WO2024051641A1

WO2024051641A1 - Anti-egfr and cmet bispecific chimeric antigen receptor and use thereof

Info

Publication number: WO2024051641A1
Application number: PCT/CN2023/116708
Authority: WO
Inventors: 高明明; 沈浩; 靳利远; 张伟伟
Original assignee: 复星凯特生物科技有限公司
Priority date: 2022-09-09
Filing date: 2023-09-04
Publication date: 2024-03-14
Also published as: CN117305248A

Abstract

The present invention provides an anti-EGFR and cMet bispecific chimeric antigen receptor and use thereof. Specifically, the present invention provides an immune cell expressing a first CAR and a second CAR, the first CAR targets a first tumor cell marker, and the second CAR targets a second tumor cell marker. The first CAR and the second CAR of the present invention recognize the corresponding tumor cell markers at the same time, so that the immune cell can be fully activated to enhance the targeting specificity and exert the anti-tumor effect, and the on-target/off-tumor toxicity is reduced, thereby improving the safety of cell therapy.

Description

Anti-EGFR and cMet bispecific chimeric antigen receptors and their applications

Technical field

The invention belongs to the field of biotechnology. In particular, the present invention relates to anti-EGFR and cMet bispecific chimeric antigen receptors and their uses.

Background technique

T cells genetically engineered to express chimeric antigen receptors (CAR) have been shown to have a therapeutic effect on patients with certain B-cell leukemias or lymphoma subtypes, and have also shown potential efficacy in patients with multiple myeloma. Currently, three CAR T drugs have been approved, namely Yescarta for the treatment of large B-cell lymphoma and follicular lymphoma, Tecartus for the treatment of mantle cell lymphoma, and Kymriah for the treatment of large B-cell lymphoma and Acute lymphoblastic leukemia in children and young adults. CAR molecules include three main structural components, namely the extracellular antigen-binding functional domain; the transmembrane domain to transmit antigen recognition signals; and the intracellular signaling domain. The first generation of CAR only contains CD3ζ activation signal. Due to the lack of costimulatory signal functional domain, it cannot fully activate CAR T cells, shows limited cytotoxicity, and cannot effectively expand or persist in the body. The second-generation CAR also introduced the costimulatory functional domain of CD28 or 4-1BB, which enhanced the cytotoxicity of CAR T and improved its persistence. Currently, all CAR T cell products on the market adopt second-generation designs. By adding another costimulatory domain, such as OX40, ICOS, CD27, etc., the third generation CAR further expands on the second generation. Although it is reported to have stronger persistence, it may induce excessive release of cytokines. At present, its clinical efficacy has not been verified. In order to expand the efficacy of CAR T and apply it to a wider range of malignant tumors, especially solid tumors, innovative engineering construction of CAR T is needed.

Various adverse reactions may occur during CAR T cell therapy, such as cytokine release syndrome (CRS), neurological toxicity, on-target off-tumor toxicity, and tumor lysis syndrome. levy. Among them, CRS is systemic and can affect various organs of the body. It is the most frequent and symptomatic acute adverse reaction. Effective clinical management and intervention of CRS is required. As tumor-related antigens are highly expressed in tumor cells, they are also expressed at non-specific low levels in normal tissues or cells, such as CD19, Her2, ROR1, Muc1, etc. Target toxicity means that CAR T cells are activated by recognizing antigens expressed by normal tissues and cause damage to normal tissues. CAR T treats CD19-positive malignant lymphoma because there are CD19-expressing B cell progenitor cells in the bone marrow. cells, which are eliminated together with tumor cells, resulting in B cell dysplasia and reduced hemoglobin. Clinically, immunoglobulin is infused to replace the antibody function produced by B cells. In a clinical trial of CAR T cells targeting carbonic anhydrase IX to treat renal cancer, several patients developed liver enzyme abnormalities. These adverse reactions were attributed to CAR T cell infiltration and action on carbonic anhydrase-expressing cells. Bile duct epithelium. A patient with colorectal cancer developed fatal lung damage after receiving an infusion of Her2-targeting CAR T cells because lung epithelial cells also expressed low levels of Her2. In addition, similar pulmonary edema toxicity also occurred in clinical trials of EGFRvIII-targeted CAR T therapy for glioma. Since single-targeting CAR T cells may cause target toxicity, it is currently difficult to accurately judge through in vitro screening methods such as IHC or tissue chips.

Therefore, there is an urgent need in this field to develop a new type of chimeric antigen receptor T cell, which can recognize multiple antigens at the same time, enhance targeting specificity, exert anti-tumor efficacy, and can reduce on-target/off-tumor toxicity and improve cell therapy. security.

Contents of the invention

The purpose of the present invention is to provide a new type of chimeric antigen receptor T cell that can recognize multiple antigens at the same time, enhance targeting specificity, exert anti-tumor efficacy, and reduce on-target/off-tumor (on target/off-tumor) off tumor) toxicity and improve the safety of cell therapy.

A first aspect of the present invention provides an engineered immune cell, the engineered immune cell expresses a first CAR and a second CAR, the first CAR targets a first tumor cell marker, and the third Two CARs target a second tumor cell marker, and the first tumor cell marker is selected from the following group: cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47, or a combination thereof; the second tumor cell The marker is selected from the group consisting of: EGFR, EpCAM, Her2, Her3, or a combination thereof.

In another preferred embodiment, the immune cell contains a first CAR and a second CAR.

In another preferred embodiment, the immune cells are NK cells, macrophages or T cells, preferably T cells.

In another preferred embodiment, the first CAR and the second CAR are located on the cell membrane of the immune cell.

In another preferred embodiment, the first CAR and the second CAR contain antigen-binding domains targeting tumor cell markers.

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

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

In another preferred example, the structure of the first CAR is shown in Formula I:
L1-S1-H1-TM1-C1-CD3ζ (I)

In the formula, the "-" is a connecting peptide or peptide bond;

L1 is none or the first signal peptide sequence;

S1 is an antigen-binding domain targeting a first tumor cell marker selected from the group consisting of: cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47, or a combination thereof;

H1 is none or the first hinge area;

TM1 is the first transmembrane domain;

C1 is no or the first costimulatory signal molecule;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

In another preferred embodiment, the antigen-binding domain targeting the first tumor cell marker includes an antibody single-chain variable region sequence targeting the tumor cell marker.

In another preferred embodiment, the structure of the single-chain variable region sequence of the antibody targeting the first tumor cell marker is shown in Formula A1 or A2:
V _H1 -V _L1 (A1); or
V _L1 -V _H1 (A2);

Wherein, V _L1 is the light chain variable region of the anti-first tumor cell marker antibody; V _H1 is the heavy chain variable region of the anti-first tumor cell marker antibody; "-" is the connecting peptide (or flexible linker) or Peptide bonds.

In another preferred embodiment, the V _L1 and V _H1 are connected through a flexible joint.

In another preferred embodiment, the flexible linker is 1-5 (preferably, 2-4) consecutive sequences represented by GGGGS.

In another preferred embodiment, the amino acid sequence of the flexible linker is shown in positions 120-134 of SEQ ID NO.: 1.

In another preferred embodiment, the amino acid sequence of V _L1 is shown at positions 135-247 of SEQ ID NO.: 1, and the amino acid sequence of V _H1 is shown at positions 1-119 of SEQ ID NO.: 1.

In another preferred embodiment, the single-chain variable region sequence of the antibody targeting the first tumor cell marker is shown in SEQ ID NO.: 1.

In another preferred embodiment, the amino acid sequence of the first CAR is as shown in any one of SEQ ID NO.: 2-4.

In another preferred example, the structure of the second CAR is shown in Formula II:
L2-S2-H2-TM2-C2-Z2 (II)

In the formula, the "-" is a connecting peptide or peptide bond;

L2 is none or the second signal peptide sequence;

S2 is an antigen-binding domain targeting a second tumor cell marker selected from the group consisting of: EGFR, EpCAM, Her2, Her3, or a combination thereof;

H2 is no or second hinge region;

TM2 is the second transmembrane domain;

C2 is the second costimulatory signal molecule;

Z2 is a cytoplasmic signaling sequence that is absent or derived from CD3ζ.

In another preferred embodiment, the antigen-binding domain targeting the second tumor cell marker includes an antibody single-chain variable region sequence targeting the tumor cell marker.

In another preferred embodiment, the structure of the single-chain variable region sequence of the antibody targeting the second tumor cell marker is shown in Formula A3 or A4:
V _H2 -V _L2 (A3); or
V _L2 -V _H2 (A4);

Wherein, V _L2 is the light chain variable region of the anti-second tumor cell marker antibody; V _H2 is the heavy chain variable region of the anti-second tumor cell marker antibody; "-" is the connecting peptide (or flexible linker) or Peptide bonds.

In another preferred embodiment, the V _L2 and V _H2 are connected through a flexible joint.

In another preferred embodiment, the amino acid sequence of the flexible linker is as shown at positions 120-134 in SEQ ID NO.: 5.

In another preferred embodiment, the amino acid sequence of the flexible linker is as shown at positions 120-134 in SEQ ID NO.: 6.

In another preferred embodiment, the amino acid sequence of V _L2 is shown in positions 135-241 of SEQ ID NO.:5, and the amino acid sequence of V _H2 is shown in positions 1-119 of SEQ ID NO.:5.

In another preferred embodiment, the amino acid sequence of V _L2 is shown at positions 135-241 of SEQ ID NO.:6, and the amino acid sequence of V _H2 is shown at positions 1-119 of SEQ ID NO.:6.

In another preferred embodiment, the single-chain variable region sequence of the antibody targeting the second tumor cell marker is shown in SEQ ID NO.: 5 or 6.

In another preferred embodiment, the amino acid sequence of the second CAR is as shown in any one of SEQ ID NO.: 7-14.

In another preferred embodiment, the single-chain variable region of the antibody targeting the first or second tumor cell marker The sequences are murine, human, human and murine chimeric, or fully humanized single-chain antibody variable region fragments.

In another preferred embodiment, each of L1 and L2 is independently a signal peptide selected from the following group of proteins: CD8a, CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

In another preferred embodiment, the L1 is a signal peptide derived from GM-CSF.

In another preferred example, the L2 is a signal peptide derived from CD8a.

In another preferred embodiment, the amino acid sequences of L1 and L2 are independently as shown in SEQ ID NO.: 15 and 16.

In another preferred embodiment, the H1 and H2 are each independently a hinge region of a protein selected from the following group: CD8, Ig (immunoglobulin) hinge, or a combination thereof.

In another preferred example, H1 and H2 are each independently a hinge region derived from CD8.

In another preferred embodiment, the amino acid sequences of H1 and H2 are independently as shown in SEQ ID NO.: 17.

In another preferred example, the TM1 and TM2 are each independently a transmembrane region of a protein selected from the following group: CD8, CD28, CD8a, CD33, CD37, CD8α, CD5, CD16, ICOS, CD9, CD22, CD134 , CD137, CD154, CD19, CD45, CD4, CD3ε, or combinations thereof.

In another preferred example, the TM1 and TM2 are transmembrane regions derived from CD8.

In another preferred example, the TM2 is a transmembrane region derived from CD28.

In another preferred example, the amino acid sequences of TM1 and TM2 are shown in SEQ ID NO.: 18.

In another preferred embodiment, the amino acid sequence of TM2 is shown in SEQ ID NO.: 19.

In another preferred example, the C1 and C2 are each independently a costimulatory signal molecule selected from the following group of proteins: CD28, 4-1BB (CD137), CD30, CD40, CD70, CD134, LIGHT, DAP10, CDS , ICAM-1, OX40, or combinations thereof.

In another preferred example, the C1 is none; or a costimulatory signal molecule derived from CD28 and/or 4-1BB.

In another preferred example, the C2 is a costimulatory signal molecule derived from CD28 and/or 4-1BB.

In another preferred embodiment, the amino acid sequences of C1 and C2 are independently as shown in SEQ ID NO.: 20.

In another preferred embodiment, the amino acid sequences of C1 and C2 are independently as shown in SEQ ID NO.: 21.

In another preferred example, the amino acid sequence of CD3ζ is shown in SEQ ID NO.: 22.

A second aspect of the present invention provides a method for preparing the engineered immune cells described in the first aspect of the present invention, comprising the following steps:

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

(B) Modify the immune cells so that the immune cells express a first CAR and a second CAR, the first CAR targets a first tumor cell marker, and the second CAR targets a first tumor cell marker. Two tumor cell markers, thereby obtaining the engineered immune cells described in the first aspect of the present invention.

In another preferred embodiment, step (A) also includes isolating and/or activating immune cells to be modified.

In another preferred embodiment, step (B) includes (B1) introducing a first expression cassette expressing a first CAR targeting the first tumor cell marker into the immune cell; and (B2) introducing A second expression cassette expressing a second CAR targeting a second tumor cell marker is introduced into the immune cell; wherein step (B1) can be performed before, after, simultaneously, or alternately with step (B2).

In another preferred embodiment, 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 embodiment, when the immune cells to be modified in step (A) already express the first CAR and the second CAR, step (B) can be omitted.

In another preferred embodiment, the immune cells are NK cells, macrophages or T cells.

In another preferred embodiment, the first expression cassette contains a nucleic acid sequence encoding the first CAR.

In another preferred embodiment, the second expression cassette contains a nucleic acid sequence encoding the second CAR.

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 first expression cassette and the second expression cassette are located in different vectors.

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

In another preferred embodiment, the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or combinations thereof.

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

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

A third aspect of the present invention provides a preparation, which contains the engineered immune cells described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

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

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

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

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

The fourth aspect of the present invention provides a use of the engineered immune cells as described in the first aspect of the present invention for preparing drugs or preparations that selectively kill tumors.

In another preferred embodiment, the tumors include tumors that highly express tumor cell markers (such as EGFR, cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47, EpCAM).

In another preferred embodiment, the tumors include tumors that simultaneously express tumor cell markers (such as EGFR, cMet, Her2, and HER3).

In another preferred embodiment, the tumor includes a tumor that expresses both EGFR and cMet.

In another preferred embodiment, the tumor is selected from the group consisting of hematological tumors, solid tumors, or combinations thereof. Preferably, the tumor is a solid tumor.

In another preferred embodiment, the blood tumor is selected from the following group: acute myelogenous leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute myeloid-monocytic leukemia, chronic lymphocytic leukemia, Chronic myeloid leukemia, chronic myelogenous leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma (MM), myelodysplastic syndrome, or combinations thereof.

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

In another preferred embodiment, the solid tumor is selected from the group consisting of prostate cancer, liver cancer, head and neck cancer, melanoma, non-Hodgkin lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, and chondrosarcoma. , Thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, small cell lung cancer, colorectal cancer, Breast cancer, medulloblastoma, breast cancer, nasopharyngeal cancer, thymic cancer, or combinations thereof.

The fifth aspect of the present invention provides a kit for selectively killing tumors. The kit contains a container, and located in the container:

(1) A first nucleic acid sequence containing a first expression cassette for expressing a first CAR targeting a first tumor cell marker selected from the group consisting of: cMet , Her2, Her3, Muc1, ROR1, PD-L1, CD47, or combinations thereof; and

(2) A second nucleic acid sequence containing a second expression cassette of a second CAR targeting a second tumor cell marker selected from the group consisting of: EGFR, EpCAM, Her2, Her3, or combinations thereof.

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

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

The sixth aspect of the present invention provides a method for selectively killing tumors, including:

A safe and effective amount of the engineered immune cells described in the first aspect of the present invention or the preparation described in the third aspect of the present invention is administered to the subject in need of treatment.

In another preferred embodiment, the subject includes humans or non-human mammals.

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

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

The seventh aspect of the present invention provides a method for treating diseases, which includes administering a safe and effective amount of the engineered immune cells described in the first aspect of the present invention or the preparation described in the third aspect of the present invention to a subject in need of treatment.

In another preferred embodiment, the method further includes administering other drugs for treating cancer or tumors to the subject in need of treatment.

In another preferred embodiment, the other drugs include CAR-T drugs.

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

In another preferred embodiment, the tumors include tumors that highly express tumor cell markers (such as EGFR, cMet, HER2, HER3, MUCl, ROR1, PD-L1, CD47).

In another preferred embodiment, the tumors include tumors that simultaneously express tumor cell markers (such as EGFR, cMet, EpCAM, HER2, HER3).

In another preferred embodiment, the tumor is selected from the following group: hematological tumors, solid tumors, or combinations thereof. Preferably, the tumor is a solid tumor.

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

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

An eighth aspect of the present invention provides a fusion protein, the fusion protein comprising a first CAR targeting a first tumor cell marker and a second CAR targeting a second tumor cell marker, the first tumor cell marker The material is selected from the following group: cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47, or a combination thereof; the second tumor cell marker is selected from the following group: EGFR, EpCAM, Her2, Her3, or a combination thereof .

In another preferred embodiment, the first CAR and the second CAR are connected through a connecting peptide.

In another preferred embodiment, the connecting peptide includes a self-cleaving protein.

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

In another preferred embodiment, the self-cleaving protein includes P2A.

In another preferred example, the structure of the fusion protein is shown in the following formula III:
L1-S1-H1-TM1-C1-CD3ζ-Z3-L2-S2-H2-TM2-C2-Z2 (III)

In the formula,

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

L1 is none or the first signal peptide sequence;

L2 is none or the second signal peptide sequence;

H1 is none or the first hinge area;

H2 is no or second hinge region;

TM1 is the first transmembrane domain;

TM2 is the second transmembrane domain;

C1 is no or the first costimulatory signal molecule;

C2 is the second costimulatory signal molecule;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

Z2 is a cytoplasmic signaling sequence that is absent or derived from CD3ζ;

Z3 is the connecting peptide.

In another preferred embodiment, the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NO.: 23-26.

The ninth aspect of the present invention provides a polynucleotide encoding the fusion protein according to the eighth aspect of the present invention.

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

(a) A polynucleotide encoding a fusion protein as shown in any one of SEQ ID NO.: 23-26;

(b) A polynucleotide whose sequence is shown in any one of SEQ ID NO.: 27-30;

(c) A polynucleotide whose nucleotide sequence has a homology of ≥75% (preferably ≥80%) to the sequence shown in (b);

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

(e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

In another preferred embodiment, the polynucleotide sequence is as shown in any one of SEQ ID NO.: 27-30.

A tenth aspect of the present invention provides a vector, which includes the polynucleotide described in the ninth aspect of the present invention.

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

In another preferred embodiment, the vector is selected from the following group: plasmids, viral vectors, transposons, or combinations thereof.

In another preferred embodiment, the vector includes DNA virus and retroviral vector.

In another preferred embodiment, the vector is selected from the following group: lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, or combinations thereof.

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

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

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

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

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

Description of the drawings

Figure 1 shows the And logic BiCAR targeting EGFR and cMet. The composition includes two independent CAR molecules, the CAR1 molecule is an anti-cMet scFv tandem CD3ζ activation signal domain, and the CAR2 molecule is an anti-EGFR scFv tandem CD28 or 4-1BB costimulatory signal domain; Figure 1B shows " and "Schematic diagram of the molecular construction of logical BiCAR expression, CAR1 molecules and CAR2 molecules are connected through P2A; Figure 1C shows the construction diagram of the second generation fully functional CAR targeting cMet, anti-cMET scFv tandem CD28 or 4-1BB costimulatory signal domain and CD3ζ activation signaling domain.

Figure 2 shows the flow cytometry analysis of CAR transduction efficiency of Mock T (2A), O-28z (2B) CAR and "And" logic BiCAR T (2C) cells.

Figure 3 shows that Mock T, O-28z CAR and BiCAR T cells prepared from peripheral blood mononuclear cells (PBMC) of different healthy donors #1 (3A) and #2 (3B) have different effects on targets in vitro. Compared with the lytic toxicity to target cells after incubation with NCI-H1975 human lung adenocarcinoma cell line co-expressing EGFR/cMet for 24 hours.

Figure 4 shows that Mock T, O-28z CAR and BiCAR T cells prepared from peripheral blood mononuclear cells (PBMC) of different healthy donors #1 (4A) and #2 (4B) were compared with each other at different effect-target ratios. The lytic toxicity of the EBC-1 human lung squamous cell carcinoma cell line co-expressing EGFR/cMet to target cells after 24 hours of co-culture in vitro.

Figure 5 shows the lytic toxicity of Mock T and BiCAR-T cells to target cells after incubation for 24 hours with the human gastric cancer cell line SNU5 cells co-expressing EGFR/cMET under different effective-target ratio conditions.

Figure 6 shows the comparison of Mock T and BiCAR-T cells under different effective target ratio conditions with control 293T-cells, 293T stable transfection strain expressing cMET alone, 293T stably transducing strain expressing EGFR alone and 293T co-expressing EGFR/cMet. Lytic toxicity of the stably transformed strains to target cells after co-incubation for 24 hours.

Figure 7 shows the line graph of tumor growth size in tumor-bearing immunodeficient mice after tail vein injection of CAR T cells. The results show that O-28z CAR and BiCAR T cells significantly killed or even eliminated tumor cells in the mice, and were small. No abnormalities were observed in the rats.

Detailed ways

After extensive and in-depth research, the inventor unexpectedly discovered for the first time a first CAR targeting a first tumor cell marker (such as cMet, Her2, Her3, Muc1, ROR1, PD-L1, CD47) and a second targeting tumor cell marker. The immune cells of the second CAR of tumor cell markers (such as EGFR, EpCAM, Her2, Her3) can be fully activated, enhance targeting specificity, exert anti-tumor efficacy, and reduce on-target/off-tumor toxicity, improving cell therapy. security. On this basis, the inventor completed the present invention.

Terminology

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

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

As used herein, the term "contains" 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, and at least one cellular domain. inner domain. "Chimeric antigen receptor (CAR)" is also known as "chimeric receptor", "T-body" or "chimeric immune receptor (CIR)". The "extracellular domain capable of binding antigen" refers to any oligopeptide or polypeptide capable of binding a certain antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known as a domain that transmits signals to activate or inhibit biological processes within 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, 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. Treatment of cells includes contact of reagents with cells, contact of reagents with fluids, and contact of fluids with cells. "Administration" and "treatment" also mean in vitro and ex vivo treatment of cells by a reagent, a diagnostic, a binding composition or by another cell. "Treatment" when applied to humans, animals or research subjects, means therapeutic treatment, prophylactic or prophylactic measures, research and diagnostics.

As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including any CAR of the invention and compositions thereof, to a patient with one or more symptoms of a disease for which the therapeutic agent is known to be effective. These symptoms are therapeutic. Typically, a therapeutic agent is administered to a patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

As used herein, the terms "optionally" or "optionally" mean that the subsequently described event or circumstance may occur but does not necessarily occur. For example, "optionally including 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may be present but are not required to be present, and may be 1, 2 or 3.

"Sequence identity" as used herein means the degree of identity between two nucleic acids or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, deletions and other mutations. The sequence identity between the sequence described in the present invention and its identical sequence 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%.

EGFR

Epidermal factor growth receptor (EGFR) belongs to the tyrosine kinase family, which includes three other members: ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4. It is anchored in the cytoplasmic membrane and includes an extracellular ligand-binding functional domain, a hydrophobic transmembrane region and an intracellular tyrosine kinase domain. After ligands of EGFR (such as EGF, TGF-a, etc.) bind to it, they will form homodimers or heterodimers with other family members, leading to autophosphorylation of tyrosine residues, thereby activating multiple A downstream signaling pathway that regulates cell proliferation, survival and apoptosis. The EGFR signaling network plays an important role in the maintenance and growth of epithelial tissue, and its abnormal activation and activity are related to the occurrence, progression and poor prognosis of various cancers.

EGFR is overexpressed in a variety of tumors, including 25-77% of colorectal cancers, 80-100% of head and neck cancers, 40-80% of non-small cell lung cancer (NSCLC), 50-90% of renal cancers, 30- 50% of pancreatic cancers, 14-91% of breast cancers, 40-63% of glioblastomas, 35-70% of ovarian cancers, etc. Gene mutations and overexpression of EGFR abnormally activate downstream pathways and are closely related to carcinogenesis and cancer progression. Current drugs targeting EGFR include tyrosine kinase inhibitors (TKIs) (such as erlotinib, gefitinib, osimertinib, etc.) and monoclonal antibodies (cetuximab and panitumumab). ), has been approved for the treatment of non-small cell lung cancer, colorectal cancer and head and neck cancer respectively. EGFR is also widely expressed in normal human organs and tissues, including skin, lungs, liver, kidneys, etc. The real-world adverse reactions of approved monoclonal antibody drugs and the clinical on-target toxicity of CAR T cell therapy drugs developed for EGFR are mainly manifested as skin toxicity. EGFR proto-oncogene driver mutations are an important factor in NSCLC carcinogenesis and disease progression. They are detected in 50-60% of Asian patients and are clinically used as biomarkers for prognosis and disease prediction. EGFR-TKI drugs (gefitinib, erlotinib, etc.) have good efficacy in patients with advanced NSCLC with EGFR-sensitive mutations. However, after a median treatment time of 6-12 months, most patients have no response to EGFR-TKI. TKI resistance develops. The resistance mechanism mainly includes acquired mutations of EGFR. About 50% of drug-resistant patients have the T790M mutation. The resistance to third-generation TKI drugs such as osimertinib is due to the C797S mutation; and the activation of the alternative pathway cMet and Her2 signaling pathways. About 30% of patients treated with osimertinib will develop acquired cMet gene amplification, thus avoiding the target EGFR, leading to TKI resistance. NSCLC is a typical case of multiple driver gene mutations. Genomic instability, tumor heterogeneity, and primary or acquired drug resistance generated during disease progression have created bottlenecks in the application of targeted therapy for advanced NSCLC. Therefore, multi-target Targeted therapy will have opportunity to achieve better clinical outcomes.

cMet

Mesenchymal epidermal transformation factor (cMet) is a type of receptor tyrosine kinase. After binding to the ligand hepatocyte factor (HGF), it leads to dimerization and phosphorylation of the receptor, activating a variety of different cell signaling pathways. Involved in cell proliferation, movement, migration, invasion and angiogenesis. cMet is normally expressed in a variety of human tissues and organs, including liver, esophagus, stomach, colorectum, skin, ovary and endometrium. cMet is very important for controlling cell homeostasis under normal physiological conditions. When cMet is overactivated, it will initiate the transformation of normal cells into tumor cells, resulting in epithelial-mesenchymal transition. Gene mutation, gene amplification or overexpression of cMet can lead to abnormal activation of its signaling pathway and promote tumor progression.

cMet is overexpressed in a variety of cancers, including lung, breast, ovarian, renal, colorectal, thyroid, liver, and gastric cancers. Overexpression of cMet is directly related to poor survival. In some cancers, such as lung cancer, colorectal cancer, ovarian cancer, etc., cMet and/or its ligand HGF are used as clinical prognostic indicators. Among cancers with abnormal cMet genes, gastric cancer and colorectal cancer are typical. In gastric cancer, about 10-20% of cMet gene copy number amplifications and about 25% of cMet gene mutations; in colorectal cancer patients, the cMet mutation rate is about 15%. cMet is overexpressed in 25%-75% of NSCLC. In EGFR-TKI-resistant NSCLC, 15%-30% have acquired amplification of the cMet gene, and cMet is also used as a detection marker for TKI resistance in NSCLC patients with EGFR mutations.

Targeted drugs for cMet all inhibit the HGF/cMet signaling pathway. Those in clinical trials mainly include receptor enzyme inhibitors (such as Tivantinib, Capmatinib, etc.), monoclonal antibodies (such as Onartuzumab), and antibody drug conjugates ( For example Telisotuzumab vedotin). Due to cMet mutations in many types of cancer leading to its overexpression, acquired gene amplification caused by EGFR-TKI resistance in NSCLC, and resistance in the development of cMet inhibitors, multiple targets for specific indications and patient groups Developing or combining treatments will have the opportunity to improve clinical efficacy.

antigen binding domain

In the present invention, the antigen-binding domain of the chimeric antigen receptor CAR specifically binds to tumor cell markers, such as EGFR, cMet, Her2, Her3, Mucl, ROR1, or PD-L1, etc. The antigen-binding domain of CAR is a single-chain variable fragment (scFv) formed by connecting the heavy chain and light chain of a monoclonal antibody through flexible linkers of different lengths. svFv sequences are usually derived from mouse, humanized or fully human monoclonal antibodies. In addition, the antigen-binding domain also includes smaller, alpaca-derived heavy chain Nanobodies due to the lack of light chains. (VH/nanobody). The affinity of scFv or VH to the target antigen is crucial for regulating CAR T cell function, but too high affinity may cause overactivation of CAR T cells and lead to cell death. In addition to affinity, the density and epitope of the target antigen are also important factors affecting antigen recognition and efficacy. In addition, the high expression of antigen-independent scFv and CAR molecules on the cell membrane will induce the aggregation of CAR molecules, resulting in antigen-independent signal transduction (tonic signaling) and off-target activation, which may ultimately lead to early exhaustion of CAR T cells. .

hinge region and transmembrane region

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

The transmembrane domain may be derived from natural or synthetic sources. In natural sources, this domain can be derived from any membrane-bound or transmembrane protein. Preferably, the hinge region in the CAR of the present invention is the hinge region of CD8, and the transmembrane region of the present invention is the transmembrane region of CD8 or CD28.

intracellular domain

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 specialized function. For example, the effector function of a T cell may be cytolytic activity or auxiliary activity including cytokine secretion. The term "intracellular signaling domain" therefore refers to the portion of a protein that transduces effector function signals and directs the cell to perform specialized functions. Although typically the entire intracellular signaling domain can be used, in many instances it is not necessary to use the entire chain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain as long as it transduces an effector function signal. The term "intracellular signaling domain" generally refers to any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

Preferred examples of intracellular signaling domains for use in CARs of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to initiate signal transduction upon antigen receptor binding, as well as these sequences any derivative or variant and any synthetic sequence having the same functional capabilities.

In preferred embodiments, the cytoplasmic domain of the CAR can be designed to include CD3ζ signaling itself domain, or may be combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of a CAR may include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules required for an effective lymphocyte response to antigen, rather than antigen receptors or their ligands. Preferably, 4-1BB (CD137), CD28, etc. are included.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be connected to each other randomly or in a prescribed 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 4-1BB, and/or the signaling domain of CD28 (costimulatory molecule) and the signaling domain of CD3ζ.

Chimeric Antigen Receptor (CAR)

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

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

CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. Such a high response rate has been unachievable by any previous treatment method, triggering an upsurge in clinical research in countries around the world.

Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes target-specific binding elements (also called antigen-binding domains). cell The endodomain includes costimulatory signaling regions and/or zeta chain portions. A costimulatory signaling domain refers to the portion of the intracellular domain that includes costimulatory molecules. Costimulatory molecules are cell surface molecules that are required for effective lymphocyte response to antigen, rather than antigen receptors or their ligands.

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

When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to fail to grow, be driven to death, or otherwise affected, and cause the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecules and/or zeta chains. Preferably, the antigen binding domain is fused to an intracellular domain in combination with a 4-1BB signaling domain and/or a CD3ζ signaling domain.

As used herein, "antigen-binding domain" and "single-chain antibody fragment" all refer to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment with antigen-binding activity. Fv antibodies are the smallest antibody fragments that contain an antibody heavy chain variable region, a light chain variable region, but no constant region, and have all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1/6 of a complete antibody. Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain. As a preferred mode of the present invention, the scFv includes specific recognition of tumor cell markers with high expression (such as PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin18.2, Mucin 1 (MUC1 ), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138), preferably single chain antibodies.

In a preferred embodiment, the antigen-binding portion of the CAR of the present invention targets the first tumor cell marker and the second tumor cell marker. In a preferred embodiment, the antigen-binding portion of the CAR of the present invention is a first scFv targeting cMet and a second scFv targeting EGFR.

In a preferred embodiment, the structure of the first scFv is shown in Formula A1 or A2:
V _H1 -V _L1 (A1); or
V _L1 -V _H1 (A2);

In a preferred embodiment, the amino acid sequence of V _L1 is such as positions 135-247 in SEQ ID NO.:1 is shown, and the amino acid sequence of V _H1 is shown in positions 1-119 of SEQ ID NO.: 1.

In a preferred embodiment, the structure of the second scFv is shown in formula A3 or A4:
V _H2 -V _L2 (A3); or
V _L2 -V _H2 (A4);

In a preferred embodiment, the amino acid sequence of V _L2 is shown in positions 135-241 of SEQ ID NO.:5, and the amino acid sequence of V _H2 is shown in positions 1-119 of SEQ ID NO.:5.

In a preferred embodiment, the first scFV and the second scFv comprise variant forms, and the variants have ≥80%, ≥85%, ≥90%, ≥ 95%, ≥98% or ≥99% homology.

In the present invention, the first scFV and the second scFV of the invention also include conservative variants thereof, which means that compared with the amino acid sequences of the first scFV and the second scFV of the invention respectively, there are at most 10, preferably At most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.

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

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

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

In the present invention, the intracellular domain in the first CAR of the present invention includes the transmembrane region of CD8 and the signaling domain of CD3ζ.

In the present invention, the intracellular domain in the second CAR of the present invention includes the transmembrane region of CD8 or CD28, and the costimulatory factor of 4-1BB or CD28.

In a preferred embodiment of the present invention, the amino acid sequence of the first CAR is as shown in any one of SEQ ID NO.: 2-4.

In a preferred embodiment of the present invention, the amino acid sequence of the second CAR is shown in any one of SEQ ID NO.: 7-14.

In a preferred embodiment of the present invention, the amino acid sequence of the fusion protein containing the first CAR and the second CAR (ie, logic BiCAR) is as shown in any one of SEQ ID NO.: 23-26.

Among them, in SEQ ID NO.: 23, positions 1-22 are the first signal peptide; positions 23-269 are the antigen-binding domains targeting the first tumor cell marker (for example, the antibody single chain targeting cMet can variable region sequence); positions 270-314 are the hinge region; positions 315-338 are the transmembrane region (such as the transmembrane region of CD8); positions 339-450 are CD3ζ, and positions 451-472 are the connecting peptide (such as self-cleaving protein), positions 473-493 are the second signal peptide, and positions 494-734 are the antigen-binding domain targeting the second tumor cell marker (such as the single-chain variable region sequence of an antibody targeting EGFR) ; Positions 735-779 are the hinge region; positions 780-806 are the transmembrane region (such as the transmembrane region of CD28); positions 807-847 are costimulatory elements (such as CD28).

Among them, in SEQ ID NO.: 24, positions 1-22 are the first signal peptide; positions 23-269 are the antigen-binding domains targeting the first tumor cell marker (for example, the antibody single chain targeting cMet can variable region sequence); positions 270-314 are the hinge region; positions 315-338 are the transmembrane region (such as the transmembrane region of CD8); positions 339-450 are CD3ζ, and positions 451-472 are the connecting peptide (such as self-cleaving protein), positions 473-493 are the second signal peptide, and positions 494-734 are the antigen-binding domain targeting the second tumor cell marker (such as the single-chain variable region sequence of an antibody targeting EGFR) ; Positions 735-779 are the hinge region; positions 780-806 are the transmembrane region (such as the transmembrane region of CD28); positions 807-847 are costimulatory elements (such as CD28).

Among them, in SEQ ID NO.: 25, positions 1-22 are the first signal peptide; positions 23-269 are the antigen-binding domains targeting the first tumor cell marker (for example, the antibody single chain targeting cMet can variable region sequence); positions 270-314 are the hinge region; positions 315-338 are the transmembrane region (such as the transmembrane region of CD8); positions 339-450 are CD3ζ, and positions 451-472 are the connecting peptide (such as self-cleaving protein), positions 473-493 are the second signal peptide, and positions 494-734 are the antigen-binding domain targeting the second tumor cell marker (such as the single-chain variable region sequence of an antibody targeting EGFR) ; Positions 735-779 are the hinge region; positions 780-803 are the transmembrane region (such as the transmembrane region of CD8); positions 804-845 are costimulatory elements (such as 4-1BB).

Among them, in SEQ ID NO.: 26, positions 1-22 are the first signal peptide; positions 23-269 are the antigen-binding domains targeting the first tumor cell marker (for example, the antibody single chain targeting cMet can variable region sequence); positions 270-314 are the hinge region; positions 315-338 are the transmembrane region (such as the transmembrane region of CD8); positions 339-450 are CD3ζ, and positions 451-472 are the connecting peptide (such as self-cleaving protein), positions 473-493 are the second signal peptide, and positions 494-734 are the antigen-binding domain targeting the second tumor cell marker (such as the single-chain variable region sequence of an antibody targeting EGFR) ; Positions 735-779 are the hinge region; Positions 780-803 are the transmembrane region region (such as the transmembrane region of CD8); positions 804-845 are costimulatory elements (such as 4-1BB).

Chimeric Antigen Receptor T Cells (CAR-T Cells)

As used herein, the terms "CAR-T cells", "CAR-T" and "CAR-T cells of the present invention" all refer to the CAR-T cells of the present invention. The CAR-T cells of the present invention can target tumor surface antigens. (such as PSMA), used to treat tumors with high expression or positive PSMA, especially solid tumors.

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

In the present invention, the fusion protein containing the first CAR and the second CAR (i.e., logic BiCAR) of the present invention includes (i) an extracellular domain, which includes an antigen targeting a first tumor cell surface antigen; (ii) a third a hinge region; (iii) a first transmembrane domain; (iv) an optional first costimulator; and (iv) a signaling domain of CD3ζ; and; (v) a linking peptide (e.g., a self-cleaving protein ); (vi) an extracellular domain comprising an antigen targeting a second tumor cell surface antigen; (ii) a second hinge region; (iii) a second transmembrane domain; and (iv) a second costimulatory factor .

Chimeric Antigen Receptor Macrophages (CAR-M cells)

As used herein, the terms "CAR-M cells", "CAR-M" and "CAR-M cells of the present invention" all refer to the CAR-M cells of the present invention. The CAR-M cells of the present invention can target tumor surface antigens. (such as cMet and EGFR), used to treat tumors with high expression or positivity of tumor antigens (such as cMet and EGFR), especially solid tumors.

Macrophages are the main effectors and regulators of the innate immune system. They have phagocytic ability, can secrete pro-inflammatory factors, and present antigens to T cells to activate the immune system.

Compared with CART, CAR-M can directly kill antigen-specific tumor cells in vitro, inhibit tumor growth in vivo, reshape the tumor microenvironment, and has good anti-tumor activity. In addition, CAR-M also has the ability to present antigens. , present tumor cell antigens and activate endogenous T cells.

Chimeric Antigen Receptor NK Cells (CAR-NK Cells)

As used herein, the terms "CAR-NK cells", "CAR-NK" and "CAR-NK cells of the present invention" all refer to the CAR-NK cells of the present invention. The CAR-NK cells of the present invention can target tumor surface antigens (such as cMet and EGFR) and are used to treat tumors with high cMet and EGFR expression or positivity, especially solid tumors.

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

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

exogenous T cell antigen receptor

As used in this article, exogenous T cell antigen receptor (TCR) refers to the α chain and β chain of TCR cloned from tumor reactive T cells through gene transfer technology, and through genetic engineering means, lentivirus or Retrovirus is used as a vector to transfer exogenous TCR into T cells.

Exogenous TCR-modified T cells can specifically recognize and kill tumor cells, and by optimizing the affinity of TCR and tumor-specific antigens, the affinity of T cells with tumors can be increased and the anti-tumor effect can be improved.

carrier

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

preparation

The invention provides an engineered immune cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 ³ -1×10 ⁸ cells/Kg body weight, more preferably 1×10 ⁴ -1×10 ⁷ cells/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; proteins; polypeptides or amino acids such as glycine ; antioxidant; chelating agent such as EDTA or glutathione; adjuvant agents (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The present invention encompasses the therapeutic use of cells (eg, T cells) transduced with a retroviral vector (RVV) encoding an expression cassette of the invention. The transduced T cells can target tumor cell marker proteins (such as cMet and EGFR), cooperatively activate T cells, trigger cellular immune responses, and thereby selectively kill tumor cells, such as tumor cells with high expression of cMet and EGFR.

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

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

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

Although the data disclosed herein specifically disclose including an antigen-binding domain targeting a first tumor cell surface antigen, the hinge and transmembrane regions, and the CD3ζ signaling domain, P2A, an antigen-binding domain targeting a second tumor cell surface antigen, hinge and transmembrane regions, and 4-1BB/CD28 retroviral vectors, but the invention should be construed to include any number of changes in each part of the construct composition.

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

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

Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include prostate cancer, liver cancer, fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

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

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

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

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

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

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

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

When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered can be determined by the physician, who takes into account the patient (subject) ) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease. It may generally be stated that pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight (including all integers within those ranges). value) application. T cell compositions can also be administered multiple times at these dosages. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.

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

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

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

fusion protein

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

As used herein, the term "fusion protein" also includes variant forms of any of the sequences shown in SEQ ID NO.: 23-26 having the above-mentioned activities. These variant forms include (but are not limited to): deletion, insertion and/or substitution of 1-3 (usually 1-2, more preferably 1) amino acids, and addition of or One or several (usually within 3, preferably within 2, and more preferably within 1) amino acids are missing. For example, in the art, substitutions with amino acids with similar or similar properties generally do not alter the function of the protein. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. Furthermore, 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 (such as cyclic peptides).

The present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion proteins. As used herein, the terms "fragment," "derivative" and "analogue" refer to polypeptides that substantially retain the function or activity of the fusion proteins of the invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) a polypeptide in which one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in which one or more conservative amino acid residues are substituted. A polypeptide with a substituent group in an amino acid residue, or (iii) a polypeptide formed by fusion of a polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a fusion of additional amino acid sequences A polypeptide formed from this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, secretory sequence or tag sequence such as 6His). Such fragments, derivatives and analogs are within the scope of those skilled in the art in light of the teachings herein.

A preferred type of active derivative refers to one in which at most 3, preferably at most 2, and more preferably at most 1 amino acid is replaced by an amino acid with similar or similar properties compared to the amino acid sequence of the present invention to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1

The invention also provides analogs of the fusion proteins of the invention. The difference between these analogs and the polypeptides shown in any of SEQ ID NO.: 23-26 can be differences in amino acid sequences, differences in modified forms that do not affect the sequence, or both. Analogues also include analogs with residues that differ from natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified forms (which generally do not alter the primary structure) include chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of the polypeptide during its synthesis and processing or during further processing steps. This modification can 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 (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility properties.

In one embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NO.: 23-26.

coding sequence

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

The polynucleotides of the invention may be in DNA form or RNA form. DNA can be a coding strand or a non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence encoding the polypeptide shown in any one of SEQ ID NO.: 23-26 or may be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding a polypeptide shown in any one of SEQ ID NO.: 23-26, but with different sequences in the corresponding coding regions.

The full-length nucleotide sequence of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the polypeptide of the present invention (or its fragment, or its derivative) can be obtained entirely through chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the vectors or polypeptide coding sequences of the invention. The polynucleotides, vectors or host cells described above may be isolated.

As used herein, "isolated" means that a substance has been separated from its original environment (in the case of a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state within living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified if it is separated from other substances existing in the natural state.

The polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be the substitution, deletion or insertion of one or more nucleotides, but does not substantially change its encoding of the fusion protein of the invention. function.

The full-length nucleotide sequence encoding the fusion protein of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the relevant published nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as the primer. Template, amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

In one embodiment of the present invention, the polynucleotide sequence encoding the fusion protein is as shown in any one of SEQ ID NO.: 27-30.

Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.

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

The present invention also relates to vectors containing the polynucleotides of the present invention, as well as host cells genetically engineered using the vectors or protein coding sequences of the present invention, and methods for expressing the fusion proteins of the present invention on the NK cells using recombinant technology.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to obtain NK cells expressing the fusion protein of the present invention. Generally speaking, it includes the steps of: transducing the first expression cassette and/or the second expression cassette of the present invention into NK cells, thereby obtaining the NK cells.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of the invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate 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.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture. Fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli.

Vectors containing appropriate DNA sequences as described above and appropriate promoter or control sequences can be used to transform appropriate host cells to enable expression of proteins.

The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: bacterial cells of Escherichia coli, Bacillus subtilis, and Streptomyces; fungal cells such as Pichia pastoris and Saccharomyces cerevisiae cells; plant cells; insect cells of Drosophila S2 or Sf9; CHO, NS0, COS7, or 293 cells animal cells etc. In a preferred embodiment of the present invention, NK cells are selected as host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl ₂ The procedures used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformants can be cultured using conventional methods to express the protein encoded by the gene of the present invention. Depending on the host cells used, the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells grow to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

The protein in the above method can be expressed within the cell, on the cell membrane, or secreted outside the cell. If desired, 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 these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The main advantages of the present invention include:

1. The present invention discovered for the first time a first CAR targeting a first tumor cell marker (such as cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47) and a second tumor cell marker (such as EGFR , EpCAM, Her2, Her3), the engineered immune cells of the second CAR can be fully activated, enhance target specificity, exert anti-tumor efficacy, and reduce on-target/off-tumor toxicity and improve cell Safety of treatment.

2. The present invention discovered for the first time that the immune cells of the present invention can simultaneously recognize two different antigens, EGFR and cMet, and enhance the specificity and selectivity of CAR T cells in targeting tumors, as well as their homing in tumors. In addition, it can inhibit two pathogenic signaling pathways at the same time, thereby inhibiting disease progression and improving survival.

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

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

general method

Retroviral particle preparation

Phoenix GP cells in the logarithmic growth phase were seeded into T75 culture flasks at a density of 4E6/flask, and cultured overnight in a 37°C, 5% _CO2 incubator for transfection. The culture medium was DMEM containing 10% FBS. culture medium. The next day, the culture medium was replaced with fresh DMEM before transfection. The transfection steps are as follows: Add the target gene plasmid, such as pMSGV-CAR, and the envelope plasmid pMD2.G to the Opti-MEM medium and mix well; then add DNA transfection reagent and mix well; add it dropwise into the culture bottle and culture for 18 hours. ; Replace the DMEM medium containing 10% FBS; collect the supernatant after 72 hours, centrifuge, filter, aliquot, store at -80°C for later use or directly infect PG13 cells. PG13 cells in the logarithmic growth phase were seeded into a 6-well plate at a density of 7E4/well and cultured overnight in a 37°C, 5% _CO2 incubator for transfection. The culture medium was DMEM containing 10% FBS. base. The next day, the culture medium in the 6-well plate was replaced with the aforementioned filtered culture supernatant and the virus infection enhancer Polybrene was added. After 24 h of culture, the culture medium was replaced with fresh DMEM. After 72 hours of culture, the transduction efficiency of PG13-CAR was detected to construct a PG13-CAR retrovirus production cell line, which was amplified and cultured before cryopreservation. PG13-CAR cells in the logarithmic growth phase were inoculated into T75 culture bottles at a density of 2E7/flask, and cultured in a 37°C, 5% _CO2 incubator for 24 hours. The supernatant containing retrovirus was collected, centrifuged, filtered, and infected. cell.

CAR T cell preparation

Mononuclear cells were isolated from peripheral blood of healthy individuals by Ficoll-Hypaque density gradient centrifugation. Add TransAct activator and recombinant human IL-2 to the lymphocyte culture medium to stimulate, activate and expand T cells; at the same time, use Retronectin to coat a 6-well plate, add the retrovirus supernatant to the 6-well plate and centrifuge; After centrifugation is completed, discard the supernatant, add the activated T cells to the 6-well plate, and centrifuge for transduction; after culturing for 24 hours in a 37°C, 5% _CO2 incubator, transfer the T cells from the 6-well plate to the culture chamber. Amplification culture was carried out in bottles.

Detection of CAR molecule expression by flow cytometry

When the CAR T cells are cultured to the 7th day, take 1E6 cells and wash the cells twice with buffer; add 1ug MET-His and 1ug EGFR-Fc antigen in the cell suspension and incubate for 30 minutes at 4°C; wash the cells twice with buffer and then add Anti-His-PE and Allophycocyanin AffiniPure Goat Anti-Human IgG antibodies were incubated in the cell suspension at 4°C in the dark for 30 minutes; the cells were washed twice and then analyzed by flow cytometry. Measure the expression of CAR molecules.

Determination of cytotoxicity by HiBiT tag protein detection method (HiBiT Extracellular Detection System, Promega)

Collect the target cells carrying the HaloTag-HiBiT label (including NCI-H1975-Halotag-HiBiT, EBC-1-HaloTag-HiBiT) in the logarithmic growth phase and the CAR T cells cultured to day 7, and prepare cells with different cell concentrations. Suspension; target cells were seeded in a 96-well plate (3 multiple wells) at 5000 cells/well, then CAR T cell suspension was added according to different effect-to-target ratios, and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. Add a certain final concentration of digitonin to the maximum-release positive control group and incubate for 30 minutes. Then remove the well plate from the incubator. After returning to room temperature, add an equal volume of pre-prepared detection Buffer (containing 1:100 dilution of LgBiT Protein). and diluted 1:50 HiBiT Extracellular Substrate), mix on a shaker, use a microplate reader to detect the luminescence value, and calculate the killing activity of CAR T cells.

Cytotoxicity assay via CCK8

Toxicity assay of CAR-T cells was performed using CCK8 kit (Doren Chemical Research Institute). Collect the target cells in the logarithmic growth phase and the CAR T cells cultured to day 7 to prepare cell suspensions with different cell densities. Take 50ul target cells and plate them at 5000 cells/well (3 replicate wells), and then use 3:1, Add 50ul of CAR T cell suspension at the effect-to-target ratio of 1:1, 1:3 and 1:10, and incubate for 24hr in a 5% _CO2 incubator at 37°C and saturated humidity, then add 10ul of CCK8 to each well and react for 4hr. Finally, the absorbance at a wavelength of 450 nm was measured using a microplate reader (Thermo Varioskan LUX).

Mouse CDX model to detect anti-tumor activity of CAR T cells

NCI-H820 tumor cells in the logarithmic growth phase were collected and inoculated subcutaneously on the backs of immune-deficient mice. When the tumors grew to approximately 120 ^mm3 , CAR T cells were infused into the tail vein, and the mice's status was subsequently observed every week. The size of the tumor mass was measured twice and the mouse tumor growth curve was drawn to evaluate the in vivo anti-tumor activity of CAR T cells.

Example 1

Virus preparation: Add retrovirus target gene plasmid pMSGV-CAR and envelope plasmid pMD2.G Mix it into X-tremeGENE 9 DNA Transfection Reagent (Roche), add it to the culture dish cultivating Phoenix GP cells, mix gently, collect the supernatant after 72 hours, centrifuge at 1000g at low speed, filter with 0.45um filter membrane, and filter the supernatant Add it to PG13 cells and add Polybrene transfection enhancer to prepare the retrovirus production cell line PG13-CAR. Inoculate PG13-CAR at 2E7/flask density into T75 cell culture flasks. Collect the supernatant after 24 hours of culture. Centrifuge at 400g at low speed and filter with a 0.45um filter. Use it directly or aliquot and freeze at -80°C.

CAR T cell preparation: Obtain mononuclear cells from healthy human peripheral blood by Ficoll-Hypaque density gradient centrifugation and inoculate them into lymphocyte culture medium (Gibco), add TransAct (Miltenyi Biotec) and IL-2 (GE Healthcare) for stimulation activation and expansion culture. At the same time, use Retronectin to coat a 6-well plate, add the retrovirus supernatant to the coated 6-well plate, and centrifuge at 2000g for 2 hours; after centrifugation is completed, discard the supernatant, and add the activated T cells to the 6-well plate. Centrifuge at 1000g for 20 minutes for transduction, place in a 37°C, 5% CO2 incubator, and culture. After 24 hours, transfer the cells to a T75 culture flask for culture and expansion. The results are shown in Figure 1. The results show that Figure 1A shows the composition of the "AND" logic BiCAR targeting EGFR and cMet, including 2 independent CAR molecules. The CAR1 molecule is an anti-cMet scFv tandem CD3ζ activation signal domain ( As shown in SEQ ID NO.: 2, SEQ ID NO.: 3-4 is a fully functional 2G single CAR against cMet), and the CAR2 molecule is an anti-EGFR scFv tandem CD28 or 4-1BB costimulatory signaling domain (such as Any one of SEQ ID NO.: 7-10 is shown, wherein SEQ ID NO.: 11-14 is a fully functional 2G single CAR against EGFR); Figure 1B shows a schematic diagram of the molecular construction of "AND" logic BiCAR expression, CAR1 The molecule and the CAR2 molecule are linked through P2A (as shown in any of SEQ ID NO.: 23-26); Figure 1C shows the composition of a second-generation fully functional CAR targeting cMet, scFv tandem CD28 or 4-1BB Stimulation signal domain and CD3ζ activation signal domain (as shown in any one of SEQ ID NO.: 3-4).

Example 2

Detect the expression of CAR molecules by flow cytometry: When the CAR T cells are cultured on the 7th day, take the 1E6 cells, wash them twice with washing buffer, and add 1ug cMet-His (Acrobiosystem) and 1ug EGFR-Fc antigen (Acrobiosystem) , after incubating at 4°C for 30 minutes, wash twice with wash buffer, then add Anti-His-PE (Miltenyi Biotec) and Allophycocyanin (APC) AffiniPure Goat Anti-Human IgG, Fcγfragment specific (Jackson ImmunoResearch) and incubate at 4°C in the dark for 30 minutes . After washing the cells, flow cytometry (CytoFLEX LX, Beckman Coulter) was used to detect the expression of CAR molecules.

The results are shown in Figure 2. The expression of CAR molecules in CAR T cells was detected by flow cytometry. As shown in Figure 2A, Mock T cells did not detect the expression of the first CAR (anti-cMet-scFv-CD3ζ/O-z) or the second CAR molecule (anti-EGFR-scFv-CD28/C-28); Figure As shown in Figure 2B, the fully functional second-generation CAR T (anti-cMet-scFv-CD28-CD3ζ/O-28z) targeting the cMet target has a transduction rate of 67%; as shown in Figure 2C, the "AND" logic BiCAR In T cells, the transduction rate of co-expression of the first CAR molecule O-z and the second CAR molecule C-28 was 76%.

Example 3

HiBiT Extracellular Detection System (Promega) method to detect cytotoxicity: collect the lung cancer cell line NCI-H1975-HaloTag-HiBiT carrying the HaloTag-HiBiT label in the logarithmic growth phase and CAR T cells cultured to day 7, and prepare different cell concentrations of cell suspension. Inoculate target cells into a 96-well plate at 5000 cells/well (3 multiple wells), then add CAR T cell suspension according to the effect-to-target ratio of 1:10, 1:3, 1:1 and 3:1, 37°C, 5 Incubate for 24 hours in a % CO ₂ incubator. Add a certain final concentration of digitonin to the maximum-release positive control group and incubate for 30 minutes. Then take out the 96-well plate from the incubator. After returning to room temperature, add an equal volume of pre-prepared test solution. Buffer (contains 1:100 diluted LgBiT Protein and 1:50 diluted HiBiT Extracellular Substrate), mix on a shaker, use a microplate reader to detect the luminescence value, and calculate the killing activity of CAR T cells.

As shown in Figure 3, the results show that BiCAR T cells from Donor#1 (Figure 3A) and Donor#2 (Figure 3B) can effectively kill NCI-H1975 tumor cells in vitro, and this cytotoxicity is positively correlated with cell dose. relationship.

Example 4

HiBiT Extracellular Detection System (Promega) method to detect cytotoxicity: collect the lung cancer cell line EBC-1-Halotag-HiBiT carrying the HaloTag-HiBiT label in the logarithmic growth phase and CAR T cells cultured to day 7, and prepare different cell concentrations of cell suspension. The target cells were seeded into a 96-well plate (3 multiple wells) at 5000cells/well, and then the CAR T cell suspension was added according to the effect-to-target ratio of 1:10, 1:3, 1:1 and 3:1, at 37°C. Incubate for 24 hours in a 5% CO ₂ incubator. Add a certain final concentration of digitonin to the maximum-release positive control group and incubate for 30 minutes. Then take out the 96-well plate from the incubator and add an equal volume of pre-prepared detection Buffer (containing 1: 100 diluted LgBiT Protein and 1:50 diluted HiBiT Extracellular Substrate), mix on a shaker, use a microplate reader to detect the luminescence value, and calculate the killing activity of CAR T cells.

As shown in Figure 4, the results show that BiCAR T cells from Donor#1 (Figure 4A) and Donor#2 (Figure 4B) can effectively kill EBC-1 tumor cells in vitro, and this cytotoxicity is positively correlated with cell dose. relationship.

Example 5

Toxicity assay of CAR-T cells was performed using CCK8 kit (Doren Chemical Research Institute). Collect the human gastric cancer cell line SNU5 in the logarithmic growth phase and the CAR T cells cultured to day 7 to prepare cell suspensions with different cell densities. Take 50ul of target cells and plate them at 5000 cells/well (3 replicate wells), and then use 3 :1, 1:1, 1:3 and 1:10, add 50ul CAR T cell suspension and incubate it in a 5% _CO2 incubator at 37°C and saturated humidity for 24hr, then add 10ul CCK8 to each well. , after 4 hours of reaction, use a microplate reader (Thermo Varioskan LUX) to measure the absorbance at a wavelength of 450 nm.

As shown in Figure 5, the results show that BiCAR-T cells can significantly inhibit the growth of SNU5 tumor cells, and this cytotoxicity is positively correlated with cell dose.

Example 6

HiBiT Extracellular Detection System (Promega) method to detect cytotoxicity: collect stably transfected cell lines 293-Halotag-HiBiT, 293-Halotag-HiBiT-MET, 293-Halotag-HiBiT-EGFR carrying HaloTag-HiBiT tags in logarithmic growth phase , 293-Halotag-HiBiT-MET&EGFR, and CAR T cells cultured to day 7 to prepare cell suspensions with different cell concentrations. The target cells were seeded into a 96-well plate (3 multiple wells) at 5000cells/well, and then the CAR T cell suspension was added according to the effect-to-target ratio of 1:10, 1:3, 1:1 and 3:1, at 37°C. Incubate for 24 hours in a 5% CO ₂ incubator. Add a certain final concentration of digitonin to the maximum-release positive control group and incubate for 30 minutes. Then take out the 96-well plate from the incubator and add an equal volume of pre-prepared detection Buffer (containing 1: 100 diluted LgBiT Protein and 1:50 diluted HiBiT Extracellular Substrate), mix on a shaker, use a microplate reader to detect the luminescence value, and calculate the killing activity of CAR T cells.

Figure 6 shows the response of BiCAR T cells to the 293T cell line co-expressing EGFR and cMet (6A), the 293T cell line expressing cMet alone (6B), the 293T cell line expressing EGFR alone (6C), and the control 293T cell line. (6D) In vitro cytolytic toxicity after 24 h of co-incubation. The ratios of CAR-T effector cells to target cells are 1:10, 1:3, 1:1 and 3:1 respectively. The results showed that BiCAR T cells exhibited a dose-dependent and specific in vitro killing function against stably transduced cell lines expressing cMet as a single target or cMet/EGFR as both targets.

Example 7

Mouse CDX model was used to detect the anti-tumor activity of CAR T cells: NCI-H820 cells in the logarithmic growth phase were collected and inoculated subcutaneously on the backs of immunodeficient mice. When the tumor volume reaches about ^120mm3 , mice with similar tumor burdens are randomly divided into groups, and Mock T, O-28z CAR and BiCAR T cells are infused into the tail vein. The size of subcutaneous tumors was measured twice a week and the status of mice was observed, and tumor growth curves were drawn to evaluate the in vivo anti-tumor activity of CAR T cells.

As shown in Figure 7, the results showed that both O-28z CAR and BiCAR-T cells significantly inhibited tumor growth and completely eliminated tumor cells in mice 20 days after CAR T cell infusion.

sequence list

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

Claims

An engineered immune cell, characterized in that the engineered immune cell expresses a first CAR and a second CAR, the first CAR targets a first tumor cell marker, and the second CAR targets a first tumor cell marker. Two tumor cell markers, the first tumor cell marker is selected from the following group: cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47, or a combination thereof; the second tumor cell marker is selected from the following group Group: EGFR, EpCAM, Her2, Her3, or combinations thereof.
The immune cell according to claim 1, wherein the structure of the first CAR is as shown in Formula I:
L1-S1-H1-TM1-C1-CD3ζ (I)

In the formula, the "-" is a connecting peptide or peptide bond;

L1 is none or the first signal peptide sequence;

S1 is an antigen-binding domain targeting a first tumor cell marker selected from the group consisting of: cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47, or a combination thereof;

H1 is none or the first hinge area;

TM1 is the first transmembrane domain;

C1 is no or the first costimulatory signal molecule;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.
The immune cell according to claim 1, wherein the structure of the second CAR is shown in Formula II:
L2-S2-H2-TM2-C2-Z2 (II)

In the formula, the "-" is a connecting peptide or peptide bond;

L2 is none or the second signal peptide sequence;

S2 is an antigen-binding domain targeting a second tumor cell marker selected from the group consisting of: EGFR, EpCAM, Her2, Her3, or a combination thereof;

H2 is no or second hinge region;

TM2 is the second transmembrane domain;

C2 is the second costimulatory signal molecule;

Z2 is a cytoplasmic signaling sequence that is absent or derived from CD3ζ.
A method for preparing engineered immune cells according to claim 1, characterized by comprising the following steps:

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

(B) Modify the immune cells so that the immune cells express a first CAR and a second CAR, the first CAR targets a first tumor cell marker, and the second CAR targets a first tumor cell marker. Two tumor cell markers, thereby obtaining the engineered immune cells described in claim 1.
A preparation, characterized in that the preparation contains the engineered immune cells of claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.
The use of engineered immune cells according to claim 1, characterized in that it is used to prepare drugs or preparations that selectively kill tumors.
The use according to claim 6, wherein the tumor includes a tumor expressing both EGFR and cMet.
A kit for selectively killing tumors, characterized in that the kit contains a container, and located in the container:

(1) A first nucleic acid sequence containing a first expression cassette for expressing a first CAR targeting a first tumor cell marker selected from the group consisting of: cMet , Her2, Her3, Muc1, ROR1, PD-L1, CD47, or combinations thereof; and

(2) A second nucleic acid sequence containing a second expression cassette of a second CAR targeting a second tumor cell marker selected from the group consisting of: EGFR, EpCAM, Her2, Her3, or combinations thereof.
A fusion protein, characterized in that the fusion protein includes a first CAR targeting a first tumor cell marker and a second CAR targeting a second tumor cell marker, the first tumor cell marker being selected from the group consisting of The lower group: cMet, Her2, Her3, Mucl, ROR1, PD-L1, CD47, or a combination thereof; the second tumor cell marker is selected from the lower group: EGFR, EpCAM, Her2, Her3, or a combination thereof.
The fusion protein of claim 9, wherein the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NO.: 23-26.
A polynucleotide encoding the fusion protein of claim 9.
A vector, characterized in that the vector includes the polynucleotide of claim 11.