CN116199787A - Preparation and application of chimeric antigen receptor immune cells constructed based on GFD structural domain of uPA - Google Patents

Abstract

The invention provides preparation and application of chimeric antigen receptor immune cells constructed based on a growth factor-like domain (GFD) of uPA. In particular, the invention provides a GFD engineered Chimeric Antigen Receptor (CAR) comprising an extracellular binding domain capable of specifically targeting GFD binding proteins (including uPAR). The CAR immune cell has stronger specificity and more proper target affinity, so the killing capacity to the target cell is stronger and the safety is high.

Description

Preparation and application of chimeric antigen receptor immune cells constructed based on GFD structural domain of uPA

Technical Field

The invention belongs to the technical field of biological medicines for tumor immunotherapy, relates to a specific chimeric antigen receptor immune cell, and in particular relates to a chimeric antigen receptor of a specific targeting GFD binding protein, a modified immune response cell thereof, and a preparation method and application thereof.

Background

Chimeric antigen receptor T cell therapy (CAR-T) is an immunotherapy in which T cells are artificially engineered in vitro to be targeted, and then returned to the patient, specifically killing tumor cells. In CAR-T therapy, CAR engineered T cells combine the specificity of antibodies with the homing and killing ability of T cells, specifically exerting an effect. In addition, other characteristic modifications (e.g., migration, homeostasis proliferation, resistance inhibition, etc.) may be embedded into the CAR-T cell to allow it to better exert its effects.

Urokinase-type plasminogen activator receptor (uPAR), a GPI-anchored cell membrane receptor, consists of three homologous domains (DI, DII), whose main function is to activate urokinase (uPA) proteolytic activity, regulating extracellular matrix (ECM) component degradation. Both pro-uPA and uPA bind uPAR, and a portion of uPAR is cleaved by protease hydrolysis upon ligand binding, yielding soluble uPAR (suPAR). There is a great deal of literature demonstrating the importance of uPAR in the progression of most tumors, over-expression in most tumor cells and tumor stroma, including solid tumors such as breast, colorectal, prostate, pancreatic, ovarian, lung and glioma, and hematological malignancies such as acute leukemia and myeloma. Whereas normal tissue detects low uPAR expression in only a subset of sternal epithelium, monocytes, macrophages and neutrophils. Studies have shown that (1) elevated uPAR expression is associated with adverse outcomes (invasion, metastasis and recurrence) in different types of cancer patients; (2) uPAR is closely related to tumor metabolism; (3) uPAR expression is associated with RAS gene mutation in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) patients; (4) uPAR is a key participant in regulating the transition between tumor cell dormancy and proliferation; (5) uPAR is associated with multi-drug resistance (MDR) of tumor cells; (6) uPAR is associated with tumor angiogenesis.

Aging (aging) is a complex process that is affected by both genetic and environmental factors, is closely related to permanent, progressive deterioration of physiological cells, and is one of the causes of aging of the population that is becoming increasingly severe. Aging can significantly improve the prevalence of the organism, namely aging-related diseases such as diabetes, alzheimer's disease, cardiovascular diseases, neurodegenerative diseases and the like. Individual aging is associated with aging cells, but is quite different. Senescent cells are cytokines associated with cells in a stable cell cycle arrest and secretion regulating tissue microenvironment. Physiologically, senescent cells are a mechanism of tumor inhibition that can prevent expansion of precancerous cells and play a beneficial role in the wound healing response. As an individual ages, aging cells accumulate within the tissue. The proportion of senescent cells in the tissues of elderly individuals is small, but the accumulation of senescent cells promotes aging in individuals and creates an inflammatory environment, resulting in chronic tissue damage, such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis. The elimination of senescent cells in elderly individuals can ameliorate these pathological conditions and even promote longevity. Studies have demonstrated that uPAR is an important surface marker of cellular senescence and that senescent cells secrete soluble uPAR (suPAR) as part of the senescence-associated secretory phenotype (SASP).

In view of the important role of uPAR in the development of tumor and aging, there have been a great deal of research into the role of uPAR in the diagnosis, prognosis of tumor and aging, and aging-related diseases; and exploring the role of targeting uPAR in related therapies using uPAR as a therapeutic target for malignancy, aging, and aging-related diseases.

The uPA protein contains an ATF domain at the N-terminus (Amino-Terminal Fragment) and a catalytic domain at the C-terminus, which in turn can be divided into two small building blocks: GFD (Growth Factor-like Domain) and Kringle domains. Although the GFD domain is necessary for uPA to bind uPAR, ATF has been used mostly to study uPA binding to uPAR, and ATF domain and EGF (epidermal growth factor) have been used to construct bispecific antibodies targeting EGFR and uPAR, and ATF domain has been used to construct uPAR-targeted CAR-T. The Kringle domain in ATF can bind to other proteins such as integrins (integrins) and the like, potentially causing T cells to kill integrin positive cells, resulting in side effects. It is not clear whether a smaller GFD domain can be used to construct CAR-T.

Thus, although CAR-T targeting uPAR has been reported, there is still a need to develop more CAR-T technologies to target uPAR for tumor and aging-related disease therapies. There is an urgent need in the art to develop chimeric antigen receptor T cells that target the uPA receptor (uPAR).

Disclosure of Invention

The invention aims to provide a chimeric antigen receptor immune cell which is constructed based on a growth factor-like domain (GFD) of uPA and takes uPAR as a target point, and a preparation method and an application method thereof.

In a first aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR), said CAR comprising an extracellular binding domain comprising a structure based on a uPA growth factor-like domain (GFD) or fragment thereof, and said extracellular binding domain being capable of specifically binding to a GFD binding protein.

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

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

In another preferred embodiment, the GFD or fragment thereof is capable of specifically binding to a GFD binding protein, preferably in the form of a ligand receptor.

In another preferred embodiment, the GFD binding protein is a protein capable of binding GFD, including but not limited to uPAR.

In another preferred embodiment, the uPAR is located on a cell membrane.

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

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

In another preferred embodiment, the uPAR is of human or monkey origin.

In another preferred embodiment, the uPAR is of human origin.

In another preferred embodiment, the growth factor-like domain or fragment thereof has the amino acid sequence at positions 21 to 68 of the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the growth factor-like domain or fragment thereof is selected from the group consisting of:

(i) A sequence shown in positions 21 to 68 of the sequence shown in SEQ ID NO. 1; and

(ii) An amino acid sequence obtained by performing substitution, deletion, alteration or insertion of one or more amino acid residues, or adding 1 to 10 amino acid residues, preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues, to the N-terminus or C-terminus thereof based on the sequence shown at positions 21 to 68 of the sequence shown in SEQ ID NO. 1; and the amino acid sequence obtained has a sequence identity of ≡85% (preferably ≡90%, more preferably ≡95%, for example ≡96%,. Gtoreq.97%,. Gtoreq.98% or ≡99%) with the sequence shown at positions 21 to 68 of the sequence shown in SEQ ID NO. 1; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

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

L-EB-H-TM-C-CD3ζ-RP (I)

in the method, in the process of the invention,

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

l is an absent or signal peptide sequence;

EB is an extracellular binding domain;

h is a no or hinge region;

TM is a transmembrane domain;

c is an absent or co-stimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

RP is absent or reporter.

In another preferred embodiment, the reporter protein RP further comprises a self-cleaving recognition site, preferably a T2A sequence, at its N-terminus.

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

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

In another preferred example, the amino acid sequence of the mKate2 red fluorescent protein is shown as SEQ ID NO. 2.

In another preferred embodiment, said L includes, but is not limited to, a signal peptide of a protein selected from the group consisting of: CD8, CD28, uPA, GM-CSF, CD4, CD137, or a combination thereof.

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

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

In another preferred embodiment, the H is a hinge region of a protein including, but not limited to, the group consisting of: CD8, CD28, CD137, or a combination thereof.

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

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

In another preferred embodiment, the TM is a transmembrane region comprising a protein selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, the TM is a CD 8-derived transmembrane region.

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

In another preferred embodiment, said C is a costimulatory signaling molecule comprising but not limited to proteins from the group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), NKG2D, GITR, TLR2, or combinations thereof.

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

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

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

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

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

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

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

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

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

In another preferred embodiment, the carrier includes, but is not limited to, a carrier selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.

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

In another preferred embodiment, the carrier includes, but is not limited to, a carrier selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM, pHCMV, pLBS.CAG, pHR, pLV, etc.

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

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

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

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

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

In another preferred embodiment, the engineered immune cells include, but are not limited to, those selected from the group consisting of: t cells, NK cells, NKT cells, or macrophages.

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

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

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

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

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

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

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

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

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

In another preferred embodiment, the uPA receptor includes, but is not limited to uPAR.

In another preferred embodiment, the diseases associated with abnormal expression of uPA receptor include, but are not limited to, tumors, aging, and aging-related diseases.

In another preferred embodiment, the disorder associated with aberrant expression of uPA receptor comprises a disorder associated with aberrant expression of uPAR.

In another preferred embodiment, the disorder associated with abnormal expression of uPAR comprises: tumors, aging-related diseases, and the like.

In another preferred embodiment, the disease is aging or a related disease in the individual caused by aging cells.

In another preferred embodiment, the abnormal expression of uPAR refers to high uPAR expression.

In another preferred embodiment, the high uPAR expression means that the ratio (F1/F0) of the uPAR expression level (F1) to the normal cell or tissue uPAR expression level (F0) is not less than 1.5, preferably not less than 2, more preferably not less than 2.5.

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

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

In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), myeloma, and the like, or a combination thereof.

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

In another preferred embodiment, the tumor is selected from the group consisting of: colorectal cancer, brain tumor, breast cancer, endometrial cancer, bladder cancer, prostate cancer, pancreatic cancer.

In another preferred embodiment, the aging-related disorder is selected from the group consisting of: type II diabetes, autoimmune diseases, fatty liver, liver cirrhosis, liver fibrosis, pulmonary fibrosis, osteoarthritis, infection, cardiovascular and cerebrovascular diseases, and the like.

In another preferred embodiment, the use comprises: killing tumor cells, clearing senescent cells, or a combination thereof.

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

In another preferred embodiment, the aging-related disorder is selected from the group consisting of: type II diabetes, autoimmune diseases, fatty liver, liver cirrhosis, liver fibrosis, pulmonary fibrosis, osteoarthritis, infection, cardiovascular and cerebrovascular diseases, and the like.

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

In another preferred embodiment, the disease is a disease associated with aberrant expression of the uPA receptor.

In another preferred embodiment, the disease is cancer, tumor or aging related disease.

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

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

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

In another preferred embodiment, the disease is cancer, tumor, and the other treatment methods include chemotherapy, radiation therapy, targeted therapy, and the like.

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

Drawings

FIG. 1 shows a schematic of GFD-CAR and other vector construction.

Wherein A is a uPA sequence diagram, wherein 1-18AA is a signal peptide, and 19-182AA is an extracellular domain; b is a schematic diagram of the structure of a control group CD19-CAR, GFD-CAR, SMB-CAR, clone20-CAR and ATF-CAR, wherein the signal peptide, the hinge region and the transmembrane region are all derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3 zeta is derived from human CD3, mKate2 is a fluorescent label and is used for detecting CAR expression; c is the HindIII digestion identification of the CD19-CAR, GFD-CAR, SMB-CAR, clone20-CAR and ATF-CAR vectors of the control group.

Figure 2 shows the results of CAR transfection efficiency assays.

Wherein A is the result of cell fluorescence expression of a control group CD19-CAR, GFD-CAR, SMB-CAR, clone20-CAR and ATF-CAR after T cells are infected for 72 hours, wherein Bright is Bright field, and mKate2 is CAR fluorescence expression; b is the result of flow detection fluorescence expression.

Figure 3 shows the results of a targeted uPAR CAR-Ts screening-killing assay.

Wherein, a, b. Pancreatic cancer cell lines; C. a human normal cell line. ( * P < = 0.05; * P < = 0.01; * P < = 0.001; * P < = 0.0001; ns, no significant difference. )

FIG. 4 shows the results of a targeted uPAR CAR-Ts screening-TNF- α release assay.

Wherein, a, b. Pancreatic cancer cell lines; C. a human normal cell line. ( * P < = 0.05; * P < = 0.01; * P < = 0.001; * P < = 0.0001; ns, no significant difference. )

Figure 5 shows the results of GFD-CAR vs ATF-CAR infection efficiency.

Figure 6 shows the result of a broad-spectrum cytotoxicity-killing assay of GFD-CAR.

Wherein A, B is a non-small cell lung cancer cell line; C. d, E is a gastric cancer cell line. ( * P < = 0.05; * P < = 0.01; * P < = 0.001; * P < = 0.0001; ns, no significant difference. )

FIG. 7 shows the results of a TNF- α release assay for the broad-spectrum cytotoxic effect of GFD-CAR.

Wherein A, B is a non-small cell lung cancer cell line; C. d, E is a gastric cancer cell line. ( * P < = 0.05; * P < = 0.01; * P < = 0.001; * P < = 0.0001; ns, no significant difference. )

Figure 8 shows the results of the effective target-specific gradient dependent assay-killing assay for GFD-CAR.

Wherein A, B, C is a pancreatic cancer cell line; D. e, F is a breast cancer cell line. ( * P < = 0.05; * P < = 0.01; * P < = 0.001; * P < = 0.0001; ns, no significant difference. )

FIG. 9 shows the results of cytokine TNF- α release assays following a GFD-CAR gradient dependent assay.

Wherein A, B, C is a pancreatic cancer cell line; D. e, F is a breast cancer cell line. ( * P < = 0.05; * P < = 0.01; * P < = 0.001; * P < = 0.0001; ns, no significant difference. )

Figure 10 shows that GFD-CAR is dependent on target uPAR specificity for cytotoxic effects.

Wherein A is the result of a cytotoxicity-killing test of GFD-CAR on HEK-293T-Vector (Vector control) and HEK-293T-uPAR (HEK-293T cell overexpressing uPAR); b is the detection result of cytokine TNF-alpha release in the supernatant of the killing test cells; c is RNA level to verify the over-expression efficiency; d is flow cytometry to verify overexpression efficiency.

Figure 11 shows a cytotoxicity assay of GFD-CAR on senescent cells.

Wherein A is the direct killing effect of each group on aging cells HEL1-P16 observed under the lens; b is the counting result of the counting under the mirror; c is the release detection result of IFN-gamma in the cell supernatant during killing.

Detailed Description

Through extensive and intensive studies, the present inventors have developed, for the first time, a chimeric antigen receptor immune cell constructed based on the GFD domain of uPA through a large number of experiments. Experimental results show that the CAR-T targeting the uPA receptor has a remarkable killing effect on target cells with high expression of uPAR, and has no or almost no killing effect on cells with no expression or low expression of uPAR, so that the CAR-T targeting the uPA receptor has higher specificity. The present invention has been completed on the basis of this finding.

Terminology

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Plasminogen activation system

The Plasminogen Activator (PA) system is an extracellular proteolytic enzyme system associated with a variety of physiological and pathophysiological processes. The primary physiological function of the PA system is the conversion of inactive plasminogen to plasmin, which can be mediated by two types of PA: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). PA systems were originally thought to play a role in clot dissolution of fiber formation. Later studies, however, showed that the PA system has additional functions in other biological processes, such as embryogenesis, angiogenesis, cell migration, wound healing, inflammatory reactions, and apoptotic cell death. In cancer, the PA system plays a dominant role in tumor growth, angiogenesis, tumor cell invasion, migration and metastasis.

uPAR association with tumor: almost 90% of human cancer-related deaths are caused by metastatic spread of tumor cells. One of the major events behind metastasis is the proteolytic, degradation of the extracellular matrix (ECM) to promote tumor cell invasion, migration and homing to distant organs. Although several protease systems are involved in this process, there is a great deal of evidence that the uPA-uPAR system is a core participant in the mediation of proteolysis during cancer invasion and metastasis. The function of the uPA-uPAR system is not limited to proteolysis, but rather plays a broader role in several links from tumorigenesis to metastatic cancer.

Urokinase-type plasminogen activator receptor (uPAR), a GPI-anchored cell membrane receptor, consists of three homologous domains (DI, DII). Its main function is focusing on the proteolytic activity of urokinase (uPA) on the cell surface responsible for the degradation of extracellular matrix (ECM) components. There is a great deal of literature demonstrating the importance of uPAR in most tumor progression, over-expression in a vast number of tumor cells and tumor stroma, including solid tumors such as breast, colorectal, prostate, pancreatic, ovarian, lung and glioma, and hematological malignancies such as acute leukemia and myeloma. Whereas normal tissue detects low uPAR expression in only a subset of sternal epithelium, monocytes, macrophages and neutrophils. Studies have shown that (1) elevated uPAR expression is associated with adverse outcomes (invasion, metastasis and recurrence) in different types of cancer patients; (2) uPAR is closely related to tumor metabolism; (3) uPAR expression is associated with RAS mutations in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) patients; (4) uPAR is a key participant in regulating the transition between single cell tumor dormancy and proliferation; (5) uPAR is associated with multi-drug resistance (MDR) of tumor cells; (6) uPAR is associated with tumor angiogenesis. Given the important role of uPAR in the development of tumorigenesis, a great deal of research has been done: (1) Exploring the role of uPAR in the diagnosis and prognosis of tumors; (2) The role of targeting uPAR in tumor therapy was explored using uPAR as a therapeutic target for malignant tumors.

Connection of uPAR to aging and related diseases: aging (aging) is a complex process that is affected by both genetic and environmental factors, is closely related to permanent, progressive deterioration of physiological cells, and is one of the causes of aging of the population that is becoming increasingly severe. Aging can significantly improve the prevalence of the organism, namely aging-related diseases such as diabetes, alzheimer's disease, cardiovascular diseases, neurodegenerative diseases and the like.

Cell senescence is characterized by stable cell cycle arrest and regulation of senescence-associated secretory processes in the tissue microenvironment. Physiologically, aging is a mechanism that inhibits tumors, prevents expansion of precancerous cells, and plays a beneficial role in the wound healing response. Pathologically, abnormal accumulation of aging cells produces an inflammatory environment, causes chronic tissue damage, and causes diseases such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis. The elimination of senescent cells from damaged tissues of the body can ameliorate these pathological conditions and even promote longevity.

It has been demonstrated that uPAR is an important surface marker for senescent cells. A portion of uPAR is cleaved by protease hydrolysis upon binding to the ligand, yielding soluble uPAR (suPAR). Senescent cells secrete suPAR as part of the senescence-associated secretory phenotype (SASP).

Therefore, the uPAR-targeted CAR-T of the present invention is of great value for the treatment of solid tumors, hematological tumors, aging-related disorders or other disorders associated with abnormal uPAR expression.

Urokinase type plasminogen activator

Urokinase-type plasminogen activator (urokinase, uPA) is a key serine protease involved in the conversion of inactive plasminogen to active plasmin, which in turn plays a role in a series of events in the transfer cascade. It was first discovered in 1947 by MacFarlane and piling, in which a novel "unnamed" protein with fibrinolytic activity was reported. Five years later, sobel and colleagues named this "unnamed" protein as "urokinase". Further studies have shown that urokinase is also present in plasma, and many tissues of the ECM.

pro-uPA (411 amino acids) is a precursor structure of uPA, which consists of three domains: a growth factor-like domain (GFD), a Kringle Domain (KD) and a serine protease domain homologous to Epidermal Growth Factor (EGF). GFD (from 21 to 68 amino acids) and KD (69-151 amino acids) are located at the N-terminus, while the catalytic serine protease domain (179-431 amino acids) is located at the "C-terminus". There is a linker region (151-179 amino acids) between the N-terminal and C-terminal. Once pro-uPA is required for secretion, the peptide bond between Lys179 and IIe180 in the linker region is cleaved to yield disulfide-linked double-stranded forms of uPA. After another round of proteolysis at the peptide bond between Lys135 and Lys136, the double-stranded form of uPA can be further cleaved into two parts: (1) A catalytically active low molecular weight form of uPA with serine protease domain and (2) an inactive Amino Terminal Fragment (ATF) consisting of GFD and KD. Studies have shown that the GFD domain is a critical region for the specific binding of uPA to uPAR, and have demonstrated that either the ATF fragment, the double-stranded form of uPA, or pro-uPA can specifically bind to uPAR with nearly uniform affinity.

The specific binding of uPA and uPAR depends on the GFD domain, and the GFD domain is used as a recognition region of CAR-T to recognize target (uPAR) positive cells, thereby exerting an effect. The CAR-T constructed based on the GFD domain of uPA of the present invention has a better affinity and better target specificity than the CAR-T targeting uPAR that exists today (antibody derived CAR-T; CAR-T constructed based on the full length of the uPA extracellular domain) (Wang L, et al biomed pharmacother.2019sep;117:109173;Amor C,et al.Nature.2020Jul;583 (7814): 127-132).

Chimeric Antigen Receptor (CAR) of the invention

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

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

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

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

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

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

In the present invention, the extracellular domain of the chimeric antigen receptor is a uPA protein or fragment thereof that specifically binds to the uPAR target of the CAR of the present invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence at positions 21 to 68 of the sequence shown as SEQ ID NO. 1.

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

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the CD8 hinge region, the transmembrane region, the 4-1BB costimulatory domain, and the CD3ζ signaling domain.

In the present invention, the extracellular binding domain of the CAR of the invention also includes sequence-based conservative variants, meaning that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence at positions 21 to 68 of SEQ ID NO. 1 to form a polypeptide.

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

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

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

The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands. The intracellular domains in the CARs of the invention include a 4-1BB costimulatory domain and a signaling domain of cd3ζ.

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

Chimeric antigen receptor immune cells (CAR-immune cells)

In the present invention, a chimeric antigen receptor immune cell is provided, comprising a chimeric antigen receptor of the present invention having a specific targeting uPA receptor (preferably uPAR).

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

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

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

As used herein, the terms "CAR-NK cells", "CAR-NK cells of the invention" all refer to CAR-NK cells of the fifth aspect of the invention. The CAR-NK cells of the invention can be used for tumors that express high levels of uPA receptor, preferably uPAR.

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

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

Carrier body

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Formulations

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

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

Therapeutic applications

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The main advantages of the invention include:

(a) Target specificity: uPAR is only low expressed on the cell membrane of very few normal cells, but expression in tumor, tumor stroma and senescent cells is significantly up-regulated, so that the CAR of the invention is directed only against target cells whose cell membranes are highly expressing uPAR, without substantial killing of normal cells.

(b) The uPAR is not only overexpressed in tumor cells, but also highly expressed in tumor stroma, and has important effect on tumor angiogenesis. Therefore, the uPAR serving as a target can target not only tumor cells but also tumor microenvironment, and the anti-tumor effect is exerted in various aspects.

(c) The uPAR is a target related to invasion and metastasis of tumors, plays an important role in invasion and metastasis of tumors, and can be targeted not only to tumor in-situ foci but also to metastatic tumors by taking the uPAR as the target. Targeting metastatic tumors improves tumor invasion and metastasis may be of more practical significance.

(d) uPAR is associated with chemotherapy drug resistance, and targeting uPAR may be beneficial in patients resistant to chemotherapy.

(e) The present invention utilizes the mode of action of ligand binding to receptor, rather than scfv in the traditional sense.

(f) The CAR of the invention based on a specific segment of a natural ligand receptor, safety tests in animals, in particular primates, are of more reference value for clinical applications.

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

Table a summary of amino acid sequences to which the invention relates

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Example 1: preparation of uPAR-targeted CAR vectors

The corresponding nucleotide sequence is obtained by artificial synthesis based on the nucleotide sequence of uPAR ligand uPA (PLAU) (NM_ 002658.6), or uPAR ligand VTN (NM_ 000638.4), or uPAR binding polypeptide Clone20 (PMID: 8041758), or anti-CD19 scFv, and the human CD8 signal peptide, human CD8 alpha hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3 zeta intracellular region gene sequence information. The CD8 signal peptide and the target recognition region of the CAR (e.g., GFD region of GFD CAR) were synthesized at the well-known biology company and the nucleotide sequence was cloned and inserted into lentiviral vector pTomo into which CD8TM, 4-1BB, cd3ζ had been inserted by means of seamless cloning. The domains of Pro-uPA and VTN are shown in FIG. 1A, the full length of the CAR is shown in FIG. 1B, where CD19-CAR is the vector control, designed based on anti-CD19 scFv; ATF-CAR is a positive control, is a CAR designed based on the ATF domain of uPA disclosed in the prior art (Wang L, et al biomed pharmacothers.2019 Sep; 117:109173); GFD-CAR is a uPA-based GFD domain design; the SMB-CAR is designed for a VTN-based SMB domain; clone20-CAR was designed based on uPAR specific binding polypeptide Clone 20.

The recombinant plasmid is subjected to HindIII digestion identification, sequenced, and the digested electrophoresis bands and sequencing results are compared to confirm whether the plasmid is correct. The results indicate that the coding sequence of the CAR was correctly inserted into the predetermined position of the plasmid (fig. 1C).

All plasmids were extracted with QIAGEN endotoxinfree megapump kit and purified plasmids were lentivirally packaged with Biyundian lipo8000 transfected HEK-293T cells.

Example 2: lentivirus package

HEK-293T cells were cultured in 15cm dishes for virus packaging. Preparing 2ml of OPTI-MEM dissolved plasmid mixture (core plasmid 20ug, pCMV delta R8.9 ug, PMD2.G 4 ug) after transfection of HEK-293T cells with confluence of about 80% -90%; in another centrifuge tube 2ml OPTIMEM and 68ul lipo 6000. After standing at room temperature for 5min, the plasmid complex was added to the liposome complex, and standing at room temperature for 20min. The mixture was added dropwise to 293T cells, and the medium was removed after incubation at 37℃for 6 hours. The preheated complete medium was re-added. After collecting the virus supernatant for 48 hours and 72 hours, it was centrifuged at 3000rpm at 4℃for 20 minutes. After filtration through a 0.45um filter, the virus was concentrated by centrifugation at 25000rpm for 2.5 hours at 4 ℃. After the concentrated virus was solubilized with 30ul of virus lysate overnight, the virus titer was detected by QPCR. The results show that the virus titer meets the requirements.

Example 3: CAR-T cell preparation

Monocytes were isolated from human peripheral blood using Ficol isolation and purified CD3+ T cells were obtained from RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3/CD28 magnetic beads (Life technology), 200U/ml IL2 (PeproTech) was added, and after 48 hours of stimulated incubation, virus infection was performed. Lentiviruses were used to prepare CAR-T cells by infecting T cells in the presence of lentiboost at moi=100, and the medium was changed one day after infection.

Example 4: detection of positive rate of infected CAR-T cells by flow cytometry

CAR-T cells, CD19-CAR control and NTD cell control after 72 hours of virus infection were collected separately by centrifugation, the supernatant was washed once with PBS, the cells were resuspended in PBS containing 2% fbs, and the positive rate was detected by flow.

Results: the results of transfection efficiency are shown in FIG. 2. Wherein NTD is an uninfected T cell, and CD19-CAR is infected with a CD 19-targeted control virus T cell. Fig. 2A represents the observation result under a fluorescence microscope, and fig. 2B represents the flow type detection result. The CAR cells co-express the CAR-mKate2 fusion protein, the expressed fusion protein is cut by T2A, and the formed mKate2 protein shows red fluorescence in cells. The results indicate that GFD-CAR is able to effectively infect T cells.

Example 5: construction of target cells carrying luciferases

The luciferase fragment was PCR amplified from pGL3-luciferase plasmid, and then ligated into pTomo vector by XbaI and BamHI to construct pTomo-EGFP-T2A-luciferase plasmid. IRES and puromycin fragments were amplified from pTomo and PLkO.1 plasmids, respectively. The pTomo-EGFP-T2A-luciferase-IRES-Puro plasmid was successfully constructed by three fragment ligation. Lentiviral packaging and titre assays As previously described, human PANC1 ASPC1 BXPC3 MCF7 MDA-MB-468MDA-MB-231HEK-293T cell lines were infected with MOI=100, respectively, and after 48 hours, PANC1, ASPC1, BXPC3, MCF7, MDA-MB-468, MDA-MB-231, HEK-293T-luciferase cells were obtained, respectively, by screening with puromycin (1 ug/ml) for 1 week.

Example 6: screening-killing test of targeting uPAR CAR-Ts

In this example, screening targeted uPAR CAR-Ts. The target cells used include: target cells expressing uPAR: pancreatic cancer cell line: ASPC1 and BXPC3; and target cells that express low or no uPAR: human embryonic kidney cell line: HEK-293T.

Target cells carrying luciferases were digested and counted and cell densities were adjusted to 2 x 10 x 4/ml. 100ul of cells carrying luciferases were seeded in black 96-well plates and CAR-T/NT cells were adjusted to a cell density of 1X 10≡5 according to 5:1 into a black 96-well plate, 100ul per well. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours. Cell supernatants were collected and frozen at-80℃for detection of cytokine release. Cell killing was detected with promega fluorescence detection kit, first cells were treated with 30ul 1 x plb lysate for 30 min, and immediately after addition of 30ul substrate per well were detected with BioTek microplate reader.

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

The results are shown in FIG. 3, where targeting uPAR CAR-Ts has a different degree of killing on these cells, where the GFD-CAR of the invention has significant killing on target cells expressing uPAR (ASPC 1 and BXPC 3) and substantially no killing on target cells that are low or not expressing uPAR (HEK-293T), embodying the specificity of the GFD-CAR of the invention.

Example 7: targeting uPAR CAR-Ts screening-TNF-alpha Release assay

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

The method comprises the following steps: the cell supernatant of example 6, in which the CAR-T cells of the present invention were incubated with target cells, was used to detect TNF-alpha according to the human TNF-alpha double antibody sandwich ELISA assay kit (proteontech).

The standard was dissolved with Sample Diluent PT and diluted in a gradient to 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0 pg/ml.

100ul of test sample, standard substance or Sample Diluent PT6 (serving as blank hole) is added into each hole, and after uniform mixing, the sealing film is covered for incubation for 120 minutes at 37 ℃.

Then sequentially operating according to the following steps:

washing the wells with 1 x Wash Buffer for 4 times, and staying for 1 minute each time;

100ul of 1 Xdetection antibody is added to each hole, a sealing film is covered, and incubation is carried out for 1h at 37 ℃;

repeating the steps (1)

100ul of 1 XHRP-labeled secondary antibody was added to each well and incubated at 37℃for 40 min.

Repeating the steps (1)

Each well was incubated with 100ul of TMB color development solution at 37℃for 15min in the absence of light.

100ul of Stop solution was added to each well and mixed well.

Absorbance was measured at 450 nm.

The results are shown in FIG. 4: targeting uPAR CAR-Ts had varying degrees of TNF- α release from these cells, indicating that the killing effect of CAR-T was associated with TNF- α release.

Example 8: comparison of GFD-CAR vs ATF-CAR infection efficiency

GFD-CAR and ATF-CAR were individually T-cell infected at different MOI, CAR-T cells were prepared, CAR-T cells PBS after 72 hours of virus infection were individually collected by centrifugation, the disposable supernatants were washed, cells were resuspended in PBS containing 2% fbs, and positive rate was flow tested.

The results are shown in figure 5, where GFD-CAR exhibited higher infection efficiency than ATF-CAR under comparable conditions.

Example 9: broad-spectrum cytotoxicity-killing assay of GFD-CAR

In this example, the effectiveness of the GFD-CAR was further demonstrated by verifying its broad-spectrum cytotoxic effect. The target cells used include: target cells expressing uPAR: non-small cell lung cancer cell line: a549 and H1299; gastric cancer cell line: AGS, MKN28, and NuGC4. Target cells carrying luciferases were digested and counted and cell densities were adjusted to 2 x 10 x 4/ml. 100ul of cells carrying luciferases were seeded in black 96-well plates, CAR-T/NTD cells were adjusted to a cell density of 1 x 10≡5, and were seeded in black 96-well plates at a 5:1 effective target ratio of 100ul per well. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours. Cell supernatants were collected and frozen at-80℃for detection of cytokine release. Cell killing was detected with promega fluorescence detection kit, cells were first treated with 30ul of 1 x plb lysate for 30 min, and immediately after addition of 30ul of substrate per well were detected with BioTek microplate reader.

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

The results are shown in FIG. 6: the target GFD-CAR has better killing effect on the tumor cells. A. B is a non-small cell lung cancer cell line; C. d, E is a gastric cancer cell line.

Example 10: broad-spectrum cytotoxicity of GFD-CAR-TNF-alpha Release assay

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

The method comprises the following steps: the cell supernatant of example 6, in which the CAR-T cells of the present invention were incubated with target cells, was used to detect TNF-alpha according to the human TNF-alpha double antibody sandwich ELISA assay kit (proteontech).

The standard was dissolved with Sample Diluent PT and diluted in a gradient to 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0 pg/ml.

100ul of test sample, standard substance or Sample Diluent PT6 (serving as blank hole) is added into each hole, and after uniform mixing, the sealing film is covered for incubation for 120 minutes at 37 ℃.

Then sequentially operating according to the following steps:

(1) Washing the wells with 1 x Wash Buffer for 4 times, and staying for 1 minute each time;

(2) 100ul of 1 Xdetection antibody is added to each hole, a sealing film is covered, and incubation is carried out for 1h at 37 ℃;

(3) Repeating the steps (1)

(4) 100ul of 1 XHRP-labeled secondary antibody was added to each well and incubated at 37℃for 40 min.

(5) Repeating the steps (1)

(6) Each well was incubated with 100ul of TMB color development solution at 37℃for 15min in the absence of light.

(7) 100ul of Stop solution was added to each well and mixed well.

(8) Absorbance was measured at 450 nm.

The results are shown in FIG. 7: targeting GFD-CARs has higher TNF- α release to these tumor cells. A. B is a non-small cell lung cancer cell line; C. d, E is a gastric cancer cell line.

Example 11: gradient dependency assay for GFD-CAR

In this example, the gradient killing ability of CAR-T cells of the invention against different target cells was tested. The target cells used include: pancreatic cancer cell line: PANC1, ASPC1, and BXPC3; breast cancer cell line: MCF7, MDA-MB-468 and MDA-MB-231.

The cell density was adjusted to 2 x 10. Sup..sup.4/ml after digestion and counting of the cells carrying luciferases. 100ul of cells carrying luciferases were seeded in black 96-well plates and CAR-T/NT cells were adjusted to a cell density of 1X 10≡5 according to E:T of 0.5: 1. 1: 1. 2: 1. 4:1 or 1: 1. 2: 1. 4: 1. 8:1 was seeded into a black 96-well plate with 100ul of seed per well. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours. Cell supernatants were collected and frozen at-80℃for detection of cytokine release. Cell killing was detected with promega fluorescence detection kit, first cells were treated with 30ul 1 x plb lysate for 30 min, and immediately after addition of 30ul substrate per well were detected with BioTek microplate reader.

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

The results are shown in FIG. 8. The results show that the killing effect of GFD-CAR-T cells on tumor cells gradually increases with the increase of the effective target ratio (E: T). Wherein A, B, C is a pancreatic cancer cell line; D. e, F is a breast cancer cell line.

Example 12: cytokine TNF-alpha release assay following GFD-CAR gradient dependent assays

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

The method comprises the following steps: the cell supernatant of example 11, in which the CAR-T cells of the present invention were incubated with target cells, was used to detect TNF-alpha according to the human TNF-alpha double antibody sandwich ELISA detection kit (proteontech).

The standard was dissolved with Sample Diluent PT and diluted in a gradient to 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0 pg/ml.

100ul of test sample, standard substance or Sample Diluent PT6 (serving as blank hole) is added into each hole, and after uniform mixing, the sealing film is covered for incubation for 120 minutes at 37 ℃.

Then sequentially operating according to the following steps:

(1) Washing the wells with 1 x Wash Buffer for 4 times, and staying for 1 minute each time;

(2) 100ul of 1 Xdetection antibody is added to each hole, a sealing film is covered, and incubation is carried out for 1h at 37 ℃;

(3) Repeating the steps (1)

(4) 100ul of 1 XHRP-labeled secondary antibody was added to each well and incubated at 37℃for 40 min.

(5) Repeating the steps (1)

(6) Each well was incubated with 100ul of TMB color development solution at 37℃for 15min in the absence of light.

(7) 100ul of Stop solution was added to each well and mixed well.

(8) Absorbance was measured at 450 nm.

The results are shown in FIG. 9. The results indicate that the killing effect of GFD-CAR-T cells on different tumor cells is related to IFNgamma and TNF alpha release. Wherein A, B, C is a pancreatic cancer cell line; D. e, F is a breast cancer cell line.

Example 13: GFD-CAR target specificity verification

HEK-293T is a normal cell line negative for uPAR, and GFD-CAR-T has no obvious killing effect on HEK-293T. In this example, the invention synthesizes the uPAR coding region in vitro and constructs pTomo-CMV-uPAR-luciferase-IRES-puro lentiviral plasmid by enzymatic ligation. Lentiviruses were packaged in vitro and HEK-293T cells were infected. A stable overexpressed HEK-293T cell line (termed HEK-293T-uPAR) was obtained.

As shown in FIG. 10, HEK-293T-uPAR cells stably overexpress uPAR.

In vitro killing and cytokine TNF- α Release assay as described in examples 6,7 the killing of HEK-293T-uPAR cells by GFD-CAR-T was assayed by fluorescence of luciferases as described above

The results are shown in FIG. 10. The results indicate that for the cell line HEK-293T-uPAR with overexpression of uPAR on cell membrane (fig. 10c, d), the CAR-T cells of the invention had significant killing effect on GFD-CAR-T, whereas the CAR-T cells of the invention had no significant killing effect on HEK-293T-Vector stably transferring the carrier scaffold (fig. 10A); and cytokine TNF- α secretion was significantly up-regulated after co-culture of GFD-CAR-T cells with HEK-293T-uPAR cells (fig. 10B).

The results of the example demonstrate that the GFD-CAR-T of the invention specifically kills the uPAR target and has excellent specificity.

Example 14 cytotoxicity assay of GFD-CAR on senescent cells

HEL1-P16 is a senescent cell line, stabilized by green fluorescent protein (EGFP), HEL1-P16 cells were compared to the CAR-T cells and controls of the invention (NTD and CD 19-CAR-T) cells at 2:1, after co-culturing the effective target ratio for 20 hours, observing the killing effect under a mirror, and counting the number of cells for statistics; cell supernatants were collected simultaneously to detect release of cytokine IFN-gamma.

The results are shown in FIG. 11, which shows a significant killing effect of GFD-CAR-T on HEL1-P16 cells (FIGS. 11A, B) and a higher cytokine IFN- γ release (FIG. 11C) compared to the control group. The results of this example demonstrate that GFD-CAR-T of the present invention is capable of effecting clearance of senescent cells by targeting uPAR.

Discussion of the invention

Compared with normal tissues, the uPAR molecule plays an important role in the development process of tumorigenesis, and compared with normal tissues, the expression of the uPAR molecule in various tumor tissues is obviously up-regulated, so that the uPAR molecule becomes an ideal target for treating uPAR high-expression tumors. In addition, uPA can bind to multiple receptors, thus the inventors have intercepted the specific domain growth factor-like domain (GFD) that binds to uPAR as the recognition domain for CARs, making the constructed CARs more specific and suitable for targeting uPAR positive tumor cells and senescent cells.

In addition, the present application also demonstrates the performance of a number of CARs constructed based on uPAR-binding elements, including ATF-CAR, SMB-CAR, clone20-CAR, and GFD-CAR of the present invention. The results show that not all uPAR binding elements constructed CARs can have similar properties, and that the killing activity, transfection efficiency and specificity of GFD-CARs of the invention are exceptionally excellent.

Therefore, the chimeric antigen receptor immune cells constructed based on the GFD domain of uPA can well recognize the uPAR, have very high specificity and killing activity on tumor cells with high expression of the uPAR, have no killing effect on normal cells without or with low expression of the uPAR, and can be used for treating tumors with high expression of the uPAR and aging.

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

Claims (10)

1. A Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular binding domain comprising the structure of a uPA growth factor-like domain (GFD) or fragment thereof based on the amino acid sequence set forth in SEQ ID No. 1.

2. The CAR of claim 1, wherein the extracellular binding domain comprises GFD or a fragment thereof having the amino acid sequence set forth in SEQ ID No. 1 or having the amino acid sequence set forth in SEQ ID No. 1 at positions 21 to 68.

3. The CAR of claim 1 or 2, wherein the CAR has the structure of formula I:

L-GFD-H-TM-C-CD3ζ-RP (I)

in the method, in the process of the invention,

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

l is an absent or signal peptide sequence;

GFD is the GFD domain of uPA;

h is a no or hinge region;

TM is a transmembrane domain;

c is an absent or co-stimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

RP is absent or reporter.

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

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

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

7. An engineered immune cell comprising the vector of claim 5 or the nucleic acid molecule of claim 4 or the CAR of claim 1 integrated into a chromosome.

8. A method of preparing the engineered immune cell of claim 7, comprising the steps of: transduction of the nucleic acid molecule according to claim 4 or the vector according to claim 5 into an immune cell, thereby obtaining said engineered immune cell.

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

10. Use of a CAR according to claim 1, a nucleic acid molecule according to claim 4, a vector according to claim 5, or a host cell according to claim 6, and/or an engineered immune cell according to claim 7, for the preparation of a medicament or formulation for the prevention and/or treatment of diseases associated with abnormal expression of uPAR receptors.

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