WO2023083192A1

WO2023083192A1 - Engineered immune cell for combined expression of ccr2b and cd40l, and preparation and application thereof

Info

Publication number: WO2023083192A1
Application number: PCT/CN2022/130696
Authority: WO
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: 广州百吉生物制药有限公司
Priority date: 2021-11-10
Filing date: 2022-11-08
Publication date: 2023-05-19
Also published as: CN113755448B; CN113755448A

Abstract

Provided are an engineered immune cell for combined expression of CCR2b and CD40L, and preparation thereof and an application thereof. By simultaneously expressing, in a CAR-T cell in a combined manner, a CAR molecule containing an NKG2D extracellular domain, and CCR2b and CD40L, the sensitivity of the CAR-T cell to a chemokine and the migration capability of the CAR-T cell to a solid tumor lesion can be improved by means of CCR2b; by means of CD40L, the endogenous natural and adaptive immune response of an organism can also be activated, the tumor treat effect is improved, and the relapse risk is reduced; and by means of an NKG2D CAR molecule, various target antigens on the surfaces of malignant solid tumor cells can further be identified, and the risk of reduction in therapeutic effect caused due to tumor heterogeneity or target antigen loss is reduced, thereby improving the tumor treatment effect. The engineered immune cell can synergistically and remarkably improve the in-vitro killing effect on tumor cells.

Description

Engineered immune cells jointly expressing CCR2b and CD40L and its preparation and application

technical field

The invention belongs to the field of tumor immunity and cell therapy, and in particular relates to an engineered immune cell jointly expressing CCR2b and CD40L.

Background technique

Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture, modify and amplify immune cells collected from patients in vitro, and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors.

T cells are an important class of lymphocytes involved in cellular immunity, and can specifically recognize and kill tumor cells through the signal transmission of antigen-presenting cells. However, tumor cells can also be affected by the reduction or loss of antigenic epitopes, immunosuppression, and tumor heterogeneity (that is, the same malignant tumor exists in different patients or between tumor cells in different parts of the same patient from genotype to phenotype). Differences) and other ways to hinder the specific recognition of T cells, thereby evading the body's immune response.

Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) therapy was developed to address this problem. Specifically, the CAR molecule is an artificially designed and constructed receptor molecule, which consists of a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane region, a co-stimulatory domain, and an intracellular signaling domain. Therefore, CAR molecules have the functions of specifically recognizing tumor surface antigens, activating T cell killing activity, and stimulating T cell proliferation. By collecting and culturing T cells from tumor patients and artificially transducing the gene encoding CAR molecules, the patient's own T cells can express CAR molecules. After reinfusion into the patient's body, T cells can efficiently and specifically recognize and kill tumor cells through CAR molecules, achieving the effect of cancer treatment.

Since the concept of CAR-T therapy was first proposed in 1989, it has experienced 30 years of development and multiple rounds of technological changes (Figure 1). The first-generation CAR-T only has a single-chain antibody as the extracellular antigen-binding domain and CD3ζ as the intracellular signaling domain, which cannot fully activate the activity of T cells, and the therapeutic effect is not good. The second-generation CAR-T introduced a co-stimulatory domain on the basis of the first-generation CAR-T, which improved the in vitro proliferation ability and cytokine release level of T cells. The third-generation CAR-T adds a co-stimulatory domain to the second-generation CAR-T. Although it can improve the killing activity of T cells, it may induce excessive release of cytokines. Therefore, on the basis of the second-generation CAR-T, the new-generation CAR-T jointly expresses other auxiliary factors, such as the joint expression of IL-12 or the STAT3/5 binding domain in the IL-2Rβ cell, which helps to improve tumor killing Effects such as activity and safety.

Although CAR-T therapy has achieved satisfactory results in hematological tumors, there is still a lot of room for improvement in the therapeutic effect of CAR-T on solid tumors. The reasons are as follows: (1) Many solid tumors are difficult to be detected in the early stage, and have the characteristics of high malignancy, high recurrence rate, and poor prognosis. For example, 83% of colorectal cancer patients were in the middle and late stage when they were first diagnosed, and 44% of the patients had metastases to the liver, lung and other parts, and nearly half of the patients survived for less than 5 years; about 70% of ovarian cancer When patients are diagnosed, the cancer cells have already metastasized, and it is difficult to be cured by surgery, chemotherapy, and radiotherapy. The recurrence rate after treatment is still as high as 70%; 90% of pancreatic cancer patients are diagnosed at an advanced stage, and the 5-year survival rate is only 7%. (2) During the treatment of solid tumors, tumor tissues often have an immunosuppressive microenvironment, which can hinder the migration and infiltration of CAR-T cells. (3) Many malignant solid tumors are also characterized by high heterogeneity, and a single target antigen often cannot achieve the best therapeutic effect, and there is a risk of recurrence. Therefore, CAR-T cell therapy for patients with malignant solid tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer needs to further improve its efficiency and effectiveness.

To sum up, further research is still needed in this field to develop an engineered immune cell with higher efficiency and better therapeutic effect for malignant tumors (especially solid tumors).

Contents of the invention

The purpose of the present invention is to provide an engineered immune cell (such as CAR-T cell) with higher efficiency and better therapeutic effect for malignant tumors (especially solid tumors).

Another object of the present invention is to provide an engineered immune cell (such as CAR-T cell) jointly expressing CCR2b and CD40L and its preparation method and application.

The first aspect of the present invention provides an engineered immune cell, the engineered immune cell is a T cell or NK cell, and the immune cell has the following characteristics:

(a) the immune cell expresses a chimeric antigen receptor (chimeric antigen receptor, CAR), wherein the CAR targets surface markers of tumor cells, wherein the antigen-binding domain of the CAR includes the extracellular domain of NKG2D ;

(b) said immune cell expresses an exogenous CCR2b protein; and

(c) The immune cells express exogenous CD40L protein.

In another preferred embodiment, the T cells include αβT, γδT cells, NKT cells, MAIT cells, or combinations thereof.

In another preferred example, the engineered immune cells are selected from the following group:

(i) chimeric antigen receptor T cells (CAR-T cells);

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

In another preferred example, the CCR2b protein and/or CD40L can be expressed constitutively or inducibly.

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

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

(b) When the CAR-T cells are exposed to an inducer, the CAR-T cells are induced to express CCR2b and/or CD40L proteins.

In another preferred example, in the CAR cells, CAR, CCR2b and CD40L proteins are expressed in series.

In another preferred example, in the CAR cells, CAR, CCR2b and CD40L proteins are independently expressed.

In another preferred embodiment, the "activation" refers to the binding of the CAR to surface markers of tumor cells.

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

In another preferred example, the chimeric antigen receptor CAR is located on the cell membrane of the engineered immune cells.

In another preferred example, the chimeric antigen receptor CAR is located on the cell membrane of the CAR-T cell.

In another preferred example, the CCR2b protein is located on the cell membrane of the CAR-T cells.

In another preferred example, the structure of the CAR is shown in Formula I:

L-NKG2D-H-TM-C-CD3ζ (I)

In the formula,

L is nothing or a signal peptide sequence;

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

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory signal domain;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutants/modifiers thereof);

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

In another preferred example, said L is respectively selected from signal peptides of proteins in the following group: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modifications thereof, or combinations thereof.

In another preferred example, the H is selected from the hinge region of proteins in the following group: CD8, CD28, CD137, IgG, or a combination thereof.

In another preferred embodiment, the TM is selected from the transmembrane region of the following group of proteins: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 , CD137, CD154, CD278, CD152, CD279, CD233, CD314, or a mutation/modification thereof, or a combination thereof.

In another preferred example, the C is selected from the co-stimulatory domains of proteins in the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10 , CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, or mutants/modifications thereof, or combinations thereof.

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

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

In another preferred example, in addition to the first CAR shown in formula I, the CAR cell also contains a second CAR for targeting the second antigen, and the structure of the second CAR is shown in formula II:

L-scFv-H-TM-C-CD3ζ(II)

In the formula,

L is nothing or a signal peptide sequence;

scFv is an antigen-binding domain;

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory domain;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ or a mutation/modification thereof;

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

In another preferred example, the scFv is an antibody single-chain variable region sequence targeting a tumor antigen.

In another preferred example, the scFv is an antibody single-chain variable region sequence targeting an antigen selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133 , CD44, CD166, CD133, CD276, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands (ligands), LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1 , Claudin18.2, CEA, TAG-72, TROP2, or combinations thereof.

In another preferred example, the amino acid sequence of the NKG2D is shown in SEQ ID NO: 1, wherein the extracellular domain is 73-216.

In another preferred example, the CCR2b protein includes a full-length CCR2b protein or an active fragment thereof (ie, an active fragment retaining the function of binding to a corresponding ligand). In another preferred example, the amino acid sequence of the CCR2b protein is shown in SEQ ID NO:2.

In another preferred embodiment, the CD40L protein includes full-length CD40L protein or its active fragment (ie, the active fragment retains the function of binding to CD40).

In another preferred example, the amino acid sequence of the CD40L protein is shown in SEQ ID NO:5.

In another preferred example, the first CAR shown in formula I and the second CAR shown in formula II can be combined into one to form a CAR shown in formula IIIa or IIIb:

L-NKG2D-scFv-H-TM-C-CD3ζ (IIIa)

L-scFv-NKG2D-H-TM-C-CD3ζ (IIIb)

In the formula,

L is nothing or a signal peptide sequence;

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

scFv is an antigen-binding domain;

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory domain;

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

The 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) transforming the immune cells so that the immune cells express CAR molecules and exogenous CCR2b protein and exogenous CD40L protein, thereby obtaining the engineered immune cells described in the first aspect of the present invention, Wherein the CAR targets surface markers of tumor cells, wherein the antigen binding domain of the CAR includes the extracellular domain of NKG2D.

In another preference, in step (B), including:

(B1) introducing the first expression cassette expressing the CAR into the immune cells; (B2) introducing the second expression cassette expressing CCR2b into the immune cells; (B3) introducing the third expression cassette expressing CD40L into the immune cells Immune cells; wherein the steps (B1), (B2) and (B3) can be performed in any order.

In another preferred example, the step (B1) can be performed before, after, simultaneously or alternately with the step (B2).

In another preferred example, the step (B1) can be performed before, after, simultaneously, or alternately before the step (B3).

In another preferred example, the step (B2) can be performed before, after, simultaneously or alternately with the step (B3).

In another preferred example, the steps (B1), (B2) and (B3) are performed simultaneously or alternately.

In another preferred embodiment, a method for preparing the CAR-T cells of the present invention is provided, comprising the following steps:

(A) providing a T cell to be modified;

(B) transforming the T cells so that the T cells express the CAR molecule and exogenous CCR2b protein and exogenous CD40L protein, thereby obtaining the engineered immunity described in the first aspect of the present invention cell.

In another preferred embodiment, in step (B), it includes: introducing the first expression cassette expressing the CAR into the T cell; and introducing the second expression cassette expressing CCR2b and the third expression cassette expressing CD40L The T cells; wherein the introducing steps can be performed in any order.

In another preferred example, for any two expression cassettes in the first, second and third expression cassettes (taking the first expression cassette and the second expression cassette as an example), their transcription directions are in the same direction ( →→), opposite (→←) or opposite (←→).

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

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

In another preferred example, when two or three of the CAR molecule, the exogenous CCR2b protein and the exogenous CD40L protein are expressed in series, a connecting peptide is also included between the two proteins.

In another preferred example, the connecting peptide is P2A or T2A.

In another preferred example, the vector is a viral vector, preferably the viral vector contains the first and second expression cassettes in tandem form.

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

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

The 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 contains the CAR-T cells of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

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

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

In another preferred example, the concentration of the engineered immune cells (such as CAR-T cells) in the preparation is 1×10 ³ -1×10 ⁸ cells/ml, preferably 1×10 ⁴ -1 ×10 ⁷ cells/ml.

The fourth aspect of the present invention provides the use of the engineered immune cell according to the first aspect of the present invention for preparing a drug or preparation for preventing and/or treating cancer.

In another preferred embodiment, the use of the CAR-T cells according to the first aspect of the present invention is provided for the preparation of drugs or preparations for preventing and/or treating cancer or tumors.

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

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

In another preferred example, the tumor is selected from the group consisting of colon cancer, rectal cancer, ovarian cancer, or pancreatic cancer.

In another preferred example, the tumor is a tumor with high expression of NKG2D ligand and/or high expression of chemokines and/or high expression of CD40.

In another preferred example, the tumor is a tumor with high expression of NKG2D ligands and high expression of chemokines.

In another preferred example, the tumor is a tumor with high expression of NKG2D ligand and high expression of CD40.

In another preferred embodiment, the chemokine is selected from the group consisting of CCL2, CCL7, or a combination thereof.

In another preferred example, the tumor is a tumor with high expression of NKG2D ligand, high expression of chemokines CCL2 and/or CCL7, and high expression of CD40.

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

In another preferred example, the tumor has high expression of NKG2D ligands (including any one of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof) and/or chemokines ( Including tumors with high expression of CCL2, CCL7, CCL8, CCL12, CCL13, CCL16, or their combination) and/or high expression of CD40.

The fifth aspect of the present invention provides a kit for preparing the engineered immune cell described in the first aspect of the present invention, the kit includes 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 NKG2D;

(2) a second nucleic acid sequence containing a second expression cassette for the combined expression of CCR2b; and

(3) A third nucleic acid sequence containing a third expression cassette for co-expressing CD40L.

In another preferred embodiment, a kit for preparing the engineered immune cell according to the first aspect of the present invention is provided, the kit includes a container, and in the container:

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

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

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

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

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

In another preferred example, when two or three of the first, second and third nucleic acid sequences are located on the same vector, a connecting peptide expression for expressing the connecting peptide is also included between them. box.

In another preferred example, the connecting peptide is P2A or T2A.

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

The sixth aspect of the present invention provides a method for treating tumors, comprising the step of: administering a safe and effective amount of the preparation described in the third aspect of the present invention to a subject in need, thereby treating the subject's tumor.

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 in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the structures of CAR molecules of different generations.

Figure 2 shows the structure of the CAR molecule.

Figure 3 shows the expression rate of NKG2D CAR molecules of each CAR-T cell detected by flow cytometry.

Figure 4 shows the expression rate of CCR2b of each CAR-T cell detected by flow cytometry.

Figure 5 shows the expression rate of CD40L of each CAR-T cell detected by flow cytometry.

Figure 6 shows the expression rate of NKG2D ligands (MICA/MICB) in target cells detected by flow cytometry.

Figure 7 shows the expression rate of NKG2D ligand (ULBP-1) in target cells detected by flow cytometry.

Figure 8 shows the expression rate of NKG2D ligands (ULBP-2/5/6) in target cells detected by flow cytometry.

Figure 9 shows the expression rate of NKG2D ligand (ULBP-3) in target cells detected by flow cytometry.

Figure 10 shows the expression rate of NKG2D ligand (ULBP-4) in target cells detected by flow cytometry.

Figure 11 shows the expression rate of CD40 in target cells detected by flow cytometry.

Figure 12 shows the results of Incucyte's real-time detection of the chemotaxis and migration ability of BN009.

Figure 13 shows the killing effect of each NKG2D CAR-T cell on tumor cells detected by EuTDA.

Figure 14 shows the IFN-γ release level of each NKG2D CAR-T cell detected by ELISA.

Detailed ways

After extensive and in-depth research and a large number of screenings, the inventors co-expressed specific CAR, CCR2b and CD40L proteins for the first time in engineered immune cells, that is, combining CAR containing NKG2D extracellular domain (ED) with CCR2b and CD40L Expressed in CAR-T and other immune cells. The CAR-T cells of the present invention simultaneously have the multi-target recognition ability of NKG2D, the enhanced chemotaxis and migration ability of CCR2b, and the immune activation ability of CD40L, and unexpectedly exhibit a synergistic killing effect on tumor cells in vitro. The present invention has been accomplished on this basis.

Experiments suggest that, compared with the prior art, the immune cells of the present invention can improve the sensitivity of CAR-T cells to chemokines such as CCL2 and CCL7 through CCR2b, and promote tumor lesions of solid tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer. Efficient migration to improve treatment efficiency; at the same time, NKG2D CAR molecules can recognize various target antigens on the surface of malignant tumor cells such as colorectal cancer, ovarian cancer, and pancreatic cancer (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 , ULBP6), reducing the risk of decreased efficacy due to tumor heterogeneity or loss of target antigens. In addition, co-expressed CD40L can effectively activate the body's endogenous natural and adaptive immune responses, thereby helping T cells overcome the immunosuppressive tumor microenvironment, improve tumor therapy, and reduce the risk of tumor recurrence.

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

the term

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

The term "about" can refer to a value or composition within an acceptable error range for a 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 "administration" refers to the physical introduction of a 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 or other parenteral routes of administration, for example by injection or infusion.

Antibody

As used herein, the term "antibody" (Ab) shall include, but not be limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds , or an antigen-binding portion thereof. Each H chain is comprised of 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 CH3. 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 one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

antigen binding domain

As used herein, "antigen-binding domain" and "single-chain antibody fragment" all refer to a Fab fragment, Fab' fragment, F(ab') ₂ fragment, or a single Fv fragment having antigen-binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant region, and the smallest antibody fragment with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. The antigen binding domain is usually a scFv (single-chain variable fragment). A single-chain antibody is preferably a sequence of one amino acid chain encoded by one nucleotide chain.

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

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

Chimeric Antigen Receptor (CAR)

As used herein, a Chimeric antigen 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 known as an antigen binding domain). The intracellular domain includes the co-stimulatory domain and the CD3ζ chain portion. When CAR is expressed in T cells, the extracellular segment 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 such as IL-2 and IFN-γ etc., affecting tumor cells so that they do not grow, are induced to die, or are otherwise affected, and result in a reduction or elimination of the patient's tumor burden. The antigen binding domain is preferably fused to an intracellular domain from one or more of the co-stimulatory molecule and the CD3zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain and the CD3ζ signaling domain.

In one embodiment, the CAR of the present invention targets NKG2D ligands and can specifically bind to MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

Chimeric Antigen Receptor T Cells (CAR-T Cells)

As used herein, the terms "CAR-T cell", "CAR-T" and "CAR-T cell of the present invention" all refer to the CAR-T cell described in the first aspect of the present invention. The CAR-T cells of the present invention can be used to treat tumors with high expression of NKG2D ligands, 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 CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigen, it can target 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) using the patient's own 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.

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 described in the first aspect of the present invention. The CAR-NK cells of the present invention can be used to treat tumors with high expression of NKG2D ligands, such as colorectal cancer, ovarian cancer, pancreatic cancer and the like.

Natural killer (NK) cells are a major type of immune effector cells, which protect the body from virus infection and tumor cell invasion through non-antigen-specific pathways. NK cells through engineering (gene modification) may obtain new functions, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxicity.

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

protein NKG2D

In the present invention, NKG2D includes wild type or its mutant or its derivative form or its active fragment. Preferred NKG2Ds are NKG2Ds from mammals such as humans and non-human primates.

The accession number of the amino acid sequence of the 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 as follows:

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

Chemokine

Chemokines are a special class of cytokines, including more than 50 members. According to the structure, it is divided into four types: CC, CXC, CX3C and XC; chemokine receptors are correspondingly divided into four types: CCR, CXCR, CX3CR and XCR, with about 20 members.

In the engineered immune cells of the present invention, the expressed chemokine receptor 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:

protein CD40L

Leukocyte differentiation antigen 40 ligand (cluster of differentiation 40 ligand, CD40L), also known as CD154 or tumor necrosis factor-associated activation protein (tumor necrosis factor-associated activation protein, TRAP).

The accession number of the amino acid sequence of human CD40L protein is NP_000065.1, and the accession number of the nucleotide amino acid sequence is NM_000074.3. The full-length amino acid sequence of human CD40L is as follows:

In the present invention, suitable CD40L includes wild-type and mutant-type CD40L, as long as the mutant-type CD40L has basic functions of wild-type CD40L. Furthermore, in the present invention, preferred CD40L is from mammals, such as humans and non-human mammals.

CD40L is mainly expressed in activated CD4+T lymphocytes, activated CD8 ⁺ T cells, basophils, mast cells and NK cells. CD40L and its receptor CD40 are a pair of co-stimulatory molecules in the inflammatory and immune response systems in vivo. In innate immunity, the CD40L/CD40 co-stimulatory pathway is an important trigger for the maturation process of monocytes, which mainly drives the differentiation of monocytes into M1 lineage macrophages and DC cells. At the same time, this pathway can also promote the release of cytokines and chemokines from DC cells, induce the expression of other co-stimulatory molecules, and promote the cross-presentation of antigens. In humoral immunity, this pathway is also involved in the T cell-dependent B lymphocyte response process, the formation of germinal centers, the generation of long-term memory B cells, antibody production, and antibody class switching. In cellular immunity, this pathway can promote T cell activation and amplify T cell-mediated immune response, play an important role in the process of CD4 ⁺ T cell differentiation, and can also promote the expansion and pluripotency of CD8 + T cells, It is the basis for generating memory CD8+ T cells.

In the anti-tumor immune response, the CD40L/CD40 co-stimulatory pathway also plays multiple roles, such as activating the proliferation of T cells and the release of cytokines, and inducing the M2 lineage macrophages to M1 lineage macrophages with anti-tumor activity. shift etc. In tumors with loss of some target antigens but high expression of CD40, this pathway can also mediate the killing effect of T cells on tumor cells.

In the present invention, when CD40L is co-expressed with a specific CAR molecule and CCR2b, CD40L, as an auxiliary factor of CAR-T cells, can activate the endogenous immune response of the body while CAR-T cells kill tumor cells, enhance and Prolongs the healing effect. In addition, unexpectedly, CD40L can also play a synergistic effect with CCR2b, thereby synergistically significantly improving the killing effect against tumor cells in vitro.

expression cassette

As used herein, an "expression cassette" or "expression cassette of the invention" includes a first expression cassette, a second expression cassette and a third expression cassette. The expression cassette of the present invention is as described in the fifth aspect of the present invention, the first expression cassette comprises the nucleic acid sequence encoding the CAR. The second expression cassette expresses exogenous CCR2b protein. The second expression cassette expresses exogenous CD40L protein.

In the present invention, CCR2b and CD40L proteins can be expressed constitutively or inducibly.

In the case of induced expression, when the CAR-T cells are activated by the corresponding inducer, the second expression cassette expresses the CCR2b protein, and the third expression cassette expresses the CD40L protein; thus, when the CAR-T cells of the present invention are not exposed to the corresponding induced When the agent is used, the second expression cassette does not express the CCR2b protein, and the third expression cassette does not express the CD40L protein.

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

carrier

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

Usually, the expression cassette or nucleic acid sequence of the present invention can be connected downstream of the promoter through routine operations, and incorporated into an expression vector. The vector can be integrated into the genome of eukaryotic cells and then replicated. A typical cloning vector contains transcriptional and translational terminators, an initial sequence and a promoter useful for regulating the expression of the desired nucleic acid sequence.

The expression vectors of the invention can 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, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated 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 such vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, and the like.

Furthermore, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and 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, herpesviruses, and lentiviruses. Generally, suitable vectors contain at least one origin of replication functional in the organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and US Patent No. 6,326,193).

A number of virus-based systems have been developed and used for gene transduction in mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In one embodiment, lentiviral vectors are used. Many DNA viral 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, can regulate the frequency of transcription initiation. Typically, these elements are located in a region of 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible in order to preserve promoter function 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 before activity begins to decline. Depending on the promoter, it appears that individual elements can act 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 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus (Epstein-Barr virus, EBV) immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters, such as but not limited to protein 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 capable of turning on expression of the polynucleotide sequence linked to the inducible promoter when desired, or turning 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 the identification and selection of expressing cells from the transfected or infected cell population by the viral vector. In other aspects, selectable markers can be carried on a single 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 the host cell. Useful selectable marker genes include, for example, antibiotic resistance genes such as neomycin and the like.

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 easily introduced into host cells, eg, mammalian (eg, human T cells), bacterial, yeast or insect cells, by any method 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, 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, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001). Preferred methods for introducing polynucleotides into host cells are liposome transfection and cationic complex polyethylenimine transfection.

Biological methods for introducing polynucleotides 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, eg human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, eg, 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 lipid-based systems. plastid. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).

Where a non-viral delivery system is 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. Lipid-associated nucleic acids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides To liposomes, entrapped in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexes with micelles, or otherwise associated with lipids. 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 solution, possibly forming aggregates of non-uniform size or shape. Lipids are lipid substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm as well as compounds comprising long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

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

It should be understood that in the present invention, in addition to using multiple lentiviruses for transduction, direct transfection of mRNA or plasmids, or expression of artificial transcription factors, etc., can jointly express CCR2b, CD40L and T cells in immune cells such as T cells. NKG2D CAR molecule.

preparation

The present invention provides an engineered immune cell (such as CAR-T cell) according to the first aspect of the present 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/ml, more preferably 1×10 ⁴ -1×10 ⁷ cells/ml.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives. The formulations of the invention are preferably formulated for intravenous administration.

therapeutic application

The invention includes the therapeutic use of cells (eg, T cells) transduced with a vector (eg, a lentiviral vector) comprising an expression cassette of the invention. The transduced T cells can target the surface markers of tumor cells and express CCR2b protein, synergistically and significantly improving their killing efficiency against tumor cells.

Therefore, the present invention also provides a method for stimulating an immune response mediated by T cells targeting mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to mammals.

In one embodiment, the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient. In this way, the probability of graft-versus-host reaction is extremely low, and the antigen is recognized by T cells without MHC restriction. Furthermore, a single CAR-T can treat all cancers that express that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

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

Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors. Cancer types treated with the CAR of the present 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 present 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 present invention.

The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions 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.

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

When an "immunologically effective amount", "antitumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the precise amount of a composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) with individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. Pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably at a dose of 10 ⁵ to 10 ⁷ cells/kg body weight (including all integer values within the range). T cell compositions can also be administered multiple times at these doses. 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 art by monitoring the patient for signs of disease, and adjusting treatment accordingly.

Administration of the compositions to a subject may be by any convenient means, including by spraying, injection, swallowing, infusion, implantation or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, 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 composition of the invention is preferably administered by intravenous injection. Compositions of T cells can be injected directly into tumors, lymph nodes or sites 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., previously , simultaneously or subsequently) to the patient in a form of treatment including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients. In a further embodiment, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants 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 in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents 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 peripheral blood stem cell transplantation. 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.

Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1×10 ⁵ to 1×10 ¹⁰ modified T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion.

Main advantages of the invention

(1) The present invention uses the CCR2b protein to enable the immune cells of the present invention to migrate to the tumor site more efficiently, thereby significantly improving the effect of inhibiting tumors and reducing toxic and side effects. Experiments show that the present invention significantly improves the ability of CAR-T cells to migrate to places with high CCL2 concentration (such as lesion sites).

(2) The antigen binding domain of the engineered immune cells of the present invention adopts the extracellular binding domain of NKG2D, which can recognize 8 kinds of targets on the cell surface of malignant tumors (such as colorectal cancer cells, ovarian cancer, pancreatic cancer, etc.) through NKG2D CAR molecules. Spot antigens (MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6), reduce the risk of decreased efficacy due to tumor heterogeneity or loss of target antigens.

(3) The present invention uses CD40L as the second cofactor to more effectively activate the body's endogenous natural and adaptive immune responses. CD40L activates the proliferation of T cells and the release of cytokines, induces the transformation of M2 lineage macrophages into M1 lineage macrophages with anti-tumor activity, enhances the antigen presentation function of DC cells, etc., and at the same time improves the ability of T cells to certain Killing effect of tumor cells with antigen loss but high CD40 expression.

(4) When the CAR molecule of the NKG2D extracellular domain is combined with CCR2b and CD40L at the same time, it can unexpectedly significantly improve the chemotactic migration ability of CAR-T cells to high concentrations of CCL2.

(5) Unexpectedly, when the NKG2D CAR molecule of the present invention, exogenous CCR2b protein and exogenous CD40L protein are jointly expressed, the in vitro killing effect on tumor cells can be synergistically and significantly improved.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention, not to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (Sambrook et al., New York: Cold Spring Harbor Laboratory Press, 1989), or Follow the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Reagents and materials used in the examples were obtained commercially unless otherwise stated.

Materials and Methods

CAR molecule and its structure

In the embodiments, each NKG2D CAR comprises the following partial structures: human CD8 signal peptide [abbreviated as CD8 (SP)], human NKG2D extracellular domain [abbreviated as NKG2D (ED)], optimized human CD8 hinge region [abbreviated as CD8 ( hinge)], human CD8 transmembrane domain [abbreviated as CD8(TM)], human 4-1BB intracellular domain [abbreviated as 4-1BB(ID)], human CD3ζ intracellular signal transduction domain [abbreviated as CD3ζ(ID) )], self-cleaving peptide P2A, human CCR2b, self-cleaving peptide T2A, human CD40L.

The second-generation NKG2D CAR molecule used as a control was named BN001, the new-generation NKG2D CAR molecule jointly expressing CCR2 was named BN003, the new-generation NKG2D CAR molecule jointly expressing CD40L was named BN004, and the new-generation NKG2D CAR molecule jointly expressing CCR2 and CD40L was named BN004. Named BN009.

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

BN001 is composed of CD8 (SP), NKG2D (ED), CD8 (hinge), CD8 (TM), 4-1BB (ID) and CD3ζ (ID) in series from its amino terminal to carboxyl terminal.

BN003 consists of CD8 (SP), NKG2D (ED), CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3ζ (ID), P2A, and CCR2b in series from its amino terminal to carboxyl terminal.

BN004 consists of CD8 (SP), NKG2D (ED), CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3ζ (ID), P2A, and CD40L in series from its amino terminal to carboxyl terminal.

CD8(SP), NKG2D(ED), CD8(hinge), CD8(TM), 4-1BB(ID), CD3ζ(ID), P2A, CCR2b, T2A, CD40L are sequentially connected in series from the amino terminal to the carboxyl terminal of BN009 composition.

SEQ ID NO: 1 (human NKG2D amino acid sequence)

SEQ ID NO: 2 (human CCR2b amino acid sequence)

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

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

SEQ ID No:5 (human CD40L amino acid sequence)

SEQ ID No:6 (optimized human CD8 hinge region amino acid sequence)

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

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

SEQ ID No:9 (amino acid sequence of human CD3ζ intracellular signal transduction domain)

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

SEQ ID No: 11 (amino acid sequence of self-cleaving peptide T2A)

Example 1 lentivirus preparation

1.1 Obtaining lentiviral vector plasmids

The nucleotide sequences of BN001, BN003, BN004, and BN009 were synthesized from the whole gene, and then connected to the lentiviral vector pCDH-EF1-MCS-T2A-copGFP plasmid by molecular cloning, so that it was in the human EF1α promoter and Kozak sequence expression under control.

1.2 Transfection of lentiviral vector plasmid into 293T cells

Mix the above-mentioned lentiviral vector plasmids with lentiviral packaging plasmids pRSV-Rev, pMDLg/pRRE and pMD2.G with polyethyleneimine transfection reagent, and co-transfect 293T cells. After culturing for 48 hours, collect the virus supernatant, centrifuge at 4,500 rpm for 10-15 minutes at 4°C, filter through a filter membrane with a pore size of 0.5 μm, and then use a hollow fiber column ultrafiltration system to concentrate the lentivirus, and then use chromatography to extract the lentivirus. Purify, and finally filter and sterilize with a filter membrane with a pore size of 0.22 μm, and store in -80°C.

1.3 Determination of lentivirus titer

Adjust the concentration of Jurkat cells to 1×10 ⁵ cells/300 μl, mix thoroughly and take 300 μl of resuspended cells to each well of a 24-well plate. Take 70 μl lentiviral concentrate and perform 5-fold serial dilution with Opti-MEM medium. Add 200 μl/well of each diluted lentivirus into the above 24-well plate to infect Jurkat cells with the lentivirus (the Jurkat cells in the negative control group are only added with Opti-MEM medium), and culture in a cell culture incubator (Cultivation temperature is 37°C, carbon dioxide concentration is 5%). After culturing for 3 days, 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 above cells were stained with corresponding antibodies, and then the proportion of Jurkat cells successfully transduced by lentivirus was detected by flow cytometry. The lentivirus infection rate of Jurkat cells is recorded as P (%), the volume of virus liquid is recorded as V (ml), and the dilution factor of virus liquid is recorded as N, and the lentivirus titer is calculated by the following formula:

Lentivirus titer (TU/ml) = P/V×N×10 ⁵

Results: The titer of BN001 was 5.46×10 ⁸ TU/ml, the titer of BN003 was 2.46×10 ⁸ TU/ml, the titer of BN004 was 6.31×10 ⁸ TU/ml, and the titer of BN009 was 9.51×10 ⁸ TU/ml.

Example 2 Preparation and detection of CAR-T cells

(a) Preparation of T cells

Adjust the density of peripheral blood mononuclear cells from healthy donors to 2×10 ⁶ /ml, add 50ng/ml anti-CD3 antibody, 50ng/ml anti-CD28 antibody, and 200IU/ml recombinant IL-2, and place in a cell culture incubator Cultivate for 24 hours (cultivation temperature is 37° C., carbon dioxide concentration is 5%).

(b) Lentiviral transduction of T cells

The obtained T cells were washed, and the cell density was adjusted to 2×10 ⁶ /ml. Add lentivirus at MOI=1～10TU/cell for transduction, supplement 50ng/ml anti-CD3 antibody, 50ng/ml anti-CD28 antibody, and 200IU/ml recombinant IL-2 at the same time, and culture in a cell culture incubator ( The culture temperature was 37°C, and the carbon dioxide concentration was 5%). After 24 hours, the cell density was adjusted to 1.5-2×10 ⁶ /ml, and 300 IU/ml of IL-2 was supplemented. On the 4th day after transduction, the cells were washed to remove residual lentiviral particles in the supernatant, and continued to be cultured in a cell incubator for 5 days (the culture temperature was 37°C, and the carbon dioxide concentration was 5%), during which the cell density was maintained at 1～2×10 ⁶ /ml. The cells were harvested on the 10th day after transduction, and frozen in liquid nitrogen with a freezing medium (freezing medium containing 5% human serum albumin: normal saline = 1:1) for future use. The obtained CAR-T cells followed the nomenclature of the corresponding CAR molecules, namely BN001, BN003, BN004 and BN009, and the T cells not transduced with lentivirus were named Ctrl T.

(c) Detection of the expression of CAR molecules

The Ctrl T, BN001, BN003, BN004 and BN009 cells to be tested were washed twice with PBS, and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). Add APC-labeled anti-human NKG2D antibody and BV421-labeled anti-human CD3 antibody to the cell suspension to be tested according to the antibody instructions, and incubate at 4°C for 60 min. Using Ctrl T cells as a negative control, the expression rates of NKG2D CAR molecules in BN001 and BN009 cells were detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 3. The gate was set according to the APC fluorescence signal level of Ctrl T cells, and the expression rate of CAR molecules in Ctrl T cells as a negative control was regarded as 0.64%, and the expression rate of CAR molecules in BN001 cells was about 92.17%. The CAR molecule expression rate of BN003 cells is about 98.31%, the CAR molecule expression rate of BN004 cells is about 97.71%, and the CAR molecule expression rate of BN009 cells is about 85.29%.

(d) Detection of expression of CCR2b

The Ctrl T, BN001, BN003, BN004 and BN009 cells to be tested were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human CCR2b antibody and BV421-labeled anti-human CD3 antibody were added to the cell suspension to be detected, and incubated at 4°C for 60 min. Using Ctrl T cells not transfected with lentivirus as a negative control, the expression rate of CCR2b in the above CAR-T cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 4. Since T cells have a certain level of endogenous CCR2 expression, Ctrl T cells can be divided into CCR2-negative and CCR2-positive cell populations. According to the PE fluorescence signal level of the CCR2-negative Ctrl T cell population, the CCR2 expression rate of Ctrl T is about 44.20%, the CCR2b expression rate of BN001 is about 23.71%, the CCR2b expression rate of BN003 cells is about 99.37%, and the BN004 cells The expression rate of CCR2b in BN009 cells is about 24.55%, and the expression rate of CCR2b in BN009 cells is about 96.11%.

(e) Detection of CD40L expression

The Ctrl T, BN001, BN003, BN004 and BN009 cells to be tested were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human CD40L antibody and BV421-labeled anti-human CD3 antibody were added to the cell suspension to be detected, and incubated at 4°C for 60 min. Using Ctrl T cells not transfected with lentivirus as a negative control, the CD expression rate of the above CAR-T cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 5. Since T cells have a certain level of endogenous CD40L expression, Ctrl T cells can be divided into CD40L-negative and CD40L-positive cell populations. According to the PE fluorescence signal level of the CD40L-negative Ctrl T cell population, the CD40L expression rate of Ctrl T is about 2.75%, the CD40L expression rate of BN001 is about 21.43%, the CD40L expression rate of BN003 is about 41.85%, and the CD40L expression rate of BN004 is about 2.75%. The expression rate is about 86.74%, and the expression rate of BN009 cells is about 71.86%.

Example 3 Target cell detection

(a) Target cell culture conditions

Tested colorectal cancer cell lines (also known as target cells or target cell lines): HCT 116 (McCoy's 5a medium + 10% fetal bovine serum + 100U/ml penicillin + 100μg/ml streptomycin), LS174T (EMEM medium +10% fetal bovine serum+100U/ml penicillin+100μg/ml streptomycin), LoVo (F-12K medium+10% fetal bovine serum+100U/ml penicillin+100μg/ml streptomycin), SW480 (Leibovitz's L-15 Medium + 10% fetal bovine serum + 100 U/ml penicillin + 100 μg/ml streptomycin). Tested ovarian cancer cell line: SK-OV-3 (McCoy's 5a medium + 10% fetal bovine serum + 100 U/ml penicillin + 100 μg/ml streptomycin).

(b) Detection of expression of NKG2D ligands (MICA/MICB)

The above target cells were washed twice with PBS and resuspended with FACS buffer. Add APC-labeled anti-human MICA/MICB antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 min. The target cells incubated without antibody were used as negative control, and the MICA/MICB expression rate of the target cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 6, the MICA/MICB expression rates of HCT 116, LS174T and SW480 were all higher than 96%, and the MICA/MICB expression rates of LoVo and SK-OV-3 were lower than 8%.

(c) Detection of expression rate of NKG2D ligand (ULBP-1)

The above target cells were washed twice with PBS and resuspended with FACS buffer. Add PC5.5-labeled anti-human ULBP-1 antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 min. The target cells incubated without antibody were used as a negative control, and the expression rate of ULBP-2/5/6 in the target cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 7, the ULBP-1 expression rate of HCT116 is about 77.25%, the ULBP-1 expression rate of LoVo is about 9.09%, and the ULBP-1 expression rate of other cells is lower than 3%.

(d) Detection of expression rate of NKG2D ligand (ULBP-2/5/6)

The above target cells were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human ULBP-2/5/6 antibodies were added to each target cell suspension, and incubated at 4°C for 60 min. The target cells incubated without antibody were used as a negative control, and the expression rate of ULBP-2/5/6 in the target cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 8. The ULBP-2/5/6 expression rates of HCT116 and SW480 are both higher than 92%, the ULBP-2/5/6 expression rate of LoVo is about 86.82%, and the ULBP-2/5/6 expression rate of SK-OV-3 is about 86.82%. The expression rate of 2/5/6 was about 56.90%, and the expression rate of ULBP-2/5/6 in LS174T was about 33.42%.

(e) Detection of expression rate of NKG2D ligand (ULBP-3)

The above target cells were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human ULBP-3 antibody was added to each target cell suspension, and incubated at 4°C for 60 min. The target cells incubated without antibody were used as a negative control, and the expression rate of ULBP-3 in the target cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 9, the ULBP-3 expression rates of HCT116 and SW480 are both higher than 92%, the ULBP-3 expression rates of LS174T are about 46.57%, and the ULBP-3 expression rates of LoVo and SK-OV-3 are lower than 34%.

(f) Detection of the expression rate of NKG2D ligand (ULBP-4)

The above target cells were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled anti-human ULBP-4 antibody was added to each target cell suspension, and incubated at 4°C for 60 min. The target cells incubated without antibody were used as a negative control, and the expression rate of ULBP-4 in the target cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 10. The ULBP-4 expression rates of HCT 116, LS174T, LoVo and SW480 were all above 80%, and the ULBP-4 expression rate of SK-OV-3 was about 71.64%.

(g) Detect the expression rate of CD40

The above target cells were washed twice with PBS and resuspended with FACS buffer. Add APC-labeled anti-human CD40 antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 min. The target cells incubated without antibody were used as a negative control, and the CD40 expression rate of the target cells was detected by flow cytometry. Analyzed by CytExpert software.

The results are shown in Figure 11, the expression rate of CD40 in HCT 116 is about 94.56%, the expression rate of SK-OV-3 is about 86.61%, and the expression rate of other cells is lower than 3%.

Example 4 Chemotaxis migration of CAR-T cells

In this example, the Incucyte S3 Live Cell Dynamic Imaging Analyzer was used to detect the migration of T cells in real time. Pretreatment of ClearView 96-well Chemotaxis Motility Microplate: Add 20 μg/ml Protein-G protein to the upper chamber of the microplate, and place it at 37°C for 1 hour to coat the upper chamber microporous membrane; after washing with D-PBS, Add 5 μg/ml of ICAM-1 protein to the upper chamber of the microwell plate, place it at 37°C for 2 hours, and coat the upper chamber microporous membrane twice; finally use 1% BSA to coat the upper and lower surfaces of the upper chamber microporous membrane Carry out sealing for 30min. Wash and resuspend the T cells to be tested with RPMI 1640 medium containing 0.5% FBS, and adjust the cell density to 2-8×10 ⁴ cells/ml. After washing with D-PBS, 60 μl of T cell suspension to be detected (5000 cells/well) was added to the upper chamber of the microwell plate. After standing still for 1 hour, RPMI 1640 medium containing 0.5% FBS and corresponding concentration of CCL2 were added to the lower chamber, and placed in the Incucyte S3 live cell dynamic imaging analyzer, and the T cells in the upper chamber were tested for 24 hours at intervals of 30 minutes. Imaging records. The change of the total area of T cells in the upper chamber was analyzed by the Incucyte chemotaxis and migration analysis module software to reflect the chemotaxis and migration ability of T cells (the more cells migrated from the upper chamber to the lower chamber, the number of cells in the upper chamber and the corresponding The smaller the total cell area, the stronger the chemotaxis and migration ability).

The results are shown in Figure 12, within 24h, there was no significant difference in the migration efficiency of BN001, BN003, BN004 and BN009 induced by CCL2 (A in Figure 12); after adding 100ng/ml CCL2 in the lower chamber medium, The migration efficiency of BN004 to the lower chamber was not significantly different from that of BN001, but the migration efficiency of BN003 and BN009 cells was significantly higher than that of BN001 (B in Figure 12), and BN009 had the highest migration efficiency. From the migration curve, it can be seen that after adding CCL2, the degree of chemotactic migration in BN003 gradually increased slowly, while the degree of migration in BN009 began to increase significantly at about 6 hours, indicating that BN009 can respond to CCL2 in a more sensitive manner The combined expression of CCL2 and CD40L can effectively enhance the chemotactic migration ability of BN009 to high concentrations of CCL2.

In addition, although both BN009 and BN003 express CCR2b, it was originally believed that the CD40L and NKG2D ectodomain and other structures had no substantial impact on the chemotactic migration ability (in Figure 12B, the chemotactic migration ability of BN001 and BN004 to high concentrations of CCL2 is basically different), The results in Figure 12B unexpectedly show that the CAR molecule of the NKG2D ectodomain is co-expressed with CCR2b and CD40L in CAR-T cells at the same time. Data from 10-24 hours in Figure 12B).

Example 5 In vitro function of CAR-T cells

(a) Detection of killing effect by EuTDA method

Wash target cells once with AIM-V medium. Adjust the target cell density to 1×10 ⁶ /ml, add DELFIA BATDA Reagent at 2 μl/ml, mix well, and incubate at 37°C for 30 minutes. After the target cells were washed three times with AIM-V medium, the target cells were seeded in a 96-well plate at a density of 1×10 ⁴ /well. 100 μl of T cells were added, and cultured in a carbon dioxide incubator for 2 hours (the culture temperature was 37° C., and the carbon dioxide concentration was 5%). Finally, centrifuge at 500×g for 5 min, and transfer 20 μl of the supernatant to a 96-well plate added with Europium solution (200 μl/well). After incubation at room temperature for 15 min, it was detected in a microplate reader.

The results are shown in Table 1 and Figure 13. Compared with Ctrl T, BN001, BN003, BN004 and BN009 have significant killing effects on each target cell. Compared with BN001, BN003 and BN004 did not significantly increase the killing rate of each target cell, while the killing rate of BN009 was significantly higher than that of BN001 (P<0.05).

In order to analyze whether the combined expression of CCR2 and CD40L has a synergistic effect on the killing effect in the process of CAR-T cells killing tumor cells, the increase in the killing rate of BN003, BN004, and BN009 relative to BN001 was calculated (increase = corresponding CAR - T cell killing rate - BN001 killing rate). The results are shown in Table 2. The increase in the killing effect of BN009 on each target cell is higher than the sum of the increase in BN003 and BN004, indicating that the combined expression of CCL2 and CD40L in CAR-T cells has a synergistic effect on the killing effect. Significantly better than the scheme of expressing CCL2 or CD40L alone in CAR-T cells.

Table 1 The killing rate of NKG2D CAR-T cells on tumor cells detected by EuTDA (n=3)

Table 2 The killing rate improvement of each NKG2D CAR-T cell relative to BN001 (n=3)

*: When the improvement of BN009 relative to BN001 (that is, the overall improvement of CCR2b and CD40L) is greater than the sum of "the improvement of BN003 (the improvement of CCR2b) + the increase of BN004 (the improvement of CD40L)", it indicates that there is synergy effect.

(b) Detection of cytokine secretion

Ctrl T, BN001, BN003, BN004, and BN009 were co-cultured with corresponding target cells in AIM-V medium without IL-2 (effect-to-target ratio 2.5:1). After 24 hours, dissolve the INF-γ standard with ddH ₂ O, place at room temperature for 15-20 minutes to ensure full dissolution, and dilute the standard according to the recommended gradient ratio. Aspirate the supernatant of the above co-cultured cells and dilute them 2-fold and 20-fold with ddH ₂ O. The standard and experimental samples were added to the corresponding reaction wells, 100 μl per well. After incubating at room temperature for 1-3 hours, prepare 1× cleaning solution, wash each well with 360 μl cleaning solution 4 times, pat dry the liquid in the well, add 200 μl enzyme-labeled detection antibody to each well, and incubate at room temperature for 1-3 hours. Each well was washed 4 times with 360 μl of washing solution, and after the liquid in the well was patted dry, 200 μl of chromogenic substrate was added. After incubation at room temperature in the dark for 30-60 min, 50 μl of stop solution was added to each well, and the absorbance at 450 nm was measured with a microplate reader.

The results are shown in Table 3 and Figure 14. Compared with Ctrl T cells, BN001, BN003, BN004 and BN009 all had significant IFN-γ release after co-culture with each target cell. For both SW480 and SK-OV-3 cells, no increase in IFN-γ release levels was detected for BN003, BN004 and BN009 relative to BN001. For LS174T cells, the IFN-γ release level of BN004 and BN009 was significantly higher than that of BN001 (P<0.05). For HCT116 and SK-OV-3, only the IFN-γ release level of BN009 was significantly higher than that of BN001 (P<0.05). The above results indicated that, for some target cells, co-expression of CCL2 and CD40L in CAR-T cells can significantly increase the release level of IFN-γ.

Table 3 ELISA detection of IFN-γ release level of each NKG2D CAR-T cell (pg/ml)

^* Signal below detection threshold.

Example 6 In vivo tumor suppression function of CAR-T cells

Using LoVo as target cells and BN001 and BN009 as effector cells to test the inhibitory effect of subcutaneous tumor transplantation in mice. Experiments were conducted with immunodeficient B-NDG mice to observe the effect of NKG2D CAR-T cells co-expressing CCR2 on tumor infiltration and inhibition. B-NDG mice aged 6-8 weeks (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were used for subcutaneous tumor efficacy experiments. A total of 24 mice were selected for the experiment, and were randomly divided into 4 groups, 6 mice in each group, which were vehicle control group, Ctrl T control group, BN001 control group, and BN009 experimental group.

The target cells in logarithmic growth phase and in good growth state were collected by trypsinization method, washed once with normal saline, and the cell density was adjusted to 2×10 ⁷ /ml. 100 μl of cell suspension was subcutaneously injected into the right side of the B-NDG mice near the armpit, that is, each mouse was inoculated with 2×10 ⁶ target cells, and the inoculation diary was regarded as day 0.

Seven days after inoculation of target cells (or when the average tumor volume is about 100 mm ³ ), CAR-T cells (1×10 ⁷ /monkey), Ctrl T cells (1×10 ⁷ /monkey) and vehicle were respectively injected through the tail vein. (100 μl/only), the day of injection of the test substance was recorded as the 0th day of treatment. The tumor size and mouse weight were measured 2 to 3 times a week, and blood samples were collected on the 3rd, 10th, and 28th days. After adding EDTA for anticoagulation, qPCR was used to detect the retention of CAR-T cells in the blood cells in the mice. In this case, INF-γ was detected by ELISA to monitor the release level of cytokines. After 28 days of treatment, the mice were euthanized, and the tumor, heart, liver, spleen, lung, kidney, brain, ovary and other tissues were taken and weighed. The tissues of 2 mice in each group were stored in a refrigerator at 80°C for DNA extraction , to detect the infiltration of CAR-T cells in tumor tissues and their distribution in various organs; the tissues of 2 mice in each group were fixed, and the morphology of tumor cells was detected by HE staining and the tissues were detected by immunohistochemistry. The expression of antigens in the

The results are shown in Table 4. Twenty-eight days after injection of CAR-T cells, the average tumor volume of mice in vehicle control group was about 947 mm ³ , and the average tumor burden was about 1.215 g; the average tumor volume of mice in Ctrl T control group was about The average tumor volume of mice ^{in the BN001 control group was about 1178mm 3} ^, and the average tumor burden was about 1.267g; the average tumor volume of mice in the BN009 experimental group was about 623mm ³ , and the average tumor The load is about 0.860g. Compared with BN001, the average tumor burden of mice injected with BN009 cells was significantly reduced by about 32.1%.

LTR sequences in tumor tissue were detected by qPCR to measure the infiltration of CAR-T cells in tumor tissue. The results showed that the average background signal level of LTR in the tumor tissue of the mice in the vehicle control group was 8.18 copies/μg DNA; the average background signal level of LTR in the tumor tissue of the mice in the Ctrl T control group was 42.86 copies/μg DNA; The average LTR content in the tumor tissue of the mice in the BN001 control group was 2055.38 copies/μg DNA; the average LTR content in the tumor tissue of the mice in the BN009 experimental group was 3872.70 copies/μg DNA. Compared with BN001, the homing ability of BN009 to subcutaneous transplanted tumor tissue in mice was significantly improved, with an increase rate of about 88.4%.

Table 4 CAR-T antitumor effect and homing situation in the in vivo drug efficacy experiment

discuss

In CAR-T therapy for solid tumors such as colorectal cancer, the main targets identified are CD133, CEA, EGFR, HER-2, and NKG2D ligands. Among them, NKG2D ligands include MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and ULBP-6, which are widely expressed in a variety of tumor cells. NKG2D (also known as CD314) is an important activating receptor in the innate immune system, mainly expressed on the surface of natural killer cells, γδT cells and CD8+T cells. Using NKG2D as the antigen recognition domain of CAR molecules has many advantages. Unlike specific antibody CAR molecules targeting a single target, CAR molecules engineered based on NKG2D can recognize the above-mentioned 8 different NKG2D ligands, which is more conducive to the treatment of tumors with high heterogeneity or easy loss of target antigens. Additionally, these NKG2D ligands are all located on the surface of target cells. Therefore, compared with TCR-T, NKG2D CAR-T does not require the antigen presentation process of MHC molecules to directly recognize tumor cells. Importantly, NKG2D ligands are expressed at high levels in epithelial tumor cells such as colorectal cancer, ovarian cancer, pancreatic cancer, and leukemia, but are not expressed or expressed at very low levels in normal cells. ideal target. In addition, the surface of NKG2D CAR-T cells does not carry any foreign protein structures that may trigger the patient's immune response, thereby reducing the possibility of CAR-T cells being rejected by the patient's immune system.

Solid tumor cells can prevent the migration and infiltration of CAR-T cells into tumor tissue by secreting chemokines CXCL12 and CXCL5. In contrast, solid tumor cells secrete less chemokines that can promote CAR-T cell migration. These two factors make it difficult for CAR-T cells to reach solid tumor sites. Therefore, improving the specific recognition and sensitivity of CAR-T cells to tumor chemokines is one of the key factors affecting the therapeutic effect of CAR-T, and the combined expression of chemokine receptors in CAR-T cells is the solution to this problem. important method. Chemokines are a special class of cytokines, including more than 50 members. According to the structure, it is divided into four types: CC, CXC, CX3C and XC; chemokine receptors are correspondingly divided into four types: CCR, CXCR, CX3CR and XCR, with about 20 members. One of the main mechanisms of action of chemokines is to induce directional migration of immune cells by forming concentration gradients soluble or immobilized in the matrix, thereby regulating the infiltration of immune cells in tissues. At present, some CAR-T technologies have used the combined expression of chemokine receptors to promote the rapid migration of CAR-T cells to cancer cells, thereby improving the tumor therapeutic effect of CAR-T cells. Different types of solid tumors release different types and levels of chemokines, and have different immune escape mechanisms. Therefore, selecting the appropriate target antigen for a specific cancer type, combined with the expression of an appropriate chemokine receptor, is the key to improving the therapeutic effect of this type of CAR-T cells.

Through the specific combination of specific CAR molecules, specific chemokine receptors and CD40L, the present invention not only efficiently solves the problem of targeting malignant tumor lesions, but also effectively overcomes the problem of malignant tumor heterogeneity, and achieves synergistic and excellent treatment effect.

On the one hand, the present invention uses CCR2b as a co-expressed chemokine receptor to improve the ability of CAR-T cells to migrate to malignant tumor lesions, thereby improving the treatment efficiency. CCL2, a chemokine mainly recognized by CCR2b, is abnormally highly expressed in various malignant tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer, and its expression level is extremely low in normal tissue cells. At the same time, the affinity between CCR2b and CCL2 is extremely high (about 0.7nM, the smaller the IC ₅₀ value, the higher the corresponding affinity and sensitivity), and the sensitivity is much higher than the combination of other chemokine receptors and chemokines (such as CCR2a/ The IC ₅₀ value of the combination of CCL2 is about 5nM, and the C ₅₀ value of the combination of CXCR3/CXCL11 is about 8.2nM).

On the other hand, the present invention uses NKG2D as the extracellular recognition domain of the CAR molecule, which can recognize various target antigens on the surface of tumor cells (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6), and can reduce The risk of reduced efficacy due to tumor heterogeneity or antigen loss is more conducive to improving the therapeutic effect on malignant tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer. Therefore, the present invention further improves the effectiveness of treatment, and improves the ability of immune cells such as CAR-T cells to fight against the high heterogeneity of malignant tumors. At the same time, studies have also shown that NKG2D CAR-T will also target immunosuppressive cells and new blood vessels in the tumor microenvironment, helping immune cells such as T cells overcome the immunosuppressive tumor microenvironment and improve the effect of tumor treatment.

In yet another aspect, the present invention uses CD40L as a second cofactor to more effectively activate the body's endogenous natural and adaptive immune responses. Activate the proliferation of T cells and the release of cytokines through CD40L, induce the transformation of M2 lineage macrophages into M1 lineage macrophages with anti-tumor activity, etc., and at the same time improve the ability of T cells to respond to tumors with certain antigen loss but high CD40 expression cell killing. In addition, unexpectedly, in the present invention, when the NKG2D CAR molecule of the present invention, exogenous CCR2b protein and exogenous CD40L protein are jointly expressed, they can synergistically significantly improve the in vitro killing effect on tumor cells.

In summary, the present invention provides a novel and more efficient engineered immune cell (such as CAR-T cell), which can efficiently chemotaxis and migrate to malignant solid tumors such as colorectal cancer, ovarian cancer, and pancreatic cancer, and can effectively activate Endogenous immune system, and CAR-T cells that can effectively fight against tumor heterogeneity.

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

Claims

An engineered immune cell, characterized in that the engineered immune cell is a T cell or NK cell, and the immune cell has the following characteristics:

(a) the immune cell expresses a chimeric antigen receptor (chimeric antigen receptor, CAR), wherein the CAR targets surface markers of tumor cells, wherein the antigen-binding domain of the CAR includes the extracellular domain of NKG2D ;

(b) said immune cell expresses an exogenous CCR2b protein; and

(c) The immune cells express exogenous CD40L protein.
The engineered immune cell according to claim 1, wherein the structure of the CAR is as shown in formula I:

L-NKG2D-H-TM-C-CD3ζ (I)

In the formula,

L is nothing or a signal peptide sequence;

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

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory signal domain;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

The "-" is a connecting peptide or a peptide bond.
The engineered immune cell according to claim 1, characterized in that, in addition to the first CAR shown in formula I, the CAR cell also contains a second CAR for the second antigen, the second CAR The structure of CAR is shown in Formula II:

L-scFv-H-TM-C-CD3ζ (II)

In the formula,

L is nothing or a signal peptide sequence;

scFv is an antigen-binding domain;

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory domain;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ or a mutation/modification thereof;

The "-" is a connecting peptide or a peptide bond.
The engineered immune cell according to claim 1, wherein the amino acid sequence of the extracellular domain of the NKG2D is shown in positions 73-216 of SEQ ID NO:1.
The engineered immune cell according to claim 1, wherein the amino acid sequence of the CCR2b protein is as shown in SEQ ID NO: 2; and/or

The amino acid sequence of the CD40L protein is shown in SEQ ID NO:5.
The engineered immune cell according to claim 1, wherein the first CAR shown in formula I and the second CAR shown in formula II can be combined into one to form a CAR shown in formula IIIa or IIIb :

L-NKG2D-scFv-H-TM-C-CD3ζ (IIIa)

L-scFv-NKG2D-H-TM-C-CD3ζ (IIIb)

In the formula,

L is nothing or a signal peptide sequence;

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

scFv is an antigen-binding domain;

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory domain;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ or a mutation/modification thereof;

The "-" is a connecting peptide or a peptide bond.
A method for preparing the engineered immune cell according to claim 1, comprising the following steps:

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

(B) transforming the immune cell so that the immune cell expresses the CAR molecule and exogenous CCR2b protein and exogenous CD40L protein, thereby obtaining the engineered immune cell according to claim 1 .
The method according to claim 7, characterized in that, in step (B), comprising:

(B1) introducing the first expression cassette expressing the CAR into the immune cells; (B2) introducing the second expression cassette expressing CCR2b into the immune cells; (B3) introducing the third expression cassette expressing CD40L into the immune cells Immune cells; wherein the steps (B1), (B2) and (B3) can be performed in any order.
A preparation, characterized in that the preparation contains the engineered immune cell according to claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.
The preparation according to claim 9, wherein the preparation contains the CAR-T cells of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
A use of the engineered immune cell according to claim 1, characterized in that it is used to prepare a drug or preparation that can prevent and/or treat cancer.
The use according to claim 11, wherein the tumor is selected from the group consisting of colon cancer, rectal cancer, ovarian cancer, or pancreatic cancer.
The use according to claim 11, characterized in that the tumor is a tumor with high expression of NKG2D ligand and/or high expression of chemokines and/or high expression of CD40.
A kit for preparing the engineered immune cell according to claim 1, characterized in that the kit contains 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 NKG2D;

(2) a second nucleic acid sequence containing a second expression cassette for the combined expression of CCR2b; and

(3) A third nucleic acid sequence containing a third expression cassette for co-expressing CD40L.
A method for treating tumors, comprising the step of: administering a safe and effective amount of the preparation according to claim 9 to a subject in need, thereby treating the subject's tumor.