CN113621582B

CN113621582B - Engineered immune cell for combined expression of CCR2b, and preparation and application thereof

Info

Publication number: CN113621582B
Application number: CN202111109672.2A
Authority: CN
Inventors: 张曦; 黄智宏; 韩德平; 燕妮; 王调霞; 朱晓娜; 王宁; 林伟雄; 卢永
Original assignee: Guangzhou Baiji Biopharmaceutical Co ltd
Current assignee: Zhongxin Cancer Clinical Medical Research And Transformation Guangzhou Co ltd
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2022-09-02
Anticipated expiration: 2041-09-23
Also published as: CN113621582A; WO2023046110A1

Abstract

The present invention relates to engineered immune cells that co-express CCR2 b. Specifically, the engineered immune cells of the invention are T cells or NK cells. According to the invention, the CAR molecule containing NKG2D ectodomain and CCR2b are jointly expressed in the CAR-T cell, so that the sensitivity of the CAR-T cell to chemokines such as CCL2 and CCL7 can be improved through CCR2b, the CAR-T cell can migrate to focuses such as colorectal cancer, ovarian cancer and pancreatic cancer with high efficiency, and the treatment efficiency is improved; meanwhile, multiple target antigens on the cell surfaces of colorectal cancer, ovarian cancer, pancreatic cancer and the like can be identified through the NKG2D CAR molecule, the risk of curative effect reduction caused by tumor heterogeneity or target antigen loss is reduced, and the tumor treatment effect is improved.

Description

Engineered immune cell for combined expression of CCR2b, and preparation and application thereof

Technical Field

The invention belongs to the field of tumor immunity and cell therapy, and particularly relates to an engineered immune cell jointly expressing CCR2 b.

Background

The cellular immunotherapy is a new tumor treatment mode with obvious curative effect, and is a novel autoimmune anticancer treatment method. It is a method for using biological technology and biological preparation to make in vitro culture, modification and amplification of immune cell collected from patient body and then making it be returned into patient body so as to excite and enhance the self-immune function of body and attain the goal of curing tumor.

T cells are an important class of lymphocytes involved in cellular immunity, and can specifically recognize and kill tumor cells through signal transmission by antigen presenting cells. However, tumor cells can also prevent specific recognition of T cells by reducing or losing epitopes, immunosuppression, tumor heterogeneity (i.e., the difference between different individuals of the same malignancy or between different tumor cells at different locations within the same patient from genotype to phenotype), and the like, thereby evading the immune response of the body.

Chimeric antigen receptor T cell (CAR-T) therapy is responsible for this problem. Specifically, the CAR molecule is a receptor molecule that is artificially designed and constructed and consists of a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane region, a costimulatory domain, an intracellular signaling domain, and the like. Therefore, the CAR molecule has the functions of specifically recognizing tumor surface antigens, activating T cell killing activity, stimulating T cell proliferation and the like. The CAR molecule is expressed by T cells autologous to the patient by harvesting T cells from the patient from which the tumor was cultured and artificially transducing the gene encoding the CAR molecule. After being returned to a patient, the T cells can efficiently and specifically recognize and kill tumor cells through the CAR molecules, so that the effect of treating cancer is achieved.

The concept of CAR-T therapy was first introduced since 1989, undergoing thirty years of development and multiple rounds of technological change (figure 1). The first generation CAR-T only had a single chain antibody as the extracellular antigen binding domain and CD3 ζ as the intracellular signaling domain, failed to completely activate T cell activity, and had poor therapeutic effects. The second generation CAR-T introduces a costimulatory domain on the basis of the first generation CAR-T, and improves the in vitro proliferation capacity and cytokine release level of T cells. Third generation CAR-T adds a costimulatory domain to the second generation CAR-T, which, although enhancing the killing activity of T cells, may induce excessive release of cytokines. Therefore, the new generation of CAR-T jointly expresses other accessory factors on the basis of the second generation CAR-T, for example, jointly expresses STAT3/5 binding domain and the like in IL-12 or IL-2R beta cells, and is beneficial to improving the effects of tumor killing activity, safety and the like.

Although CAR-T treatment has achieved satisfactory results in hematological tumors, CAR-T has much room for improvement in the therapeutic efficacy of solid tumors. The reason is that: (1) many solid tumors are difficult to be discovered in early stage, and have the characteristics of high malignancy, high recurrence rate, poor prognosis and the like. For example, 83% of patients with colorectal cancer are already at the middle and advanced stage when first diagnosed, and 44% of patients have developed metastasis to the liver, lung, etc., with nearly half of patients surviving for less than 5 years; when about 70% of ovarian cancer patients are diagnosed, cancer cells have already been transferred, and are difficult to cure by means of operations, chemotherapy and radiotherapy, and the recurrence rate after treatment is still up to more than 70%; 90% of pancreatic cancer patients are diagnosed at an advanced stage and have a 5-year survival rate of only 7%. (2) During treatment of solid tumors, tumor tissue often has an immunosuppressive microenvironment that can impede migration and infiltration of CAR-T cells. (3) Many malignant solid tumors also have the characteristic of high heterogeneity, and a single target antigen often cannot achieve the optimal treatment effect and has the risk of relapse. Therefore, there is a need for further improvement in the efficiency and effectiveness of CAR-T cell therapy for patients with malignant solid tumors such as colorectal cancer, ovarian cancer, pancreatic cancer, and the like.

In view of the above, there is still a need in the art for further research to develop an engineered immune cell with better efficacy and therapeutic effect against malignant tumors (especially solid tumors).

Disclosure of Invention

The invention aims to provide an engineered immune cell (such as CAR-T cell) with higher efficiency and better treatment effect aiming at malignant tumors (particularly solid tumors).

It is a further object of the invention to provide an engineered immune cell (e.g., CAR-T cell) that co-expresses CCR2b and methods of making and using the same.

In a first aspect of the invention, there is provided an engineered immune cell, which is a T cell or an NK cell, and which has the following characteristics:

(a) the immune cell expresses a Chimeric Antigen Receptor (CAR), wherein the CAR targets a surface marker of a tumor cell, wherein the antigen binding domain of the CAR comprises the extracellular domain of NKG 2D; and

(b) the immune cells express exogenous CCR2b protein.

In another preferred embodiment, the T cells comprise α β T, γ δ T cells, NKT cells, MAIT cells, or a combination thereof.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

(i) a chimeric antigen receptor T cell (CAR-T cell);

(ii) chimeric antigen receptor NK cells (CAR-NK cells).

In another preferred embodiment, the CCR2b protein may be constitutively expressed or inducibly expressed.

In another preferred embodiment, there is provided a chimeric antigen receptor T cell (CAR-T cell) having one or more of the following characteristics:

(a) the cells express a chimeric antigen receptor, CAR, that targets a surface marker of a tumor cell; and

(b) when the CAR-T cell is contacted with an inducer, the CAR-T cell induces expression of CCR2b protein.

In another preferred embodiment, the CAR and CCR2b proteins are expressed in tandem in said CAR cell.

In another preferred embodiment, the CAR and CCR2b proteins are each independently expressed in the CAR cell.

In another preferred embodiment, the "activation" refers to binding of the CAR to a surface marker of a tumor cell.

In another preferred embodiment, the "surface marker of a tumor" refers to a specific antigen on the surface of the tumor.

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

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

In another preferred embodiment, the CCR2b protein is localized to the cell membrane of the CAR-T cell.

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

L-NKG2D-H-TM-C-CD3ζ (I)

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

l is a null or signal peptide sequence;

NKG2D is an NKG2D extracellular domain or an active fragment thereof;

h is a zero or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal domain;

CD3 ζ is a cytoplasmic signaling sequence (including wild-type, or mutants/modifications thereof) derived from CD3 ζ;

the "-" is a connecting peptide or a peptide bond.

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

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

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutations/modifications thereof, or combinations thereof.

In another preferred embodiment, C is a co-stimulatory domain of a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, or a mutation/modification thereof, or a combination thereof.

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

In another preferred embodiment, the amino acid sequence of the extracellular domain of NKG2D is shown in SEQ ID NO. 1, positions 73-216 or as shown in SEQ ID NO. 4.

In another preferred embodiment, the CAR cell contains, in addition to the first CAR of formula I, a second CAR for a second antigen, the second CAR having the structure according to formula II:

L-scFv-H-TM-C-CD3ζ (II)

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

l is a null or signal peptide sequence;

scFv is an antigen binding domain;

h is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory domain;

CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ or a mutation/modification thereof;

the "-" links peptides or peptide bonds.

In another preferred embodiment, the scFv is an antibody single chain variable region sequence that targets a tumor antigen.

In another preferred embodiment, the scFv is an antibody single chain variable region sequence targeting an antigen selected from the group consisting of: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133, CD44, CD166, ABCB5, ALDH1, mesothelin (mesothelin, MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands (ligands), LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin18.2, CEA or a combination thereof.

In another preferred embodiment, the amino acid sequence of NKG2D is shown in SEQ ID NO. 1, wherein the extracellular domain is from position 73 to 216.

In another preferred embodiment, the amino acid sequence of the CCR2b protein is shown as SEQ ID NO. 2.

In another preferred embodiment, the first CAR of formula I and the second CAR of formula II can be combined into one, thus constituting a CAR of formula IIIa or IIIb:

L-NKG2D-scFv-H-TM-C-CD3ζ (IIIa)

L-scFv-NKG2D-H-TM-C-CD3ζ (IIIb)

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

l is a null or signal peptide sequence;

NKG2D is an NKG2D extracellular domain or an active fragment thereof;

scFv is an antigen binding domain;

h is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory domain;

the "-" links peptides or peptide bonds.

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

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

(B) engineering the immune cell such that the immune cell expresses a CAR molecule and an exogenous CCR2b protein, thereby obtaining an engineered immune cell according to the first aspect of the invention, wherein the CAR targets a surface marker of a tumor cell, wherein the antigen binding domain of the CAR comprises the extracellular domain of NKG 2D.

In another preferred example, step (B) includes:

(B1) introducing a first expression cassette expressing the CAR into the immune cell; and (B2) introducing a second expression cassette expressing CCR2B into the immune cell;

wherein said step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

In another preferred embodiment, there is provided a method of making a CAR-T cell of the invention, comprising the steps of:

(A) providing a T cell to be engineered;

(B) engineering the T cell such that the T cell expresses the CAR molecule and an exogenous CCR2b protein, thereby obtaining the engineered immune cell of the first aspect of the invention.

In another preferred embodiment, step (B) comprises (B1) introducing into the T cell a first expression cassette expressing the CAR; and (B2) introducing a second expression cassette expressing CCR2B into the T cell; wherein said step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

In another preferred example, when the T cell to be engineered in step (a) already expresses the CAR, then in step (B) a second expression cassette comprising (B2) is introduced into said T cell.

In another preferred embodiment, the transcription directions of the first expression cassette and the second expression cassette are in the same direction (→ →), in opposite direction (→ ←), and in opposite direction (→).

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, when the first and second expression cassettes are located in the same vector, a third expression cassette for expressing the linker peptide is further included between the first and second expression cassettes.

In another preferred embodiment, the linker peptide is P2A.

In another preferred embodiment, the vector is a viral vector, preferably comprising the first and second expression cassettes in tandem.

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

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

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

In another preferred embodiment, the formulation contains a CAR-T cell of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

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

In another preferred embodiment, the formulation comprises an injection.

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

In a fourth aspect of the invention, there is provided a use of the engineered immune cell according to the first aspect of the invention for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.

In another preferred embodiment, there is provided the use of a CAR-T cell according to the first aspect of the invention for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumour.

In another preferred embodiment, the formulation contains CAR-T cells, and a pharmaceutically acceptable carrier, diluent, or excipient.

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

In another preferred embodiment, the tumor is selected from the group consisting of: colorectal cancer, colon cancer, rectal cancer, ovarian cancer, pancreatic cancer.

In another preferred example, the tumor is a tumor with high expression of NKG2D ligand (including any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof).

In another preferred example, the tumor is a tumor with high NKG2D ligand (including any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof) expression and/or high chemokine (including any one of CCL2, CCL7, CCL8, CCL12, CCL13, CCL16, or a combination thereof) expression.

In a fifth aspect of the invention, there is provided a kit for preparing the engineered immune cell of the first aspect of the invention, the kit comprising a container, and in the container:

(1) a first nucleic acid sequence containing a first expression cassette for expressing the CAR, wherein the antigen-binding domain of the CAR is the extracellular domain of NKG 2D; and

(2) a second nucleic acid sequence comprising a second expression cassette for the combined expression of CCR2 b.

In another preferred embodiment, there is provided a kit for preparing the engineered immune cell of the first aspect of the invention, the kit comprising a container, and within the container:

(1) a first nucleic acid sequence containing a first expression cassette for expressing the CAR; and

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

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

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

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

In another preferred embodiment, when the first and second nucleic acid sequences are located on the same vector, a third nucleic acid sequence is further included between the first and second nucleic acid sequences, and the third nucleic acid sequence comprises a third expression cassette for expression of the linker peptide.

In another preferred embodiment, the connecting peptide is P2A.

In another preferred embodiment, the vector is a viral vector, preferably the viral vector comprises the first and second nucleic acid sequences in tandem.

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

Drawings

Figure 1 shows the structure of a contemporary CAR molecule.

Figure 2 shows the structure of the CAR molecule.

Figure 3 shows the expression rate of the NKG2D CAR molecule as flow-detected.

Figure 4 shows flow assay expression rates of CEA CAR molecules.

FIG. 5 shows the expression rate of CCR2b as detected by flow.

FIG. 6 shows the flow-measured expression rate of NKG2D ligand (MICA/MICB) in target cells.

FIG. 7 shows the expression rate of NKG2D ligand (ULBP-2/5/6) in target cells by flow assay.

FIG. 8 shows the expression rate of NKG2D ligand (ULBP-3) in target cells by flow assay.

FIG. 9 shows the expression rate of NKG2D ligand (ULBP-4) in target cells by flow assay.

FIG. 10 shows the expression rate of CEA in target cells by flow assay.

FIG. 11 shows the killing effect of NKG2D CAR-T cells detected by the EuTDA method.

FIG. 12 shows the killing effect of CEA CAR-T cells detected by the EuTDA method.

FIG. 13 shows ELISA detection of IFN- γ release levels from CAR-T cells.

FIG. 14 shows the principle and results of Transwell's detection of the chemotactic migration ability of CAR-T cells.

Detailed Description

The present inventors have extensively and intensively studied and, for the first time, have expressed specific CAR and CCR2b proteins, i.e., NKG2D Extracellular Domain (ED) -containing CAR and CCR2b in combination in CAR-T cells, through extensive screening. Compared with the prior art, the immune cell can improve the sensitivity of CAR-T cells to CCL2, CCL7 and other chemotactic factors through CCR2b, efficiently migrate to the focus of colorectal cancer, ovarian cancer, pancreatic cancer and other solid tumors, and improve the treatment efficiency; meanwhile, multiple target antigens (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6) on the surfaces of malignant tumor cells of colorectal cancer, ovarian cancer, pancreatic cancer and the like can be recognized through the NKG2D CAR molecules, so that the risk of reduced curative effect caused by tumor heterogeneity or target antigen loss is reduced. Meanwhile, research shows that NKG2D CAR-T can also target immunosuppressive cells and new vessels in a tumor microenvironment, help T cells to overcome the immunosuppressive tumor microenvironment, and improve the tumor treatment effect. The present invention has been completed based on this finding.

The invention takes CAR-T cells as an example, and representatively describes the engineered immune cells of the invention in detail. The engineered immune cells of the invention are not limited to the CAR-T cells described above and below, and the engineered immune cells of the invention have the same or similar technical features and benefits as the CAR-T cells described above and below. Specifically, when the immune cells express the chimeric antigen receptor CAR, the NK cells are identical to T cells (or the T cells can be replaced with NK cells).

Term(s) for

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

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

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

Antibodies

As used herein, the term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

Antigen binding domains

As used herein, "antigen binding domain" and "single chain antibody fragment" each refers to a Fab fragment, Fab 'fragment, F (ab') ₂ A fragment, or a single Fv fragment. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain.

In the present invention, the scFv comprises an NKG2D extracellular domain or an active fragment thereof that specifically recognizes a tumor highly expressed antigen.

In addition, the immune cells of the present invention may also contain additional antibodies, preferably single chain antibodies or Fv antibodies, that specifically recognize antigens highly expressed by tumors.

Chimeric Antigen Receptor (CAR)

As used herein, a Chimeric immunoantigen receptor (CAR) includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes an optional signal peptide and a target-specific binding domain (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory domain and a CD3 zeta chain portion. When the CAR is expressed in T cells, the extracellular domain recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activation and proliferation of the cell, cytolytic toxicity and secretion of cytokines such as IL-2 and IFN- γ, etc., affecting the tumor cell, causing the tumor cell to not grow, to be forced to die or otherwise affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the CD3 zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain and the CD3 zeta signaling domain.

In one embodiment, the CARs of the invention target NKG2D ligand, and are capable of specifically binding MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP 6.

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to a CAR-T cell according to the first aspect of the invention. The CAR-T cell can be used for treating tumors with high NKG2D ligand expression, such as colorectal cancer, ovarian cancer, pancreatic cancer and the like.

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

Chimeric antigen receptor NK cells (CAR-NK cells)

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to a CAR-NK cell according to the first aspect of the invention. The CAR-NK cells can be used for treating tumors with high expression of NKG2D ligand, such as colorectal cancer, ovarian cancer, pancreatic cancer and the like.

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

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

In the present invention, NKG2D includes wild type or mutant or derivative forms thereof or active fragments thereof. Preferred NKG2D is from mammalian (e.g., human and non-human primates) NKG 2D.

The accession number of the amino acid sequence of human NKG2D protein is NP-031386, and the accession number of the nucleotide amino acid sequence is NM-007360. The full-length amino acid sequence of human NKG2D is shown below:

MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQD LLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV(SEQ ID No: 1)

wherein, positions 1-51 are intracellular domains; positions 52-72 are transmembrane regions; positions 73-216 are the NKG2D extracellular domain (underlined).

Chemotactic factor

Chemokines are a specific class of cytokines, comprising more than 50 members. The compounds are divided into four types of CC, CXC, CX3C and XC according to the structure; chemokine receptors are classified into the CCR, CXCR, CX3CR, and XCR4 members, with about 20 members.

In the engineered immune cell of the present invention, the chemokine receptor expressed is CCR2b protein, and the chemokines that can be bound include CCL2, CCL7, CCL8, CCL12, CCL13, CCL16, and the like.

The accession number of the amino acid sequence of the CCR2b protein is NP _001116868.1, and the accession number of the nucleotide amino acid sequence is NM _ 001123396.4. The specific sequence is as follows:

amino acid sequence:

MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGN

MLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLY

HIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTK

CQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHR

AVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCI

NPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL

(SEQ ID No: 2)

the nucleotide sequence is as follows:

ATGCTGTCCACATCTCGTTCTCGGTTTATCAGAAATACCAACGAGAGCGGTGAAGAAGTCACCACCTTTT

TTGATTATGATTACGGTGCTCCCTGTCATAAATTTGACGTGAAGCAAATTGGGGCCCAACTCCTGCCTCC

GCTCTACTCGCTGGTGTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCGTCCTCATCTTAATAAACTGC

AAAAAGCTGAAGTGCTTGACTGACATTTACCTGCTCAACCTGGCCATCTCTGATCTGCTTTTTCTTATTA

CTCTCCCATTGTGGGCTCACTCTGCTGCAAATGAGTGGGTCTTTGGGAATGCAATGTGCAAATTATTCAC

AGGGCTGTATCACATCGGTTATTTTGGCGGAATCTTCTTCATCATCCTCCTGACAATCGATAGATACCTG

GCTATTGTCCATGCTGTGTTTGCTTTAAAAGCCAGGACGGTCACCTTTGGGGTGGTGACAAGTGTGATCA

CCTGGTTGGTGGCTGTGTTTGCTTCTGTCCCAGGAATCATCTTTACTAAATGCCAGAAAGAAGATTCTGT

TTATGTCTGTGGCCCTTATTTTCCACGAGGATGGAATAATTTCCACACAATAATGAGGAACATTTTGGGG

CTGGTCCTGCCGCTGCTCATCATGGTCATCTGCTACTCGGGAATCCTGAAAACCCTGCTTCGGTGTCGAA

ACGAGAAGAAGAGGCATAGGGCAGTGAGAGTCATCTTCACCATCATGATTGTTTACTTTCTCTTCTGGAC

TCCCTATAATATTGTCATTCTCCTGAACACCTTCCAGGAATTCTTCGGCCTGAGTAACTGTGAAAGCACC

AGTCAACTGGACCAAGCCACGCAGGTGACAGAGACTCTTGGGATGACTCACTGCTGCATCAATCCCATCA

TCTATGCCTTCGTTGGGGAGAAGTTCAGAAGGTATCTCTCGGTGTTCTTCCGAAAGCACATCACCAAGCG

CTTCTGCAAACAATGTCCAGTTTTCTACAGGGAGACAGTGGATGGAGTGACTTCAACAAACACGCCTTCC

ACTGGGGAGCAGGAAGTCTCGGCTGGTTTATAA (SEQ ID No: 11)

expression cassette

As used herein, "expression cassette" or "expression cassette of the invention" includes a first expression cassette and a second expression cassette. Expression cassette according to the fifth aspect of the invention, the first expression cassette comprises a nucleic acid sequence encoding said CAR. The second expression cassette expresses an exogenous CCR2b protein.

In the present invention, the CCR2b protein may be constitutively expressed or inducibly expressed.

(ii) in the case of inducible expression, the second expression cassette expresses a CCR2b protein when the CAR-T cell is activated by the corresponding inducer; thus, the second expression cassette does not express CCR2b protein when the CAR-T cells of the invention are not contacted with the corresponding inducer.

In one embodiment, the first expression cassette and the second expression cassette each further comprise a promoter and/or a terminator. The promoter of the second expression cassette may be a constitutive or inducible promoter.

Carrier

The invention also provides a vector containing the expression cassette. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of a transgene into a cell genome and replication with replication of the daughter cell genome. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, because they can transduce non-proliferating cells and have the advantage of low immunogenicity.

In general, the expression cassette or nucleic acid sequence of the invention can be ligated downstream of a promoter by conventional procedures and incorporated into an expression vector. The vector may integrate into the genome of eukaryotic cells and replicate in response thereto. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

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

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

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

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

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

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

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

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

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

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

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

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

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

Preparation

The invention provides a pharmaceutical composition comprising an engineered immune cell according to the first aspect of the invention (e.g. a CAR-T cell), and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10 ³ -1×10 ⁸ Individual cells/ml, more preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

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

Therapeutic applications

The invention encompasses therapeutic applications of cells (e.g., T cells) transduced with vectors (e.g., lentiviral vectors) comprising expression cassettes of the invention. The transduced T cells can target the surface markers of the tumor cells and express CCR2b protein, and the killing efficiency of the T cells on the tumor cells is synergistically and remarkably improved.

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

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

In one embodiment, the CAR-T cells of the invention can undergo stable in vivo expansion and can last for a period of months to years. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells can induce a specific immune response to the highly expressed tumor cells of the antigen recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the invention elicit a specific immune response against tumor cells with high expression of NKG2D ligand.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Types of cancer treated with the CARs of the invention include, but are not limited to: colorectal cancer, ovarian cancer, and pancreatic cancer.

Generally, cells activated and expanded as described herein can be used for the treatment and prevention of diseases such as tumors. Accordingly, the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell of the invention.

The CAR-T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

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 condition of the patient, and the type and severity of the patient's disease, or may be determined by clinical trials.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). Pharmaceutical compositions comprising T cells described herein can be in the range of 10 ⁴ To 10 ⁹ Dosage of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁷ Doses (including all integer values within the range) of individual cells/kg body weight are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J. of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous 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 compositions of the invention are preferably administered by intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

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

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ⁵ To 1 × 10 ¹⁰ Personal notebookThe modified T cells of the invention are administered to a patient, for example, by intravenous infusion.

The main advantages of the invention

(1) The invention utilizes CCR2b protein to ensure that the immune cells of the invention can migrate to the tumor part more efficiently, thereby obviously improving the effect of inhibiting the tumor and reducing toxic and side effects. Experiments show that the invention obviously improves the capability of CAR-T cells to migrate to the high concentration of CCL2 (such as a focus part).

(2) The antigen binding domain of the engineered immune cell adopts the extracellular binding domain of NKG2D, can recognize 8 target antigens (MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6) on the cell surface of malignant tumors (such as colorectal cancer cells, ovarian cancer, pancreatic cancer and the like) through NKG2D CAR molecules, and reduces the risk of reduced curative effect caused by tumor heterogeneity or target antigen loss.

(3) Expression of the exogenous CCR2b protein unexpectedly promoted the expression rate of NKG2D CAR molecules in immune cells.

(4) Expression of NKG2D CAR molecules also promoted expression of exogenous CCR2 b.

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

Materials and methods

CAR molecules and structures thereof

In the examples, CEA CAR-T was used as a control for NKG2D CAR-T cells. The NKG2D CAR and CEA CAR molecules comprise the following partial structures, respectively: human CD8 signal peptide [ abbreviated as CD8(SP) ], human NKG2D extracellular domain [ abbreviated as NKG2D (ED) ], human anti-CEA single-chain antibody [ abbreviated as CEAscFv (hMN-14) ], optimized human CD8 hinge region [ abbreviated as CD8(hinge) ] and human CD8 transmembrane domain [ abbreviated as CD8(TM) ], human 4-1BB intracellular domain [ abbreviated as 4-1BB (ID) ], human CD3 zeta intracellular signal transduction domain [ abbreviated as CD3 zeta (ID) ], self-cleaving peptide P2A, human CCR2 b.

The second generation NKG2D CAR molecule as control was named BN 001;

a new generation of NKG2D CAR molecule that jointly expresses CCR2 is named BN 003;

a second generation CEA CAR molecule, as a control, was designated BC001 and a new generation CEA CAR molecule, which jointly expresses CCR2, was designated BC 006.

The specific structure of the CAR molecule is shown in fig. 2, specifically as follows:

BN001 is composed of CD8(SP), NKG2D (ED), CD8(hinge), CD8(TM), 4-1BB (ID), and CD3 zeta (ID) connected in series in sequence from the amino terminal to the carboxyl terminal.

BN003 is composed of, in sequence from the amino terminus to the carboxy terminus, CD8(SP), NKG2D (ED), CD8(hinge), CD8(TM), 4-1BB (ID), CD3 zeta (ID), P2A and CCR2b in series.

BC001 is composed of CD8(SP), CEAscFv (hMN-14), CD8(hinge), CD8(TM), 4-1BB (ID), and CD3 zeta (ID) connected in series in sequence from amino terminal to carboxyl terminal.

BC006 is composed of CD8(SP), CEAscFv (hMN-14), CD8(hinge), CD8(TM), 4-1BB (ID), CD3 zeta (ID), P2A, CCR2b connected in series in sequence from amino terminus to carboxy terminus.

Amino acid sequence

SEQ ID NO:1(NKG2D amino acid sequence)

MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

SEQ ID NO 2(CCR2b amino acid sequence)

MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL

SEQ ID No. 3 (human CD8 signal peptide amino acid sequence)

MALPVTALLLPLALLLHAARPS

SEQ ID No. 4 (human NKG2D extracellular domain amino acid sequence)

IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

SEQ ID No. 5 (human anti-CEA single-chain antibody amino acid sequence)

DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGGGTKVGIKGGGGSGGGGSGGGGSEVQLVESGGGVVQPGRSLRLSCSSSGFDFTTYWMSWVRQAPGKGLEWVAEIHPDSSTINYAPSLKDRFTISRDNSKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS

SEQ ID No. 6 (optimized amino acid sequence of hinge region of human CD 8)

TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFA

SEQ ID No. 7 (human CD8 transmembrane domain amino acid sequence)

CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR

SEQ ID No. 8 (human 4-1BB intracellular domain amino acid sequence)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

SEQ ID No. 9 (amino acid sequence of intracellular signaling domain of human CD3 ζ)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 10 (self-cleaving peptide P2A amino acid sequence)

GSGATNFSLLKQAGDVEENPGP

Example 1 Lentiviral preparation

1.1 acquisition of lentiviral vector plasmids

The nucleotide sequences of BN001, BN003, BC001 and BC006 are synthesized by the whole gene, and then are connected to a lentiviral vector pCDH-EF1-MCS-T2A-copGFP plasmid in a molecular cloning mode, so that the lentiviral vector is expressed under the control of a human EF1 alpha promoter and a Kozak sequence.

Transfection of 293T cells with lentiviral vector plasmids

The above lentiviral vector plasmids were mixed with lentiviral packaging plasmids pMD2.G, pRSV-Rev and pMDLg/pRRE using polyethyleneimine transfection reagent to cotransfect 293T cells. After culturing for 48 h, respectively collecting virus supernatants, centrifuging at 4500 rpm at 4 ℃ for 10-15 min, filtering with a filter membrane with the aperture of 0.5 mu m, concentrating lentivirus with a hollow fiber column ultrafiltration system, purifying lentivirus with chromatography, filtering with a filter membrane with the aperture of 0.22 mu m for sterilization, and subpackaging at-80 ℃ for storage.

Lentiviral titer determination

The concentration of Jurkat cells was adjusted to 1X 10 ⁵ At 300. mu.l, after mixing well, 300. mu.l of the resuspended cells were taken into each well of a 24-well plate. 70. mu.l of the lentivirus concentrate was diluted in 5-fold gradient in Opti-MEM medium. The lentivirus of each dilution gradient was added to the above 24-well plate at a rate of 200. mu.l/well, and the lentivirus-infected Jurkat cells (the Jurkat cells of the negative control group were added to Opti-MEM medium only) were cultured in a cell culture chamber (culture temperature: 37 ℃ C., carbon dioxide concentration: 5%). After 3 days of incubation, the cells in each well were gently mixed and transferred to a 1.5-ml centrifuge tube, washed twice with staining buffer (100 ml PBS + 1% BSA) and centrifuged at 800 g for 3 min each time. The cells were stained with the corresponding antibodies and the proportion of Jurkat cells successfully transduced by lentivirus was determined by flow cytometry. The lentivirus infection rate of Jurkat cells was taken as P and the lentivirus titer was calculated by the following formula:

lentiviral titer (TU/ml) = P/V × 10 ³ ×10 ⁵

As a result: BN001 titer of 5.46X 10 ⁸ TU/ml, BN003 titre 2.46X 10 ⁸ TU/ml, BC001 Titer 2.58X 10 ⁸ TU/ml, BC006 titer 5.66X 10 ⁸ TU/ml。

Example 2 preparation and detection of CAR-T cells

2.1T cell preparation

Will be outside of healthy donorThe density of mononuclear cells in the peripheral blood is adjusted to 2X 10 ⁶ And adding 50 ng/ml anti-CD 3 antibody, 50 ng/ml anti-CD 28 antibody and 200 IU/ml recombinant IL-2, and culturing in a cell culture box for 24 h (the culture temperature is 37 ℃ and the carbon dioxide concentration is 5%).

Lentivirally transduced T cells

The obtained T cells were washed and the cell density was adjusted to 2X 10 ⁶ And/ml. Lentivirus was added at an MOI = 1-10 TU/cell for transduction while supplementing 50 ng/ml anti-CD 3 antibody, 50 ng/ml anti-CD 28 antibody, and 200 IU/ml recombinant IL-2, and cultured in a cell culture chamber (culture temperature 37 ℃ C., carbon dioxide concentration 5%). After 24 hours, the cell density is adjusted to 1.5-2 x 10 ⁶ And 300 IU/ml IL-2. On day 4 after transduction, the cells were washed to remove residual lentiviral particles in the supernatant, and cultured in a cell culture chamber for 5 days (culture temperature 37 ℃ C., carbon dioxide concentration 5%) while maintaining the cell density at 1-2X 10 ⁶ And (4) the concentration is/ml. Cells were harvested on day 10 post transduction and frozen in liquid nitrogen with a freezing medium (5% human serum albumin in freezing medium: physiological saline = 1:1) for future use. The CAR-T cells obtained followed the nomenclature of the corresponding CAR molecules BN001, BN003, BC001 and BC006, respectively, and T cells not transduced with lentiviruses named Ctrl T.

Expression detection of molecules

Ctrl T, BN001 and BN003 cells to be detected were washed twice with PBS and resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). The APC-labeled anti-human NKG2D antibody and the PE-labeled anti-human CD3 antibody were added to the cell suspension to be detected according to the antibody instructions and incubated at 4 ℃ for 30 min. And (3) detecting the expression rate of the NKG2D CAR molecules of BN001 and BN003 cells by using Ctrl T cells as negative controls through a flow cytometer. Analysis was performed using CytExpert software.

As shown in fig. 3, the expression rate of the NKG2D CAR molecule in BN001 cells was about 87.2%, and the expression rate of the NKG2D CAR molecule in BN003 cells was about 92.2%. It was unexpectedly found that the combined expression of CCR2b increased the expression rate of NKG2D CAR molecules (by about 5.7%). This suggests an increased lethality of BN003, which jointly expresses CCR2b + NKG 2D.

Expression detection of molecules

The Ctrl T, BC001 and BC006 cells to be detected were washed twice with PBS and resuspended in FACS buffer. The APC-labeled CEA protein and PE-labeled anti-human CD3 antibody were added to the cell suspension to be detected according to the antibody instructions and incubated at 4 ℃ for 30 min. Ctrl T cells not transfected with lentivirus were used as negative control, and the expression rate of CEA CAR molecules in BC001 and BC006 cells was measured by flow cytometry. Analysis was performed using CytExpert software.

The results are shown in FIG. 4, where the expression rate of the CEA CAR molecule by BC001 cells was about 71.5%, and the expression rate of the CEA CAR molecule by BC006 cells was about 36.1%. The combined expression of CCR2b decreased the expression rate of CEA CAR molecules (by about 50.5%). This suggests that the killing of BC006 in combination with CCR2b would be reduced.

Detection of expression of

The Ctrl T, BN001, BN003, BC001 and BC006 cells to be detected were washed twice with PBS and resuspended in FACS buffer. PE-labeled anti-human CCR2b antibody and APC-labeled anti-human CD3 antibody were added to the cell suspension to be detected according to the antibody instructions and incubated at 4 ℃ for 30 min. Ctrl T cells which are not transfected by lentivirus are used as negative control, and the CCR2b expression rate of the CAR-T cells is detected by a flow cytometer. Analysis was performed using CytExpert software.

The results are shown in FIG. 5, wherein the expression rate of CCR2b in BC006 cells is about 26.67%, and the expression rate of CCR2b in BN003 cells is about 55.4%, which indicates that the expression level of CCR2b is reduced when the cells are combined with CEA.

Example 3 target cell detection

3.1 conditions for culturing target cells

Colorectal cancer cell lines (also known as target cells or target cell lines) to be tested: HCT116 (McCoy's 5a medium + 10% fetal calf serum + 100U/ml penicillin + 100. mu.g/ml streptomycin), HT-29(McCoy's 5a medium + 10% fetal calf serum + 100U/ml penicillin + 100. mu.g/ml streptomycin), LoVo (F-12K medium + 10% fetal calf serum + 100U/ml penicillin + 100. mu.g/ml streptomycin), SW480(Leibovitz's L-15 medium + 10% fetal calf serum + 100U/ml penicillin + 100. mu.g/ml streptomycin), T84(DMEM/F-12 medium + 5% fetal calf serum + 100U/ml penicillin + 100. mu.g/ml streptomycin), SK-OV-3(McCoy's 5a medium + 10% fetal bovine serum + 100U/ml penicillin + 100. mu.g/ml streptomycin).

Expression detection of ligand (MICA/MICB)

The target cells were washed twice with PBS and resuspended in FACS buffer. APC-labeled anti-human MICA/MICB antibody was added to each target cell suspension according to the antibody instructions and incubated at 4 ℃ for 30 min. The MICA/MICB expression rate of the target cells was measured by flow cytometry using the target cells incubated without the addition of the antibody as a negative control. Analysis was performed using CytExpert software.

As shown in FIG. 6, the MICA/MICB expression rates of HCT 166, HT-29 and SW480 cells were between 96% and 100%, that of T84 cells was about 56%, that of LoVo cells was about 7% and that of SK-OV-3 cells was about 1.8%.

Expression rate of ligand (ULBP-2/5/6)

The target cells were washed twice with PBS and resuspended in FACS buffer. PE-labeled anti-human ULBP-2/5/6 antibody was added to each target cell suspension according to the antibody instructions and incubated at 4 ℃ for 30 min. Using the target cells incubated without the added antibody as a negative control, the expression rate of ULBP-2/5/6 was measured by flow cytometry. Analysis was performed using CytExpert software.

As shown in FIG. 7, the expression rate of ULBP-2/5/6 of T84 cell was about 1.6%, the expression rate of ULBP-2/5/6 of HCT116, HT-29, LoVo and SW480 cells was 86-99%, and the expression rate of ULBP-2/5/6 of SK-OV-3 cell was about 56.9%.

Expression rate of ligand (ULBP-3)

The target cells were washed twice with PBS and resuspended in FACS buffer. PE-labeled anti-human ULBP-3 antibody was added to each target cell suspension according to the antibody instructions and incubated at 4 ℃ for 30 min. And detecting the expression rate of the ULBP-3 of the target cells by using a flow cytometer by taking the target cells which are not incubated by adding the antibody as negative controls. Analysis was performed using CytExpert software.

As shown in FIG. 8, the expression rates of ULBP-3 in HCT 166, HT-29 and SW480 cells were between 59% and 95%, the expression rate of ULBP-3 in LoVo cells was about 21%, the expression rate of ULBP-3 in T84 cells was less than 3%, and the expression rate of ULBP-3 in SK-OV-3 cells was about 33.1%.

Expression rate of ligand (ULBP-4)

The target cells were washed twice with PBS and resuspended in FACS buffer. PE-labeled anti-human ULBP-4 antibody was added to each target cell suspension according to the antibody instructions and incubated at 4 ℃ for 30 min. And detecting the expression rate of the ULBP-4 of the target cells by using a flow cytometer by taking the target cells which are not incubated by adding the antibody as negative controls. Analysis was performed using CytExpert software.

As shown in FIG. 9, the ULBP-4 expression rates of HCT116, HT29, LoVo and SW480 were all above 82.2%, the ULBP-4 expression rate of T84 was about 2.0%, and the ULBP-4 expression rate of SK-OV-3 cells was about 71.6%.

Detection of expression of

The cells to be tested were washed twice with PBS and resuspended in FACS buffer. anti-CEA primary antibody was added to the cell suspension to be tested according to the antibody instructions and incubated at 4 ℃ for 60 min. After washing with PBS, FITC-labeled secondary antibody was added and incubated at 4 ℃ for 60 min. Target cells incubated with FITC-labeled IgG alone were used as negative controls, and the CEA expression rate of the target cells was measured by flow cytometry. Analyzed using CytExpert software.

As shown in FIG. 10, the CEA expression rate of LoVo cells was about 60%, that of T84 cells was about 75%, that of HCT116, HT-29 and SW480 was between 0.06% and 6%, and that of SK-OV-3 cells was about 0.01%.

Example 4 in vitro function of CAR-T cells

4.1 detection of killing Effect by EuTDA method

According to the flow detection result of the target antigen, the cells HCT116, HT-29, SW480 and SK-OV-3 are selected to detect the killing effect of NKG2D CAR-T cells, and LoVo and T84 are selected to detect the killing effect of CEA CAR-T cells. Target cells were washed once with AIM-V medium. Adjusting the target cell density to 1 × 10 ⁶ At a concentration of 2. mu.l/ml, DELFIA BATDA Reagent was added thereto, mixed, and incubated at 37 ℃ for 30 min. After washing the target cells three times with AIM-V medium, 1X 10 ⁴ Density of pores will targetCells were seeded in 96-well plates. 100 μ l T cells (effective target ratio of 2.5:1, 5:1 and 10:1) were added and incubated in a carbon dioxide incubator for 2 h (incubation temperature 37 ℃ C., carbon dioxide concentration 5%). Finally, centrifugation was carried out at 500 g for 5 min, and 20. mu.l of the supernatant was transferred to a 96-well plate to which Europium solution (200. mu.l/well) was added. After incubation at room temperature for 15 min, detection was carried out in a microplate reader.

As shown in FIG. 11, in the conditions of the target effect ratios of 2.5:1, 5:1 and 10:1, the killing rates of BN001 to HCT116 cells are respectively 45% + -2%, 81% + -4% and 91% + -1%, the killing rates to HT-29 cells are respectively 49% + -5%, 87% + -5% and 90% + -6%, the killing rates to SW480 are respectively 55% + -9%, 70% + -9% and 66% + -1%, and the killing rates to SK-OV-3 are respectively 14% + -1%, 25% + -1% and 42% + -1%; under the conditions of target effect ratios of 2.5:1, 5:1 and 10:1, the killing rates of BN003 to HCT116 cells are respectively 50% + -1%, 71% + -5% and 84% + -6%, the killing rates to HT-29 cells are respectively 43% + -8%, 78% + -8% and 90% + -6%, the killing rates to SW480 are respectively 43% + -3%, 61% + -5% and 67% + -3%, and the killing rates to SK-OV-3 are respectively 14% + -1%, 25% + -1% and 46% + -2%. Statistical analysis shows that the killing effect of BN001 and BN003 on corresponding target cells is not statistically different and is significantly higher than that of Ctrl T cells, which indicates that the combined expression CCR2b does not have negative influence on the killing effect of NKG2D CAR-T cells (when the CAR-T cells are co-cultured, the CAR-T cells can be directly contacted with the target cells without chemotactic migration, so in an in vitro detection experiment, even if the improvement effect of the combined expression CCR2b on the killing effect of the CAR-T cells is not observed, the condition is normal).

In addition, as shown in FIG. 12, the killing rates of BC001 to LoVo cells were 56% + -2%, 89% + -1%, 96% + -4%, and the killing rates to T84 were 26% + -1%, 49% + -6%, 77% + -3%, respectively, at the targeting efficiency ratios of 2.5:1, 5:1, and 10:1, respectively; under the conditions of target effect ratios of 2.5:1, 5:1 and 10:1, the killing rates of BC006 on LoVo cells are respectively 49% + -6%, 64% + -8% and 86% + -12%, and the killing rates on T84 cells are respectively 28% + -1%, 46% + -2% and 75% + -2%. Statistical analysis shows that the killing effect of BC001 and BC006 cells on T84 cells is not statistically different, but the killing effect of BC006 cells on LoVo cells is significantly lower than that of BC001, which indicates that the combined expression of CCR2b can cause the reduction of the killing effect of CEA CAR-T cells in part cases.

Gamma secretion assay

Ctrl T, BN001, BN003, BC001 and BC006 were co-cultured with the corresponding target cells, respectively, in AIM-V medium without IL-2 (effective to target ratio of 2.5: 1). After 24 h, ddH ₂ And dissolving the IFN-gamma standard substance by using O, standing at room temperature for 15-20 min to ensure full dissolution, and diluting the standard substance according to the recommended gradient multiple ratio. The cell supernatant from the above co-culture was aspirated and washed with ddH ₂ O was diluted 2-fold and 20-fold. The standard and experimental samples were added to the corresponding reaction wells, respectively, at 100. mu.l per well. And after incubation for 1-3 h at room temperature, preparing 1 × cleaning solution, cleaning each hole for 4 times by using 360 μ l of cleaning solution, drying the liquid in each hole, adding 200 μ l of enzyme-labeled detection antibody into each hole, and incubating for 1-3 h at room temperature. Each well was washed 4 times with 360. mu.l of wash solution, and after the wells were patted dry, 200. mu.l of chromogenic substrate was added. And incubating for 30-60 min at room temperature in a dark place, adding 50 mu l of stop solution into each hole, and measuring the light absorption value at 450 nm by using an enzyme-labeling instrument.

As shown in FIG. 13, the IFN-. gamma.release levels of BN001 to HCT116 cells, HT-29 cells, SW480 cells and SK-OV-3 cells were 2425. + -. 453 pg/ml, 10066. + -. 644 pg/ml, 5083. + -. 111 pg/ml and 8309. + -. 979 pg/ml, respectively, at a target-to-effect ratio of 2.5: 1; the IFN-gamma release level of BN003 on HCT116 cells, HT-29 cells, SW480 cells and SK-OV-3 cells is 25502 +/-1756 pg/ml, 10524 +/-312 pg/ml, 7157 +/-431 pg/ml and 9055 +/-241 pg/ml respectively; the IFN-gamma release level of BC001 to LoVo cells and T84 cells is 8611 +/-480 pg/ml and 10413 +/-383 pg/ml respectively; the IFN-gamma release level of BC006 on LoVo cells and T84 cells was 4845. + -.356 pg/ml and 6279. + -.382 pg/ml, respectively. Statistical analysis shows that IFN-gamma of BN001, BN003, BC001 and BC006 cells is significantly higher than that of Ctrl T cells, which shows that the CAR-T cells can effectively release IFN-gamma after identifying target cells; wherein the levels of IFN- γ release from BN001 and BN003 on HCT116, HT-29 and SK-OV-3 cells are not significantly different, while the levels of IFN- γ release from BN003 on SW480 are significantly higher than that of BN001, indicating that under certain circumstances, the combined expression of CCR2b enhances the activity of NKG2D CAR-T cells; in contrast, BC001 showed significantly higher IFN- γ release levels on both LoVo and T84 cells than BC006, suggesting that the combined expression of CCR2b negatively affected the IFN- γ release levels on CEA CAR-T cells.

Chemotactic migration assay

600 μ l of CCL2 protein (25 ng/ml) diluted in complete medium was added to the lower chamber of a Transwell 24-well plate. 100. mu.l of each of 4X 10 density beads were placed in a Transwell plate top chamber with a pore size of 8 μm ⁵ CAR-T cells/ml, and cultured in a carbon dioxide incubator (37 ℃ C. at a carbon dioxide concentration of 5%). After 6 h, the medium in the lower chamber was aspirated, the cells therein were mixed and the number of T cells transferred to the lower chamber was counted using a hemocytometer.

As a result, as shown in FIG. 14, when CCL2 protein was not added to the lower chamber, the numbers of cells migrating to the lower chamber were about 2.14X 10 for BN001, BN003, BC001, and BC006, respectively ⁵ 1.99X 10 ⁵ 9.90 x 10 ⁴ 1.20 x 10 ⁵ A plurality of; when 25 ng/ml of CCL2 protein was added to the lower chamber, the number of cells migrating to the lower chamber was about 2.18X 10 for BN001, BN003, BC001 and BC006, respectively ⁵ 2.87X 10 ⁵ 8.61X 10 ⁴ 1.34 × 10 ⁵ And (4) respectively. The number of BN003 cells migrating to the lower chamber is remarkably higher than that of BN001 cells serving as a control, and the migration efficiency is improved by about 31.6%; although the number of cells that BC006 migrated to the lower chamber was also significantly higher than BC001 cells as a control, the migration efficiency was improved by about 55.6%, but the migration capacity of BC006 was only 46.7% of BN 003. The above results demonstrate that the combined expression of CCR2b can effectively mediate migration of CAR-T cells to CCL2 at high concentrations. Among them, NKG2D CAR-T cells, which jointly express CCR2b, had the highest ability to migrate directionally.

Unexpectedly, the chemokine concentration and the culture time used in the Transwell experiment are equivalent to those of the prior art (the experimental conditions are the same as those of patent CN111607006B), but the detected proportion of cells migrating is significantly higher (BN003 migrates about 71.8% of the cells under the condition that 25 ng/ml CCL2 induces 6 h) than that of the prior art (KD-207 cells migrate about 40% of the cells under the condition that 25 ng/ml CXCL2 induces 6 h), further indicating that the NKG2D CAR-T cells jointly expressing CCR2b can efficiently migrate directionally.

Example 5 CAR-T cells in vivo tumor suppressor function

The combined expression of CCR2b in NKG2D CAR-T cells more enhanced the ability to chemotactic migrate compared to CEA CAR-T cells. Therefore, in this example, a test for the effect of inhibiting subcutaneous transplantation tumor in mice was conducted using BN001 and BN003 as effector cells and SK-OV-3 as target cells. Experiments were performed with immunodeficient B-NDG mice to observe the effect of NKG2D CAR-T cells in combination expressing CCR2 on tumor infiltration and inhibition. The method comprises the following steps:

24B-NDG mice of 6-8 weeks old are taken to carry out subcutaneous tumor efficacy experiments, and are randomly divided into 4 groups, 6 mice in each group are respectively a solvent control group, a Ctrl T control group, a BN001 control group and a BN003 experiment group.

Collecting target cells in logarithmic growth phase and good growth state by trypsinization, washing with physiological saline for 1 time, and adjusting cell density to 2 × 10 ⁷ And/ml. The right side of B-NDG mice was injected subcutaneously near the underarm with 100. mu.l of cell suspension, i.e., each mouse was inoculated with 2X 10 cells ⁶ The day of inoculation of the target cells of (4), day 0.

Day 7 after target cell inoculation (or tumor mean volume of about 100 mm) ³ Time), CAR-T cells (1 × 10) were injected separately via tail vein ⁷ Per), Ctrl T cells (1X 10) ⁷ One) and vehicle (100 μ l/one), the day of injection of the test substance is recorded as day 0 of treatment. Measuring the size of the tumor and the weight of the mouse 2-3 times every week, collecting blood on

days

3, 10, 28 and 42, adding EDTA for anticoagulation, detecting the retention condition of CAR-T cells in blood cells in the mouse by qPCR, and detecting IFN-gamma by ELISA to monitor the release level of the cytokine. After 50 days of treatment, the mice are euthanized, tissues such as tumors, hearts, livers, spleens, lungs, kidneys, brains, ovaries and the like are weighed, tumor tissues of 2 mice in each group are stored in a refrigerator at 80 ℃ for extracting DNA, and the infiltration condition of CAR-T cells in the tumor tissues and the distribution condition of the CAR-T cells in each organ are detected; tumor tissues of 2 mice were fixed in each group, and morphology of tumor cells was detected by HE staining and tissues were detected by immunohistochemistryExpression profile of antigen.

Results As shown in Table 1, the mean tumor volume of vehicle control mice was about 1588 mm 50 days after CAR-T cell injection ³ (ii) a The mean tumor volume of Ctrl T control mice was about 1530 mm ³ (ii) a The average tumor volume of BN001 control group mice is about 409 mm ³ (ii) a The average tumor volume of the BN003 experimental group of mice is about 94 mm ³ . The in vivo tumor inhibition effect of BN003 and BN001 is obviously better than that of Ctrl T control group. Meanwhile, the tumor inhibition effect of BN003 is further obviously superior to that of BN 001. The mean tumor volume of mice injected with BN003 cells was significantly reduced compared to BN001, with a reduction of about 77%.

LTR sequences in tumor tissues were examined by qPCR to measure the CAR-T cell infiltration in tumor tissues. As a result, the mean background signal level of LTR in the tumor tissue of the vehicle control group mouse was found to be 30.5 copies/. mu.g DNA; the mean background signal level of LTR within tumor tissue of Ctrl T control mice was 25.2 copies/. mu.g DNA; the mean LTR content in the tumor tissues of the BN001 control group mice was 11852.0 copies/. mu.g DNA; the mean LTR content in tumor tissues of the mice of the BN003 experimental group was 47375.1 copies/. mu.g DNA. The degree of infiltration of BN003 cells in tumor tissue was also significantly higher than BN 001. Compared with BN001, the homing capacity of the BN003 cell to the subcutaneous transplanted tumor tissue of the mouse is obviously improved, and the improvement amplitude is close to 300 percent. The above results all indicate that the new generation of NKG2D CAR-T cells co-expressing CCR2 can migrate to the tumorigenic site with high efficiency, and are superior to the second generation of NKG2D CAR-T cells in therapeutic effect.

TABLE 1 last draw material detection for in vivo efficacy experiments

Discussion of the related Art

In CAR-T treatment of solid tumors such as colorectal cancer, CD133, CEA, EGFR, HER-2, NKG2D ligands and the like are mainly recognized targets. Among them, NKG2D (also called CD314) is an important activating receptor in the innate immune system, mainly expressed on the surface of natural killer cells, γ δ T cells and CD8+ T cells. NKG2D has numerous advantages as an antigen recognition domain of a CAR molecule. Unlike specific antibody CAR molecules directed against a single target, CAR molecules engineered based on NKG2D can recognize at least 8 different NKG2D ligands, including MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, and ULBP-6, and are more beneficial for treating tumors with high heterogeneity or susceptibility to loss of target antigen. In addition, these NKG2D ligands are located on the surface of target cells. Thus, in contrast to TCR-T, NKG2D CAR-T does not require the antigen presentation process of MHC molecules to directly recognize tumor cells. Importantly, the NKG2D ligand has high expression level on tumor cells of epithelial origin such as colorectal cancer, ovarian cancer, pancreatic cancer, leukemia and the like, but does not express or has extremely low expression level in normal cells, and is an ideal target point for tumor specific treatment. Furthermore, the NKG2D CAR-T cell surface does not carry any foreign protein structures that may elicit an immune response in a patient, thereby reducing the likelihood that the CAR-T cell will be rejected by the patient's immune system.

Solid tumor cells can prevent migration and infiltration of CAR-T cells into tumor tissue by secreting chemokines CXCL12 and CXCL 5. Conversely, solid tumor cells secrete less chemokines that can promote migration of CAR-T cells. These two factors make CAR-T cells difficult to reach the solid tumor site. Therefore, improving the specific recognition and sensitivity of CAR-T cells to tumor chemokines is one of the key factors affecting the efficacy of CAR-T therapy, and the combined expression of chemokine receptors in CAR-T cells is an important approach to solve the problem.

Chemokines are a specific class of cytokines, comprising more than 50 members. The compounds are divided into four types of CC, CXC, CX3C and XC according to the structure; chemokine receptors are divided into the CCR, CXCR, CX3CR, and XCR4 species, with about 20 members. One of the main mechanisms of action of chemokines is to modulate the infiltration of immune cells in tissues by inducing the directed migration of immune cells by forming a concentration gradient that is soluble or immobilized in a matrix. At present, partial CAR-T technology adopts a mode of jointly expressing chemokine receptors to promote rapid migration of CAR-T cells to cancer cells, so as to improve the tumor treatment effect of the CAR-T cells.

Different types of solid tumors release different types and levels of chemokines, and have different mechanisms of immune escape. Therefore, selection of appropriate target antigens for a particular cancer species, combined with expression of appropriate chemokine receptors, is critical to improving the therapeutic efficacy of this type of CAR-T cell.

According to the invention, through the specific combination of the specific CAR molecule and the specific chemokine receptor, the problem of targeting malignant tumor focus is efficiently solved, and the problem of malignant tumor heterogeneity is effectively overcome.

On one hand, the invention improves the migration capacity of CAR-T cells to malignant tumor focuses by using CCR2b as a jointly expressed chemokine receptor, thereby improving the treatment efficiency. The chemokine CCL2 mainly recognized by CCR2b is abnormally and highly expressed in various malignant tumors such as colorectal cancer, ovarian cancer, pancreatic cancer and the like, and the expression level of the chemokine CCL2 in normal tissue cells is extremely low. At the same time, CCR2b has very high affinity for CCL2 (about 0.7 nM, IC) ₅₀ The smaller the number, the higher the corresponding affinity and sensitivity), the sensitivity is much higher than for other chemokine receptor and chemokine combinations (e.g., IC for CCR2a/CCL2 combination ₅₀ A combination C of CXCR3/CXCL11 with a value of about 5 nM ₅₀ Values of about 8.2 nM).

On the other hand, the NKG2D is used as the extracellular recognition domain of the CAR molecule, so that a plurality of target antigens (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6) on the surface of a tumor cell can be recognized, the risk of reducing the curative effect caused by tumor heterogeneity or antigen loss can be reduced, and the therapeutic effect on malignant tumors such as colorectal cancer, ovarian cancer, pancreatic cancer and the like can be improved. Therefore, the invention further improves the effectiveness of treatment and the capacity of immune cells such as CAR-T cells to resist the high heterogeneity of malignant tumors.

Meanwhile, research also shows that NKG2D CAR-T can also target immunosuppressive cells and new vessels in a tumor microenvironment, and is beneficial to immune cells such as T cells and the like to overcome the immunosuppressive tumor microenvironment, so that the tumor treatment effect is improved.

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

Sequence listing

<110> Guangzhou Baiji biopharmaceutical Co., Ltd

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aatcccatca tctatgcctt cgttggggag aagttcagaa ggtatctctc ggtgttcttc 960

cgaaagcaca tcaccaagcg cttctgcaaa caatgtccag ttttctacag ggagacagtg 1020

gatggagtga cttcaacaaa cacgccttcc actggggagc aggaagtctc ggctggttta 1080

taa 1083

Claims

1. An engineered immune cell, wherein said engineered immune cell is a T cell, and wherein said immune cell has the following characteristics:

(a) the immune cell expresses a Chimeric Antigen Receptor (CAR), wherein the CAR targets a surface marker of a tumor cell, wherein the antigen-binding domain of the CAR comprises the extracellular domain of NKG 2D; and

(b) the immune cells express exogenous CCR2b protein;

wherein the CAR has the structure shown in formula I:

L-NKG2D-H-TM-C-CD3ζ (I)

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

l is a null or signal peptide sequence;

NKG2D is NKG2D extracellular domain, wherein the amino acid sequence of the extracellular domain of NKG2D is shown in SEQ ID NO. 1, 73-216;

h is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal domain;

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

the "-" is a connecting peptide or a peptide bond.

2. The engineered immune cell of claim 1,

l is a signal peptide sequence of a protein selected from the group consisting of: CD8, GM-CSF, CD4, CD28, or CD 137;

h is a hinge region of a protein selected from the group consisting of: CD8, CD28, or CD 137;

TM is a transmembrane domain of a protein selected from the group consisting of: CD28 or CD 8;

c is a co-stimulatory signaling domain from 4-1 BB.

3. The engineered immune cell of claim 1, wherein, in the CAR represented by formula I,

l is a signal peptide sequence shown in SEQ ID No. 3;

h is a hinge region shown as SEQ ID No. 6;

TM is a transmembrane domain shown in SEQ ID No. 7;

c is a costimulatory signal domain shown in SEQ ID No. 8;

CD3 zeta is the cytoplasmic signaling sequence of CD3 zeta shown in SEQ ID No. 9.

4. The engineered immune cell of claim 1, wherein the amino acid sequence of the CCR2b protein is set forth in SEQ ID No. 2.

5. A method of preparing the engineered immune cell of claim 1, comprising the steps of:

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

(B) engineering the immune cell such that the immune cell expresses the CAR of claim 1 and an exogenous CCR2b protein to obtain the engineered immune cell of claim 1.

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

7. The formulation of claim 6, wherein the concentration of said engineered immune cells in said formulation is 1 x 10 ³ -1×10 ⁸ Individual cells/ml.

8. Use of the engineered immune cell of claim 1 in the preparation of a medicament or formulation for the treatment of cancer, wherein the cancer is a cancer with high expression of the NKG2D ligand.

9. The use of claim 8, wherein the cancer is selected from the group consisting of: colon cancer, rectal cancer, ovarian cancer, pancreatic cancer.

10. The use of claim 8, wherein said cancer is a cancer with high expression of NKG2D ligand and high expression of chemokines.

11. A kit for preparing the engineered immune cell of claim 1, comprising a container, and within the container:

(1) a first nucleic acid sequence comprising a first expression cassette for expressing the CAR of claim 1, wherein the antigen binding domain of the CAR is the extracellular domain of NKG2D, wherein the extracellular domain of NKG2D has the amino acid sequence shown in SEQ ID No. 1, positions 73-216; and