CN111218426A

CN111218426A - Preparation and application of CAR-T cell for overcoming tumor microenvironment

Info

Publication number: CN111218426A
Application number: CN202010119470.5A
Authority: CN
Inventors: 江文正; 陆妍瑾; 周滢; 伍松梅; 何聪; 刘明耀; 席在喜
Original assignee: East China Normal University; Bioray Laboratories Inc
Current assignee: East China Normal University; Bioray Laboratories Inc
Priority date: 2020-02-26
Filing date: 2020-02-26
Publication date: 2020-06-02

Abstract

The invention provides a preparation method and application of CAR-T cells for overcoming tumor microenvironment. Specifically, the invention provides an engineered immune cell expressing a chimeric antigen receptor CAR that targets a tumor cell surface antigen and a chimeric cytokine receptor, including IL-4R and IL-15R. The engineered immune cells can reverse the inhibitory effect of inhibitory cytokines infiltrated in an immunosuppressive microenvironment, and can selectively kill tumor cells.

Description

Preparation and application of CAR-T cell for overcoming tumor microenvironment

Technical Field

The invention belongs to the field of biotechnology. In particular, the invention relates to preparation and application of CAR-T cells for overcoming tumor microenvironment.

Background

Chimeric Antigen Receptor (CAR) -T cell immunotherapy is a promising adoptive cellular immunotherapy, whose basic principle is to modify T lymphocytes by genetic engineering to express Chimeric antigen receptors, killing tumor cells in a non-MHC-restricted manner. Now that the development to the fourth generation, with the determination of efficacy of CAR-T in hematological tumor therapy, solid tumor researchers have also begun to attempt CAR-T therapy, but with far less than expected efficacy. Solid tumors have a unique immunosuppressive microenvironment, preventing recruitment of CAR-T cells to the tumor, with an inhibitory cytokine being one of the inhibitory factors in the tumor microenvironment. Tumors secrete a variety of inhibitory cytokines, and the immunosuppressive microenvironment obviously inhibits the proliferation and functions of T cells, so that the tumors escape.

Therefore, there is an urgent need in the art to develop a novel chimeric antigen receptor molecule that not only reverses the inhibitory effect of inhibitory cytokines infiltrated in the immunosuppressive microenvironment, but also enhances the killing effect on tumors.

Disclosure of Invention

The invention aims to provide a novel chimeric antigen receptor molecule which not only can reverse the inhibition effect of inhibitory cytokines infiltrated in an immunosuppressive microenvironment, but also can enhance the killing effect on tumors.

In a first aspect of the invention, there is provided an engineered immune cell expressing a chimeric antigen receptor CAR that targets a tumor cell surface antigen and a chimeric cytokine receptor, including IL-4R and IL-15R.

In another preferred embodiment, the tumor cell antigens include cell surface antigens of various solid and non-solid tumors.

In another preferred embodiment, the tumor cell surface antigen comprises NKG2D ligand.

In another preferred embodiment, the NKG2D ligand is selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof.

In another preferred embodiment, the immune cell is an NK cell or a T cell, preferably a T cell.

In another preferred embodiment, the chimeric antigen receptor CAR is localized to the cell membrane of the immune cell.

In another preferred embodiment, the chimeric antigen receptor CAR contains an antigen binding domain that targets a tumor cell surface antigen.

In another preferred embodiment, the antigen binding domain is an antibody or antigen binding fragment.

In another preferred embodiment, the antigen binding fragment is a Fab or scFv or a single domain antibody sdFv.

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

L-S-H-TM-C-CD3ζ(I)

wherein "-" is a linker peptide or a peptide bond;

l is a null or signal peptide sequence;

s is an antigen binding domain comprising the extracellular segment of NKG 2D;

h is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ.

In another preferred embodiment, the amino acid sequence of the extracellular domain protein of NKG2D is selected from the group consisting of:

(a) 1 amino acid sequence of the protein as shown in SEQ ID NO;

(b) 1, and (a) a protein derived from (a) and having the function of (a) a protein, which is formed by substituting, deleting or adding one or more (e.g., 1 to 10) amino acid residues in the amino acid sequence of SEQ ID NO; or

(c) And (b) a protein derived from (a) and having more than 90% (preferably more than or equal to 95%) homology with the protein sequence defined by (a) and having the protein function of (a).

In another preferred embodiment, the nucleotide sequence of the extracellular domain protein encoding NKG2D is selected from the group consisting of:

(a) the polynucleotide with the nucleotide sequence shown in SEQ ID NO. 2;

(b) a polynucleotide having homology of 70% or more (preferably 80% or more, 90% or more, 95% or more or 98% or more) with the sequence shown in SEQ ID NO. 2, and having an activity of targeting or binding to NKG2D ligand;

(c) 2, and having the activity of targeting or binding to NKG2D ligand, wherein the 5 'end and/or the 3' end of the polynucleotide is truncated by 1-60 (preferably 1-30, more preferably 1-6) nucleotides.

In another preferred embodiment, the extracellular segment of NKG2D is of human origin.

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8a, CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

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

In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of: CD8a, CD28, CD137, or a combination thereof.

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

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD3 epsilon, CD4, CD8, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3, or a combination thereof.

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

In another preferred embodiment, C is a costimulatory signal molecule for a protein selected from the group consisting of: OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, DAP10, CDS, ICAM-1, or a combination thereof.

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

In another preferred embodiment, the amino acid sequence of the CAR is as set forth in SEQ ID No. 3.

In another preferred embodiment, the chimeric cytokine receptor has the structure according to formula II:

Z1-L’-Z2(II)

wherein,

z1 is the extracellular domain of IL-4R;

l' is an optional linker peptide;

z2 is the transmembrane domain and intracellular domain of IL-15R.

In another preferred embodiment, the amino acid sequence of Z1 is shown in SEQ ID No. 4.

In another preferred embodiment, the amino acid sequence of Z2 is shown in SEQ ID NO. 5.

In another preferred embodiment, the amino acid sequence of the chimeric cytokine receptor is shown in SEQ ID No. 6.

In another preferred embodiment, the length of the linking peptide is 1 to 300aa, preferably 2 to 100aa, more preferably 3 to 50 aa.

In a second aspect, the invention provides a method of preparing an engineered immune cell according to the first aspect of the invention, comprising the steps of:

(A) providing an immune cell to be modified; and

(B) engineering the immune cell such that the immune cell expresses a chimeric antigen receptor CAR targeting a tumor cell surface antigen and a chimeric cytokine receptor comprising IL-4R and IL-15R to obtain the engineered immune cell of the first aspect of the invention.

In another preferred example, in step (a), the method further comprises isolating and/or activating the immune cells to be modified.

In another preferred embodiment, step (B) comprises (B1) introducing into the immune cell a first expression cassette expressing the CAR targeted to the tumor cell surface antigen; and (B2) introducing a second expression cassette expressing a chimeric cytokine receptor into the immune cell; wherein said step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

In another preferred example, in step (B), the first expression cassette and/or the second expression cassette is introduced into the nucleus of the immune cell.

In another preferred example, when the immune cell to be engineered in step (a) already expresses the CAR, then step (B1) may be omitted.

In another preferred embodiment, the immune cell is an NK cell or a T cell.

In another preferred embodiment, the first expression cassette comprises a nucleic acid sequence encoding the chimeric antigen receptor CAR.

In another preferred embodiment, the second expression cassette comprises a nucleic acid sequence encoding a chimeric cytokine receptor.

In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

In another preferred embodiment, the first expression cassette and the second expression cassette are located in the same vector.

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

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

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

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

In a third aspect, the invention provides a formulation comprising an engineered immune cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

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

In another preferred embodiment, the formulation comprises an injection.

In another preferred embodiment, the concentration of said engineered immune cells in said formulation is 1 × 10³-1×10⁸Individual cells/ml, preferably 1X 10⁴-1×10⁷Individual cells/ml.

In another preferred embodiment, the formulation further contains other drugs (such as emerging antibody drugs, other CAR-T drugs, or chemotherapeutic drugs) for treating cancer or tumors.

In a fourth aspect, the invention provides the use of an engineered immune cell according to the first aspect of the invention for the preparation of a medicament or formulation for selective killing of tumors.

In another preferred embodiment, the medicament or formulation is used to reverse the inhibitory effect of IL-4 on engineered immune cells in an immunosuppressive microenvironment.

In another preferred embodiment, the tumor comprises a tumor that highly expresses IL-4.

In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof, preferably the tumor is a solid tumor.

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

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

In another preferred embodiment, the solid tumor is selected from the group consisting of: liver cancer, head and neck cancer, melanoma, non-hodgkin's lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, renal cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, prostate cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, lung small cell carcinoma, colorectal cancer, breast cancer, medulloblastoma, breast cancer, or a combination thereof.

In a fifth aspect, the present invention provides a kit for preparing a medicament for selective killing of tumors, the kit comprising a container, and, in the container:

(1) a first nucleic acid sequence comprising a first expression cassette for expression of a chimeric antigen receptor, CAR, targeted to a tumor cell surface antigen; and

(2) a second nucleic acid sequence comprising a second expression cassette for expression of a chimeric cytokine receptor, including IL-4R and IL-15R.

In another preferred embodiment, the first and second nucleic acid sequences are independent or linked.

In another preferred embodiment, the first and second nucleic acid sequences are in the same or different containers.

In another preferred embodiment, the first and second nucleic acid sequences are located on the same or different vectors.

In another preferred embodiment, the first and second nucleic acid sequences are located on the same vector.

In a sixth aspect, the present invention provides a method for selectively killing tumors, comprising:

administering to a subject in need thereof a safe and effective amount of an engineered immune cell according to the first aspect of the invention, or a formulation according to the third aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In a seventh aspect, the present invention provides a method for treating a disease comprising administering to a subject in need thereof a safe and effective amount of an engineered immune cell according to the first aspect of the present invention, or a formulation according to the third aspect of the present invention.

In another preferred embodiment, the method further comprises administering to a subject in need of treatment an additional agent for treating cancer or tumor.

In another preferred embodiment, the other drug comprises a CAR-T drug.

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

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

The eighth aspect of the invention provides a fusion protein comprising a chimeric antigen receptor CAR targeting a tumor cell surface antigen and a chimeric cytokine receptor comprising IL-4R and IL-15R.

In another preferred embodiment, the CAR and the chimeric cytokine receptor are linked by a linking peptide.

In another preferred embodiment, the linker peptide comprises a self-cleaving protein.

In another preferred embodiment, the self-cleaving protein is selected from the group consisting of: T2A, P2A, E2A, F2A, or a combination thereof.

In another preferred embodiment, the self-cleaving protein comprises T2A.

In another preferred embodiment, the structure of the fusion protein is represented by formula III below:

L-S-H-TM-C-CD3ζ-(Z3-P)m(I)

in the formula,

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

l is a null or signal peptide sequence;

s is an antigen binding domain comprising the extracellular segment of NKG 2D;

h is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

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

z3 is a linker peptide;

p is a chimeric cytokine receptor;

m is 1, 2, 3, or 4.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID No. 7.

In a ninth aspect, the present invention provides a polynucleotide encoding the fusion protein of the eighth aspect of the present invention.

In another preferred embodiment, the polynucleotide is selected from the group consisting of:

(a) a polynucleotide encoding a fusion protein as set forth in SEQ ID No. 7;

(b) a polynucleotide sequence as shown in SEQ ID No. 8;

(c) a polynucleotide having a nucleotide sequence having a homology of 75% or more (preferably 80% or more) to the sequence of (b);

(d) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added to the 5 'end and/or the 3' end of the polynucleotide shown in (b);

(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).

In another preferred embodiment, the polynucleotide sequence is as shown in SEQ ID No. 8.

In a tenth aspect, the present invention provides a vector comprising a polynucleotide according to the ninth aspect of the invention.

In another preferred embodiment, the vector comprises DNA and RNA.

In another preferred embodiment, the carrier is selected from the group consisting of: a plasmid, a viral vector, a transposon, or a combination thereof.

In another preferred embodiment, the vector comprises a DNA virus or a retroviral vector.

In another preferred embodiment, the carrier is selected from the group consisting of: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

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

In another preferred embodiment, the vector comprises one or more promoters operably linked to the nucleic acid sequence, enhancer, intron, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker, nucleic acid restriction site, and/or homologous recombination site.

In another preferred embodiment, the vector is a vector containing or inserted with the polynucleotide of the ninth aspect of the present invention.

In another preferred embodiment, the vector is used for expressing the fusion protein according to the eighth aspect of the invention.

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

Drawings

FIG. 1 shows a map of pCDH-NKG2D-IL-4R/IL-15R plasmid.

FIG. 2 shows the results of enzyme cleavage identification.

FIG. 3 shows the proportion of CAR-T cells obtained after flow cytometry to determine lentivirus-infected T cells.

FIG. 4 shows the results of flow-detecting killing of CAR-T cells against Huh-7, Panc-1.

Detailed Description

The inventor of the invention has conducted extensive and intensive research, and unexpectedly found that the engineered immune cell containing the chimeric antigen receptor CAR targeting the tumor cell surface antigen and the chimeric cytokine receptor can reverse the inhibition effect of the inhibitory cytokine infiltrated in the immunosuppressive microenvironment, and can selectively kill the tumor cell with high IL-4 expression, and the higher the IL-4 expression level, the better the killing effect. On this basis, the inventors have completed the present invention.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

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

As used herein, a "Chimeric Antigen Receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are also referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". The term "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding an antigen. "intracellular domain" refers to any oligopeptide or polypeptide known to be a domain that transmits signals to activate or inhibit biological processes in a cell.

As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

As used herein, "tumor antigen" refers to an antigenic biomolecule, the expression of which results in cancer.

As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of an anti-human LAG-3 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, comprising any of the CARs of the invention and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.

"sequence identity" as referred to herein means the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between a sequence described in the present invention and a sequence with which it is identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

Chimeric cytokine receptors

In the present invention, the Chimeric cytokine receptors (CARs) are composed of an extracellular antigen recognition region, usually scFv (single-chain variable fragment), a transmembrane region, and an intracellular costimulatory signal region. The design of CARs has undergone the following development process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and, because of the single activation domain in the cell, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function of the second generation CARs is greatly improved, and the persistence of CAR-T cells and the killing capability of the CAR-T cells on tumor cells are further enhanced. On the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and the development is three-generation and four-generation CARs. The extracellular domain of CARs recognizes a specific antigen and subsequently transduces this signal through the intracellular domain, causing activated proliferation, cytolytic toxicity and cytokine secretion of the cell, thereby clearing the target cell. Autologous cells from the patient (or a heterologous donor) are first isolated, activated and genetically engineered to produce immune cells for CAR production, and then injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by immune cells in a non-MHC restricted manner.

In a preferred embodiment, the chimeric cytokine receptor includes IL-4R and IL-15R.

IL-4 is a cytokine with multifaceted efficacy, secreted by Th2 cells, mast cells, basophils IL-4 is also a key factor secreted by cancer cells, and in breast, prostate, lung, kidney and other types of cancer IL-4 has a high expression level, furthermore, IL-4 receptor (IL-4R) is overexpressed in many types of cancer, IL-4 can initially stimulate tumor cells by increasing tumor growth, increasing its apoptosis resistance, and in CAR-T cell therapy studies IL-4 is a cytokine that exerts CAR-T cell function in the tumor microenvironment.

Interleukin-15 (IL-15) is secreted by mononuclear phagocytes and other cells following viral infection and plays an important biological role in the maintenance and function of a variety of cell types. IL-15 is thought to be a cytokine that plays a major role in immune homeostasis. In the absence of antigen, IL-15 provides a signal to maintain survival of memory T cells. Mouse memory CD8 deficient in IL-15 or its receptor⁺IL-15 receptors are heterotopic trimers which include a gamma chain (CD132) shared by gamma c family cytokines such as IL-2, IL-7, IL-4, IL-9 and IL-21 receptors and β chain (IL-2/15R beta or CD122) shared with IL-2R, the α chain being its own chain.

the invention constructs an IL-4R/IL-15R chimeric receptor for the first time, IL-15 and the IL-4 receptor share a common cytokine receptor gamma chain of a heterodimer receptor thereof, and the reversal of IL-4 immunosuppression can be effectively realized by constructing the chimeric receptor by utilizing an IL-4R α chain extracellular segment and an IL-15R β chain transmembrane region and intracellular segment, and after the chimeric receptor is constructed, the chimeric receptor is expressed in an engineered immune cell, such as an NKG2D-CAR-T cell, the killing effect of the chimeric receptor on a high-expression IL-4 tumor cell is detected, and the effect of the chimeric receptor in a tumor immunosuppression microenvironment is analyzed.

Extracellular domain protein of NKG2D

The invention unexpectedly discovers that the extracellular domain protein of the specific NKG2D can be specifically combined with the NKG2D ligand.

NKG2D ((Natural Killer Group 2, memer D), a C-type lectin receptor, is the NK major activating receptor cell, its ligand is expressed in many types of cancer, including ovarian cancer, colon cancer, lung cancer, breast cancer, kidney cancer, prostate cancer, melanoma and leukemia, however, it is not expressed or low expressed in healthy tissues NKG 2D-based CAR-T cells provide good targets for tumor therapy, at present, NKG 2D-expressing CAR-T has been shown to be effective in most cancer types, such as multiple myeloma, ovarian cancer and lymphoma, among others.

In a preferred embodiment of the invention, the amino acid sequence of the extracellular domain protein of NKG2D is shown as SEQ ID No. 1.

LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQD LLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTI IEMQKGDCALYASSFKGYIENCSTPNTYIC MQRTV(SEQ IDNO.:1)

The nucleotide sequence of the extracellular domain protein of NKG2D is shown in SEQ ID No. 2.

TTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTG(SEQ ID NO.:2)

Tumor cell surface antigens

Tumor cell surface antigens of the present invention include, but are not limited to, NKG2D ligands.

In a preferred embodiment, the ligands of NKG2D of the invention comprise: MICA, MICB, ULBP1-6, the ligand of which is not expressed or is expressed in a low amount in normal cells, but the expression amount of the ligand is greatly increased when the cells are infected or cancerated, especially in many solid tumors, such as breast cancer, pancreatic cancer, prostate cancer, ovarian cancer and the like, therefore, the NKG2D receptor is designed into the CAR structure, and when the ligand is combined, the T cells are activated to generate a series of anti-tumor responses.

Antigen binding domains

In the present invention, the antigen binding domain of the chimeric antigen receptor CAR specifically binds to a tumor cell surface antigen.

In a preferred embodiment, the antigen binding domain of the chimeric antigen receptor CAR of the invention targets the NKG2D ligand.

Hinge region and transmembrane region

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

The transmembrane domain may be derived from natural sources or synthetic sources. In natural sources, the domain may be derived from any membrane bound or transmembrane protein. Preferably, the hinge region in the CAR of the invention is that of CD8a and the transmembrane region of the invention is that of CD8 a.

Intracellular domains

The intracellular domain or additional intracellular signaling domain of the CAR of the invention is responsible for the activation of at least one normal effector function of the immune cell in which the CAR has been placed. The term "effector function" refers to a cell's exclusive function. For example, the effector function of a T cell may be cytolytic activity or helper activity involving secretion of cytokines. The term "intracellular signaling domain" thus refers to a portion of a protein that transduces effector function signals and directs a cell to perform a proprietary function. Although the entire intracellular signaling domain may generally be used, in many instances, the entire strand need not be used. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the entire chain, so long as it transduces effector function signals. The term intracellular signaling domain thus refers to any truncated portion of an intracellular signaling domain that includes sufficient signal transduction of effector function.

Preferred examples of intracellular signaling domains for the CARs of the invention include cytoplasmic sequences of the T Cell Receptor (TCR) and co-receptors that act synergistically to initiate signal transduction upon antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence with the same functional capacity.

In preferred embodiments, the cytoplasmic domain of the CAR can be designed to itself include the CD3 zeta signaling domain, or can be associated with any other desired cytoplasmic domain(s) useful in the context of the CARs of the invention. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules required for effective response of lymphocytes to antigens, rather than antigen receptors or their ligands. Preferably, 4-1BB (CD137) and the like are included.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention can be linked to each other randomly or in a defined order. Optionally, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine doublets provide particularly suitable linkers.

In one embodiment, the cytoplasmic domain in the CAR of the invention is designed to include the signaling domain of 4-1BB (co-stimulatory molecule) and the signaling domain of CD3 ζ.

Chimeric Antigen Receptor (CAR)

Chimeric immune antigen receptors (CARs) consist of an extracellular antigen recognition region, usually a scFv (single-chain variable fragment), a transmembrane region, and an intracellular costimulatory signal region. The design of CARs goes through the following process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and, because of the single activation domain in the cell, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function of the second generation CARs is greatly improved, and the persistence of CAR-T cells and the killing capability of the CAR-T cells on tumor cells are further enhanced. On the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and the development is three-generation and four-generation CARs.

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

CAR-immune cell therapy has achieved very high clinical response rates in the treatment of hematological malignancies, which rates were previously unattainable by any therapeutic approach, and have triggered a hot surge of clinical research in the world.

Specifically, the Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and/or a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

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

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecules and/or the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the 4-1BB signaling domain and/or the CD3 zeta signaling domain combination.

As used herein, "antigen binding domain" and "single chain antibody fragment" each refer to an Fab fragment, Fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of scFv is typically 1/6 of that of a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. In a preferred embodiment of the present invention, the scFv comprises an antibody, preferably a single-chain antibody, that specifically recognizes a tumor-highly expressed NKG2D ligand.

In a preferred embodiment, the antigen binding portion of the CAR of the invention targets the NKG2D ligand. In a preferred embodiment, the antigen binding portion of the CAR of the invention is an extracellular domain of NKG2D that targets the NKG2D ligand.

In a preferred embodiment, the extracellular domain protein of NKG2D comprises a variant form, wherein the variant form has a homology of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more with the extracellular domain protein sequence list of its wild type NKG 2D.

In the present invention, the extracellular segment protein of NKG2D of the present invention also includes conservative variants thereof, which means that 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 the amino acid sequence of the extracellular segment protein of NKG2D of the present invention to form a polypeptide.

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

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

The extracellular domain of the CAR of the invention includes the extracellular domain of NKG2D, preferably NKG2D having a specific sequence.

In the present invention, the intracellular domains in the CAR of the invention include the transmembrane region of CD8a, the costimulatory factor of 4-1BB, and the signaling domain of CD3 zeta.

In a preferred embodiment of the invention, the amino acid sequence of the CAR is as set forth in SEQ ID No. 3:

MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

in a preferred embodiment of the invention, the amino acid sequence of the CAR is as set forth in SEQ ID No. 7:

MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSEGRGSLLTCGDVEENPGPMGWLCSGLLFPVSCLVLLQVASSGNMKVLQEPTCVSDYMSISTCEWKMNGPTNCSTELRLLYQLVFLLSEAHTCIPENNGGAGCVCHLLMDDVVSADNYTLDLWAGQQLLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTYAVNIWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSEWSPSTKWHNSYREPFEQHIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV。

in a preferred embodiment of the invention, the nucleotide sequence of the CAR is SEQ ID No. 8:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGTTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTGACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCACAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGATCCGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGGGGTGGCTTTGCTCTGGGCTCCTGTTCCCTGTGAGCTGCCTGGTCCTGCTGCAGGTGGCAAGCTCTGGGAACATGAAGGTCTTGCAGGAGCCCACCTGCGTCTCCGACTACATGAGCATCTCTACTTGCGAGTGGAAGATGAATGGTCCCACCAATTGCAGCACCGAGCTCCGCCTGTTGTACCAGCTGGTTTTTCTGCTCTCCGAAGCCCACACGTGTATCCCTGAGAACAACGGAGGCGCGGGGTGCGTGTGCCACCTGCTCATGGATGACGTGGTCAGTGCGGATAACTATACACTGGACCTGTGGGCTGGGCAGCAGCTGCTGTGGAAGGGCTCCTTCAAGCCCAGCGAGCATGTGAAACCCAGGGCCCCAGGAAACCTGACAGTTCACACCAATGTCTCCGACACTCTGCTGCTGACCTGGAGCAACCCGTATCCCCCTGACAATTACCTGTATAATCATCTCACCTATGCAGTCAACATTTGGAGTGAAAACGACCCGGCAGATTTCAGAATCTATAACGTGACCTACCTAGAACCCTCCCTCCGCATCGCAGCCAGCACCCTGAAGTCTGGGATTTCCTACAGGGCACGGGTGAGGGCCTGGGCTCAGTGCTATAACACCACCTGGAGTGAGTGGAGCCCCAGCACCAAGTGGCACAACTCCTACAGGGAGCCCTTCGAGCAGCACATTCCGTGGCTCGGCCACCTCCTCGTGGGCCTCAGCGGGGCTTTTGGCTTCATCATCTTAGTGTACTTGCTGATCAACTGCAGGAACACCGGGCCATGGCTGAAGAAGGTCCTGAAGTGTAACACCCCAGACCCCTCGAAGTTCTTTTCCCAGCTGAGCTCAGAGCATGGAGGAGACGTCCAGAAGTGGCTCTCTTCGCCCTTCCCCTCATCGTCCTTCAGCCCTGGCGGCCTGGCACCTGAGATCTCGCCACTAGAAGTGCTGGAGAGGGACAAGGTGACGCAGCTGCTCCTGCAGCAGGACAAGGTGCCTGAGCCCGCATCCTTAAGCAGCAACCACTCGCTGACCAGCTGCTTCACCAACCAGGGTTACTTCTTCTTCCACCTCCCGGATGCCTTGGAGATAGAGGCCTGCCAGGTGTACTTTACTTACGACCCCTACTCAGAGGAAGACCCTGATGAGGGTGTGGCCGGGGCACCCACAGGGTCTTCCCCCCAACCCCTGCAGCCTCTGTCAGGGGAGGACGACGCCTACTGCACCTTCCCCTCCAGGGATGACCTGCTGCTCTTCTCCCCCAGTCTCCTCGGTGGCCCCAGCCCCCCAAGCACTGCCCCTGGGGGCAGTGGGGCCGGTGAAGAGAGGATGCCCCCTTCTTTGCAAGAAAGAGTCCCCAGAGACTGGGACCCCCAGCCCCTGGGGCCTCCCACCCCAGGAGTCCCAGACCTGGTGGATTTTCAGCCACCCCCTGAGCTGGTGCTGCGAGAGGCTGGGGAGGAGGTCCCTGACGCTGGCCCCAGGGAGGGAGTCAGTTTCCCCTGGTCCAGGCCTCCTGGGCAGGGGGAGTTCAGGGCCCTTAATGCTCGCCTGCCCCTGAACACTGATGCCTACTTGTCCCTCCAAGAACTCCAGGGTCAGGACCCAACTCACTTGGTG。

wherein, the 1 st to 21 st positions in SEQ ID No. 7 are signal peptides; extracellular domain protein with NKG2D at position 22-156; hinge region at positions 157-173; positions 174-225 are transmembrane domains (e.g., the transmembrane domain of CD8 a); positions 226-267 are co-stimulatory elements (e.g., 4-1 BB); CD3 ζ at positions 268-379, a linker peptide (e.g., a self-cleaving protein) at positions 380-399, and a chimeric cytokine receptor at positions 400-942.

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T cells of the invention" all refer to CAR-T cells of the invention, which can be targeted to a tumor surface antigen (e.g., NKG2D ligand), for the treatment of tumors that are highly expressed or positive for IL-4, particularly solid tumors.

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

In the present invention, the CAR of the invention comprises (i) an extracellular domain comprising an antigen that targets a tumor cell surface antigen; (i i) a transmembrane domain; (iii) (i ii) a co-stimulatory factor; and (iv) the signaling domain of CD3 ζ; and; (v) a linker peptide (e.g., a self-cleaving protein); (vi) chimeric cytokine receptors (including IL-4R and IL-15R).

Chimeric antigen receptor NK cells (CAR-NK cells)

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to a CAR-NK cell of the invention. The CAR-NK cells of the invention can target tumor surface antigens (e.g., NKG2D ligands) for the treatment of tumors that are highly expressed or positive for IL-4, particularly solid tumors.

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

CAR-NK cells also have the following advantages compared to autologous CAR-T cells, for example: (1) directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) they release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) is easy to be amplified in vitro and can be developed into ready-made products. Otherwise, similar to CAR-T cell therapy.

Exogenous T cell antigen receptor

as used herein, a foreign T cell antigen receptor (TCR) is a TCR that is exogenously transferred into a T cell by means of genetic engineering, using lentivirus or retrovirus as a vector, by cloning the α chain and β chain of the TCR from a tumor-reactive T cell by gene transfer technique.

The exogenous TCR modified T cell can specifically recognize and kill tumor cells, and affinity of the T cell and tumor can be improved and anti-tumor effect can be improved by optimizing affinity of TCR and tumor specific antigen.

Carrier

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

The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

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

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

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

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

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

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

Another example of a suitable promoter is the extended growth factor-1 α (EF-1 α). however, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40(SV40) early promoter, mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.

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

A suitable reporter gene may include a gene encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). suitable expression systems are well known and may be prepared using known techniques or obtained commercially.typically, a construct with a minimum of 5 flanking regions that exhibits the highest level of reporter gene expression is identified as a promoter.

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

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

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

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

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

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

Preparation

The invention provides an engineered immune cell according to the first aspect of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³-1×10⁸One cell/Kg body weight, more preferably 1X 10⁴-1×10⁷One cell/Kg body weight.

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

Therapeutic applications

The invention includes therapeutic applications of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target marker (such as NKG2D ligand) proteins of tumor cells, synergistically activate the T cells, cause cellular immune response, reverse the inhibition effect of IL-4 on engineered immune cells in an immunosuppression microenvironment, and thus remarkably improve the killing efficiency of the T cells on tumor cells with high expression of IL-4.

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

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

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells that are markers of tumor cells (such as NKG2D ligand) elicit a specific immune response against cells that express markers of tumor cells (such as NKG2D ligand).

Although the data disclosed herein specifically disclose lentiviral vectors comprising an antigen binding domain targeting a tumor cell surface antigen, a hinge and transmembrane region, and 4-1BB and CD3 zeta signaling domains, T2A, chimeric cytokine receptors (including IL-4R and IL-15R), the invention should be construed to include any number of variations on each of the construct components.

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

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

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include liver cancer, pancreatic cancer, fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

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

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

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

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

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

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

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

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

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

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

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Usually, each treatment or eachFor one course of treatment, 1 × 10⁶1 to 10¹⁰A subject modified T cell (e.g., a CAR-T cell of the subject invention) is administered to a patient, for example, by intravenous infusion.

Fusion proteins

As used herein, the terms "fusion protein", "fusion protein of the invention", and "polypeptide of the invention" have the same meaning and all have the structure described in the seventh aspect of the invention.

The term "fusion protein" as used herein also includes variants of the sequence of SEQ ID No. 7 having the above-described activity. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which a polypeptide is fused with another compound (such as a compound for increasing the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused with the polypeptide sequence (a fusion protein in which a tag sequence such as a leader sequence, a secretory sequence or 6His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of the present invention. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

the analogs can differ from the polypeptide set forth in SEQ ID No. 7 by amino acid sequence differences, by modifications that do not affect the sequence, or by both.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In one embodiment of the invention, the amino acid sequence of the fusion protein is shown in SEQ ID No. 7.

Coding sequence

The invention also relates to polynucleotides encoding the fusion proteins according to the invention.

The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence encoding the polypeptide set forth in SEQ ID No. 7 or a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a polypeptide having the sequence shown in SEQ ID No. 7, but differ in the sequence of the corresponding coding region.

The full-length nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the polypeptides of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, and to genetically engineered host cells with the vector or polypeptide coding sequences of the invention. The polynucleotide, vector or host cell may be isolated.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polynucleotide encoding the fusion protein of the invention.

The full-length nucleotide sequence encoding the fusion protein of the present invention or a fragment thereof can be obtained by PCR amplification, recombinant methods, or synthetic methods. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

In one embodiment of the invention, the polynucleotide sequence encoding the fusion protein is shown in SEQ ID No. 8.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The invention also relates to a vector comprising the polynucleotide of the invention, as well as a genetically engineered host cell with the vector or protein coding sequence of the invention, and a method for expressing the fusion protein of the invention on the NK cells by recombinant techniques.

NK cells expressing the fusion protein of the present invention can be obtained by using the polynucleotide sequence of the present invention by a conventional recombinant DNA technique. Generally comprising the steps of: transducing the first expression cassette and/or the second expression cassette according to the invention into an NK cell, thereby obtaining said NK cell.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the fusion proteins of the invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells of the genera escherichia coli, bacillus subtilis, streptomyces; fungal cells such as pichia, saccharomyces cerevisiae cells; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, NS0, COS7, or 293 cells. In a preferred embodiment of the invention, the NK cell is selected as a host cell.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The main advantages of the invention include:

1. the invention discovers for the first time that the chimeric antigen receptor CAR containing the targeted tumor cell surface antigen and the engineered immune cell of the chimeric cytokine receptor can reverse the inhibition effect of the inhibitory cytokine infiltrated in an immunosuppressive microenvironment, and can selectively kill tumor cells, such as tumor cells with high IL-4 expression, and the higher the IL-4 expression level is, the better the killing effect is.

2. The invention discovers for the first time that the killing effect of the engineered immune cells on tumor cells with high expression of inhibitory cytokine IL-4 is enhanced, and the inhibition effect of a tumor microenvironment is overcome.

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

Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.

Experimental Material

1.1 cells, strains and plasmids

293T cell line, Huh-7 cell line, Panc-1 cell line, by the laboratory storage. The lentiviral vector of this study was a three plasmid system. (psPAX2, pMD2.G and pCDH-G2D-CAR2-T2A-IL-4R/IL-15R or pCDH-G2D-CAR 2). psPAX2 is a second generation lentiviral packaging plasmid (packaging vector) in which the gene HIV gag is used to encode the major structural protein of the virus, the gene pol is used to encode a virus-specific enzyme, and the gene REV is used to encode a regulatory factor. pMD2.G is a lentiviral envelope plasmid (envelope vector) containing the envelope protein gene VSV-G used to encode viral packaging.

1.2 blending of major reagents

LB Medium reagents were accurately weighed according to the following Table (Table 1), and 50mL ddH was added₂And dissolving the O. Adjusting the pH value (7.2-7.4), fixing the volume to 100mL, and sterilizing with high-pressure steam (121 ℃, 20 min). Cooled to room temperature and stored at 4 ℃.

TABLE 1 LB liquid Medium ingredient Table (100mL)

LB Amp⁺Solid plate reagents were accurately weighed according to the following table (Table 2) and 50mL ddH was added₂And dissolving the O. Adjusting the pH value (7.2-7.4), fixing the volume to 100mL, and sterilizing with high-pressure steam (121 ℃, 20 min). And cooling the culture medium to about 60 ℃, treating under aseptic conditions, adding Amp to the concentration of 100 mu g/mL, and mixing uniformly. 10mL of the medium was poured into the bacterial culture plate, and the plate was allowed to stand at room temperature. After the culture medium is solidified, the flat plate is sealed by using sealing glue, and the flat plate is inverted and stored at the temperature of 4 ℃.

TABLE 2 LB liquid solid Medium ingredient Table (100ml)

Cell complete medium DMEM complete medium (10%): DMEM cell culture solution + 10% FBS + 1% P/S; X-VIVO complete medium: X-VIVO cell culture medium + 10% Gibco FBS + 1% P/S + 10. mu.g/mL IL-7+10g/mL IL-15+ 10. mu.g/mL IL-21.

PBS buffer reagents were accurately weighed according to the following table (Table 3), and 800mL ddH was added₂And dissolving O, and metering to 1000 mL. Sterilizing with high pressure steam (121 deg.C, 20min), and storing at 4 deg.C.

TABLE 31 XPBS buffer Components Table

Transfection reagent PEI solution Linear polyethyleneimine HCl (polyethyleneimine HCl MAX, Linear, Mw 40000/PEI MAX 40000)100.0mg was weighed and dissolved in 80mL ddH 2O. The pH was adjusted (7.0) to 100 mL. Sterile processing, filtering the solution with 0.22 μm filter membrane, and subpackaging in 15mL centrifuge tubes, and storing at-20 deg.C.

Agarose gel 0.45g agarose was weighed and dissolved in 30mL 1XTAE (1.5%), heated to dissolve, cooled to 50 deg.C, added 5. mu.L EB, mixed well and poured onto a horizontal plate of electrophoresis tank (to prevent air bubbles from forming), and inserted into a spotting comb.

2. Experimental methods

2.1 construction of the plasmid PCDH-NKG2D-CAR2-T2A-IL-4R/IL-15R

2.1.1 obtaining the sequences of NKG2D and the chimeric receptor IL-4R/IL-15R

From the website https:// www.ncbi.nlm.nih.gov/nuccore/AF461811.1 through NCBI database

Obtaining an NKG2D extracellular segment sequence (788-1193): TTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTG (SEQ ID NO. 9)

obtaining a signal peptide sequence of an IL-4R α chain from a website https:// www.ncbi.nlm.nih.gov/nuccore/NM _000418.4 (199-273): ATGGGGTGGCTTTGCTCTGGGCTCCTGTTCCCTGTGAGCTGCCTGGTCCTGCTGCAGGTGGCAAGCTCTGGGAAC (SEQ ID No.: 10);

extracellular region sequence of IL-4R α chain (274-895):

ATGAAGGTCTTGCAGGAGCCCACCTGCGTCTCCGACTACATGAGCATCTCTACTTGCGAGTGGAAGATGAATGGTCCCACCAATTGCAGCACCGAGCTCCGCCTGTTGTACCAGCTGGTTTTTCTGCTCTCCGAAGCCCACACGTGTATCCCTGAGAACAACGGAGGCGCGGGGTGCGTGTGCCACCTGCTCATGGATGACGTGGTCAGTGCGGATAACTATACACTGGACCTGTGGGCTGGGCAGCAGCTGCTGTGGAAGGGCTCCTTCAAGCCCAGCGAGCATGTGAAACCCAGGGCCCCAGGAAACCTGACAGTTCACACCAATGTCTCCGACACTCTGCTGCTGACCTGGAGCAACCCGTATCCCCCTGACAATTACCTGTATAATCATCTCACCTATGCAGTCAACATTTGGAGTGAAAACGACCCGGCAGATTTCAGAATCTATAACGTGACCTACCTAGAACCCTCCCTCCGCATCGCAGCCAGCACCCTGAAGTCTGGGATTTCCTACAGGGCACGGGTGAGGGCCTGGGCTCAGTGCTATAACACCACCTGGAGTGAGTGGAGCCCCAGCACCAAGTGGCACAACTCCTACAGGGAGCCCTTCGAGCAGCAC(SEQ ID NO.:11)

the transmembrane region sequence of the IL-15R β chain obtained from the website https:// www.ncbi.nlm.nih.gov/nuccore/NM _000878.5 is (846-893): ATTCCGTGGCTCGGCCACCTCCTCGTGGGCCTCAGCGGGGCTTTTGC (SEQ ID NO.: 12);

the intracellular sequence of the IL-15 R.beta.chain is (894-1781):

TTCATCATCTTAGTGTACTTGCTGATCAACTGCAGGAACACCGGGCCATGGCTGAAGAAGGTCCTGAAGTGTAACACCCCAGACCCCTCGAAGTTCTTTTCCCAGCTGAGCTCAGAGCATGGAGGAGACGTCCAGAAGTGGCTCTCTTCGCCCTTCCCCTCATCGTCCTTCAGCCCTGGCGGCCTGGCACCTGAGATCTCGCCACTAGAAGTGCTGGAGAGGGACAAGGTGACGCAGCTGCTCCTGCAGCAGGACAAGGTGCCTGAGCCCGCATCCTTAAGCAGCAACCACTCGCTGACCAGCTGCTTCACCAACCAGGGTTACTTCTTCTTCCACCTCCCGGATGCCTTGGAGATAGAGGCCTGCCAGGTGTACTTTACTTACGACCCCTACTCAGAGGAAGACCCTGATGAGGGTGTGGCCGGGGCACCCACAGGGTCTTCCCCCCAACCCCTGCAGCCTCTGTCAGGGGAGGACGACGCCTACTGCACCTTCCCCTCCAGGGATGACCTGCTGCTCTTCTCCCCCAGTCTCCTCGGTGGCCCCAGCCCCCCAAGCACTGCCCCTGGGGGCAGTGGGGCCGGTGAAGAGAGGATGCCCCCTTCTTTGCAAGAAAGAGTCCCCAGAGACTGGGACCCCCAGCCCCTGGGGCCTCCCACCCCAGGAGTCCCAGACCTGGTGGATTTTCAGCCACCCCCTGAGCTGGTGCTGCGAGAGGCTGGGGAGGAGGTCCCTGACGCTGGCCCCAGGGAGGGAGTCAGTTTCCCCTGGTCCAGGCCTCCTGGGCAGGGGGAGTTCAGGGCCCTTAATGCTCGCCTGCCCCTGAACACTGATGCCTACTTGTCCCTCCAAGAACTCCAGGGTCAGGACCCAACTCACTTGGTGTAG(SEQ ID NO.:13)

2.1.2 construction of the PCDH-NKG2D-CAR2-T2A-IL-4/IL-15R vector

After finding out the sequences of NKG2D and IL-4R/IL-15R, artificially synthesizing an NKG2D-CAR-IL4R/IL15R sequence and inserting the sequence between the multiple cloning sites Xba I and Not I of the vector plasmid PCDH to construct a PCDH-NKG2D-CAR2-T2A-IL-4/IL-15R recombinant expression plasmid.

2.1.3 restriction enzyme identification two single restriction sites were selected after synthesis of recombinant plasmid: xba I, Not I, prepared in the following Table (Table 4) 20. mu.l of the reaction system was digested. After mixing evenly, the mixture is put into water bath with 37 ℃ for 1 to 2 hours to react.

TABLE 4 digestion system

2.1.3 agarose gel electrophoresis detection electrophoresis tank and 1xTAE buffer solution to the gel, the sample application hole is placed in the negative pole, in two holes respectively sample 5 u L sample and 1 u L6 x loading buffer mixture and 6 u L DL10000 DNA marker. After spotting, 120V electrophoresis is carried out for 40min, and ultraviolet photography is carried out for inspection.

2.1.4 plasmid is dipped into the pipette tip and taken out, and then added into 100 mul of competent cells, the operation processes of ice box freezing for 30min, water bath heating at 42 ℃ for 1.5min and ice box freezing for 5min are respectively carried out, the pipette tip is added into an LB culture medium without resistance to be shaken for recovery for 45min, 200 mul of coating plates are selected, inverted culture is carried out after the pipette tip is positively arranged in an incubator at 37 ℃ for 10min, and the colony group growing on the plates is converted successfully.

2.1.5 selecting a colony from the proliferation and amplification culture plate of the strain, adding 5mL of LB culture medium and 100 mu g/mL of ampicillin, shaking for 8h at 200r/min, adding 250mL of LB, and shaking for 12-16h to finally obtain the strain liquid. 25% glycerol was added and stored (-80 ℃).

2.1.6 Small extract of Plasmid, big extract of Plasmid Small extract using small extract Kit (TIANPrep Mini Plasmid Kit);

plasmid Large-Scale extraction an endotoxin-free Plasmid Large-Scale extraction Kit (Endofree Maxi Plasmid Kit) was used. The eluent is 65 ℃ endotoxin-free water, and 1.5mL of eluent is used for eluting and storing plasmids. The concentration of the extracted plasmid was measured and stored at-20 ℃.

3 cell culture

3.1 passage cell 293T cell is adherent cell, cultured in 10% DMEM medium. T cells were suspension cells, cultured in complete x-vivo medium. The Huh-7, Panc-1 cancer cell line was cultured with serum concentration 10% DMEM. Passaging cells involved the process of discarding the original medium, PBS wash, pancreatin digestion, DMEM stop digestion, centrifugation (1200rpm, 3min) for resuspension, and replating. All cells were incubated at 37 ℃ in 5% CO₂Culturing in an incubator.

3.2 frozen cells are prepared into frozen stock solution according to the proportion of 10 percent DMSO, 10 percent serum and 80 percent DMEM, the cells are suspended by the frozen stock solution after centrifugation, and are subpackaged, 1mL of the frozen stock solution is put into an ice box at the temperature of minus 80 ℃ for freezing, or the frozen stock solution is frozen by gradient cooling (firstly respectively put at the temperature of minus 4 ℃ for 30min and minus 20 ℃ for 40min for treatment, and finally stored at the temperature of minus 80 ℃).

3.3 resuscitating the cells, taking out the frozen cells, rapidly shaking and thawing the frozen cells in a water bath at 37 ℃, adding 7-8 mL of PBS, centrifuging at 1200rpm for 3min, washing once with PBS, resuspending the cells with 10% serum in DMEM, and then subpackaging the cells in a culture dish.

4 preparing two lentiviruses of LV-NKG2D, LV-NKG2D-IL4R/IL15R

Dilution of the three plasmids (psPAX2, pMD2.G and PCDH-G2D): serum-free DMEM was added to the three plasmid system to dilute to 500. mu.L, gently pipetted and mixed. Dilution of transfection reagent PEI: serum-free DMEM was added to the transfection reagent PEI and diluted to 500. mu.L, gently pipetted and mixed, and incubated at room temperature for 5 min. Preparation of three plasmid-PEI mixtures: dripping the incubated PEI diluent into the diluted three plasmids, lightly blowing and uniformly mixing, and incubating for 15min at room temperature to form a three plasmid-PEI mixed solution. Uniformly dripping the mixed solution of the three plasmids and the PEI into 293T cells cultured in advance, and placing the cells at 37 ℃ and 5 percent CO₂Culturing in an incubator. After liquid change, 48h and 72h of transfection, the culture supernatant of 293T cells was collected and supplemented with 5mL of 10% DMEM medium. The two lentiviruses are designated LV-NKG2D, LV-NKG2D-IL4R/IL-15R, respectively.

For measurement of viral titer, 293T cells were collected by resuspension, counted, and then counted using a counter (10-fold dilution, 500. mu.l system) or a counter (10-fold dilution, injected into wells of a counter, and counted as 4 cells/4X 10⁵). Approximately 2X 10 addition of 293T cells per well in 24-well plates⁵The first well is used as control, and then 30, 50 and 100 μ L of each virus is added into the subsequent wells, if the virus concentrate is added with 0.1, 0.5 and 1 μ L, the infection system is supplemented with 500 μ L, and the infection lasts for 48-72 h. After infection, 293T cells were digested, washed with PBS, resuspended, and 0.1. mu.L of antibody (APC-NKG2D HUMAN) was added in the dark, incubated at 4 ℃ for 30min, and control 293T cells were not treated with antibody. After staining, PBS was washed, resuspended, transferred to flow tube, and analyzed on flow cytometer. Finally, the virus titer was calculated from the assay results: titer ═ 2 × 10⁵X Positive Rate)/Virus volume TU/mL

Lentiviral infection of T cells

The concentrated virus infects T cells to prepare CAR-T cells. Stimulating and culturing T cells, closely observing, and treating infection when the growth state is good, the number of the cells is large, the cells are agglomerated in large blocks, the shape is mellow, and the cell fragments are few. T cells were counted and plated in 12-well plates at 1X 10 per well⁶Each cell was added with virus at a MOI of 10 (MOI TU/1 × 10)⁶) Making 1-2 multiple wells for each virus, setting 1 well as a control group, adding no virus, and supplementing the X-VIVO culture medium to 1 mL. After the infection system is added, the mixture is sealed, centrifuged at 1800rpm for 1h at 32 ℃, and cultured in an incubator for 12-18 h, and then the solution is changed. And (3) detecting the positive rate of the CAR-T cells in a flow mode after 24-48 h.

6 CAR-T killing tumor cells

After the CAR-T cells are prepared, target cells Huh-7 and Panc-1 are killed according to the effective target ratio of 1:1, 3:1 and 9:1 respectively, and the killing time is 14 h.

The target cell treatment method comprises the following steps: digestion, PBS washing, heavy suspension, appropriate amount of cells with 1mL PBS heavy suspension, adding apoptosis antibody CFSE 1 u l (light treatment), 37 degrees C under the conditions of staining 15min (blow the cell so that it does not precipitate in the bottom). Supplementing appropriate amount of 10% DMEM medium, standing at 4 deg.C for 20min, washing with PBS 3 times, resuspending the cell pellet with X-VIVO medium, and counting. CAR-T cell treatment method: after centrifugation, the X-VIVO was resuspended and counted. In 96-well low adsorption plates, 3X 10 of the total amount was added per well⁴And (3) adding the T cells and the two CAR-T cells into the target cells according to the effective target ratio of 1:1, 3:1 and 9:1 respectively, preparing a group of BLANK only adding the target cells, supplementing X-VIVO culture medium to 200 mu L per well, and ensuring that the final system volume of all the multiple wells is the same. And blowing and beating the uniform cells, putting the cells in an incubator at 37 ℃ for killing and culturing for 14h, and then carrying out flow detection. Flow detection treatment: centrifuging the cells after the killing experiment is finished, resuspending the cells by 100 mu Lannexin V Binding Buffer, adding 0.3 mu L Annexin V antibody (processing in a dark place), dyeing for 15min, adding PI dyeing solution, and detecting the killing efficiency by a flow cytometer after mixing uniformly.

EXAMPLE 1 construction of plasmid PCDH-G2D-CAR2-T2A-IL-4R/IL-15R

1.1 construction of plasmids

A recombinant plasmid of plasmid pCDH-G2D-CAR2-T2A-IL-4R/IL-15R was constructed. Wherein the chimeric receptor IL-4R/IL-15R is linked with NKG2D-CAR by T2A respectively at the front and back. The plasmid map is shown in FIG. 1.

1.2 enzyme digestion identification

Two single enzyme cutting sites Xba I and Not I on the recombinant plasmid are selected to carry out enzyme cutting identification on the NKG2D-IL-4R/IL-15R fragment in the recombinant plasmid, and the result obtains a DNA fragment with the length of 2792bp, which indicates that the recombinant plasmid is successfully constructed, and is shown in figure 2.

Example 2 preparation of CAR-T cells by Lentiviral infection of T cells

Two lentiviruses, LV-NKG2D, LV-NKG2D-IL4R/IL15R, were used to infect T cells (MOI 10) at a cell density of 1X 10⁶one/mL, while uninfected T cells were cultured as controls. After 48h, the infection positive rate is determined by flow cytometry, and the infection efficiency of the two viruses is higher, namely 97.5% and 98.5%, respectively, as shown in figure 3.

Example 3 CAR-T killing Effect assay

HUH7 cells (hepatoma cells) and Panc-2 cells (pancreatic cancer cells) highly expressing IL-4 were used as target cells (3X 10)⁴One), co-cultured with effector cells, two CAR-T cell killing functions were tested. The experiment was divided into four groups: target cell group (blank control group), T cell co-culture group not infected with lentivirus (negative control group), CAR-T cell co-culture group infected with virus LV-NKG2D (experimental group), CAR-T cell co-culture group infected with virus LV-NKG2D-IL4R/I-L15R (experimental group). Effector cells and target cells were co-cultured at an effective-to-target ratio of 1:1, 3:1, 9:1, respectively, with a killing time of 14 h. After killing, the cells are stained (Annexin V-PI double staining) and the killing efficiency is analyzed by a flow cytometry method, and the result is shown in figure 4, the NKG2D-CAR-T cells and the NKG2D-IL-4R/IL-15R-CAR-T cells have obvious killing effect on Huh-7 and Panc-1, but the CAR-T cells co-expressing the chimeric receptor IL-4R/IL-15R have more obvious killing effect on IL-4 high-expression cells Huh-7 and Panc-1, which indicates that the chimeric receptor IL-4R/IL-15R can reverse the inhibitory signal of IL-4 secreted by the tumor cells, so that the killing effect on the tumor cells high-expression IL-4 is enhanced.

Discussion of the related Art

On the basis of the influence of an immunosuppressive microenvironment, the construction of the chimeric receptor structurally carries out one-time updating improvement on the CAR-T cell, so that the double-targeting CAR-T cell can be combined with NKG2D ligand highly expressed by tumor cells and can reverse the inhibition effect of inhibitory cytokines infiltrated in the immunosuppressive microenvironment, and the construction of the chimeric receptor IL-4R/IL-15R is carried out on the basis of the research of the construction of IL-4R/IL-7R. At present, the construction of pCDH-NKG2D-IL4R/IL15R-CAR vector and NKG2D-IL4R/IL15R-CAR-T cell is completed in the research, and the cancer cell lines Huh-7 and Panc-1 with high IL-4 expression are detected and screened by Real Time PCR, the CAR-T cell is killed according to the effective target ratio of 1:1, 3:1, 9:1 and the killing Time of 14h, and finally the killing effect is detected. The experiment initially leads to the conclusion that: after the chimeric receptor IL-4R/IL-15R is added into the CAR-T cell taking the NKG2D ligand as the target, the killing of the liver cancer cell (HUH-7) and the pancreatic cancer cell (Panc-1) with high IL-4 expression is obviously enhanced. The co-expression of the chimeric receptor IL-4R/IL-15R can reverse the inhibition effect of IL-4 on NKG2D-CAR-T in an immunosuppressive microenvironment, thereby enhancing the killing of NKG2D-CAR-T on tumor cells and improving the performance of CAR-T cells.

The application of CAR-T cells has profound significance in the treatment of tumors, and the functions of the CAR-T cells are enhanced along with the algebraic renewal of the CAR-T cells due to the improved structural design of the CAR-T cells. Future improvements to various aspects of CAR-T design will be a focus of research and a breakthrough in its function. The study focused on breaking through the tumor-inhibiting microenvironment, thereby improving the killing efficiency of CAR-T cells.

Sequence listing

<110> university of east China

Shanghai Yao Biotech Co Ltd

<120> preparation and application of CAR-T cell for overcoming tumor microenvironment

<130>P2019-2330

<160>13

<170>SIPOSequenceListing 1.0

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Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly

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Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe

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Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser

35 40 45

Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu

50 55 60

Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro

65 70 75 80

Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn

85 90 95

Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala

100 105 110

Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr

115 120 125

Ile Cys Met Gln Arg Thr Val

130 135

<210>2

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<212>DNA

<213> Intelligent (Homo sapiens)

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ttattcaacc aagaagttca aattcccttg accgaaagtt actgtggccc atgtcctaaa 60

aactggatat gttacaaaaa taactgctac caattttttg atgagagtaa aaactggtat 120

gagagccagg cttcttgtat gtctcaaaat gccagccttc tgaaagtata cagcaaagag 180

gaccaggatt tacttaaact ggtgaagtca tatcattgga tgggactagt acacattcca 240

acaaatggat cttggcagtg ggaagatggc tccattctct cacccaacct actaacaata 300

attgaaatgc agaagggaga ctgtgcactc tatgcctcga gctttaaagg ctatatagaa 360

aactgttcaa ctccaaatac atacatctgc atgcaaagga ctgtg 405

<210>3

<211>379

<212>PRT

<213> Artificial sequence (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 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr

20 25 30

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

35 40 45

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

50 55 60

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

65 70 75 80

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly

85 90 95

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

100 105 110

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

115 120 125

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser

130 135 140

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

370 375

<210>4

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<213> Artificial sequence (artificial sequence)

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Met Gly Trp Leu Cys Ser GlyLeu Leu Phe Pro Val Ser Cys Leu Val

1 5 10 15

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

20 25 30

Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met

35 40 45

Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu

50 55 60

Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly

65 70 75 80

Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala

85 90 95

Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys

100 105 110

Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn

115 120 125

Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser

130 135 140

Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala

145 150 155 160

Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn

165 170 175

Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys

180 185 190

Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr

195 200 205

Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser

210 215 220

Tyr Arg Glu Pro Phe Glu Gln His

225 230

<210>5

<211>311

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>5

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

1 5 10 15

Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro

20 25 30

Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe

35 40 45

Phe Ser Gln Leu Ser Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu

50 55 60

Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro

65 70 75 80

Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu

85 90 95

Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn

100 105 110

His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His

115 120 125

Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr Tyr

130 135 140

Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val Ala Gly Ala Pro

145 150 155 160

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

165 170 175

Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro

180 185 190

Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser

195 200 205

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

210 215 220

Arg Asp Trp Asp Pro Gln Pro LeuGly Pro Pro Thr Pro Gly Val Pro

225 230 235 240

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

245 250 255

Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro

260 265 270

Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg

275 280 285

Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly

290 295 300

Gln Asp Pro Thr His Leu Val

305 310

<210>6

<211>543

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>6

Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val

1 5 10 15

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

20 25 30

Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met

35 40 45

Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu

50 55 60

Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly

65 70 75 80

Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala

85 90 95

Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys

100 105 110

Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn

115 120 125

Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser

130 135 140

Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala

145 150 155 160

Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn

165 170 175

Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys

180 185 190

Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr

195 200 205

Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser

210 215 220

Tyr Arg Glu Pro Phe Glu Gln His Ile Pro Trp Leu Gly His Leu Leu

225 230 235 240

Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu

245 250 255

Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys

260 265 270

Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His

275 280 285

Gly Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser

290 295 300

Phe Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu

305 310 315 320

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

325 330 335

Glu Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr

340 345 350

Asn Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu

355 360 365

Ala Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro

370 375 380

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

385 390 395 400

Gln Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg

405 410 415

Asp Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro

420 425 430

Pro Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro

435 440 445

Pro Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu

450 455 460

Gly Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro

465 470 475 480

Pro Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly

485 490 495

Pro Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly

500 505 510

Glu Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr

515 520 525

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

530 535 540

<210>7

<211>942

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>7

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 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr

20 25 30

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

35 40 45

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

50 55 60

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

65 70 75 80

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly

85 90 95

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

100 105 110

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

115 120125

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser

130 135 140

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

275 280285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val Leu

405 410 415

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

420 425 430

Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met Asn

435 440 445

Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu Val

450 455 460

Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly Gly

465 470 475 480

Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala Asp

485 490 495

Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys Gly

500 505 510

Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn Leu

515 520 525

Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser Asn

530 535 540

Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala Val

545 550 555 560

Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn Val

565 570 575

Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys Ser

580 585 590

Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr Asn

595 600 605

Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser Tyr

610 615 620

Arg Glu Pro Phe Glu Gln His Ile Pro Trp Leu Gly His Leu Leu Val

625 630 635 640

Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile

645 650 655

Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn

660 665 670

Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly

675 680 685

Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe

690 695 700

Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu

705 710 715 720

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

725 730 735

Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn

740 745 750

Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala

755 760 765

Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp

770 775 780

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

785 790 795 800

Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp

805 810 815

Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro

820 825 830

Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro

835 840 845

Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly

850 855 860

Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro

865 870 875 880

Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro

885 890 895

Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu

900 905 910

Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu

915 920 925

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

930 935 940

<210>8

<211>2826

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>8

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgttattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 120

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 180

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 240

gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 300

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 360

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 420

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtgac cacgacgcca 480

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcac agcccctgtc cctgcgccca 540

gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 600

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 660

atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 720

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 780

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac840

aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 900

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 960

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1020

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1080

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcgga 1140

tccgagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctatg 1200

gggtggcttt gctctgggct cctgttccct gtgagctgcc tggtcctgct gcaggtggca 1260

agctctggga acatgaaggt cttgcaggag cccacctgcg tctccgacta catgagcatc 1320

tctacttgcg agtggaagat gaatggtccc accaattgca gcaccgagct ccgcctgttg 1380

taccagctgg tttttctgct ctccgaagcc cacacgtgta tccctgagaa caacggaggc 1440

gcggggtgcg tgtgccacct gctcatggat gacgtggtca gtgcggataa ctatacactg 1500

gacctgtggg ctgggcagca gctgctgtgg aagggctcct tcaagcccag cgagcatgtg 1560

aaacccaggg ccccaggaaa cctgacagtt cacaccaatg tctccgacac tctgctgctg 1620

acctggagca acccgtatcc ccctgacaat tacctgtata atcatctcac ctatgcagtc 1680

aacatttgga gtgaaaacga cccggcagat ttcagaatct ataacgtgac ctacctagaa 1740

ccctccctcc gcatcgcagc cagcaccctg aagtctggga tttcctacag ggcacgggtg 1800

agggcctggg ctcagtgcta taacaccacc tggagtgagt ggagccccag caccaagtgg 1860

cacaactcct acagggagcc cttcgagcag cacattccgt ggctcggcca cctcctcgtg 1920

ggcctcagcg gggcttttgg cttcatcatc ttagtgtact tgctgatcaa ctgcaggaac 1980

accgggccat ggctgaagaa ggtcctgaag tgtaacaccc cagacccctc gaagttcttt 2040

tcccagctga gctcagagca tggaggagac gtccagaagt ggctctcttc gcccttcccc 2100

tcatcgtcct tcagccctgg cggcctggca cctgagatct cgccactaga agtgctggag 2160

agggacaagg tgacgcagct gctcctgcag caggacaagg tgcctgagcc cgcatcctta 2220

agcagcaacc actcgctgac cagctgcttc accaaccagg gttacttctt cttccacctc 2280

ccggatgcct tggagataga ggcctgccag gtgtacttta cttacgaccc ctactcagag 2340

gaagaccctg atgagggtgt ggccggggca cccacagggt cttcccccca acccctgcag 2400

cctctgtcag gggaggacga cgcctactgc accttcccct ccagggatga cctgctgctc 2460

ttctccccca gtctcctcgg tggccccagc cccccaagca ctgcccctgg gggcagtggg 2520

gccggtgaag agaggatgcc cccttctttg caagaaagag tccccagaga ctgggacccc 2580

cagcccctgg ggcctcccac cccaggagtc ccagacctgg tggattttca gccaccccct 2640

gagctggtgc tgcgagaggc tggggaggag gtccctgacg ctggccccag ggagggagtc 2700

agtttcccct ggtccaggcc tcctgggcag ggggagttca gggcccttaa tgctcgcctg 2760

cccctgaaca ctgatgccta cttgtccctc caagaactcc agggtcagga cccaactcac 2820

ttggtg 2826

<210>9

<211>405

<212>DNA

<213> Intelligent (Homo sapiens)

<400>9

ttattcaacc aagaagttca aattcccttg accgaaagtt actgtggccc atgtcctaaa 60

aactggatat gttacaaaaa taactgctac caattttttg atgagagtaa aaactggtat 120

gagagccagg cttcttgtat gtctcaaaat gccagccttc tgaaagtata cagcaaagag 180

gaccaggatt tacttaaact ggtgaagtca tatcattgga tgggactagt acacattcca 240

acaaatggat cttggcagtg ggaagatggc tccattctct cacccaacct actaacaata 300

attgaaatgc agaagggaga ctgtgcactc tatgcctcga gctttaaagg ctatatagaa 360

aactgttcaa ctccaaatac atacatctgc atgcaaagga ctgtg 405

<210>10

<211>75

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>10

atggggtggc tttgctctgg gctcctgttc cctgtgagct gcctggtcct gctgcaggtg 60

gcaagctctg ggaac 75

<210>11

<211>621

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>11

atgaaggtct tgcaggagcc cacctgcgtc tccgactaca tgagcatctc tacttgcgag 60

tggaagatga atggtcccac caattgcagc accgagctcc gcctgttgta ccagctggtt 120

tttctgctct ccgaagccca cacgtgtatc cctgagaaca acggaggcgc ggggtgcgtg 180

tgccacctgc tcatggatga cgtggtcagt gcggataact atacactgga cctgtgggct 240

gggcagcagc tgctgtggaa gggctccttc aagcccagcg agcatgtgaa acccagggcc 300

ccaggaaacc tgacagttca caccaatgtc tccgacactc tgctgctgac ctggagcaac 360

ccgtatcccc ctgacaatta cctgtataat catctcacct atgcagtcaa catttggagt 420

gaaaacgacc cggcagattt cagaatctat aacgtgacct acctagaacc ctccctccgc 480

atcgcagcca gcaccctgaa gtctgggatt tcctacaggg cacgggtgag ggcctgggct 540

cagtgctata acaccacctg gagtgagtgg agccccagca ccaagtggca caactcctac 600

agggagccct tcgagcagca c 621

<210>12

<211>47

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>12

attccgtggc tcggccacct cctcgtgggc ctcagcgggg cttttgc 47

<210>13

<211>888

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>13

ttcatcatct tagtgtactt gctgatcaac tgcaggaaca ccgggccatg gctgaagaag 60

gtcctgaagt gtaacacccc agacccctcg aagttctttt cccagctgag ctcagagcat 120

ggaggagacg tccagaagtg gctctcttcg cccttcccct catcgtcctt cagccctggc 180

ggcctggcac ctgagatctc gccactagaa gtgctggaga gggacaaggt gacgcagctg 240

ctcctgcagc aggacaaggt gcctgagccc gcatccttaa gcagcaacca ctcgctgacc 300

agctgcttca ccaaccaggg ttacttcttc ttccacctcc cggatgcctt ggagatagag 360

gcctgccagg tgtactttac ttacgacccc tactcagagg aagaccctga tgagggtgtg 420

gccggggcac ccacagggtc ttccccccaa cccctgcagc ctctgtcagg ggaggacgac 480

gcctactgca ccttcccctc cagggatgac ctgctgctct tctcccccag tctcctcggt 540

ggccccagcc ccccaagcac tgcccctggg ggcagtgggg ccggtgaaga gaggatgccc 600

ccttctttgc aagaaagagt ccccagagac tgggaccccc agcccctggg gcctcccacc 660

ccaggagtcc cagacctggt ggattttcag ccaccccctg agctggtgct gcgagaggct 720

ggggaggagg tccctgacgc tggccccagg gagggagtca gtttcccctg gtccaggcct 780

cctgggcagg gggagttcag ggcccttaat gctcgcctgc ccctgaacac tgatgcctac 840

ttgtccctcc aagaactcca gggtcaggac ccaactcact tggtgtag 888

Claims

1. An engineered immune cell expressing a chimeric antigen receptor CAR that targets a tumor cell surface antigen and a chimeric cytokine receptor comprising IL-4R and IL-15R.

2. The engineered immune cell of claim 1, wherein said tumor cell surface antigen comprises an NKG2D ligand.

3. A method of making the engineered immune cell of claim 1, comprising the steps of:

(A) providing an immune cell to be modified; and

(B) engineering the immune cell such that the immune cell expresses a chimeric antigen receptor CAR that targets a tumor cell surface antigen and a chimeric cytokine receptor comprising IL-4R and IL-15R, thereby obtaining the engineered immune cell of claim 1.

4. A formulation comprising the engineered immune cell of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

5. Use of the engineered immune cell according to claim 1 for the preparation of a medicament or formulation for selective killing of tumors.

6. A kit for preparing a composition for selective killing of tumors, the kit comprising a container, and, within the container:

7. A method of selectively killing a tumor, comprising:

administering to a subject in need thereof a safe and effective amount of the engineered immune cell of claim 1, or the formulation of claim 4.

8. A fusion protein comprising a chimeric antigen receptor CAR that targets a tumor cell surface antigen and a chimeric cytokine receptor comprising IL-4R and IL-15R.

9. A polynucleotide encoding the fusion protein of claim 8.

10. A vector comprising the polynucleotide of claim 9.