CN116178562A - Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1 - Google Patents

Abstract

The invention provides preparation and application of a chimeric antigen receptor immune cell constructed based on EFNA 1. In particular, the invention provides an EFNA1 engineered Chimeric Antigen Receptor (CAR) comprising an extracellular binding domain capable of specifically targeting the EFNA1 receptor. The CAR immune cell has stronger specificity and target affinity, so the killing capacity to the target cell is stronger and the safety is high.

Description

Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1

Technical Field

The present invention belongs to the field of biomedicine technology for tumor immunotherapy, and relates to specific chimeric antigen receptor immune cells, and specifically to a CAR that specifically targets the EFNA1 receptor, immune response cells modified therefrom, and preparation methods and applications thereof.

Background Art

Cancer is currently considered the leading cause of death in various countries around the world and the main obstacle to extending human life. According to statistics from the World Health Organization (WHO) in 2019, cancer is the first or second leading cause of death among people under the age of 70 in 183 countries, posing a serious threat to human health. By 2020, there will be an estimated 19.3 million new cases and 10 million cancer deaths worldwide. Based on the current incidence of cancer, it is estimated that by 2040, there will be 28.4 million new cases of cancer (including non-melanoma skin cancer excluding basal cell carcinoma) worldwide, an increase of 47% from 2020. Overall, the burden of cancer incidence and mortality is growing rapidly worldwide.

According to the 2020 Global Cancer Data Statistics, it is estimated that there will be 19 million new cases of colorectal cancer and 935,000 deaths from colorectal cancer in 2020; in general, colorectal cancer ranks third in incidence and second in mortality. The high incidence and mortality make colorectal cancer a serious threat to human health. At present, the treatment methods for colorectal cancer mainly include surgery for local treatment, chemotherapy for systemic treatment, targeted therapy and immunotherapy. With the improvement of medical level, significant progress has been made in the systemic treatment of colorectal cancer. Six new chemotherapy drugs have been introduced, which have increased the median overall survival of patients with metastatic colorectal cancer from less than 9 months (untreated) to about 24 months. For patients with stage III (node-positive) colon cancer, the overall survival benefit of fluorouracil-based chemotherapy has been established. The latest data show that incorporating oxaliplatin into such adjuvant treatment regimens has a higher efficacy. Although the treatment effect continues to improve, some patients still have malignant progression, so new methods need to be explored to treat colorectal cancer.

With the development of medical technology, human life expectancy has been continuously extended, and great progress has been made in the treatment of tumors. The application of comprehensive treatment methods such as surgery, chemotherapy, radiotherapy and biological therapy has continuously extended the survival of cancer patients and greatly improved their quality of life. In addition to conventional treatment methods, immunotherapy has also become an important clinical method for treating cancer. More and more immunotherapy drugs have been approved for clinical treatment, such as PD1, PDL1 monoclonal antibodies targeting immune checkpoint inhibitors and CAR-T cell therapy.

Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that have been genetically modified to recognize specific target antigens in an MHC-unrestricted manner and to continuously activate and proliferate. Its main structures include three categories, namely, the extracellular ScFv recognition domain, which is used to recognize and bind to targets on tumor cells; the hinge region and transmembrane domain, which are mainly derived from CD8 or CD28, anchor the CAR to the cell membrane and connect the extracellular recognition domain with the intracellular signal; the intracellular domain is the activation domain, and the difference in its number and length will affect the anti-tumor effect of CAR-T. Currently, one activation domain plus one or more costimulatory domains are commonly used. CD3ζ is a common feature of the intracellular part of CAR, which can initiate signals to drive the killing effect of T cells, and the costimulatory domain is mainly derived from the CD28 receptor family or the TNF receptor family such as 4-1BB, OX40 or CD27, which also improves the killing effect of CAR-T by enhancing cytokine secretion or promoting proliferation and persistence.

Normal T cells rely on the combination of T cell surface receptors (TCR) and the major histocompatibility complex (MHC) on the surface of tumor cells to recognize tumor cells, while CAR-T cells recognize tumor associated antigens (TAA) on the surface of tumor cells in a way that is independent of MHC and only relies on the CAR structure. They have the specificity of CAR for antigen recognition and the killing characteristics of T cells.

Over the past few years, with the rapid development of gene editing, immunology and other disciplines, CAR-T cell therapy has become a new and effective treatment for blood diseases. CAR-T treatment of malignant tumors is still a hot topic of research. From the first use of tumor infiltrating lymphocytes (TILs) to treat tumors in 1986, to the development of the first generation of CAR in 1993, to the first FDA approval of CAR-T for the treatment of relapsed/refractory acute lymphoblastic leukemia in 2017, CAR-T treatment has gone through several years. Currently, there are three types of CAR-T used to treat tumors, including Kymriah and Yescarta approved by the FDA in August and October 2017 for the treatment of relapsed/refractory acute lymphoblastic leukemia and certain types of large B-cell lymphoma, and Tecartus approved in July 2020 for the treatment of adult mantle cell lymphoma (MCL). CAR-T therapy has made great progress in the treatment of blood diseases, but there are still certain limitations in the treatment of solid tumors, mainly due to the selection of effective targets and the infiltration of CAR-T cells into tumors.

Summary of the invention

The purpose of the present invention is to provide a chimeric antigen receptor immune cell with EFNA1 receptor as target and a preparation and application method thereof.

In the first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, characterized in that the CAR contains an extracellular binding domain, the extracellular binding domain includes the structure of EFNA1 or a fragment thereof based on the amino acid sequence shown in SEQ ID NO:1, and the extracellular binding domain can specifically bind to the EFNA1 receptor in a ligand receptor manner.

In another preferred embodiment, the EFNA1 receptor is selected from the following group: EphA2.

In another preferred embodiment, the extracellular binding domain has an amino acid sequence derived from EFNA1.

In another preferred embodiment, the extracellular binding domain includes EFNA1 protein or a fragment thereof.

In another preferred embodiment, the EFNA1 protein fragment includes the extracellular region of the EFNA1 protein.

In another preferred embodiment, the extracellular binding domain includes EFNA1 protein or a fragment thereof.

In another preferred embodiment, the extracellular binding domain segment includes the extracellular region of the EFNA1 protein, and the amino acid sequence of the extracellular region corresponds to positions 19-182 of the sequence shown in SEQ ID NO:1.

In another preferred embodiment, the extracellular domain has the amino acids shown at positions 19-182 of SEQ ID NO:1.

In another preferred embodiment, the EFNA1 receptor is selected from the following group: EphA2.

In another preferred embodiment, the extracellular binding domain of the CAR contains, in addition to the first extracellular domain targeting the EFNA1 receptor, a second extracellular domain targeting an additional target.

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

In another preferred embodiment, the EFNA1 protein or fragment thereof specifically binds to EFNA1 receptors including EphA2.

In another preferred embodiment, the binding molecule is selected from the following group: EphA2.

In another preferred embodiment, the EphA2 is located on the cell membrane.

In another preferred embodiment, the EphA2 is derived from human or non-human mammals.

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

In another preferred embodiment, the EphA2 is of human or monkey origin.

In another preferred embodiment, the EphA2 is of human origin.

In another preferred embodiment, the extracellular domain has an amino acid sequence as shown in SEQ ID NO: 1, or has an amino acid sequence from positions 1 to 182 (preferably from positions 19 to 182) of the sequence as shown in SEQ ID NO: 1.

In another preferred embodiment, the EFNA1 protein or its fragment corresponds to the amino acid sequence at positions 19-182 of the sequence shown in SEQ ID NO:1.

In another preferred embodiment, the amino acid sequence of the EFNA1 protein or its fragment is selected from the following group:

(i) the sequence shown in positions 19 to 182 of the sequence shown in SEQ ID NO: 1; and

(ii) on the basis of the sequence shown at positions 19 to 182 of the sequence shown as SEQ ID NO: 1, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are added at its N-terminus or C-terminus to obtain an amino acid sequence; and the obtained amino acid sequence has ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98% or ≥99%) sequence identity with the sequence shown at positions 19 to 182 of the sequence shown as 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 structure of the CAR is shown in Formula I below:

L-EB-H-TM-C-CD3ζ-RP (I)

In the formula,

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

L is none or a signal peptide sequence;

EB is the extracellular binding domain;

H is no or hinge region;

TM is the transmembrane domain;

C is no or co-stimulatory signaling molecule;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

RP is none or reporter protein.

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

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

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

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

In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, EFNA1, GM-CSF, CD4, CD137, or a combination thereof.

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

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

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

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

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

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

In another preferred embodiment, the TM is a transmembrane region derived from CD8.

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

In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: 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, the C is a co-stimulatory signal molecule derived from 4-1BB.

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

In another preferred embodiment, the amino acid sequence of the cytoplasmic signal transduction sequence derived from CD3ζ is shown as SEQ ID NO:7.

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

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

In the third aspect of the present invention, a vector is provided, characterized in that the vector contains the nucleic acid molecule as described in the second aspect of the present invention.

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

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

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

In another preferred example, the vector comprises the nucleotide sequence shown in SEQ ID NO:9.

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

In the fifth aspect of the present invention, an engineered immune cell is provided, wherein the immune cell contains the vector as described in the third aspect of the present invention or a chromosome in which an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated or expresses the CAR as described in the first aspect of the present invention.

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

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

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

In the sixth aspect of the present invention, a method for preparing the engineered immune cells as described in the fifth aspect of the present invention is provided, comprising the following steps: transducing the nucleic acid molecule as described in the second aspect of the present invention or the vector as described in the third aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.

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

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

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

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

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

In the eighth aspect of the present invention, a use of the CAR as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, or the host cell as described in the fourth aspect of the present invention, and/or the engineered immune cell as described in the fifth aspect of the present invention is provided for preparing a drug or preparation for preventing and/or treating diseases related to abnormal expression of the EFNA1 receptor.

In another preferred embodiment, the EFNA1 receptor includes but is not limited to EphA2.

In another preferred embodiment, the abnormal expression of the EFNA1 receptor refers to overexpression of the EFNA1 receptor.

In another preferred embodiment, the overexpression of the EFNA1 receptor means that the expression level of the EFNA1 receptor is ≥1.5 times, preferably ≥2 times, and more preferably ≥2.5 times the expression level under normal physiological conditions.

In another preferred embodiment, the diseases associated with abnormal expression of EFNA1 receptor include but are not limited to tumors, aging, obesity, cardiovascular diseases, diabetes, neurodegenerative diseases, infectious diseases, etc.

In another preferred embodiment, the diseases associated with abnormal expression of EFNA1 receptor include diseases associated with abnormal expression of EphA2.

In another preferred embodiment, the diseases associated with abnormal expression of EphA2 include: tumors, aging, cardiovascular diseases, obesity, etc.

In another preferred embodiment, the disease is a malignant tumor with high expression of EphA2.

In another preferred embodiment, the tumor includes blood tumors and solid tumors.

In another preferred embodiment, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), lymphoma, or a combination thereof.

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

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

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

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

In another preferred embodiment, the disease is a disease associated with abnormal expression of EFNA1 receptor.

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

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

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

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

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a schematic diagram of the construction of the EFNA1-CAR vector.

Among them, A is a schematic diagram of the EFNA1 sequence, in which 1-18AA is the signal peptide and 19-182AA is the extracellular domain; B is a schematic diagram of the control group plasmid MOCK-CAR and EFNA1-CAR structure, in which the signal peptide, hinge region, and transmembrane region are all derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3ζ is derived from human CD3, and mKate2 is a fluorescent marker for detecting CAR expression; C is HindIII enzyme digestion identification of the pTomo-EFNA1-CAR vector.

Figure 2 shows the cell expansion fold and total number of EFNA1 CAR. Figure 2A. Proliferation fold of T cells 12 days after infection; Figure 2B. Total cell count 12 days after infection.

FIG3 shows the detection of CAR infection efficiency by fluorescence microscopy and flow cytometry.

Among them, A is the cell fluorescence expression result after T cells were infected with MOCK-CAR and EFNA1-CAR for 72 hours, in which the upper row is bright field and the lower row is CAR fluorescence expression; B is the fluorescence expression result of flow cytometry detection.

Figure 4 shows the results of phenotypic detection of CAR-T cells, and the proportion of CD4 and CD8 positive T cells was detected on d3 and d6 after infection.

Figure 5 shows the killing effect of EFNA1 CAR-T on different tumor cell lines (effect-target ratio 5:1).

Among them, A. bladder cancer cell line; B. prostate cancer cell line; C. glioma cell line; D. breast cancer cell line; E, F. pancreatic cancer cell line; G, H. colorectal cancer cell line.

FIG6 shows the results of EphA2 expression detection in colorectal cancer cell lines DLD1, HCT116 and normal cell lines 293T and COS7.

Among them, A is RNA level detection; B is protein level detection; C is the membrane localization result of immunofluorescence detection.

Figure 7 shows the results of in vitro detection of EFNA1-CAR against EphA2-positive prostate cancer cell line PC-3; bladder cancer cell lines 5637, RT4, J82; colorectal cancer tumor cell lines DLD1, HCT116; and EphA2-negative normal cells HEK293T, COS7.

FIG8 shows the results of IFNγ and TNFα release detection after killing the colorectal cancer cell lines DLD1 and HCT116 at a ratio of 2:1.

FIG. 9 shows the results of efficiency detection after overexpression of EphA2.

Among them, A is RNA level; B is protein level; C is immunofluorescence membrane localization detection.

FIG10 shows the results of the killing test of EFNA1 CAR-T cells on cells overexpressing EphA2. .

DETAILED DESCRIPTION

After extensive and in-depth research and a large number of screenings, the inventors have developed for the first time a chimeric antigen receptor immune cell preparation based on EFNA1 and its application. The experimental results show that the CAR-T targeting the EFNA1 receptor of the present invention has a significant killing effect on target cells with high expression of EphA2, but has no or basically no killing effect on cells that do not express or low express EphA2, so it has high specificity. On this basis, the present invention was completed.

The experiments of the present invention show that although different erythropoietin hepatocyte receptors (such as EphA1, EphA2, EphA3 and EphA4) are expressed on multiple cells, and there are multiple ligands in the Eph receptor protein family (including at least 9 ligands such as EphrinA1-6 and EphrinB1-3), and there is a certain cross-reactivity between Eph receptor proteins and ligands, the chimeric antigen receptor immune cells of the present invention have obvious high specificity and high killing activity against tumors with high expression of EphA2, but do not show obvious killing activity against normal cells that do not express EphA2. Therefore, they have high specificity and safety and are suitable for targeting EphA2-positive tumors.

In addition, in tumor cells, several other Eph receptors besides EphA2 may also have abnormally high expression levels that exceed physiological expression levels. Therefore, based on the ability of EFNA1 to bind to these Eph receptors, it also has the potential to prevent immune escape caused by target loss during treatment and prevent tumor recurrence.

the term

In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific 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 therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

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

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

"Transduction", "transfection", "transformation" or other terms used in this article refer to the process of transferring exogenous polynucleotides to host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to introduce exogenous polynucleotides into host cells (such as Escherichia coli).

"Gene expression" or "expression" refers to the process of gene transcription, translation and post-translational modification 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 analogs thereof. A polynucleotide may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to single-stranded and double-stranded molecules interchangeably. Unless otherwise indicated, the polynucleotides in any embodiment described herein include double-stranded forms and two complementary single strands that are known or predictable to constitute the double-stranded form.

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

Those skilled in the art will readily understand the meaning of all parameters, dimensions, materials and configurations described herein. Actual parameters, dimensions, materials and/or configurations depend on the specific application for which the invention is used. Those skilled in the art will appreciate that the embodiments or claims are given by way of example only, and within the scope of equivalents or claims, the scope of the embodiments of the invention is not limited to the scope specifically described and required.

Definitions and all definitions used herein should be understood to control over dictionary definitions or definitions in documents incorporated by reference.

All references, patents, and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, in some cases including in their entirety.

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

In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. 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 or other parenteral routes of administration, such as by injection or infusion.

EphA2 and EphrinA1

Erythropoietin-producing hepatocellular receptors A2 (EphA2) are members of the receptor tyrosine kinase family. Receptor tyrosine kinase family proteins play an important role in the signaling pathway of tumor cells and are involved in the occurrence and development of tumors.

Ephrins (EFN) are ligands of the Eph receptor protein family and are divided into two subclasses: EphrinA (EphrinA1-6) and EphrinB (EphrinB1-3).

EphrinA1 (EFNA1) is the most widely studied ligand of EphA2 in tumors and the most important ligand that binds with high affinity. The total length of this protein is 205 amino acids (aa). It binds to the extracellular domain of EphA2. The interaction between the two is involved in the occurrence and development of various solid tumors. In addition to EphA2, EFNA1 can also bind to EphA1, EphA3 and EphA4. EphrinA1 binds to EphA1 with lower affinity. The most important high-affinity ligands of EphA3 are EphrinA2 (Kd = 1nM) and EphrinA3 (Kd = 5nM), and the most important high-affinity ligands of EphA4 are EphrinA2 (Kd = 10nM) and EphrinA4 (Kd = 5nM).

The structure of Eph family proteins includes a conserved extracellular N-terminal ligand-binding domain, a cysteine-rich domain containing an epidermal growth factor-like motif, and two fibronectin type III repeat sequences.

EphA2 is located on the cell membrane and has 25-35% sequence homology with other Eph receptor family members. The tyrosine residues in the juxtamembrane domain and the kinase domain are conserved.

The expression of EphA2 is involved in the occurrence and development of various tumors such as colorectal cancer. Compared with paired normal tissues, colorectal cancer clinical specimens showed significantly higher EphA2 expression levels. In addition, in univariate and multivariate analyses, high EphA2 mRNA and protein expression in stage II/III colorectal cancer tissues was found to be associated with poor overall survival. Data show that EphA2 is highly expressed in KRAS-mutated colorectal cancer cells, and the level of EphA2 is regulated by KRAS-driven MAPK and RalGDS-RalA pathways. EpHA2 has a poor prognosis in stage II/III CRC, which may be due to its ability to promote cell migration and invasion. Therefore, EphA2 can be used as a very valuable therapeutic target for CAR-T design, and its response characteristics and ability to treat colorectal cancer have not yet been reported.

As a tumor antigen target, the current inhibitors against EphA2 are mainly antibodies. Coffman et al. screened two antibodies, EA2 and B233, which promoted the phosphorylation and degradation of EphA2 in tumor cells and inhibited the growth of lung cancer and breast cancer tumors in vivo. A new type of EphA2 humanized monoclonal antibody DS-8895a was purified in 2016, and Burvenich et al. proved that it could inhibit the growth of breast and intestinal tumors in vivo. Using EphA2 single-chain antibody as the CAR sequence, CAR-T cells targeting EphA2 for the treatment of glioma were initiated by Beijing Xuanwu Hospital in 2018. There are also other CAR-T targeting EphA2 in the preclinical research stage for the treatment of lung cancer and esophageal cancer. In general, the existing CAR-T targeting EphA2 are designed based on EphA2 antibodies. However, if the antibody affinity is too low, the ability to target and bind to tumor cells is poor, and if the antibody affinity is too high, excessive immune response is likely to occur, and patient tolerance is poor.

Therefore, receptors/ligands that naturally bind to the target molecule are selected, and the conservative binding characteristics of the two evolved under natural conditions are used to design the CAR sequence, whose more suitable affinity can better overcome the problem of inappropriate affinity of artificially designed antibodies; at the same time, the research of the present invention proves that the use of the natural ligand of EphA2 as the extracellular recognition domain does not affect the proliferation and CD4/CD8 expression of CAR-T cells.

Based on this, the present invention integrates the EFNA1 fragment into the CAR vector for the first time through genetic engineering, and modifies the relevant immune cells, thereby achieving specific killing of EFNA1 receptor-positive cells, which can be used for the treatment of related diseases.

Chimeric Antigen Receptor (CAR) of the Present Invention

Chimeric antigen receptor (CAR) is composed of an extracellular antigen recognition region, a transmembrane region, and an intracellular co-stimulatory signal region.

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

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

CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. This high response rate cannot be achieved by any previous treatment method and has triggered a wave of clinical research around the world.

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

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

In the present invention, the extracellular domain of the chimeric antigen receptor is an EFNA1 protein or a fragment thereof that can specifically bind to the EphA2 target of the CAR of the present invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has an amino acid sequence from positions 19 to 182 of the sequence shown in SEQ ID NO:1.

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

The CAR of the present invention, when expressed in T cells, can recognize antigens based on antigen binding specificity. When it is combined with its associated antigen, it affects tumor cells, causing tumor cells not to grow, to be prompted to die or otherwise affected, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from costimulatory molecules and ζ chains. Preferably, the antigen binding domain is fused with the intracellular domain of the combination of CD8 hinge region and transmembrane region, 4-1BB costimulatory domain and CD3ζ signaling domain.

In the present invention, the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which means that compared with the amino acid sequence of positions 19 to 182 of SEQID NO: 1, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.

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

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

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

The intracellular domain includes a co-stimulatory signaling region and a ζ chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain that includes a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands. The intracellular domain in the CAR of the present invention includes a 4-1BB co-stimulatory domain and a CD3ζ signaling domain.

In one embodiment of the present invention, the CAR is a CAR that can specifically target EphA2.

Chimeric Antigen Receptor Immune Cells (CAR-Immune Cells)

In the present invention, a chimeric antigen receptor immune cell is provided, which comprises the chimeric antigen receptor of the present invention which specifically targets the EFNA1 receptor (preferably EphA2).

The chimeric antigen receptor immune cell of the present invention can be a CAR-T cell, or a CAR-NK cell, or a CAR-macrophage. Preferably, the chimeric antigen receptor immune cell of the present invention is a CAR-T cell.

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

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

As used herein, the terms "CAR-NK cells", "CAR-NK", and "CAR-NK cells of the present invention" all refer to the CAR-NK cells described in the fifth aspect of the present invention. The CAR-NK cells of the present invention can be used for tumors with high expression of EFNA1 receptors (preferably EphA2).

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

Compared with CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are very easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

Carrier

The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

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

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

The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present 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 such vectors, which include but are 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, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, 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 slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01/96584; WO01/29058; and U.S. Patent number 6,326,193).

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

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

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

In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify cells of potential transfection and to evaluate the functionality of regulatory sequences. Typically, reporter genes are genes that are not present in or expressed by receptor organisms or tissues, and that encode polypeptides whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into receptor cells, the expression of reporter genes is measured at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). In one embodiment of the invention, reporter genes are genes encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared or commercially available using known techniques. Typically, constructs with a minimum of 5 flanking regions showing the highest level of reporter gene expression are identified as promoters. Such promoter regions may be connected to reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

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

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

Chemical means for 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. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid/DNA or lipid/expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in the solution, and may form aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as 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 present invention, the vector is a lentiviral vector.

preparation

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

In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

Therapeutic applications

The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the tumor cell marker EphA2, synergistically activate T cells, and induce immune cell immune responses, thereby significantly improving their efficiency in killing tumor cells.

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

In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach has a very low probability of graft-versus-host disease, and antigens are recognized by T cells in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, EphA2 CAR-T cells induce a specific immune response against EphA2 cells.

Although the data disclosed herein specifically disclose lentiviral vectors comprising EFNA1 protein or fragments thereof, hinge and transmembrane regions, and 4-1BB and CD3ζ signaling domains, the present invention should be construed to include any number of variations to each of the construct components.

Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors. Cancers include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma). The types of cancer treated with the CAR of the present invention include but are not limited to cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas. Also included are adult tumors/cancers and childhood tumors/cancers.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

The CAR-modified T cells of the present invention can 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 administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and/or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

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

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

The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

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

When an "effective amount", "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, which takes into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight (including all integer values within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered by using injection techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by a person skilled in the medical field by monitoring the patient for signs of disease and adjusting the treatment accordingly.

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

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

The dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 ⁶ to 1×10 ¹⁰ CAR-T cells of the present invention can be administered to the patient, for example, by intravenous reinfusion, per treatment or per course of treatment.

The main advantages of the present invention include:

(a) Target specificity: Taking EphA2 as an example, EphA2 is basically not expressed on the cell membrane of normal cells, but its expression is upregulated under stress conditions (such as tumors). Therefore, the CAR of the present invention only targets malignant cells with high expression of EphA2 on the cell membrane, and has basically no killing effect on normal cells.

(b) The present invention utilizes the binding mode of ligand to receptor rather than the traditional scFv.

(c) The safety testing of the CAR based on the specific segment of the natural ligand receptor of the present invention in animals, especially primates, is more valuable for clinical application.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Table A Summary of the amino acid sequences involved in the present invention

Example 1: Preparation of EFNA1-CAR vector

Based on the nucleotide sequence (NM_004428.3) of the EphA2 ligand EphrinA1 (EFNA1), the human CD8α hinge region, the human CD8 transmembrane region, the human 4-1BB intracellular region, and the human CD3ζ intracellular region gene sequence information, the corresponding nucleotide sequence was obtained by artificial synthesis or PCR. The CD8 signal peptide and the extracellular region of EphrinA1 (EFNA1) were synthesized at BGI, and the nucleotide sequence of the CAR molecule was double-digested by XbaI (Thermo) and NheI (Thermo), and connected by T4 DNA ligase (NEB) into the lentiviral vector pTomo into which CD8TM, 4-1BB, and CD3ζ were inserted. The schematic diagram of the EFNA1 extracellular domain is shown in Figure 1A, and the schematic diagram of the full-length CAR is shown in Figure 1B, where MOCK is the control group.

The recombinant plasmid was sequenced and the sequencing results were compared to confirm whether the plasmid was correct. The results showed that the coding sequence of CAR was correctly inserted into the predetermined position of the plasmid (Figure 1C).

All plasmids were extracted using QIAGEN's endotoxin-free extraction kit, and the purified plasmids were transfected into 293T cells using Beyotime Lipo6000 for lentiviral packaging.

Example 2: Virus packaging

HEK-293T cells were cultured in 15cm culture dishes for virus packaging. When the confluence of HEK-293T cells was about 80%-90%, transfection was performed, and 2ml of OPTI-MEM-dissolved plasmid mixture (core plasmid 20ug, pCMVΔR8.9 10ug, PMD2.G 4ug) was prepared; 2ml OPTIMEM and 68ul of lipo 6000 were placed in another centrifuge tube. After standing at room temperature for 5min, the plasmid complex was added to the liposome complex and stood at room temperature for 20min. The above mixture was added dropwise to 293T cells, and the culture medium was removed after incubation at 37℃ for 6 hours. Preheated complete culture medium was added again. After collecting the viral supernatant at 48 hours and 72 hours, centrifuge at 3000rpm at 4℃ for 20 minutes. After filtering with a 0.45um filter membrane, the virus was concentrated by centrifugation at 25000rpm at 4℃ for 2.5 hours. The concentrated virus was dissolved with 30ul virus lysis solution overnight, and the virus titer was detected by QPCR. The results showed that the virus titer met the requirements.

Example 3: CAR-T cell preparation

Monocytes were isolated from human peripheral blood using Ficool separation medium, and purified CD3+T cells were obtained by RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3/CD28 magnetic beads (Life technology), and then 200U/ml IL2 (PeproTech) was added, and viral infection was performed after 48 hours of stimulation and culture. Lentivirus was used to infect T cells at an MOI of 20 in the presence of lentiboost to prepare CAR-T cells, and the culture medium was replaced one day after infection.

The obtained CAR-T cell proliferation multiples (Figure 2A) and total number results (Figure 2B) are shown. The results show that the use of EFNA1 to construct EphA2-targeted CAR does not affect the proliferation of CAR-T cells.

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

CAR-T cells, Mock control group and NTR cell control group after 72 hours of virus infection were collected by centrifugation, washed once with PBS and the supernatant was discarded. The cells were resuspended in PBS containing 2% FBS, and the positive rate was detected by flow cytometry.

The results of transfection efficiency are shown in Figure 3. Among them, NTR is uninfected T cells, and MOCK is T cells infected with a control virus without the CD7 extracellular binding domain.

Results: The results of transfection efficiency are shown in Figure 3, where Figure 3A represents the results of observation under a fluorescence microscope, and the results show that because CAR cells co-express CAR-mKate2 fusion protein, the fusion protein after expression is cut by T2A, and the mKate2 protein formed exhibits red fluorescence in the cell. Figure 3B represents the results of flow cytometry, and the results show that the positive rate of CAR-T expressing the CAR of the present invention is about 80%.

At the same time, CD4/CD8 flow cytometry was performed on T cells on days 3 and 6 after infection. The results are shown in Figure 4: EFNA1 as a target recognition domain does not affect the phenotype of CAR-T, that is, it does not affect the proportion of CD4 and CD8 positive T cells.

Example 5: Killing effect of EFNA1 CAR-T on various tumor cell lines

The killing effect of the CAR-T cells of the present invention on various tumor cell lines was detected at an effector-target ratio of 5:1.

The results are shown in Figure 5: EFNA1 CAR-T has a significant killing effect on tumor cells including bladder cancer cell line (RT4), prostate cancer cell line (PC3), glioma cell line (U87), breast cancer cell line (MCF7), pancreatic cancer cell line (BXPC3, PANC1) and colorectal cancer cell line (DLD1, HCT116).

Example 6: Detection of EphA2 expression in colorectal cancer cells and normal cells

The expression of EphA2 in colorectal cancer cells DLD1, HCT116 and normal cells 293T and COS7 was detected by RNA level, protein level and immunofluorescence. The RNA expression level was detected by RT-PCR (Figure 6A), the protein level was detected by Western Blot (Figure 6B), and the molecular localization was performed by immunofluorescence (Figure 6C). COS7 cells were used as negative control.

The results are shown in Figure 6, which show that both colorectal cancer cell lines DLD1 and HCT116 express EphA2 and localize it on the cell membrane, with HCT116 expressing it at a higher level. However, normal cells 293T and COS7 do not express EphA2.

Example 7: Construction of target cells carrying luciferase

The luciferase fragment was PCR amplified from the pGL3-luciferase plasmid, and then connected into the pTomo vector through XbaI and BamHI to construct the pTomo-EGFP-T2A-lucferase plasmid. The IRES and puromycin fragments were amplified from the pTomo and PLkO.1 plasmids, respectively. The pTomo-EGFP-T2A-luciferase-IRES-Puro plasmid was successfully constructed by three-fragment ligation. Lentivirus packaging and titer determination As described above, human PC-3, 5637, RT4, J82, DLD1 and HCT116 cell lines were infected at MOI = 100, and after 48 hours, puromycin (1ug/ml) was used for screening for 1 week to obtain a cell line that stably expressed luciferase, which was used in the cell killing detection experiment of Example 8.

Example 8: CAR-T cells kill tumor cells with gradient effector-target ratio

In this example, the killing ability of the CAR-T cells of the present invention on different target cells was detected. The target cells used included: target cells expressing EphA2: prostate cancer cells PC-3; bladder cancer tumor cells 5637, RT4, J82; colorectal cancer cells DLD1, HCT116; target cells that do not express or substantially do not express EphA2: 293T, COS7.

After digesting and counting the above cells carrying luciferase, the cell density was adjusted to 2*10^4/ml. 100ul of cells carrying luciferase were inoculated in a black 96-well plate, and the cell density of CAR-T/NT cells was adjusted to 1*10^5. They were inoculated into black 96-well plates according to E:T of 0.5:1, 1:1, 2:1, 4:1, and 8:1, with 100ul inoculated in each well. The above target cells and T cells were mixed and incubated in an incubator for 24 hours. The cell supernatant was collected and frozen at -80℃ to detect the amount of IFNγ released. Cell killing was detected using a promega fluorescence detection kit. First, the cells were treated with 20ul1*PLB lysate for 20 minutes, and 100ul substrate was added to each well and immediately detected with a BioTek microplate reader.

Cytotoxic killing cell % = (1-target cell fluorescence value with effector cells/target cell fluorescence value without effector cells) * 100%

The results are shown in Figure 7. The results showed that the killing effect of EFNA1-CAR-T cells on tumor cells gradually increased with the increase of effector-target ratio (E:T).

Example 9: IFNγ cytokine release

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

Taking IFNγ as an example, the method is as follows: take the cell supernatant of the CAR-T cells of the present invention co-incubated with colorectal cancer DLD1, HCT116 target cells (ET ratio of 2:1) in Example 7, and detect IFNγ according to IFN gamma Human ELISA Kit (lifetechnology).

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

Add 50ul Incubation buffer, 50ul test sample, and 50ul IFNγbiotinconjugated solution to each well, mix well, and let stand at room temperature for 90 minutes.

Then follow these steps in order:

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

(2) Add 100 μl of 1*Streptavidin-HRP solution to each well and incubate at room temperature for 45 minutes.

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

(4) Add 100ul of stabilized chromogen and let stand at room temperature for 30 minutes.

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

(6) Detect the absorbance at 450 nm.

Similarly, detection of TNFα was performed using Human TNFα ELISA Kit (BD Bioscience).

The results are shown in Figures 8A and B. When co-incubated with colorectal cancer tumor cells, the CAR-T cells of the present invention secreted significantly increased amounts of cytokines IFNγ and TNFα. This result suggests that the killing effect of EFNA1-CAR-T cells on colorectal cancer tumor cells is related to the release of IFNγ and TNFα.

Example 10: Effect of overexpression of EphA2 on the tumor killing effect of EFNA1-CAR-T

293T and COS7 are normal cell lines that are negative for EphA2, and EFNA1-CAR-T has no killing effect on 293T and COS7 that do not express EphA2. In this example, the EphA2 coding region was synthesized in vitro and pTomo-CMV-EphA2-luciferase-IRES-EGFP was constructed by enzyme ligation. Lentivirus was packaged in vitro and infected with 293T cells and COS7 cells. A 293T cell line that stably overexpressed EphA2 (called 293T-EphA2 over) and a COS7 cell line that stably overexpressed EphA2 (called COS7-EphA2 over) were obtained.

The results are shown in FIG9 . Both 293T-EphA2 over and COS7-EphA2 over cell lines can stably overexpress EphA2.

An in vitro killing experiment was performed according to the method of Example 8, and the killing effect of EFNA1-CAR-T on 293T-EphA2 over and COS7-EphA2 over cells was detected by luciferase fluorescence.

The results are shown in Figure 10. The results show that the CAR-T cells of the present invention have a significant killing effect on the cell lines 293T-EphA2over and COS7-EphA2over in which EphA2 is overexpressed on the cell membrane, while the CAR-T cells of the present invention have no killing effect on normal 293T and COS7 cells (Figure 10A). Moreover, when co-incubated with the cell line overexpressing EphA2, the IFNγ secretion of the CAR-T cells of the present invention was significantly upregulated, while the normal cells were basically the same as the control group (Figure 10B). The above results all indicate that the CAR-T cells of the present invention have a specific killing effect on EphA2 overexpressing cells, but have basically no killing power on cells that do not express EphA2, and have good safety.

discuss

Different hepatocyte receptors for erythropoietin (such as EphA1, EphA2, EphA3 and EphA4) are expressed on a variety of cells, and the Eph receptor protein family has a variety of ligands (including at least 9 ligands such as EphrinA1-6 and EphrinB1-3).

The EphA2 molecule plays an important role in the process of tumor formation. Compared with normal tissues, its expression is significantly upregulated in various tumor tissues, making it an ideal target for treating tumors with high EphA2 expression. In addition, although previous studies have shown that the EFNA1 ligand binds to four Eph receptor family members to varying degrees, the inventors have found through research that the CAR immune cells constructed based on EFNA1 have obvious high specificity and high killing activity against tumors with high EphA2 expression, but do not show obvious killing activity against normal cells that do not express EphA2. Therefore, it has high specificity and safety and is suitable for targeting EphA2-positive tumors.

In addition, in tumor cells, several other Eph receptors besides EphA2 may also have abnormally high expression levels that exceed physiological expression levels. Therefore, based on the ability of EFNA1 to bind to these Eph receptors, it also has the potential to prevent immune escape caused by target loss during treatment and prevent tumor recurrence.

Therefore, the chimeric antigen receptor immune cells we constructed based on EFNA1 can recognize the EFNA1 receptor very well, have very high specificity and killing activity against tumor cells with high expression of EphA2, but have no killing effect on normal cells that do not express EphA2. They can be used to treat tumors with high expression of EphA2.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

SEQUENCE LISTING

<110> West China Hospital, Sichuan University

<120> Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1

<130> P2021-1799

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 205

<212> PRT

<213> Artificial Sequence

<400> 1

Met Glu Phe Leu Trp Ala Pro Leu Leu Gly Leu Cys Cys Ser Leu Ala

1 5 10 15

Ala Ala Asp Arg His Thr Val Phe Trp Asn Ser Ser Asn Pro Lys Phe

20 25 30

Arg Asn Glu Asp Tyr Thr Ile His Val Gln Leu Asn Asp Tyr Val Asp

35 40 45

Ile Ile Cys Pro His Tyr Glu Asp His Ser Val Ala Asp Ala Ala Met

50 55 60

Glu Gln Tyr Ile Leu Tyr Leu Val Glu His Glu Glu Tyr Gln Leu Cys

65 70 75 80

Gln Pro Gln Ser Lys Asp Gln Val Arg Trp Gln Cys Asn Arg Pro Ser

85 90 95

Ala Lys His Gly Pro Glu Lys Leu Ser Glu Lys Phe Gln Arg Phe Thr

100 105 110

Pro Phe Thr Leu Gly Lys Glu Phe Lys Glu Gly His Ser Tyr Tyr Tyr

115 120 125

Ile Ser Lys Pro Ile His Gln His Glu Asp Arg Cys Leu Arg Leu Lys

130 135 140

Val Thr Val Ser Gly Lys Ile Thr His Ser Pro Gln Ala His Asp Asn

145 150 155 160

Pro Gln Glu Lys Arg Leu Ala Ala Asp Asp Pro Glu Val Arg Val Leu

165 170 175

His Ser Ile Gly His Ser Ala Ala Pro Arg Leu Phe Pro Leu Ala Trp

180 185 190

Thr Val Leu Leu Leu Pro Leu Leu Leu Leu Gln Thr Pro

195 200 205

<210> 2

<211> 233

<212> PRT

<213> Artificial Sequence

<400> 2

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

1 5 10 15

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

<210> 3

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 3

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

<210> 4

<211> 43

<212> PRT

<213> Artificial Sequence

<400> 4

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

1 5 10 15

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

20 25 30

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40

<210> 5

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 5

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

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 6

<211> 43

<212> PRT

<213> Artificial Sequence

<400> 6

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

1 5 10 15

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

<210> 7

<211> 111

<212> PRT

<213> Artificial Sequence

<400> 7

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> 406

<212> PRT

<213> 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 Asp Arg His Thr Val Phe Trp Asn Ser Ser Asn

20 25 30

Pro Lys Phe Arg Asn Glu Asp Tyr Thr Ile His Val Gln Leu Asn Asp

35 40 45

Tyr Val Asp Ile Ile Cys Pro His Tyr Glu Asp His Ser Val Ala Asp

50 55 60

Ala Ala Met Glu Gln Tyr Ile Leu Tyr Leu Val Glu His Glu Glu Tyr

65 70 75 80

Gln Leu Cys Gln Pro Gln Ser Lys Asp Gln Val Arg Trp Gln Cys Asn

85 90 95

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

100 105 110

Arg Phe Thr Pro Phe Thr Leu Gly Lys Glu Phe Lys Glu Gly His Ser

115 120 125

Tyr Tyr Tyr Ile Ser Lys Pro Ile His Gln His Glu Asp Arg Cys Leu

130 135 140

Arg Leu Lys Val Thr Val Ser Gly Lys Ile Thr His Ser Pro Gln Ala

145 150 155 160

His Asp Asn Pro Gln Glu Lys Arg Leu Ala Ala Asp Asp Pro Glu Val

165 170 175

Arg Val Leu His Ser Ile Gly His Ser Thr Pro Ala Pro Arg Pro Pro

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

355 360 365

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

370 375 380

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

385 390 395 400

Gln Ala Leu Pro Pro Arg

405

<210> 9

<211> 1218

<212> DNA

<213> Artificial Sequence

<400> 9

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccggatcgcc acaccgtctt ctggaacagt tcaaatccca agttccggaa tgaggactac 120

accatacatg tgcagctgaa tgactacgtg gacatcatct gtccgcacta tgaagatcac 180

tctgtggcag acgctgccat ggagcagtac atactgtacc tggtggagca tgaggagtac 240

cagctgtgcc agccccagtc caaggaccaa gtccgctggc agtgcaaccg gcccagtgcc 300

aagcatggcc cggagaagct gtctgagaag ttccagcgct tcacaccttt caccctgggc 360

aaggagttca aagaaggaca cagctactac tacatctcca aacccatcca ccagcatgaa 420

gaccgctgct tgaggttgaa ggtgactgtc agtggcaaaa tcactcacag tcctcaggcc 480

catgacaatc cacaggagaa gagacttgca gcagatgacc cagaggtgcg ggttctacat 540

agcatcggtc acagtacgcc agcgccgcga ccaccaacac cggcgcccac catcgctagc 600

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 660

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 720

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 780

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 840

agctgccgatttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 900

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 960

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1020

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1080

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 1140

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1200

caggccctgc cccctcgc 1218

<210> 10

<211> 660

<212> PRT

<213> 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 Asp Arg His Thr Val Phe Trp Asn Ser Ser Asn

20 25 30

Pro Lys Phe Arg Asn Glu Asp Tyr Thr Ile His Val Gln Leu Asn Asp

35 40 45

Tyr Val Asp Ile Ile Cys Pro His Tyr Glu Asp His Ser Val Ala Asp

50 55 60

Ala Ala Met Glu Gln Tyr Ile Leu Tyr Leu Val Glu His Glu Glu Tyr

65 70 75 80

Gln Leu Cys Gln Pro Gln Ser Lys Asp Gln Val Arg Trp Gln Cys Asn

85 90 95

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

100 105 110

Arg Phe Thr Pro Phe Thr Leu Gly Lys Glu Phe Lys Glu Gly His Ser

115 120 125

Tyr Tyr Tyr Ile Ser Lys Pro Ile His Gln His Glu Asp Arg Cys Leu

130 135 140

Arg Leu Lys Val Thr Val Ser Gly Lys Ile Thr His Ser Pro Gln Ala

145 150 155 160

His Asp Asn Pro Gln Glu Lys Arg Leu Ala Ala Asp Asp Pro Glu Val

165 170 175

Arg Val Leu His Ser Ile Gly His Ser Thr Pro Ala Pro Arg Pro Pro

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

355 360 365

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

His His Phe Lys Cys Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly

450 455 460

Thr Gln Thr Met Arg Ile Lys Ala Val Glu Gly Gly Pro Leu Pro Phe

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr

530 535 540

Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met

545 550 555 560

Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr Glu Thr Leu Tyr Pro

565 570 575

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

580 585 590

Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys

595 600 605

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

610 615 620

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

625 630 635 640

His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly

645 650 655

His Lys Leu Asn

660

<210> 11

<211> 1983

<212> DNA

<213> Artificial Sequence

<400> 11

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccggatcgcc acaccgtctt ctggaacagt tcaaatccca agttccggaa tgaggactac 120

accatacatg tgcagctgaa tgactacgtg gacatcatct gtccgcacta tgaagatcac 180

tctgtggcag acgctgccat ggagcagtac atactgtacc tggtggagca tgaggagtac 240

cagctgtgcc agccccagtc caaggaccaa gtccgctggc agtgcaaccg gcccagtgcc 300

aagcatggcc cggagaagct gtctgagaag ttccagcgct tcacaccttt caccctgggc 360

aaggagttca aagaaggaca cagctactac tacatctcca aacccatcca ccagcatgaa 420

gaccgctgct tgaggttgaa ggtgactgtc agtggcaaaa tcactcacag tcctcaggcc 480

catgacaatc cacaggagaa gagacttgca gcagatgacc cagaggtgcg ggttctacat 540

agcatcggtc acagtacgcc agcgccgcga ccaccaacac cggcgcccac catcgctagc 600

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 660

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 720

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 780

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 840

agctgccgatttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 900

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 960

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1020

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1080

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 1140

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1200

caggccctgc cccctcgcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac 1260

gtcgaggaga atcctggccc aatgagcgag ctgattaagg agaacatgca catgaagctg 1320

tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag 1380

ccctacgagg gcacccagac catgagaatc aaggcggtcg agggcggccc tctccccttc 1440

gccttcgaca tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc 1500

cagggcatcc ccgacttctt taagcagtcc ttccccgagg gcttcacatg ggagagagtc 1560

accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc 1620

tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg 1680

cagaagaaaa cactcggctg ggaggcctcc accgagaccc tgtaccccgc tgacggcggc 1740

ctggaaggca gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac 1800

ttgaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac 1860

tatgtggaca gaagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag 1920

cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caagcttaat 1980

tag 1983

Claims (10)

1. A Chimeric Antigen Receptor (CAR), comprising an extracellular binding domain comprising the structure of EFNA1 or a fragment thereof based on the amino acid sequence set forth in SEQ ID No. 1, wherein said extracellular binding domain is capable of specifically binding to the EFNA1 receptor in a ligand receptor manner.

2. The chimeric antigen receptor according to claim 1, wherein the extracellular binding domain comprises an EFNA1 protein or a fragment thereof, said EFNA1 protein or fragment thereof having the amino acid sequence shown in SEQ ID No. 1 or having the amino acid sequence from position 1 to 182 (preferably from position 19 to 182) of the sequence shown in SEQ ID No. 1.

3. The chimeric antigen receptor according to claim 1 or 2, wherein the chimeric antigen receptor has the structure according to formula I:

L-EB-H-TM-C-CD3ζ-RP(I)

in the method, in the process of the invention,

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

l is an absent or signal peptide sequence;

EB is an extracellular binding domain;

h is a no or hinge region;

TM is a transmembrane domain;

c is an absent or co-stimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

RP is absent or reporter.

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

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

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

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

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

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

10. Use of a chimeric antigen receptor according to claim 1, a nucleic acid molecule according to claim 4, a vector according to claim 5, or a host cell according to claim 6, and/or an engineered immune cell according to claim 7, for the preparation of a medicament or formulation for the prevention and/or treatment of diseases associated with aberrant expression of the EFNA1 receptor.

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