell immune response, so that the killing efficiency of the transduced T cells on the tumor cells is remarkably improved.

Accordingly, the present invention also provides a method of stimulating a T cell mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering the CAR-cells of the invention to a mammal.

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

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-modified T cells induce an immune response specific for an antigen binding domain in the CAR. For example, CAR-T cells of the IL34 receptor elicit a cell-specific immune response against the IL34 receptor.

Although the data disclosed herein specifically disclose lentiviral vectors comprising IL34 protein or fragments thereof, hinge and transmembrane regions, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of variations to each of the construct components.

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

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

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

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

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

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

In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

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

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

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

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

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

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

The main advantages of the invention include:

1) Target specificity: the IL34 receptor (especially CSF 1R) is not expressed on the cell membrane of normal cells, but is highly 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 the 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. The conservation of receptor-ligand interactions determines that safety assays in animals, particularly primates, are more responsive to their safety in humans.

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

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

TABLE 1 summary of the sequences to which the invention relates

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Table 2 shows the cell lines used in the examples.

TABLE 2 cell lines

Cell lines	Type(s)
		PANC1	Pancreatic cancer cells
BXPC3	Pancreatic cancer cells
		ASPC1	Pancreatic cancer cells
MCF-7	Breast cancer cells

Example 1: preparation of IL34-CAR vectors

Based on the nucleotide sequence information of IL34 (NM_ 001172772.2), 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, the corresponding nucleotide sequence is obtained by artificial synthesis method or PCR method. The CD8 signal peptide and the extracellular domain of IL34 are synthesized or, alternatively, the nucleotide sequence of the CAR molecule is double digested by AgeI (Thermo) and NheI (Thermo), and inserted into the lentiviral vector pTomo into which the CD8 transmembrane region, 4-1BB costimulatory domain, CD3 zeta signaling region have been inserted via a T4 DNA ligase (NEB) ligation. Competent E.coli (Stbl 3) was transformed.

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

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

Example 2: virus package

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

Example 3: CAR-T cell preparation

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

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

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

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

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

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

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

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

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

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

Results: the results of the CSF1R expression assay for each cell line are shown in FIG. 3. The immunofluorescence detection of the expression of the target cell CSF1R shows that BXPC3 and ASPC1 express the CSF1R in a high mode and PANC1 expresses the CSF1R in a low mode.

Example 6: construction of target cells carrying luciferases

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

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

Example 7: CAR-T cell killing

In this example, the killing ability of CAR-T cells of the invention against different target cells was tested. The target cells used include: target cells highly expressing CSF 1R: BXPC3, ASPC1; target cells that do not express or underexpress CSF 1R: PANC1; CSF1R ⁺ /Syndecan-1 ⁺ Is selected from the group consisting of MCF7 cells.

Cell density was adjusted to 2X 10 after digestion and counting of MCF7, PANC1-luciferase cells ⁴ /ml. Mu.l of luciferase cells were seeded in 96-well plates and the CAR-T and control cells were adjusted to a cell density of 1X 10 ⁵ Per ml, 100 μl per well was plated in black 96-well plates at 5:1 E:T. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours.

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

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

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

Results: the results of gradient killing of IL34-CAR against different pancreatic cancer cell lines are shown in figures 4 and 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), and the IL34-CART cells have no killing effect on the tumor cells which do not basically express CSF1R and have no killing effect on CSF1R ⁺ /Syndecan-1 ⁺ Also MCF7 cells of (a) have a significant killing effect.

Example 8: IFNgamma cytokine release

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

The method comprises the following steps: ifnγ was detected according to IFN gamma Human ELISA Kit (life technology) from cell supernatants of CAR-T cells of the invention of example 7 incubated with target cells such as PANC1 and ASPC 1.

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

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

Then sequentially operating according to the following steps:

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

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

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

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

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

(6) Absorbance was measured at 450 nm.

Results: the results are shown in FIG. 5. The cytokine IFNγ increases significantly after IL34-CAR-T kills ASPC 1. The right panel control results of fig. 7 show that IL34-CAR does not release ifnγ to CSF1R negative PANC1 cells. The results show that the killing effect of IL34-CAR-T cells on tumor cells is accompanied by IFN gamma release, suggesting that the killing effect is related to IFN gamma release.

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

Specific primers are designed according to CDS region sequences of CSF1R, and 293T cell cDNA is used as a template to amplify CSF1R and enzyme-cut and connect to construct pTomo-CMV-CSF1R-T2A-luciferase-IRES-puro vector. Lentiviral packaging As described in example 2, PANC1-CSF1R-luc cells were obtained 2 weeks after viral infection of PANC1 cells by screening with puromycin (1. Mu.g/ml).

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

Results: the results of killing of IL34-CAR-T cells against pancreatic cancer cell line PANC1 after over-expressing CSF1R are shown in FIGS. 6 and 7. FIG. 6 shows the detection of over-expression of CSF1R by PANC1 cells. FIG. 7 shows IL34-CAR-T killing effect and IFN gamma release after PANC1 over-expression of CSF 1R. The results show a significant increase in both the killing rate and ifnγ release of the IL34-CAR-T cells on PANC1-CSF1R cells over-expressing CSF1R compared to the control group PANC 1-con. The result shows that the killing effect of IL34-CAR-T cells on CSF1R over-expression tumor cells is obviously enhanced.

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

The CDS region-verified shRNAs were selected from the CSF1R shRNA sequence library provided by Sigma, and each shRNA selected by BLAST at NCBI, ensuring target specificity. The shRNA was ligated into the pLKO.1 vector and the correct vector was confirmed by restriction enzyme identification and sequencing.

shRNA virus packaging: the day before transfection, 100 ten thousand HEK-293T cells per dish were seeded into 6cm dishes for culture. Before transfection, 6cm dishes were replaced with 5ml fresh medium (serum-containing, antibiotic-free); two clean sterile centrifuge tubes were taken and 250 μl each was addedMedium, then 5. Mu.g shRNA plasmid, 2.5. Mu.g pCMV DeltaR 8.9, 1. Mu.g PMD2.G are added into one tube, and gently blown and mixed by a gun; another tube was filled with 17. Mu.l Lipo6000 ^TM The transfection reagent was gently beaten and mixed with a gun. After standing at room temperature for 5 minutes, the culture solution containing DNA was gently added with a gun to the culture solution containing Lipo6000 ^TM In the culture medium of the transfection reagent, the centrifuge tube is gently inverted or gently applied with a gunBlowing and mixing uniformly, standing at room temperature for 20min, adding into a 6cm dish and mixing uniformly, respectively collecting supernatant after 48h and 72h, centrifuging at 3000r/min for 20min at 4 ℃ and filtering with a 0.45 mu m filter membrane to obtain a virus-containing supernatant.

shRNA virus infection: the day before infection, 50 ten thousand ASPC1 cells per well were seeded into six well plates for culture. Before infection, six well plates were replaced with 1ml fresh culture medium (containing serum, no antibiotics) per well, and 1ml virus supernatant, 2 μl polybrane (10 mg/ml) was added; and replacing the complete culture medium after 24 hours, detecting the shRNA knocking-down efficiency after 96 hours, and carrying out IL34-CAR-T cell killing detection.

Preparation of ASPC1-shCOO2, ASPC1-shCSF1r#1, ASPC1-shCSF1r#2-luciferase cells by infection of ASPC1 cells with plko.1-shCSF1R-1#, plko.1-shCSF1R-2# lentivirus, and adjustment of cell density to 2×10 after 48 hours of digestion and counting ⁴ /ml. Mu.l of luciferase cells were seeded in 96-well plates and CAR-T cells were adjusted to a cell density of 8X 10 ⁴ Per ml, inoculated into black 96-well plates at E: T of 4:1, 2:1, 1:1, 0.5:1, 100 μl per well. And uniformly mixing the target cells and the T cells, incubating the mixture in an incubator for 24 hours, and 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. .

Results: the results are shown in FIG. 8. FIG. 8-A is a phenotype of knockdown CSF1R cells in ASPC1 cells. FIG. 8-B shows qPCR detection of CSF1RmRNA levels. FIG. 8-C is the post-silencing CSF1R killing effect of IL34-CAR-T on ASPC 1. The results show that IL34-CAR-T cells knocked down the killing rate and significantly decreased for ASPC1-shCSF1R#1 and ASPC1-shCSF1R#1 cells of CSF1R compared to control ASPC1-shCOO2 cells. This result indicates that knocking down CSF1R expression reduces the killing effect of IL 34-CAR-T.

Example 11: IL34-CAR-T inhibition effect on ASPC1-luc nude mice transplantation tumor

ASPC1-luc cells were as described in example 6. ASPC1-luc cell line digestion count was followed by addition of 30% matrigel to adjust cell density to 5X 10 ⁶ /ml. Female NCG mice of 6 weeks old were purchased from Nanjing Ji Kangyaokang biotechnology Co., ltd, and 100. Mu.l of cell suspension was inoculated subcutaneously into each mouse, and CART cells were returned after 7 daysCell preparation was as described in example 7. Nude mice were imaged 1 day prior to CART reinfusion: 200 mu l D-fluorescein plus salt (15 mg/ml) was intraperitoneally injected after 0.025% Avertin (300. Mu.l/20 g) anesthetized mice, and after 10 minutes, live-animal imaging was performed, and NTD, CD19-CAR, IL34-CAR were grouped according to fluorescence value size. Tail vein reinfusion 1 x 10 per mouse ⁷ 200 μl CART cells. The nude mice were imaged every 7 days thereafter.

Results: the results are shown in FIG. 10. The result shows that IL34-CAR-T has remarkable inhibition effect on ASPC1 nude mice transplantation tumor.

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

Sequence listing

<110> Huaxi Hospital at university of Sichuan

<120> preparation of IL 34-based chimeric antigen receptor immune cell and use thereof

<130> P2022-0323

<160> 9

<170> PatentIn version 3.5

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

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

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

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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

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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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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Pro Ser Lys Leu Gly His Lys Leu Asn

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

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

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

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

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

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

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

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

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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

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

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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

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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

340 345 350

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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<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), characterized in that the chimeric antigen receptor has the structure according to formula I:

L-EB-H-TM-C-CD3ζ-RP(I)

in the method, in the process of the invention,

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

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

EB is an extracellular binding domain that specifically binds to the IL34 receptor and has the amino acid sequence shown in positions 21 to 242 of SEQ ID NO. 1;

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

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

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

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

RP is a null or reporter protein, which is a fluorescent protein.

2. The chimeric antigen receptor according to claim 1,

l is a CD8 derived signal peptide;

h is a CD8 derived hinge region;

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

C is a costimulatory signaling molecule of 4-1BB origin.

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

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

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

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

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

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

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

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

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

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

13. Use of a CAR according to claim 1, a nucleic acid molecule according to claim 5, a vector according to claim 7, or a host cell according to claim 8, and/or an engineered immune cell according to claim 9, for the preparation of a medicament or formulation for the treatment of a disease in which IL34 receptor CSF1R is highly expressed, said disease being 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, esophageal cancer, glioma, astrocytoma, prostate cancer, nasopharyngeal carcinoma, melanoma, or a combination thereof.

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