WO2023246908A1 - Preparation and use of chimeric antigen receptor immune cell targeting csf1r - Google Patents

Abstract

Provided are preparation and use of a chimeric antigen receptor immune cell targeting CSF1R. Specifically, provided is a chimeric antigen receptor (CAR) modified on the basis of a natural CSF1R ligand, the CAR comprising an extracellular binding domain capable of specifically targeting a CSF1 receptor. The CAR immune cell has high specificity and highly efficient killing capability, and in-vivo tests show that it has excellent tumor suppression capability.

Description

Preparation and application of chimeric antigen receptor immune cells targeting CSF1R Technical field

The invention belongs to the field of immune cell therapy, and specifically relates to the preparation and application of chimeric antigen receptor immune cells targeting CSF1R.

Background technique

Cancer is the second largest disease threatening human health. In 2018, there were 18.1 million new cancer patients and 9.5 million cancer deaths worldwide. It is estimated that by 2040, there will be 29.5 million new cancer cases and 16.4 million deaths every year. Although traditional cancer treatments such as radiotherapy, chemotherapy, and surgical resection can delay the survival of cancer patients, the patient's declining quality of life and easy recurrence still restrict traditional cancer treatments.

Biological immunotherapy has become the fourth cancer treatment method after surgery, radiotherapy, and chemotherapy, and will become a necessary method of cancer treatment in the future. Chimeric Antigen Receptor-T cell (CART) T cells refer to T cells that have been genetically modified to recognize specific target antigens in an MHC-unrestricted manner and to continuously activate and expand. The structure of CAR includes a tumor-associated antigen binding region, extracellular hinge region, transmembrane region and intracellular signaling region. Cells Currently, CART therapy has shown strong killing ability in hematological malignancies. However, the application of CART therapy in solid tumors is limited by tumor heterogeneity, lack of tumor-specific antigens, and tumor immunosuppressive microenvironment.

Colony-stimulating factor 1 receptor (CSF1R) belongs to the platelet-derived growth factor family. In addition to being expressed in myeloid cells such as macrophages, Langerhans cells, and osteoclasts, CSF1R is also overexpressed in various tumors such as breast cancer, gastric cancer, and colorectal cancer. Inhibiting or knocking down CSFIR can increase the apoptosis of T-cell lymphoma cells. At the same time, inhibiting CSF1R activity can inhibit tumor growth in mouse transplant tumor models. In addition, studies have demonstrated that activation of the CSF1R paracrine pathway in osteosarcoma can promote tumor invasion, and autocrine activation of CSF1R in breast cancer is associated with tumor metastasis and growth and implies poor prognosis.

CSF1R also exists widely in the tumor microenvironment (Tumor Microenvironment, TME). The CSF1R signaling pathway activates a variety of proteins by regulating tyrosine phosphorylation, promoting the differentiation of myeloid cells, the orientation of monocytes, and the survival, proliferation, and chemotaxis of macrophages. In the TME, CSF1R regulates the function and survival of tumor-associated macrophages (TAMs), which play a crucial role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression, and therapy. In addition to TAMs, CSF1R expression can also be detected on tumor-associated dendritic cells, tumor-associated neutrophils, and myeloid-derived suppressor cells. CSF1 is the ligand of CSF1R.

Therefore, CSF1R is an important target for tumor treatment. Targeting CSF1R may inhibit tumor growth, invasion, metastasis and drug resistance, thereby prolonging the survival rate of patients.

Therefore, there is an urgent need in this field to develop chimeric antigen receptor immune cells and their therapeutic methods targeting CSF1R.

Contents of the invention

The purpose of the present invention is to provide a chimeric antigen receptor immune cell targeting the CS1 receptor (CSF1R) and its preparation and application methods.

In a first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, the CAR contains an extracellular binding domain, and the extracellular binding domain includes:

1) The structure of CSF1 or its fragment based on the amino acid sequence shown in SEQ ID NO:1; or

2) The structure of IL34 or its fragment based on the amino acid sequence shown in SEQ ID NO.10,

Moreover, the extracellular binding domain can specifically bind to the CSFl receptor.

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

In another preferred embodiment, the CSFl receptor includes CSF1R located on the cell membrane.

In another preferred embodiment, the CSF1R is derived from humans or non-human mammals.

In another preferred example, the non-human mammals include: rodents (such as rats, mice), primates (such as monkeys); preferably primates.

In another preferred embodiment, the extracellular binding domain of the CAR, in addition to the first extracellular domain targeting CSF1R, also includes a second extracellular domain targeting additional targets.

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 CSFl.

In another preferred embodiment, the extracellular binding domain includes CSFl protein or a fragment thereof.

In another preferred embodiment, the extracellular binding domain includes wild-type or mutant CSFl protein domain.

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

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

(i) The sequence represented by positions 33 to 496 of the sequence represented by SEQ ID NO:1; and

(ii) On the basis of the sequence shown in positions 33 to 496 of the sequence shown in SEQ ID NO:1, one or more amino acid residues are replaced, deleted, changed or inserted, or at its N-terminus or C 1 to 30 amino acid residues are added to the end, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence is identical to SEQ ID NO. : The sequence shown at positions 33 to 496 of the sequence shown in 1 has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) Sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

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

In another preferred embodiment, the extracellular binding domain has an amino acid sequence derived from IL34.

In another preferred embodiment, the extracellular binding domain includes IL34 protein or a fragment thereof.

In another preferred embodiment, the extracellular binding domain includes wild-type or mutant IL34 protein domain.

In another preferred example, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO: 10, preferably the amino acid sequence at positions 21 to 242 of the sequence shown in SEQ ID NO: 10.

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

(i) The sequence shown in bits 21 to 242 of the sequence shown in SEQ ID NO:10; and

(ii) On the basis of the sequence shown at positions 21 to 242 of the sequence shown in SEQ ID NO:10, one or more amino acid residues are replaced, deleted, changed or inserted, or at its N terminus or C 1 to 30 amino acid residues are added to the end, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence is the same as SEQ ID NO : The sequence shown at positions 21 to 242 of the sequence shown in 10 has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) Sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

In another preferred example, the structure of the CAR is as shown in Formula I below:
L-EB-H-TM-C-CD3ζ-RP (I)

In the formula,

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

L is none or signal peptide sequence;

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

H is the null or hinge region;

TM is the transmembrane domain;

C is no or co-stimulatory signaling molecule;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

RP is a null or reporter protein.

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

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

In another preferred example, the red fluorescent reporter protein RP (mKate2) also includes a self-cleavage recognition site located at its N-terminus, preferably a T2A sequence. In another preferred embodiment, the amino acid sequence of the mKate2 red fluorescent protein is shown in SEQ ID NO: 2.

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

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

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

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

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

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

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

In another preferred embodiment, the TM is a transmembrane region derived from CD28.

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

In another preferred embodiment, the C is a costimulatory signal molecule selected from the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS(CD278), NKG2D, GITR, TLR2, or combinations thereof.

In another preferred example, the C is a costimulatory signal molecule derived from 4-1BB.

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 CD3ζ 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 another preferred embodiment, the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO: 11.

In a second aspect of the invention, a nucleic acid molecule encoding a chimeric antigen receptor as described in the first aspect of the invention is provided.

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

In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 12.

In a third aspect of the present invention, a vector is provided, said vector containing the nucleic acid molecule according to the second aspect of the present invention.

In another preferred embodiment, the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

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

In another preferred embodiment, the vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

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

In another preferred embodiment, the vector also includes a promoter selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.

In another preferred embodiment, the vector includes the nucleotide sequence shown in SEQ ID NO:9 or SEQ ID NO:12.

In the fourth aspect of the present invention, a host cell is provided, the host cell contains the vector as described in the third aspect of the present invention or the exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated into the chromosome. Or express the CAR as described in the first aspect of the invention.

In the fifth aspect of the present invention, an engineered immune cell is provided, the immune cell contains the vector as described in the third aspect of the present invention or the exogenous DNA as described in the second aspect of the present invention is integrated into the chromosome. Nucleic acid molecules or expressions of a CAR as described in the first aspect of the invention.

In another preferred embodiment, the engineered immune cells are selected from the following group: T cells, NK cells, NKT cells, macrophages, or combinations thereof.

In another preferred embodiment, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

In another preferred embodiment, the engineered immune cells are CAR-T cells.

In the sixth aspect of the present invention, a method for preparing engineered immune cells as described in the fifth aspect of the present invention is provided, comprising the following steps: converting the nucleic acid molecule as described in the second aspect of the present invention or the nucleic acid molecule as described in the second aspect of the present invention. The vectors described in the three aspects are transduced into immune cells, thereby obtaining the engineered immune cells.

In another preferred embodiment, the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.

In the seventh aspect of the present invention, a pharmaceutical composition is provided, which pharmaceutical composition contains the CAR as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, and the third aspect of the present invention. The vector described in the aspect, the host cell as described in the fourth aspect of the present invention, and/or the engineered immune cell as described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

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

In another preferred embodiment, the dosage form of the preparation is an injection.

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

In the eighth aspect of the present invention, there is provided a CAR as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, or a vector as described in the third aspect of the present invention. The host cells described in the fourth aspect, and/or the use of the engineered immune cells described in the fifth aspect of the present invention, are used to prepare drugs or preparations for preventing and/or treating diseases with high expression of CSFl receptors.

In another preferred embodiment, the diseases associated with high CSF1R expression include but are not limited to tumors, aging, obesity, cardiovascular disease, diabetes, neurodegenerative diseases, infectious diseases, etc.

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

In another preferred embodiment, the disease is a malignant tumor with high expression of CSF1R.

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

In another preferred embodiment, the tumors include solid tumors and hematological tumors.

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

In another preferred embodiment, the blood tumor is selected from the following group: T-cell lymphoma, acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL) ), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

In another preferred embodiment, the tumor is pancreatic cancer.

In the ninth aspect of the present invention, there is provided a use of the engineered immune cells as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention, for preventing and/or treating cancer. or tumors.

In another preferred embodiment, the tumor is pancreatic cancer.

In a tenth aspect of the present invention, a method for treating diseases is provided, comprising administering to a subject in need of treatment an effective amount of engineered immune cells as described in the fifth aspect of the present invention, or as described in the seventh aspect of the present invention. pharmaceutical compositions.

In another preferred embodiment, the disease is a disease with high expression of CSFl receptor.

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

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

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

In another preferred embodiment, the method can be used in combination with other treatment methods.

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of the drawings

Figure 1 shows a schematic diagram of CSF1-CAR vector construction. Among them, A is a schematic diagram of the CSF1 sequence, 1-32AA in CSF1 is the signal peptide, and the 33-554AA sequence is the mature protein. B is a schematic structural diagram of the CD19-CAR control vector and CSF1-CAR vector. The signal peptide, hinge region, and transmembrane region are all derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3ζ is derived from human CD3, and mKate2 is a fluorescent marker. , used to detect CAR expression. C is the enzyme digestion identification of pTomo-CSF1-CAR vector HindIII and PstI.

Figure 2 shows the CAR infection efficiency assay. Among them, A is the fluorescence expression of T cells after CD19-CAR and CSF1-CAR infection for 72 hours, BF is the bright field, and mKate2 is the fluorescence expression of CAR. B is flow cytometric detection of fluorescence expression.

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

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

Figure 5 shows the IFNγ release results after CSF1-CAR kills ASPC1 pancreatic cancer cell line.

Figure 6 shows overexpression of CSF1R in the pancreatic cancer cell line PANC1. Among them, A is a schematic diagram of the overexpression vector structure. B is immunofluorescence detection of CSF1R expression in PANC1 cells. C is flow cytometric detection of CSF1R expression.

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

Figure 8 shows that knocking down CSF1R expression in ASPC1 cells reduces the killing effect of CSF1-CAR. Among them, A is the cell phenotype of ASPC1 cells 96 hours after knocking down CSF1R. B is qPCR detection of CSF1RmRNA level. C is the detection of CSF1-CAR killing of ASPC1-shCSF1R. D is the detection of IL34-CAR killing of ASPC1-shCSF1R.

Figure 9 shows the inhibitory effects of CSF1-CAR and IL34-CAR on transplanted tumors in ASPC1 nude mice. Among them, A shows the in vivo imaging of ASPC1 nude mouse transplanted tumors at different time periods after CART reinfusion. B is a statistical graph of fluorescence intensity of transplanted tumors.

Figure 10 shows a schematic diagram of IL34-CAR vector construction. Among them, A is a schematic diagram of the IL34 sequence, 1-20AA in IL34 is the signal peptide, and the 21-242AA sequence is the mature polypeptide. B is a schematic structural diagram of the control plasmids CD19-CAR and IL34-CAR. The signal peptide, hinge region, and transmembrane region are all derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3ζ is derived from human CD3, and mKate2 is a fluorescent marker. , used to detect CAR expression. C is the enzyme digestion identification of pTomo-IL34-CAR vector HindIII and PstI.

Figure 11 shows the CAR transfection efficiency assay. Among them, A is the fluorescence expression of T cells after CD19-CAR and IL34-CAR infection for 72 hours, where BF (upper row) is the bright field, and mKate2 (lower row) is the fluorescence expression of CAR. B is flow cytometric detection of fluorescence expression.

Figure 12 shows the gradient killing results of IL34-CAR on different pancreatic cancer cell lines.

Figure 13 shows the IFNγ release results after IL34-CAR kills ASPC1 pancreatic cancer cell line.

Figure 14 shows the killing effect and IFNγ release of IL34-CAR on PANC1 overexpressing CSF1R.

Figure 15 shows the cytotoxic effect of IL34-CAR on CSF1R ⁺ /Syndecan-1 ⁺ MCF7 cells.

Figure 16 shows the results of ligand screening suitable for constructing chimeric antigen receptors.

Detailed ways

After extensive and in-depth research and extensive screening, the inventors developed chimeric antigen receptor immune cells targeting CSF1R based on the receptor-ligand binding mode for the first time. The present invention has constructed a total of two CAR immune cells targeting CSF1R, whose extracellular binding domains are partial fragments of full-length CSF1 (i.e., amino acid sequence 33 to 496) and partial fragments of full-length IL34 (i.e., amino acid sequences 21 to 242). amino acid sequence). In vitro experiments indicate that the CAR-T cells of the present invention have high specificity and excellent cell killing power, and in vivo experiments also indicate that they have in vivo inhibitory capabilities. On this basis, the present invention was completed.

the term

In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before the present invention is described, it is to be understood that this invention is not limited to the specific methods 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, and that the scope of the invention will be limited only by the appended claims.

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

As used herein, the terms "optionally" or "optionally" mean that the subsequently described event or circumstance may occur but does not necessarily occur.

As used herein, the term "contains" or "includes" can mean open, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of."

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

"Transduction", "transfection", "transformation" or terms used herein refer to the process of delivering exogenous polynucleotides into host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to convert exogenous polynucleotides into host cells. The polynucleotide is introduced into a host cell (eg, E. coli).

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

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

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

Colony-stimulating factor receptor (CSF1R) CSF1R is a receptor tyrosine kinase, and after binding to a ligand, it can induce phosphorylation of proteins in the cytoplasm of CSF1R and form dimers, thereby phosphorylating a series of other proteins (such as ERK1 /2 or AKT). CSF1R is overexpressed in various tumors such as breast cancer, gastric cancer, and colorectal cancer. In T-cell lymphoma, inhibiting or knocking down CSFIR can increase tumor cell apoptosis. At the same time, in mouse transplant tumor models, inhibiting CSF1R activity can inhibit tumor growth. At the same time, studies have proven that activation of the CSF1R paracrine pathway in osteosarcoma can promote tumor invasion, and autocrine activation of CSF1R in breast cancer is associated with tumor metastasis and growth, and implies poor prognosis.

CSF1R widely exists in the tumor microenvironment (Tumor Microenvironment, TME). The CSF1R signaling pathway activates a variety of proteins by regulating tyrosine phosphorylation, promoting the differentiation of myeloid cells, the orientation of monocytes, and the survival, proliferation, and chemotaxis of macrophages. In the TME, CSF1R regulates the function and survival of tumor-associated macrophages (TAMs), which play a crucial role in tumor growth, invasion, metastasis, angiogenesis, immunosuppression, and therapy. In addition to TAMs, CSF1R expression can also be detected in tumor-associated dendritic cells, tumor-associated neutrophils, and myeloid-derived suppressor cells.

CSF1R has two ligands: colony-stimulating factor-1 (CSF-1) and IL34. The binding of CSF1 to CSF1R is mainly through salt bonds, while the binding of IL34 to CSF1R requires Hydrophobic amino acids and hydrophobic interactions bond, and CSF1 binds to one CSF1R and IL34 binds to two CSF1Rs.

CSF1

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

IL-34

Interleukin-34 (IL-34) is a cytokine discovered in 2008. It is found in spleen, thymus, heart, brain, lung, liver, kidney, testis, prostate, ovary, small intestine, colon and other tissues. Express. IL34 is a secreted homodimeric glycoprotein consisting of 242 amino acids in the human body with a molecular weight of 39kD. It is highly conserved between humans and chimpanzees (99.6% similarity) and 72% similarity between humans and mice.

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 the phosphorylation of multiple signaling pathways and promotes tumor metastasis.

In macrophages, compared with CSF1, IL34 binding to CSF1R can cause stronger ERK1/2 and AKT phosphorylation in a short period of time, thereby affecting cell morphology and phenotype. When cancer cells are exposed to stress, such as during chemotherapy, internal survival signaling cascades are triggered to prevent cell death and defend against future insults. In this regard, accumulating evidence has revealed the importance of the IL-34/CSF1R axis in cancer chemoresistance. IL-34 derived from cancer cells activates ERK1/2 and AKT downstream of CSF1R in an autocrine manner, thereby providing a critical survival signal for CSF1R-expressing cancer cells.

It has been found that IL34 has three receptors: CSF1R, tyrosine phosphatase zeta receptor (the receptor-type protein-tyrosine phosphatase zeta, PTP-ζ), and Syndecan-1.

In addition to the above-mentioned CSF1R, another representative IL34 receptor is PTP-ζ. The 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). Blocking PTP-ζ can inhibit glioblastoma. Blastoma tumors grow and extend survival in mice. Currently, there are multiple antibodies targeting PTP-ζ (7E4B11-SAP, SCB4380) that have shown very effective anti-tumor effects in vitro and in transplanted tumor models.

Another representative IL34 receptor is Syndecan-1. Syndecan-1 is highly expressed in various tumors such as myeloma, melanoma, liver cancer, lung cancer, and pancreatic cancer. The migration of bone marrow cells depends on the interaction between IL34 and Syndecan-1. Mechanistically, the binding of PTP-ζ and Syndecan-1 to IL34 depends on chondroitin sulfate. Both are transmembrane proteoglycans, with glycosaminoglycans outside the cell and PDZ domains at the intracellular C-terminus. In addition, IL34 has the strongest binding ability to CSF1R, followed by PTP-ζ and Syndecan-1. Specifically, the Kd of IL34 and CSF1R is 10 ^-12 M, and the Kd of IL34 and PTP-ζ is 10 ^-7 M. Kd with Syndecan-1 is approximately 10 ^-8 M. Therefore, the IL34-CAR of the present invention also has the potential to recognize PTP-ζ and Syndecan-1. Experiments have confirmed that IL34-CART also has a killing effect on CSF1R ⁺ /Syndecan-1 ⁺ MCF7 cells.

Chimeric Antigen Receptor (CAR) of the Invention

At present, the main way to construct CAR-T targeting specific tumor antigens is to design CAR based on related antibodies. However, if the antibody affinity is too low, the ability to target and bind to tumor cells is poor, and if the antibody affinity is too high, excessive immune responses may occur, which may lead to patient tolerance. Poor sex.

Therefore, the present invention selects a receptor/ligand that naturally binds to the target molecule, and takes advantage of the conservative characteristics of the binding evolved by the two under natural conditions to design a CAR sequence whose affinity is more suitable and can better overcome artificially designed antibodies. Affinity inappropriateness issues. The research of the present invention proves that CAR-T cells constructed using the natural ligand of CSF1R as the extracellular recognition domain can express well in vivo and produce tumor suppressive effects.

Based on this, the present invention integrates the CSF1 fragment or IL34 fragment targeting CSF1R into a CAR vector for the first time through genetic engineering, and modifies related immune cells, thereby achieving specific killing of CSF1R-positive cells and can be used for the treatment of related diseases.

Chimeric Antigen Receptor (CAR) consists of an extracellular antigen recognition region, a transmembrane region, and an intracellular costimulatory signal region.

The design of CAR has gone through the following process: the first generation CAR only has one intracellular signaling component, CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause short-term T cell proliferation and less cytokine secretion. , but cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical efficacy. The second-generation CAR introduces a co-stimulatory molecule, such as CD28, 4-1BB, OX40, and ICOS, based on the original structure. Compared with the first-generation CAR, its function is greatly improved, further enhancing the persistence of CAR-T cells and its ability to target tumor cells. of lethality. On the basis of second-generation CAR, some new immune costimulatory molecules such as CD27 and CD134 are connected in series to develop into third- and fourth-generation CAR.

The extracellular segment of CAR can recognize a specific antigen and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines, thereby eliminating target cells. First, the patient's autologous cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR immune cells, and then injected into the same patient. In this way, the probability of developing graft-versus-host disease is extremely low, and the antigen is recognized by immune cells in a non-MHC-restricted manner.

CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. Such a high response rate has not been achieved by any previous treatment method, triggering an upsurge in clinical research around the world.

Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.

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

In one embodiment of the invention, the extracellular domain of the chimeric antigen receptor is a CSFl protein or a fragment thereof that can specifically bind to the CSF1R target site. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence from positions 33 to 496 of the sequence shown in SEQ ID NO:1.

In another embodiment of the invention, the extracellular domain of the chimeric antigen receptor is an IL34 protein or a fragment thereof that can specifically bind to the CSF1R target. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence at positions 21 to 242 of the sequence shown in SEQ ID NO: 10.

The intracellular domain includes costimulatory signaling regions and zeta chain portions. A costimulatory signaling domain refers to the portion of the intracellular domain that includes costimulatory molecules. Costimulatory molecules are cell surface molecules that are required for effective lymphocyte response to antigen, rather than antigen receptors or their ligands.

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

When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to fail to grow, be driven to death, or otherwise affected, and cause the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of the CD28 signaling domain and the CD3ζ signaling domain.

In the present invention, the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which means that compared with the amino acid sequence of positions 33 to 496 of SEQ ID NO: 1, there are at most 10, preferably At most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide; or compared with the amino acid sequence at positions 21 to 242 of SEQ ID NO: 10, At most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.

In the present invention, the number of added, deleted, modified and/or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, and more preferably 1-33%. More preferably, it is 5-30%, more preferably, it is 10-25%, and even more preferably, it is 15-20%.

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

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

The intracellular domain in the CAR of the present invention includes the 4-1BB costimulatory domain and the signaling domain of CD3ζ.

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

Chimeric Antigen Receptor Immune Cells (CAR-Immune Cells)

In the present invention, a chimeric antigen receptor immune cell is provided, which contains the chimeric antigen receptor of the present invention that specifically targets CSF1R.

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

As used herein, the terms "CAR-T cell", "CAR-T" and "CAR-T cell of the present invention" all refer to the CAR-T cell described in the fifth aspect of the present invention.

CAR-T cells have the following advantages over other T cell-based treatments: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, targeting a certain Once the CAR gene construction of tumor markers is completed, it can be widely used; (3) CAR can use both tumor protein markers and glycolipid non-protein markers, expanding the target range of tumor markers; ( 4) Using the patient’s autologous cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

As used herein, the terms "CAR-NK cells", "CAR-NK" and "CAR-NK cells of the present invention" all refer to the CAR-NK cells described in the fifth aspect of the present invention. The CAR-NK cells of the present invention can be used for tumors with high expression of CSF1R.

Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

Compared with CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells of the body; (2) they release very A small amount of cytokines thus reduces the risk of cytokine storm; (3) It is easy to amplify in vitro and develop into "off-the-shelf" products. Otherwise, it is similar to CAR-T cell therapy.

carrier

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

The invention also provides vectors comprising the nucleic acid molecules of the invention. Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia virus, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

Briefly summarized, the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

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

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

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

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

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

An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , myosin promoter, heme promoter and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of a CAR polypeptide or portion thereof, the expression vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification of populations of cells that are transfected or infected by the viral vector. Identify and select expressing cells. In other aspects, the selectable marker can be carried on a separate stretch of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at appropriate times after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82). In one embodiment of the invention, the reporter gene is a gene encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, the construct with a minimum of 5 flanking regions that shows the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be ligated to a reporter gene and used to evaluate the ability of an agent to regulate promoter-driven transcription.

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

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

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenovirus and adeno-associated virus, among others. See, for example, US Patent Nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. plastid. Exemplary colloidal systems useful as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).

Where non-viral delivery systems are used, an exemplary delivery vehicle is liposomes. Consider the use of lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated into the aqueous interior of the liposomes, dispersed within the lipid bilayer of the liposomes, attached via linker molecules associated with both the liposomes and the oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexed with micelles, or otherwise associated with lipids. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in solution. For example, they may exist in bilayer structures, as micelles or have a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include lipid droplets that occur naturally in the cytoplasm as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

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

preparation

The invention provides a method containing the chimeric antigen receptor CAR according to the first aspect of the invention, the nucleic acid molecule according to the second aspect of the invention, the vector according to the third aspect of the invention, or the fourth aspect of the invention. Host cells or engineered immune cells according to the fifth aspect of the present invention, and pharmaceutically acceptable carriers, diluents or excipients. In one embodiment, the formulation is a liquid formulation. Preferably, the preparation is injection Injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 ³ -1×10 ⁸ cells/ml, more preferably 1×10 ⁴ -1×10 ⁷ cells/ml.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of cells (eg, T cells) transduced with lentiviral vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target the tumor cell marker CSF1R, synergistically activate T cells, and induce immune cell immune responses, thus significantly improving their killing efficiency against tumor cells.

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

In one embodiment, the present invention includes a type of cell therapy in which a patient's autologous T cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient. This method has a very low probability of suffering from graft-versus-host disease, and the antigen is recognized by T cells in an MHC-free manner. In addition, one 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 sustained tumor control.

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

Although the data disclosed herein specifically disclose lentiviral vectors including CSFl protein/IL34 protein or fragments thereof, hinge and transmembrane regions, and 4-1BB and CD3ζ signaling domains, the invention should be construed to include the use of constructs Any number of variations of each of the components.

Treatable cancers include tumors that are not vascularized or substantially unvascularized, as well as tumors that are vascularized. Cancer includes non-solid tumors (such as hematological tumors, such as leukemias and lymphomas) and solid tumors. Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also includes adult neoplasms/cancers and pediatric neoplasms/cancers.

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

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia leukemias and myeloblastoid, promyelocytic, myelomonocytic, monocytic and erythroleukemias), chronic leukemias such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia , heavy chain diseases, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

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

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into the mammal: i) expanding the cells, ii) introducing the CAR-encoding nucleic acid 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 human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic relative 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 methods of treating tumors comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. 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; proteins; polypeptides or amino acids such as glycine; antioxidants; chelates Adjuvants such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

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

When an "effective amount,""immunologically effective amount,""anti-tumor effective amount,""tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered can be determined by the physician, It takes into account the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease. It may generally be stated that pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight (including all integers within those ranges). value) application. T cell compositions can also be administered multiple times at these dosages. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). best for a specific patient Optimal dosages and treatment regimens can be readily determined by those skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.

Administration of subject compositions may be 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, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels, are combined with any number of relevant treatment modalities (e.g., before , simultaneously or subsequently) administered to a patient, such forms of treatment include, but are not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as for ARA-C) or natalizumab treatment in patients with MS or elfalizumab treatment in patients with psoriasis or other treatments in patients with specific tumors. In further embodiments, the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cellular compositions of the invention are administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, use of a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, following transplantation, the subject receives an infusion of expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

The dosage of the above treatments administered to a patient will vary depending on the precise nature of the condition being treated and the recipient of the treatment. Dosage proportions for human administration may be implemented in accordance with art-accepted practice. Generally, 1×10 ⁶ to 1×10 ¹⁰ CAR-T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, by intravenous infusion.

The main advantages of the present invention include:

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

(b) The present invention utilizes the binding mode of ligand and receptor instead of the binding mode of single chain variable region (ScFv) and antigen. The conservation of receptor-ligand interactions determines that safety tests in animals, especially primates, can better reflect their safety in humans.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

The reagents, plasmids, and cells in the examples of this application are all commercially available unless otherwise stated. Table 1 summarizes the sequences of the invention.

Table 1 Sequences of the present invention

Table 2 shows the cell lines used in the examples.

Table 2 Cell lines

Example 1: Preparation of CSFl-CAR vector

CSF1-CAR vector construction: artificially synthesized based on gene sequence information of CSF1 (NM_000757.6), human CD8 signal peptide, human CD8α hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3ζ intracellular region method or PCR method to obtain the corresponding nucleotide sequence. The CD8 signal peptide and CSF1 extracellular region were synthesized, and the nucleotide sequence of the CAR molecule was digested with AgeI (Thermo) and NheI (Thermo), and the CD8 transmembrane region, The 4-1BB costimulatory domain and CD3ζ signaling region were inserted into the lentiviral vector pTomo. Transform competent E. coli (Stbl3).

IL34-CAR vector construction: The same method was used to construct the IL34-CAR vector based on the nucleotide sequence of IL34 (NM_001172772.2).

Results: The recombinant plasmid was sequenced, and the sequencing results were compared to confirm whether the plasmid was correct. The sequencing primers were universal sequencing primers. Both sequencing and enzyme digestion identification results showed that the CAR coding sequence was correctly inserted into the predetermined position of the plasmid (Figure 1C and Figure 10C).

All plasmids were extracted using QIAGEN's endotoxin-free large extraction kit, and the purified plasmids were transfected into HEK-293T cells using Beyotime lipo6000 for lentivirus packaging.

Example 2: Virus packaging

HEK-293T cells were cultured in 15 cm culture dishes for virus packaging. When the HEK-293T cell confluence is about 80%-90% for transfection, prepare 2ml OPTIMEM-dissolved plasmid mixture (core plasmid 20μg, pCMVΔR8.9 10μg, PMD2.G 4μg); in another centrifuge tube, add 2ml OPTIMEM and 68 μl of lipo 6000. After standing at room temperature for 5 minutes, add the plasmid complex to the liposome complex and let stand at room temperature for 20 minutes. The above mixture was added dropwise to HEK-293T cells, incubated at 37°C for 6 hours and then the medium was removed. Re-add pre-warmed complete medium. After collecting the virus supernatant at 48 hours and 72 hours, centrifuge at 3000 rpm at 4°C for 20 minutes. After filtering with a 0.45 μm filter membrane, centrifuge at 25,000 rpm and 4°C for 2.5 hours to concentrate the virus. After the concentrated virus was dissolved in 30 μl of virus lysis solution overnight, the virus titer was detected by QPCR. The results showed that the virus titer met the requirements.

Example 3: CAR-T cell preparation

Mononuclear cells were isolated from human peripheral blood using Ficoll separation solution, and purified CD3+ T cells were obtained by RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3/CD28 magnetic beads (Life technology) and cultured with RPMI1640+10% FBS+1% PS+200 U/ml IL2 (PeproTech) for 48 hours before virus infection. Lentivirus infects T cells at MOI=100 in the presence of Lentiboost to prepare CAR-T cells. feel Change the medium after one day of staining.

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

CAR-T cells and NTD cells (control group) 72 hours after virus infection were collected by centrifugation respectively. After washing once with PBS, the supernatant was discarded and the cells were resuspended in PBS containing 2% FBS. The positive rate was detected by flow cytometry.

Results: The results of transfection efficiency are shown in Figure 2 and Figure 11.

As shown in Figure 2A and Figure 11A, after the CAR-T2A-mKate2 fusion protein expressed by CAR-T cells is cleaved, the mKate2 protein formed exhibits red fluorescence in the cell.

Figure 2B and Figure 11B show that flow cytometry was used to detect, indicating that the positive expression rate of CAR or mKate2CAR-T was approximately 50%.

Example 5: Detection of CSF1R expression in each target cell

(1) Cell immunofluorescence: Spread the target cells on a disc in a 24-well plate, fix the cells with 4% paraformaldehyde (PFA) for 20 minutes after 24 hours, wash three times with PBST, 5 minutes each time; use 10% goat serum Block for 1 hour at room temperature and incubate overnight with an antibody that specifically recognizes CSF1R at four degrees. The next day, wash three times with PBST for five minutes each time. Incubate with CY5-labeled secondary antibody that specifically recognizes the primary antibody for 1 hour at room temperature. After washing three times with PBS, the nuclei were stained with DAPI. Confocal microscopy imaging.

(2) Flow cytometry: Collect 1 million cells, fix the cells with 4% PFA at room temperature for 15 min, and centrifuge and wash with 1×PBS; permeabilize the cells with 100% methanol on ice for 15 min, centrifuge and wash with 1×PBS; 100 μl of diluted Resuspend cells in primary antibody (1:300) and incubate at room temperature for 1 hour; centrifuge and wash with 1×PBS. Discard the supernatant. Repeat. Resuspend the cells with 100 μl diluted fluorescent substance-conjugated secondary antibody (Cy5 anti-rabbit), incubate at room temperature for 30 minutes in the dark, and centrifuge and wash with 1×PBS. Discard the supernatant. Repeat. Resuspend cells in 300 μl 1×PBS and analyze by flow cytometry.

(3) qPCR: Collect the cells in the 6-well plate, remove the culture medium, add 1ml Trizol to lyse the cells, and let stand at room temperature for 5 minutes. Add 200 μl/1ml Trizol in chloroform, invert and mix 6-8 times, and let stand at room temperature for 5 minutes; 12000g, 4 Centrifuge at 15°C for 15 minutes, and pipet the supernatant into another centrifuge tube; add an equal volume of isopropanol, mix upside down, and leave at room temperature for 10 minutes; centrifuge at 12000g for 10 minutes at 4°C and discard the supernatant; add 1 ml of 70% ethanol (use RNase free H ₂ O), centrifuge at 7500g for 5 minutes at room temperature; discard the supernatant, dry the RNA at room temperature for 10 minutes, add 30 μl RNase free water to dissolve the RNA; measure the concentration of RNA with Nandrop 2000, and use 1% agarose gel electrophoresis to detect the concentration of RNA. Completeness and quantitative accuracy. cDNA was synthesized according to the instructions of RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific), and mRNA levels were detected.

Results: The results of CSF1R expression detection in each cell line are shown in Figure 3. The expression of CSF1R in target cells was detected by immunofluorescence. The results consistently showed that BXPC3 and ASPC1 expressed high levels of CSF1R, and PANC1 expressed low levels of CSF1R.

Example 6: Construction of target cells carrying luciferase

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

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

Example 7: CAR-T cell killing

In this example, the killing ability of the CAR-T cells of the present invention on different target cells was tested. The target cells used include: target cells BXPC3 and ASPC1 that highly express CSF1R; target cells PANC1 that do not express or have low expression of CSF1R; and CSF1R ⁺ /Syndecan-1 ⁺ MCF7 cells.

MCF7 and PANC1-luciferase cells were digested and counted and then the cell density was adjusted to 2×10 ⁴ /ml. Inoculate 100 μl of luciferase cells into a 96-well plate. Adjust the cell density of CAR-T and control cells to 1×10 ⁵ /ml and inoculate them into a black 96-well plate according to E:T ratio of 5:1. Inoculate 100 μl in each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.

BXPC3 and ASPC1-luciferase cells were digested and counted and then the cell density was adjusted to 2×10 ⁴ /ml. Inoculate 100 μl BXPC3 and ASPC1-luciferase cells in a 96-well plate. Adjust the cell density of CAR-T and control cells to 8×10 ⁴ /ml. According to E:T, it is 0.5:1, 1:1, 2:1, 4:1 was inoculated into a black 96-well plate, and 100 μl was inoculated into each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.

The cell supernatant was collected and frozen at -80°C to detect IFNγ release (see Example 8). Cell killing was detected using the promega fluorescence detection kit. First, the cells were treated with 20 μl of 1×PLB lysis buffer for 20 minutes. After adding 100 μl of substrate to each well, the cells were immediately detected using a BioTek microplate reader.

% Cytotoxic Killing Cells = (1-target cell fluorescence value when containing effector cells/target cell fluorescence value when there are no effector cells) × 100%

Results: The gradient killing results of CSF1-CAR on different pancreatic cancer cell lines are shown in Figure 4. The results showed that the killing effect of CSF1-CART cells on tumor cells with high CSF1R expression basically gradually increased as the effect-to-target ratio (E:T) increased. However, it has basically no killing effect on cell lines with negative or low expression of CSF1R.

The killing results of IL34-CAR on different pancreatic cancer cell lines are shown in Figure 12 and Figure 15. The results show that the killing effect of IL34-CART cells on tumor cells with high expression of CSF1R gradually increases with the increase of the effect-to-target ratio (E:T). It has basically no killing effect on tumor cells that basically do not express CSF1R. It has no killing effect on CSF1R ⁺ / Syndecan-1 ⁺ MCF7 cells also had a significant killing effect.

Example 8: IFNγ Cytokine Release

In this example, the release of cytokines when the CAR-T cells of the present invention are co-incubated with target cells is detected. The cell supernatant incubated in the cell killing experiment was used for detection.

The method is as follows: take the cell supernatant of the CAR-T cells of the present invention co-incubated with target cells in Example 7 and detect IFNγ according to IFN gamma Human ELISA Kit (life technology).

Dissolve the standard in Standard Dilution Buffer and perform gradient dilution into standards of 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, and 0pg/ml. .

Add 50 μl Incubation buffer, 50 μl detection sample, and 50 μl IFNγbiotin conjugated solution to each well, mix well, and let stand at room temperature for 90 minutes.

Then follow these steps:

(1) Wash the wells 4 times with 1×Wash Buffer and stay for 1 minute each time.

(2) Add 100μl 1× Streptavidin-HRP solution to each well and let stand at room temperature for 45 minutes.

(3) Wash the wells 4 times with 1×Wash Buffer, leaving for 1 minute each time.

(4) Add 100μl Stabilized chromogen and let stand at room temperature for 30 minutes.

(5) Add 100μl Stop solution to each well and mix well.

(6) Detect the absorbance value at 450nm.

Results: The results are shown in Figure 5 and Figure 13. Cytokines increased significantly after CSF1-CAR-T and IL34-CAR-T killed ASPC1. The results of the control group on the right of Figure 13 show that IL34-CAR did not release IFNγ from CSF1R-negative PANC1 cells. The above results suggest that the killing effect is related to the release of IFNγ.

Example 9: Effect of overexpressing CSF1R on the killing effect of CAR-T cells

Primers were designed based on the CDS region sequence of CSF1R, and the CDS sequence of CSF1R was amplified using 293T cell cDNA as a template and digested and ligated to construct the pTomo-CMV-CSF1R-T2A-luciferase-IRES-puro vector. Lentivirus was packaged as described in Example 2, and PANC1 cells were infected and selected with puromycin (1 μg/ml) for 2 weeks to obtain PANC1-CSF1R-luc cells.

PANC1-luc cells and PANC1-CSF1R-luc cells were digested and counted, and then the cell density was adjusted to 2×10 ⁴ /ml. Inoculate 100 μl of luciferase cells into a 96-well plate, adjust the cell density of CAR-T/NTD cells to 1×10 ⁵ /ml, and inoculate into a black 96-well plate according to E:T ratio of 5:1, with 100 μl in each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.

Results: The killing results of CSF1-CAR-T cells after overexpressing CSF1R in the pancreatic cancer cell line PANC1 are shown in Figure 7. Figure 6 shows the detection of overexpression of CSF1R in PANC1 cells. The results show that PANC1 overexpressing CSF1R was successfully constructed. Figures 7 and 14 respectively show the killing effect and IFNγ release of CSF1-CAR-T and IL34-CAR-T on PANC1 after overexpression of CSF1R. The results showed that compared with the control group PANC1-con, the killing rate and IFNγ release of CSF1-CAR-T cells and IL34-CAR-T cells on PANC1-CSF1R cells overexpressing CSF1R were significantly increased. This result shows that the killing effect of CSF1-CAR-T cells and IL34-CAR-T on CSF1R-overexpressing tumor cells is significantly enhanced.

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

Based on the shRNA sequence library targeting CSF1R provided by Sigma, select shRNAs that have been verified in the CDS region, and BLAST each shRNA selected on NCBI to ensure the specificity of the target. The shRNAs are constructed into the pLKO.1 vector and passed Enzyme digestion identification and sequencing ensure the correctness of the knockdown vector.

shRNA virus packaging: One day before transfection, 1 million HEK-293T cells per dish were inoculated into a 6cm dish for culture. Before transfection, replace the 6cm dish with 5ml of fresh culture medium (containing serum, without antibiotics); take two clean sterile centrifuge tubes and add 250μl to each. Medium, then add 5μg shRNA plasmid, 2.5μg pCMVΔR8.9, 1μg PMD2.G plasmid to one tube, and gently pipet and mix with a gun; add 17μl Lipo6000 ^TM transfection reagent to the other tube, and gently pipet and mix with a gun. . After standing at room temperature for 5 minutes, use a gun to gently add the culture medium containing DNA to the culture medium containing Lipo6000 ^TM transfection reagent. Gently invert the centrifuge tube or gently pipe and mix with a gun. Let it stand at room temperature for 20 minutes before adding. Mix well in a 6cm dish, collect the supernatant after 48h and 72h, centrifuge at 3000r/min for 20min at 4°C and filter through a 0.45μm filter membrane to obtain the virus-containing supernatant.

shRNA virus infection: One day before infection, 500,000 ASPC1 cells per well were seeded into a six-well plate for culture. Before infection, replace each well of the six-well plate with 1 ml of fresh culture medium (containing serum, without antibiotics), then add 1 ml of virus supernatant and 2 μl of polybrane (10 mg/ml) to prepare ASPC1-shCOO2 and ASPC1-shCSF1R cells; 24 hours later The complete culture medium was replaced, and shRNA knockdown efficiency was measured 96 hours later, and CAR-T cell killing test was performed.

Infect ASPC1 cells with pLKO.1-shCOO2, pLKO.1-shCSF1R-1#, pLKO.1-shCSF1R-2# lentivirus to prepare ASPC1-shCOO2, ASPC1-shCSF1R#1, ASPC1-shCSF1R#2-luciferase cells, 48 After digestion and counting, the cell density was adjusted to 2×10 ⁴ /ml.

CSF1-CAR-T test: Inoculate 100 μl luciferase cells in a 96-well plate, adjust the cell density of CAR-T/NT cells to 1×10 ⁵ , and inoculate them into a black 96-well plate according to E:T ratio of 4:1. Inoculate 100 μl per well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours before detecting the killing effect. As mentioned before, the killing of ASPC1 and ASPC1-shCSF1R by CSF1-CAR-T cells was detected by changes in fluorescence values.

IL34-CAR-T test: Inoculate 100 μl luciferase cells into a 96-well plate, adjust the cell density of CAR-T cells to 8×10 ⁴ /ml, and follow the E:T ratio of 4:1, 2:1, and 1:1 , 0.5:1 was inoculated into a black 96-well plate, and 100 μl was inoculated into each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours to detect the killing effect. The killing of ASPC1 and ASPC1-shCSF1R by IL34-CAR-T cells is detected by changes in fluorescence value.

The results are shown in Figure 8. Figure 8-A is a phenotypic diagram of knockdown CSF1R cells in ASPC1 cells. Figure 8-B shows qPCR detection of CSF1RmRNA levels. Figure 8-C shows the killing effect of CSF1-CAR-T on ASPC1 after silencing CSF1R. Figure 8-D shows the killing effect of IL34-CAR-T on ASPC1 after silencing CSF1R. The result shows showed that compared with control group ASPC1-shCOO2 cells, the killing rates of CSF1-CAR-T and IL34-CAR-T cells against CSF1R knockdown ASPC1-shCSF1R#1 and ASPC1-shCSF1R#2 cells were significantly reduced, and their The cell killing rate further decreased as the degree of knockdown increased. This result shows that knockdown of CSF1R on the cell membrane significantly reduces the killing effect of CSF1-CAR-T, suggesting that the CSF1-CAR-T of the present invention is highly specific for CSF1R.

Example 11: Inhibitory effect of CAR-T on ASPC1-luc nude mouse transplanted tumors

ASPCl-luc cells were constructed as described in Example 6. After digestion and counting of the ASPC1-luc cell line, 30% matrigel was added to adjust the cell density to 5×10^6/ml. Six-week-old female NCG mice were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. Each mouse was subcutaneously inoculated with 100 μl of cell suspension, and CART cells were reinfused 7 days later. CART cells were prepared as described in Example 3. Nude mouse imaging was performed 1 day before CART reinfusion: the mice were anesthetized with 0.025% Avertin (300μl/20g), intraperitoneally injected with 200μl D-luciferin plus salt (15mg/ml), and 10 minutes later, small animal in vivo imaging was performed, and according to the fluorescence Value size groups NTD, CD19-CAR, CSF1/IL34-CAR. Each mouse was infused with 1×10^7/200μl CART cells through the tail vein. Thereafter, nude mice were imaged every 7 days.

The results are shown in Figure 9. The results showed that both CSF1-CAR-T and IL34-CAR-T had significant inhibitory effects on transplanted tumors in ASPC1 nude mice.

Comparative ratio

In order to verify whether ligands with receptor binding ability in nature can play a targeting role as the antigen-binding domain of chimeric antigen receptors, the applicant used the above three target cells ASPC1, BXPC3 and PANC1 (all highly expressed CSF1R and GRP78 receptors), testing whether a variety of natural ligands can retain their target-binding ability after being constructed into chimeric antigen receptors. All chimeric antigen receptors were constructed using the methods of Examples 1-3, in which Sigmarl, Gmbp1, VAP, WDL, GIR, Vaspin, GRP78, TF-ED, SAC, Cop35 and PEP42 targeted GRP78, CSFl and IL34 targets Towards CSF1R.

The results are shown in Figure 16. Most ligands, although naturally possessing receptor binding capabilities, are unable to function as the antigen-binding domain of chimeric antigen receptors. Among the more than 10 CARs constructed based on ligands tested, only Pep42, CSF1 and IL34-CAR showed the ability to bind to the corresponding receptors and kill target cells.

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (15)

1. A chimeric antigen receptor (CAR), characterized in that the CAR contains an extracellular binding domain, and the extracellular binding domain includes:

   1) The structure of CSF1 or its fragment based on the amino acid sequence shown in SEQ ID NO:1; or

   2) The structure of IL34 or its fragment based on the amino acid sequence shown in SEQ ID NO.10,

   Moreover, the extracellular binding domain can specifically bind to the CSFl receptor.
2. The chimeric antigen receptor according to claim 1, wherein the extracellular binding domain has an amino acid sequence as shown in SEQ ID NO: 1, preferably a sequence as shown in SEQ ID NO: 1 The amino acid sequence at positions 33 to 554, more preferably the amino acid sequence at positions 33 to 496 of the sequence shown in SEQ ID NO:1.
3. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the extracellular binding domain is selected from the following group:

   (i) The sequence represented by positions 33 to 496 of the sequence represented by SEQ ID NO:1; and

   (ii) On the basis of the sequence shown in positions 33 to 496 of the sequence shown in SEQ ID NO:1, one or more amino acid residues are replaced, deleted, changed or inserted, or at its N-terminus or C 1 to 30 amino acid residues are added to the end, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence is the same as SEQ ID NO : The sequence shown at positions 33 to 496 of the sequence shown in 1 has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) Sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).
4. The chimeric antigen receptor according to claim 1, wherein the extracellular binding domain has an amino acid sequence as shown in SEQ ID NO: 10, preferably a sequence as shown in SEQ ID NO: 10 Amino acid sequence from positions 21 to 242.
5. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the extracellular binding domain is selected from the group consisting of:

   (i) The sequence shown in bits 21 to 242 of the sequence shown in SEQ ID NO:10; and

   (ii) On the basis of the sequence shown at positions 21 to 242 of the sequence shown in SEQ ID NO: 10, one or more amino acid residues are replaced, deleted, changed or inserted, or at its N-terminus or C 1 to 30 amino acid residues are added to the end, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence is identical to SEQ ID NO. : The sequence shown at positions 21 to 242 of the sequence shown in 10 has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) Sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).
6. The chimeric antigen receptor according to claim 1, wherein the structure of the chimeric antigen receptor is as shown in the following formula I:
   L-EB-H-TM-C-CD3ζ-RP (I)

   In the formula,

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

   L is none or signal peptide sequence;

   EB is the extracellular binding domain;

   H is the null or hinge region;

   TM is the transmembrane domain;

   C is no or co-stimulatory signaling molecule;

   CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

   RP is a null or reporter protein.
7. The chimeric antigen receptor as claimed in claim 1, wherein the amino acid sequence of the chimeric antigen receptor CAR is as shown in SEQ ID NO: 8.
8. The chimeric antigen receptor as claimed in claim 1, wherein the amino acid sequence of the chimeric antigen receptor CAR is as shown in SEQ ID NO: 11.
9. A nucleic acid molecule encoding the chimeric antigen receptor as claimed in claim 1.
10. A vector, characterized in that the vector contains the nucleic acid molecule according to claim 9.
11. An engineered immune cell, the immune cell contains the vector of claim 10 or the exogenous nucleic acid molecule of claim 9 integrated into the chromosome or expresses the chimeric antigen of claim 1 receptor.
12. The engineered immune cell according to claim 11, wherein the engineered immune cell is a chimeric antigen receptor T cell or a chimeric antigen receptor NK cell.
13. A method for preparing engineered immune cells as claimed in claim 11, comprising the following steps: transducing the nucleic acid molecule as claimed in claim 9 or the vector as claimed in claim 10 into immune cells, thereby obtaining the Engineered immune cells.
14. A pharmaceutical composition containing the chimeric antigen receptor as claimed in claim 1, the nucleic acid molecule as claimed in claim 9, the vector as claimed in claim 10, and/or as claimed in claim 1 The engineered immune cells described in 11, and pharmaceutically acceptable carriers, diluents or excipients.
15. Use of a chimeric antigen receptor as claimed in claim 1, a nucleic acid molecule as claimed in claim 9, a vector as claimed in claim 10, and/or an engineered immune cell as claimed in claim 11 , used to prepare drugs or preparations for preventing and/or treating diseases with high expression of CSF1 receptors.