CN114716564A

CN114716564A - Preparation and application of chimeric antigen receptor immune cells constructed based on SECTM1

Info

Publication number: CN114716564A
Application number: CN202111566838.3A
Authority: CN
Inventors: 赵旭东; 魏文文
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-07-08
Anticipated expiration: 2041-12-20
Also published as: CN114716564B

Abstract

The invention provides a preparation method and application of a chimeric antigen receptor immune cell constructed based on SECTM 1. Specifically, the invention provides a Chimeric Antigen Receptor (CAR) based on the modification of SECTM1, said CAR comprising an extracellular binding domain capable of specifically targeting SECTM1 receptor (CD 7). The CAR immune cell has high specificity and target affinity, can effectively amplify the CAR-T cell targeting CD7 without reducing the expression of CD7 on the surface of the T cell by gene editing or CD7 blocking molecules and other technologies, and the obtained CAR-T cell is mainly a CD7 negative or low-expression T cell and contains more memory T cells, so that the CAR immune cell has longer high-efficiency killing capability in vivo. Based on the properties of the CAR-T, it is reasonable to speculate that it also eliminates tumor cell contamination during CAR-T production. The test in non-human primates shows that the composition has higher safety.

Description

Preparation and application of chimeric antigen receptor immune cells constructed based on SECTM1

Technical Field

The invention belongs to the technical field of tumor immunotherapy biomedicine, relates to a specific chimeric antigen receptor immune cell, and particularly relates to a CAR specifically targeting CD7, a modified immune response cell thereof, and a preparation method and application thereof.

Background

Cancer is currently considered to be the leading cause of death and a major obstacle to extending human life in various countries of the world. According to the World Health Organization (WHO)2019, cancer is the first or second leading cause of death among 183 countries in the population before the age of 70 years, seriously threatening the health of humans. By 2020, 1930 ten thousand new cases and 1000 cancer patients die globally, and by 2040 years estimated from the current cancer incidence, 2840 new cases of cancer (including non-melanoma skin cancer except basal cell carcinoma) will occur globally, 47% more than 2020, and overall, the incidence and mortality burden of cancer is rapidly increasing worldwide. According to the global tumor data statistics in 2020, 1,101,958 new cases, which account for 5.7% of new tumor cases, can occur when blood diseases comprise leukemia, Hodgkin lymphoma and non-Hodgkin lymphoma; 594,763 death cases account for 5.9% of cancer death cases, and blood diseases still seriously threaten human health from the statistical data.

With the development of medical technology, the life of human beings is continuously prolonged, great progress is made in the treatment of tumors, and the life cycle of cancer patients is continuously prolonged and the life quality is greatly improved by applying comprehensive treatment means such as surgery, chemotherapy, radiotherapy, biotherapy and the like at present. In addition to conventional therapeutic approaches, immunotherapy has also become a clinically important approach to cancer treatment, and more immunotherapeutic drugs are approved for clinical treatment, such as PD1, PDL1 monoclonal antibodies against immune checkpoint inhibitors, and CAR-T cell therapy. Chimeric Antigen antibody Receptor-T cell (CAR-T) T cell refers to a T cell that is genetically modified to recognize a specific Antigen of interest in an MHC non-limiting manner and to continuously activate expanded T cells. The main structure of the polypeptide comprises three types, namely an extracellular ScFv recognition structural domain which is used for recognizing and combining a target spot on a tumor cell; a hinge region and transmembrane domain, derived primarily from CD8 or CD28, anchor the CAR to the cell membrane and link the extracellular recognition domain with the intracellular signal; the endodomain, which is an activation domain, the number and length of which varies to affect the anti-tumor effect of CAR-T, is now commonly used as an activation domain plus one or more costimulatory domains, CD3 ζ being a common feature of the intracellular part of CARs, which initiates signals to drive killing of T cells, whereas costimulatory domains, mainly derived from the CD28 receptor family or TNF receptor family such as 4-1BB, OX40 or CD27, also enhance the killing effect of CAR-T by enhancing cytokine secretion or promoting proliferation and persistence. Normal T cells recognize Tumor cells by relying on T cell surface Receptor (TCR) binding to Major Histocompatibility Complex (MHC) on the surface of Tumor cells, whereas CAR-T cells recognize Tumor Associated Antigens (TAA) on the surface of Tumor cells by relying only on the structure of CAR in an MHC-independent manner, with the specificity of CAR for Antigen recognition and the killing properties of T cells.

Over the past years, with the rapid development of gene editing, immunization and other disciplines, CAR-T cell therapy has become a new and well-established therapeutic approach to the treatment of hematological disorders. CAR-T treatment of malignant tumors remains a current research hotspot, and CAR-T treatment has gone through a course of years since the first treatment of tumors with Tumor Infiltrating Lymphocytes (TILs) in 1986, through the development of the first generation CARs in 1993, until the FDA first approved CAR-T for the treatment of relapsing/refractory acute lymphoblastic leukemia in 2017. Three CAR-ts are currently used for the treatment of tumors, including kymeriah and yescata approved by the FDA at 2017 and

month

8 and 10 for the treatment of relapsed/refractory acute lymphoblastic leukemia and specific types of large B-Cell Lymphoma, and Tecartus approved at month 2020 and month 7 for the treatment of adult Mantle Cell Lymphoma (MCL). CAR-T therapy is used primarily for the treatment of hematological disorders such as acute lymphoblastic leukemia, chronic lymphoblastic leukemia and multiple myeloma, the most commonly used and clinically beneficial target in hematological disorders is CD19, and CD19 CAR-T cells are designed and expanded in vitro and then injected into patients for killing CD19 positive cells. In the current CAR treatment research, CD19 has good progress in ALL, CLL and NHL, and the CR (completed response) rate of CAR-T treatment of ALL in adults reaches 83-93% and the CR rate in children reaches 67-90%; the CR rate in Diffuse Large B Cell Lymphoma (DLBCL) reaches 43-54%; the CLL reaches 21-29 percent, and has good curative effect mainly in B cell malignant tumor. Despite the high rate of complete remission, there are still some patients who relapse after CAR-T therapy targeting CD19 because of the inhibitory effect of the tumor microenvironment and the phenomenon of antigen escape.

Therefore, there is an urgent need in the art to develop new safe and effective therapeutic targets for treating diseases such as tumor and the like, and corresponding therapeutic protocols.

Disclosure of Invention

The invention aims to provide a chimeric antigen receptor immune cell taking a SECTM1 receptor as a target spot and a preparation method and an application method thereof.

In a first aspect of the invention there is provided a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular binding domain, and the extracellular binding domain comprises the structure of SECTM1 or a fragment thereof based on the amino acid sequence shown in SEQ ID NO:1,

and, the extracellular binding domain is capable of specifically binding to SECTM1 receptor in the manner of a ligand receptor.

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

In another preferred embodiment, the extracellular binding domain comprises SECTM1 protein or a fragment thereof.

In another preferred embodiment, the fragment of SECTM1 protein comprises the extracellular domain of SECTM1 protein.

In another preferred embodiment, the extracellular binding domain comprises an extracellular portion of SECTM1 protein.

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

In another preferred embodiment, the amino acid sequence of the extracellular binding domain is as shown in positions 29-145 of the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the fragment of SECTM1 protein comprises the extracellular domain of SECTM1 protein, the amino acid sequence of the extracellular domain is corresponding to positions 29-145 of the sequence shown in SEQ ID NO. 1. In another preferred embodiment, the protein SECTM1 or fragment thereof specifically binds to SECTM1 receptors including CD 7.

In another preferred embodiment, said receptor for SECTM1 is selected from the group consisting of: CD 7.

In another preferred embodiment, the binding molecule is selected from the group consisting of: CD 7.

In another preferred embodiment, the CD7 is CD7 located on the cell membrane.

In another preferred embodiment, the CD7 is derived from a human or non-human mammal.

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

In another preferred embodiment, the extracellular binding domain of the CAR comprises, in addition to the first extracellular domain directed to SECTM1 receptor, a second extracellular domain directed to an additional target.

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

In another preferred embodiment, the SECTM1 protein or fragment thereof has the amino acid sequence shown in SEQ ID NO. 1, or has the amino acid sequence from position 1 to 145, or preferably from position 29 to 145, of the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of SECTM1 protein or fragment thereof is selected from the group consisting of:

(i) 1, as shown in 29 th to 145 th positions of the sequence shown in SEQ ID NO; and

(ii) an amino acid sequence obtained by substitution, deletion, alteration or insertion of one or more amino acid residues on the basis of the sequence shown at positions 29 to 145 of the sequence shown in SEQ ID NO. 1, or addition of 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues at the N-terminus or C-terminus thereof; and the obtained amino acid sequence has a sequence identity of more than or equal to 85 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent, such as more than or equal to 96 percent, more than or equal to 97 percent, more than or equal to 98 percent or more than or equal to 99 percent) with the sequence shown in the 29 th to 145 th positions of the sequence shown in SEQ ID NO. 1; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

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

L-EB-H-TM-C-CD3ζ-RP (I)

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

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

l is a null or signal peptide sequence;

EB is an extracellular binding domain that specifically binds to SECTM1 receptor;

h is a none or hinge region;

TM is a transmembrane domain;

c is a no or co-stimulatory signal molecule;

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

RP is a null or reporter protein.

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

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

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

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, SECTM1, or a combination thereof.

In another preferred embodiment, L is a signal peptide derived from CD 8.

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

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

In another preferred embodiment, said H is a hinge region from CD 8.

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

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, 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 28-derived transmembrane region.

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

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

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

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

In another preferred embodiment, the amino acid sequence of the cytoplasmic signaling sequence derived from CD3 ζ is as set forth in SEQ ID NO 7.

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

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

In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence of SEQ ID No. 9.

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

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

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

In another preferred embodiment, the carrier is selected from the group consisting of: pTomo lentivirus vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV and the like.

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

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

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

In a fourth aspect of the invention there is provided a host cell comprising a vector or chromosome according to the third aspect of the invention into which has been integrated an exogenous nucleic acid molecule according to the second aspect of the invention or which expresses a CAR according to the first aspect of the invention.

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

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

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

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

In another preferred embodiment, the engineered immune cell is CD7 negative or low expressing CD 7.

In another preferred embodiment, the engineered immune cell is a CD7 negative T cell.

In another preferred embodiment, the engineered immune cell is a CAR-T cell having a phenotype of a memory cell.

In another preferred embodiment, the engineered immune cells are not treated with a CD7 blocking.

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

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

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

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

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

In another preferred embodiment, the concentration of said engineered immune cells in said formulation is 1 × 10³-1×10⁸Individual cells/ml, preferably 1X 10⁴-1×10⁷Individual 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, in the manufacture of a medicament or formulation for the prevention and/or treatment of a disease associated with aberrant expression of the SECTM1 receptor.

In another preferred embodiment, the receptor for SECTM1 includes, but is not limited to, CD 7.

In another preferred embodiment, the diseases related to the abnormal expression of SECTM1 receptor include, but are not limited to, tumor, aging, obesity, cardiovascular diseases, diabetes, neurodegenerative diseases, infectious diseases, etc.

In another preferred embodiment, the diseases related to the abnormal expression of SECTM1 receptor comprise: abnormal expression of CD7 is associated with diseases.

In another preferred embodiment, the diseases associated with the abnormal expression of CD7 include: tumor, aging, cardiovascular diseases, obesity, etc.

In another preferred embodiment, the disease is a high CD7 expressing malignancy.

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

In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), lymphoma, or a combination thereof.

In another preferred embodiment, the hematologic tumor is a hematologic tumor that recurs after treatment.

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

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

In another preferred embodiment, the disease is a disease associated with abnormal expression of the SECTM1 receptor.

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

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

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

In another preferred embodiment, the method may be used in combination with other therapeutic methods.

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic diagram of the construction of the SECTM1-CAR vector.

Wherein A is a sequence schematic diagram of SECTM1, wherein 1-28AA is a signal peptide, and 29-145AA is an extracellular domain; b is a structural schematic diagram of control group plasmids MOCK-CAR and SECTM1-CAR, wherein a signal peptide, a hinge region and a transmembrane region are all derived from a human CD8 molecule, 4-1BB is derived from human CD137, CD3 zeta is derived from human CD3, and mKate2 is a fluorescent marker and used for detecting CAR expression; c is the HindIII enzyme cutting identification of pTomo-SECTM1-CAR vector.

Figure 2 shows CAR-T cell proliferation fold and total, CD7 expression change detection, and cytokine release. A.T fold proliferation after 15 days post-infection; B. detecting the secretion level of the cell factor after 3 days of infection; C. the total number of cells was counted 15 days after infection.

FIG. 3 shows phenotypic testing 6 days after T cell infection. CD4, CD8 and memory phenotype CD45RO, CCR7 expression were examined. Among them, CD45RO + CCR7- (effector memory cells, EM), CD45RO + CCR7+ (central memory cells, CM), CD45RO-CCR7- (terminal effector memory cells, EMRA), CD45RO-CCR7+ (Naive cells, Naive).

Figure 4 shows CAR transfection efficiency assay results.

Wherein A is a cell fluorescence expression result of T cells infected by MOCK-CAR and SECTM1-CAR for 72 hours, BF is a bright field, and mKate2 is CAR fluorescence expression; and B is the flow detection fluorescence expression result.

FIG. 5 shows the difference in tumor cell CCRF-CEM and normal T cell CD7 expression and killing. A, flow detection of target cell CCRF-CEM and T cell CD7 expression; and B, killing detection after co-incubation.

FIG. 6 shows the results of CD7 expression measurements from different tumor cell lines. Wherein CCRF-CEM, MOLT4 are CD7 positive ALL tumor cells; KG-1a, Kasumi-3 are AML tumor cells positive for CD 7; k562 is a CD7 negative control cell.

FIG. 7 shows the results of in vitro assays for killing of different tumor cell lines by SECTM 1-CAR. Wherein K562 is a CD7 negative control cell line, CCRF-CEM, MOLT4 is a CD7 positive acute lymphoblastic leukemia cell line, KG-1a and Kasumi-3 are CD7 positive acute myelogenous leukemia cell lines.

Fig. 8 shows CD7 positive target cells at 2: detecting the cell factors IFN gamma and TNF alpha after killing by 1-effect target ratio.

Figure 9 shows the effect of over-expression of CD7 on cell killing. Wherein the K562 cells are erythroleukemia cell lines. A, CD7 level detection, B, killing effect detection and C, cytokine secretion detection.

FIG. 10, FIG. 10 (continuation-1), FIG. 10 (continuation-2) show signs and indications detected after infusion of SECTM1 CART cells in primate monkeys. A. Monkey T cell infection efficiency flow assay. B. Cytokine release assay. C. And (4) cell counting detection. D. Basic vital sign monitoring. E. Blood is routine.

Detailed Description

The inventor of the invention has extensively and deeply studied and screened a large number of times, and developed a chimeric antigen receptor immune cell constructed based on SECTM1 for the first time. The inventors have unexpectedly found that the use of a specific segment of the ectodomain of SECTM1 (i.e. amino acid sequence 29-145) as the extracellular binding domain of CAR not only has suitable affinity, but also that while high-expressing T cells of CD7 are killed during the preparation of CAR-T cells, CD 7-negative or low-expressing T cells (including CAR-T cells) can be successfully expanded to prepare desired CAR-T cells, and the prepared CAR-T cells are rich in memory T cells and thus can function in vivo for a longer period of time. This phenomenon in the CAR-T production process should also be effective in eliminating the inevitable contamination of tumor cells when isolating patient T cells. The present invention has been completed based on this finding.

Term(s) for

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

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

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

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

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

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

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

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

The inclusion 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 will depend upon the specific application for which the invention is being used. It will be understood by those skilled in the art that the embodiments or claims are given by way of example only and that within the scope of equivalents or claims, the embodiments of the invention may be covered without limitation to the specifically described and claimed scope.

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

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

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

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

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

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

CD7 molecule

The CD7 molecule is a 40kD type I transmembrane glycoprotein, a lineage specific antigen, normally found predominantly on T/NK cells and their progenitors, with only 9% of peripheral single nuclear cells (PBMCs) in peripheral blood being CD7 negative, and its primary function being to act as co-stimulatory receptors for T and B lymphocyte interactions during lymphoid development.

In addition to expression in normal T/NK cells, CD7 is also expressed on > 95% of lymphoid leukemias and lymphomas and part of peripheral blood lymphomas, and also on about 30% of AML.

The Ig superfamily molecule SECTM1 secreted by epithelial cells is the primary ligand for the CD7 molecule. The SECTM1 gene is located near the upstream 5' end of the CD7 gene, which is the only pair of molecular ligand receptors adjacent to each other in the genome, and the SECTM1 gene encodes a protein of unknown function.

The inventor unexpectedly finds that the specific segment (namely amino acid sequences at 29-145 positions) of the ectodomain of SECTM1 as the extracellular binding domain of CAR not only has proper affinity, but also has certain influence on the proliferation of CAR-T in the preparation process of CAR-T cells, but also can successfully prepare the required CAR-T cells in large quantity, and the prepared CAR-T cells have the phenotype of memory cells, so that the CAR-T cells have long-term high-efficiency killing capability and high safety on CD7 positive target cells (such as tumor cells).

Based on the fact, the invention integrates the SECTM1 specific segment into the CAR carrier by means of genetic engineering for the first time, and modifies the related immune cells, thereby realizing specific killing of cells positive to SECTM1 binding protein, and being applicable to treatment of related diseases, in particular to CD7 positive blood tumor which recurs after treatment.

Chimeric Antigen Receptors (CAR) of the invention

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

The design of the CAR goes through the following process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and due to the single intracellular activation domain, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CAR introduces a costimulatory molecule such as CD28, 4-1BB, OX40, ICOS on the basis of the original structure, and compared with the first generation CAR, the function is greatly improved, and the persistence of CAR-T cells and the killing capability to tumor cells are further enhanced. On the basis of the second generation CAR, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and the development of the second generation CAR and the fourth generation CAR is realized.

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

CAR-immunocyte therapy achieves very high clinical response rate in the treatment of hematologic malignancies, which cannot be achieved by any conventional treatment means, and causes a hot tide of clinical research in the world.

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

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

In the present invention, the extracellular domain of the chimeric antigen receptor is a SECTM1 protein or fragment thereof that specifically binds to the CD7 target of the CAR of the invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence from position 29 to 145 of the sequence shown in SEQ ID NO: 1.

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

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

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its associated antigen, the tumor cells are affected, resulting in the tumor cells not growing, being forced to die or otherwise affected, and resulting in a reduction or elimination of the tumor burden in the patient. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of a CD28 signaling domain, and a CD3 zeta signaling domain.

In the present invention, the extracellular binding domain of the CAR of the invention also includes sequence-based conservative variants, which refer to polypeptides formed by substituting up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having similar or similar properties, as compared to the amino acid sequence at positions 29 to 145 of SEQ ID No. 1.

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

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

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

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

In one embodiment of the invention, the CAR is a CAR that can specifically target CD 7.

Chimeric antigen receptor immune cells (CAR-immune cells)

In the present invention, there is provided a chimeric antigen receptor immune cell comprising the chimeric antigen receptor of the present invention having a specific targeting to SECTM1 receptor (preferably CD 7).

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

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

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

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to a CAR-NK cell according to the fifth aspect of the invention. The CAR-NK cells of the invention can be used for tumors with high expression of SECTM1 receptor (preferably CD 7).

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

CAR-NK cells also have the following advantages compared to CAR-T cells, for example: (1) directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) they release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) is easy to be amplified in vitro and can be developed into 'existing' products. Otherwise, similar to CAR-T cell therapy.

Carrier

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

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

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

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

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

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

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

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

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

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or reporter gene to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector. In other aspects, selectable markers 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 a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). In one embodiment of the invention, the reporter gene is a gene encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

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

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

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

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

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

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

Preparation

The invention provides a chimeric antigen receptor CAR comprising the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention, or a host cell of the fourth aspect of the invention or an engineered immune cell of the fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or carrierOr an excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³-1×10⁸Individual cells/ml, more preferably 1X 10⁴-1×10⁷Individual cells/ml.

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

Therapeutic applications

The invention includes therapeutic applications of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target a marker CD7 of tumor cells, and the T cells are synergistically activated to cause immune cell immune response, so that the killing efficiency of the T cells on the tumor cells is remarkably improved.

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

In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or heterologous donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This approach has a very low probability of graft versus host disease, and the antigen is recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are capable of replicating in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells of CD7 elicit specific immune responses against CD7 cells.

Although the data disclosed herein specifically disclose lentiviral vectors comprising SECTM1 protein or a fragment thereof, a hinge and transmembrane region, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.

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

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

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

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

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

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

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

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

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

When pointing toThe precise amount of a composition of the invention to be administered can be determined by a physician considering the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject), given an "effective amount", "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount". It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

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

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

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be used for each treatment or each course of treatment ⁶1 to 10¹⁰The CAR-T cells of the invention are administered to a patient, for example, by intravenous infusion.

The main advantages of the invention include:

(a) and (3) specific target spots: the CAR immune cells of the invention are only directed against malignant cells or parts of normal T cells whose cell membranes highly express CD7, and have no killing effect on other cells or tissues that do not express CD 7.

(b) The present invention utilizes the mode of action of the ligand in combination with the receptor rather than scfv in the traditional sense. Receptor/ligand binding has an affinity selected by natural evolution, and when the extracellular fragment of the ligand SECTM1 is used as an extracellular binding domain, although the suicide phenomenon of CD7 CAR-T cells is caused, a group of CAR-T cells with low expression or negative CD7 are expanded, and the cells also have the function of killing target cells.

(c) The CART cells of the invention, prepared by in vitro expansion (with partial suicide), have a phenotype that is more biased towards memory cells than the control group of CAR-T cells, which suggests a longer duration and longer duration of action in vivo.

(d) The CAR-T cell of the invention does not kill T cells negative or low-expression CD7, and can retain necessary immunity of patients when applied clinically; meanwhile, the tumor cells with higher expression of CD7 can be killed efficiently, and the problem of tumor cell pollution in the process of T cell separation and CAR-T preparation can be solved.

(e) The CAR based on the specific segment of the natural ligand receptor has higher clinical reference value for safety evaluation in primates.

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

Reagents, plasmids, and cells in the examples of the present application are commercially available unless otherwise specified.

TABLE A sequences

Wherein the underlined part is the amino acids 29-1445 of the SECTM1 protein.

Example 1: preparation of SECTM1-CAR vector

Based on the nucleotide sequence (NM-003004.3) of SECTM1/K12, the human CD8 alpha hinge region, the human CD8 transmembrane region, the human 4-1BB intracellular region and the human CD3 zeta intracellular region gene sequence information, the corresponding nucleotide sequence was obtained by an artificial synthesis method or a PCR method. After synthesizing the CD8 signal peptide and the gene fragment of the SECTM1 extracellular region, the nucleotide sequence of the CAR molecule is double digested by XbaI (thermo) and NheI (thermo), and is inserted into a lentiviral vector pTomo into which CD8TM, 4-1BB and CD3 zeta are inserted by T4 DNA ligase (NEB) ligation, and competent Escherichia coli (Stbl3) is transformed, wherein the SECTM1 schematic diagram is shown in FIG. 1A, and the full-length CAR schematic diagram is shown in FIG. 1B.

Sequencing of the recombinant plasmid revealed that the CAR coding sequence was correctly inserted into the plasmid at the predetermined position (figure 1C).

All plasmids were extracted using a QIAGEN endotoxin-free macroextraction kit, and purified plasmids were lentivirally packaged by transfecting 293T cells with Biluo Tianipo 6000.

Example 2: virus package

Packaging in 15cm culture dish with 293T cells used for less than 20 generations, and preparing 2ml OPTIMEM dissolved plasmid mixture (core plasmid 20ug, pCMV delta R8.910ug, PMD2. G4 ug) after 293T cells are transfected with a degree of confluence of about 80% -90%; in another centrifuge tube 2ml OPTIMEM and 68ul lipo 6000. Standing at room temperature for 5min, adding the plasmid complex into the liposome complex, and standing at room temperature for 20 min. The mixture was added dropwise to 293T cells, and the medium was removed after incubation at 37 ℃ for 6 hours. The pre-warmed complete medium was added again. After collecting the virus supernatants for 48 hours and 72 hours, they were centrifuged at 3000rpm at 4 ℃ for 20 minutes, and then filtered through a 0.45um filter. The virus was concentrated by centrifugation at 25000rpm at 4 ℃ for 2.5 hours. After the concentrated virus was dissolved overnight in 30ul of virus lysis solution, the virus titer was measured by QPCR. The results show that the virus titer meets the requirements.

Example 3: CART cell preparation

Mononuclear cells were isolated from Human peripheral blood using Ficool separation, and purified CD3+ T cells were obtained from RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3/CD28 magnetic beads (Life technology), and then 200U/ml IL2(PeproTech) was added to the cells, and virus infection was performed 48 hours after stimulation culture. Lentiviruses CAR-T cells were prepared by infecting T cells in the presence of lentiboost at MOI ═ 20. The medium was changed one day after infection.

The obtained CAR-T cell proliferation fold and total number results are shown in figure 2. The results show that the cell count indicates that the cell number is significantly lower on the third day after infection than in the control group (FIG. 2A), and that a significant increase in the secretion of the cytokine IFN γ can be detected (FIG. 2B), which indicates that SECTM1 CAR is indeed "suicide". Notably, unlike the ligand form of SECTM1 CAR, in this study it was observed that the proliferation fold of SECTM1 CAR was significantly lower than the control group on the first three days due to the suicide phenomenon, whereas the cell proliferation counted again on day 6 was comparable to the control group, and thereafter counted every 3 days without significant difference from the control group (fig. 3A), and the total cell count statistics showed that although T-cell prophase proliferation was affected by the suicide phenomenon, a sufficient number of T-cells could be obtained for in vivo experiments by later proliferation (fig. 2C).

The inventors have performed the detection of CD7 molecules on CAR-T cells that proliferate later, and the data indicate that the population of cells is mainly a population of CD7 negative or weakly positive T cells, while there is a report in the literature that the presence of a population of CD7 negative T cells in normal blood increases the proportion of the population of cells with age, and that the population of cells is a population of memory T cells that are biased towards CD4 positive and CD45RO positive and CD45RA negative. The inventors performed CD4, CD8 and CD45RO, CCR7 molecular assays on the resulting CD7 negative CAR-T cells, indicating that the population of CD7 negative T cells of the inventors was more positive for CD4 than the control, and also a population biased towards effector memory and central memory cell populations (fig. 3). Then having a memory phenotype for this population of CD7 negative CD7 CAR-T cells may allow this population of T cells to survive longer in vivo and thus exert a longer killing effect.

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

And respectively centrifugally collecting the CART cells and the control group T cells after virus infection for 72 hours, washing the CART cells and the control group T cells once by PBS, discarding supernatant, re-suspending the cells by PBS containing 2% FBS, and detecting the positive rate by flow.

The results of transfection efficiency are shown in FIG. 4, in which FIG. 4A represents the results of observation under a fluorescence microscope and FIG. 4B represents the results of flow assay. The results show that, as shown in the above figure, the CAR cell is the co-expressed CAR-mKate2 fusion protein, so the expressed fusion protein is cut by T2A, and the formed mKate2 protein shows red fluorescence in the cell.

FIG. 4 shows that the CAR-T positivity of the CAR of the invention expressed on the cell membrane was around 70% when the PE-CY5 channel was flow-detected using the red fluorescent mkate2 against SECTM 1.

Example 5: differential expression and killing of tumor cell and normal T cell CD7

Target cells CCRF-CEM and normal T cells are respectively marked by two fluorescent dyes of CFSE and violet, and the density of the target cells is adjusted to be 20 ten thousand/ml respectively and 40 ten thousand/ml in total. 100ul of CFSE cells were seeded in 96-well plates, CART/NK cells were adjusted to a cell density of 80 ten thousand/ml, according to E: T of 1: 1. 2: 1. 4: 1. 8: 1 into 96-well plates, 100ul per well. The target cells and T cells were mixed well and incubated in an incubator for 24 hours. After the cells are collected, the cells are stained by 7-AAD for 10min, and then the killing effect is detected by flow.

The results are shown in fig. 5, wherein fig. 5A is the result of CD7 detection of target cells and T cells, and the results show that the CD7 abundance of tumor cells is higher than that of normal T cells; fig. 5B shows the killing results of target cells and T cells, with the target cells killing effect being higher than T cells.

Example 6: detection of expression of CD7 in target cells

2 x 10 each of the target cells⁶Dividing the collection into two EP tubes, adding 1ml PBS containing 2% FBS for resuspension, and adding CD7 isotype control antibody and human CD7 antibody respectively for incubation for 1h at 4 ℃; after centrifugation and supernatant discarding, the cells were washed twice with 1ml of PBS containing 2% FBS, and the positive rate of CD7 expression in the target cells was detected on a flow-type machine after 300ul of resuspension.

The results are shown in FIG. 6. The inventor selects the T-ALL tumor cells CCRF-CEM and MOLT4 which are positive for CD7 and the AML tumor cells KG-1a, Kasumi-3 and K562 which are negative for the CD7 to detect the expression of CD7, and each cell shows different expression levels of CD 7.

Example 7: construction of target cells carrying luciferase

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-lucferase plasmid. IRES and puromycin fragments were amplified from pTomo and PLkO.1 plasmids, respectively. The pTomo-EGFP-T2A-luciferase-IRES-Puro plasmid is successfully constructed by three-segment connection. Lentiviral packaging and titre determination human CCRF-CEM and KG-1a cell lines were infected at MOI 100 as described above and screened with puromycin (1ug/ml) for 1 week 48 hours to give CCRF-CEM-luciferase and KG-1a-luciferase cells, respectively.

Example 8: CART cell killing

In this example, the killing ability of CART cells of the invention to different target cells was tested. Target cells employed included: target cells expressing CD 7: CCRF-CEM, MOLT4, KG-1a and Kasumi-3; target cells that do not express or do not substantially express CD 7: K562.

the method comprises the following steps: collecting each target cell, staining with CFSE, adding PBS containing 5% FBS in 3 times volume at 37 deg.C for 10min to stop staining for 5min, centrifuging to remove supernatant, washing cells twice, resuspending with T cell culture medium, and adjusting target cell density to 40 ten thousand/ml. 100ul of CFSE cells were seeded in 96-well plates with a ratio of E: T of 0.5: 1. 1: 1. 2: 1. 4: 1 into 96-well plates, 100ul per well. The target cells and T cells were mixed well and incubated in an incubator for 24 hours. Collecting cell supernatant, freezing and storing at-80 deg.C to detect cell factor release amount. After the cells are collected, the cells are stained by 7-AAD for 10min, and then the killing effect is detected by flow.

The results are shown in FIG. 7. The results show that the killing effect of SECTM1-CART cells on tumor cells expressing CD7 is gradually enhanced along with the increase of the effective target ratio (E: T), and the killing effect is positively correlated with the expression level of CD7 of target cells; has no obvious killing effect on a CD7 negative cell strain K562.

Example 9: cytokine release

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

Taking IFN γ as an example, the method is as follows: cell supernatants of CART cells of the invention of example 8 incubated with CD7 positive target cells CCRF-CEM, MOLT4, KG-1a and Ksumi-3(ET ratio 2: 1) were assayed for IFN γ according to the IFN gamma Human ELISA Kit (life technology).

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

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

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

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

(2) 100ul 1 × Streptavidin-HRP solution was added to each well and allowed to stand at room temperature for 45 minutes.

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

(4) 100ul of Stabilized chromogen was added and allowed to stand at room temperature for 30 minutes.

(5) 100ul of Stop solution was added to each well and mixed well.

(6) Absorbance at 450nm was measured.

Similarly, TNF α detection was performed with Human TNF α ELISA Kit (BD Bioscience) Kit.

The results are shown in FIG. 8. The results show that the killing effect of SECTM1-CART cells on tumor cells is accompanied by the release of IFN γ (FIG. 8A) and TNF α (FIG. 8B).

Example 10: effect of over-expression of CD7 on SECTM1-CART tumor killing

K562 is a CD7 negative erythroleukemia cell line, and SECTM1-CART has no killing effect on K562. In this example, a vector that overexpresses the CDS region of CD7 was packaged in vitro by lentivirus, K562 cells were infected with this lentivirus, and after 7 days of selection with puromycin (1ug/ml), a K562 CD7 over cell line that stably overexpresses CD7 was obtained and CD7 expression was detected by flow assay, as shown in FIG. 9A.

The killing effect of SECTM1-CAR on K562 CD7 over cells was tested by flow assay according to the in vitro killing method of example 8, and the results are shown in FIG. 9B. The results show that the K562 CD7 over cell line, which overexpresses CD7 on the cell membrane, can be effectively killed by SECTM1-CART of the invention. Simultaneously detecting the cytokine IFN gamma, the secretion of the cytokine is obviously up-regulated compared with K562 wild cells and over-expression groups (figure 9C)

Example 11: post-infusion safety validation of SECTM1C CAR-T cell monkeys

T cells isolated from monkey blood were infected in vitro at MOI 100 and the infection efficiency of T cells was examined by flow cytometry, indicating that the infection efficiency of SECTM1 CAR was around 35% (fig. 10A). Simultaneous collection of cell supernatants three days prior to infection for IFN γ, IL2, IL6, and TNF α cytokine assays, showed a different increase in cytokines of SECTM1 CAR relative to control T cells (fig. 10B), counting two days for infected T cells showed a 2 nd, day 4 SECTM1 CAR-T cells that were slightly lower than control T cells, but remained at comparable levels after day 6 (fig. 10C), indicating that: the SECTM1 CAR designed in the study can recognize monkey T cells and show suicide phenomenon and cytokine release, and can well simulate clinical process in primate monkeys.

Meanwhile, the weight, anal temperature, blood pressure, heart rate (figure 10D) and blood routine test (figure 10E) of the monkey are tested before and after CAR-T cell infusion, the changes are within the range, no serious toxic or side effect is observed, and the in vivo safety of the SECTM1 CAR is proved.

Discussion of the related Art

The CD7 molecule is a 40kD type I transmembrane glycoprotein, a lineage specific antigen, normally found predominantly on T/NK cells and their progenitors, with only 9% of peripheral single nuclear cells (PBMCs) in peripheral blood being CD7 negative, i.e. most PBMCs are CD7 positive.

Thus prior to the present invention, the art believed that CD7 did not appear to be a suitable target for CAR immune cell therapy, as CAR immune cells (such as CAR-T cells) directed against the CD7 target would also kill these CD7 positive cells.

In addition, some T cells are also CD7 positive during CAR-T cell production, and thus there is severe suicide of T cells, resulting in an inefficient production of CAR-T cells. Thus, some researchers developed CAR-T cells based on CD7 blocking technology. However, the CD7 blocking molecule is adopted to block the CART cell from expressing CD7, so that the defects of complex process and the like exist.

In the present invention, the inventors unexpectedly found that, by using a specific segment of ectodomain of SECTM1 (i.e. amino acid sequence 29-145) as the extracellular binding domain of CAR, not only has suitable affinity, but also has some effect on CAR-T proliferation during CAR-T cell preparation, the desired CAR-T cells can be successfully prepared in large quantities, and the prepared CAR-T cells contain more memory T cells, thus having longer high killing ability against target cells positive for CD7 (such as tumor cells). And because the expression abundance of the tumor cell CD7 is higher than that of a normal T cell, the CAR-T cell can preferentially eliminate the tumor cell with higher expression of CD7, so that the problem of tumor cell pollution in the processes of T cell separation and CAR-T cell preparation can be solved.

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

Sequence listing

<110> Sichuan university Hospital in Huaxi

<120> preparation and application of chimeric antigen receptor immune cell constructed based on SECTM1

<130> P2021-1798

<160> 11

<170> PatentIn version 3.5

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<213> Intelligent (Homo sapiens)

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Met Gln Thr Cys Pro Leu Ala Phe Pro Gly His Val Ser Gln Ala Leu

1 5 10 15

Gly Thr Leu Leu Phe Leu Ala Ala Ser Leu Ser Ala Gln Asn Glu Gly

20 25 30

Trp Asp Ser Pro Ile Cys Thr Glu Gly Val Val Ser Val Ser Trp Gly

35 40 45

Glu Asn Thr Val Met Ser Cys Asn Ile Ser Asn Ala Phe Ser His Val

50 55 60

Asn Ile Lys Leu Arg Ala His Gly Gln Glu Ser Ala Ile Phe Asn Glu

65 70 75 80

Val Ala Pro Gly Tyr Phe Ser Arg Asp Gly Trp Gln Leu Gln Val Gln

85 90 95

Gly Gly Val Ala Gln Leu Val Ile Lys Gly Ala Arg Asp Ser His Ala

100 105 110

Gly Leu Tyr Met Trp His Leu Val Gly His Gln Arg Asn Asn Arg Gln

115 120 125

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

130 135 140

Gly Phe Trp Pro Val Pro Ala Val Val Thr Ala Val Phe Ile Leu Leu

145 150 155 160

Val Ala Leu Val Met Phe Ala Trp Tyr Arg Cys Arg Cys Ser Gln Gln

165 170 175

Arg Arg Glu Lys Lys Phe Phe Leu Leu Glu Pro Gln Met Lys Val Ala

180 185 190

Ala Leu Arg Ala Gly Ala Gln Gln Gly Leu Ser Arg Ala Ser Ala Glu

195 200 205

Leu Trp Thr Pro Asp Ser Glu Pro Thr Pro Arg Pro Leu Ala Leu Val

210 215 220

Phe Lys Pro Ser Pro Leu Gly Ala Leu Glu Leu Leu Ser Pro Gln Pro

225 230 235 240

Leu Phe Pro Tyr Ala Ala Asp Pro

245

<210> 2

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Ser Lys Leu Gly His Lys Leu Asn

225 230

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

1 5 10 15

His Ala Ala Arg Pro

20

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

1 5 10 15

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

20 25 30

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

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

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Ser Leu Val Ile Thr Leu Tyr Cys

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

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

35 40

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 8

<211> 361

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

His Ala Ala Arg Pro Gln Asn Glu Gly Trp Asp Ser Pro Ile Cys Thr

20 25 30

Glu Gly Val Val Ser Val Ser Trp Gly Glu Asn Thr Val Met Ser Cys

35 40 45

Asn Ile Ser Asn Ala Phe Ser His Val Asn Ile Lys Leu Arg Ala His

50 55 60

Gly Gln Glu Ser Ala Ile Phe Asn Glu Val Ala Pro Gly Tyr Phe Ser

65 70 75 80

Arg Asp Gly Trp Gln Leu Gln Val Gln Gly Gly Val Ala Gln Leu Val

85 90 95

Ile Lys Gly Ala Arg Asp Ser His Ala Gly Leu Tyr Met Trp His Leu

100 105 110

Val Gly His Gln Arg Asn Asn Arg Gln Val Thr Leu Glu Val Ser Gly

115 120 125

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

130 135 140

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

145 150 155 160

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

165 170 175

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

245 250 255

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

260 265 270

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

275 280 285

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

290 295 300

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

305 310 315 320

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

325 330 335

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

340 345 350

Leu His Met Gln Ala Leu Pro Pro Arg

355 360

<210> 9

<211> 1080

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccgaatgaag gctgggacag ccccatctgc acagaggggg tagtctctgt gtcttggggc 120

gagaacaccg tcatgtcctg caacatctcc aacgccttct cccatgtcaa catcaagctg 180

cgtgcccacg ggcaggagag cgccatcttc aatgaggtgg ctccaggcta cttctcccgg 240

gacggctggc agctccaggt tcagggaggc gtggcacagc tggtgatcaa aggcgcccgg 300

gactcccatg ctgggctgta catgtggcac ctcgtgggac accagagaaa taacagacaa 360

gtcacgctgg aggtttcagg tgcagaaccc cagtccgccc ccgacactgg gaccacgacg 420

ccagcgccgc gaccaccaac accggcgccc accatcgcta gccagcccct gtccctgcgc 480

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 540

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 600

gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 660

tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 720

gaagaagaag gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg 780

tacaagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 840

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 900

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 960

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1020

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1080

<210> 10

<211> 615

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

His Ala Ala Arg Pro Gln Asn Glu Gly Trp Asp Ser Pro Ile Cys Thr

20 25 30

Glu Gly Val Val Ser Val Ser Trp Gly Glu Asn Thr Val Met Ser Cys

35 40 45

Asn Ile Ser Asn Ala Phe Ser His Val Asn Ile Lys Leu Arg Ala His

50 55 60

Gly Gln Glu Ser Ala Ile Phe Asn Glu Val Ala Pro Gly Tyr Phe Ser

65 70 75 80

Arg Asp Gly Trp Gln Leu Gln Val Gln Gly Gly Val Ala Gln Leu Val

85 90 95

Ile Lys Gly Ala Arg Asp Ser His Ala Gly Leu Tyr Met Trp His Leu

100 105 110

Val Gly His Gln Arg Asn Asn Arg Gln Val Thr Leu Glu Val Ser Gly

115 120 125

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

130 135 140

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

145 150 155 160

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

165 170 175

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

245 250 255

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

260 265 270

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

275 280 285

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

290 295 300

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

305 310 315 320

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

325 330 335

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

340 345 350

Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly

355 360 365

Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser

370 375 380

Glu Leu Ile Lys Glu Asn Met His Met Lys Leu Tyr Met Glu Gly Thr

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr Glu Asp

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu Pro Ser

595 600 605

Lys Leu Gly His Lys Leu Asn

610 615

<210> 11

<211> 1845

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccgaatgaag gctgggacag ccccatctgc acagaggggg tagtctctgt gtcttggggc 120

gagaacaccg tcatgtcctg caacatctcc aacgccttct cccatgtcaa catcaagctg 180

cgtgcccacg ggcaggagag cgccatcttc aatgaggtgg ctccaggcta cttctcccgg 240

gacggctggc agctccaggt tcagggaggc gtggcacagc tggtgatcaa aggcgcccgg 300

gactcccatg ctgggctgta catgtggcac ctcgtgggac accagagaaa taacagacaa 360

gtcacgctgg aggtttcagg tgcagaaccc cagtccgccc ccgacactgg gaccacgacg 420

ccagcgccgc gaccaccaac accggcgccc accatcgcta gccagcccct gtccctgcgc 480

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 540

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 600

gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 660

tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 720

gaagaagaag gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg 780

tacaagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 840

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 900

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 960

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1020

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1080

ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc 1140

ccaatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg 1200

aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag 1260

accatgagaa tcaaggcggt cgagggcggc cctctcccct tcgccttcga catcctggct 1320

accagcttca tgtacggcag caaaaccttc atcaaccaca cccagggcat ccccgacttc 1380

tttaagcagt ccttccccga gggcttcaca tgggagagag tcaccacata cgaagacggg 1440

ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc 1500

aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc 1560

tgggaggcct ccaccgagac cctgtacccc gctgacggcg gcctggaagg cagagccgac 1620

atggccctga agctcgtggg cgggggccac ctgatctgca acttgaagac cacatacaga 1680

tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga cagaagactg 1740

gaaagaatca aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc 1800

agatactgcg acctccctag caaactgggg cacaagctta attag 1845

Claims

1. A Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular binding domain comprising the structure of SECTM1 or a fragment thereof based on the amino acid sequence shown in SEQ ID NO:1,

2. The CAR of claim 1, wherein the extracellular binding domain comprises a SECTM1 protein or fragment thereof, wherein the SECTM1 protein or fragment thereof has the amino acid sequence set forth in SEQ ID No. 1, or the amino acid sequence from position 1 to 145 (preferably from position 29 to 145) of the sequence set forth in SEQ ID No. 1.

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

L-EB-H-TM-C-CD3ζ-RP (I)

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

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

l is a null or signal peptide sequence;

h is a none or hinge region;

TM is a transmembrane domain;

c is a no or co-stimulatory signaling molecule;

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

RP is a null or reporter protein.

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

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

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

7. An engineered immune cell comprising the vector or chromosome of claim 5 having integrated therein the exogenous nucleic acid molecule of claim 4 or expressing the CAR of claim 1.

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

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

10. Use of the CAR of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, or the host cell of claim 6, and/or the engineered immune cell of claim 7, for the preparation of a medicament or formulation for the prevention and/or treatment of a disease associated with aberrant expression of SECTM1 receptor.