WO2021239020A1

WO2021239020A1 - Immunotherapy method for combining chimeric antigen receptor and type i interferon and application thereof

Info

Publication number: WO2021239020A1
Application number: PCT/CN2021/096191
Authority: WO
Inventors: 张娜; 刘小红; 杜冰; 张楫钦; 刘明耀; 席在喜
Original assignee: 上海邦耀生物科技有限公司
Priority date: 2020-05-26
Filing date: 2021-05-26
Publication date: 2021-12-02
Also published as: CN113717942B; CN113717942A

Abstract

Provided are an immunotherapy method for combining a chimeric antigen receptor and a type I interferon and an application thereof. Specifically provided is an engineered immune cell expressing a chimeric antigen receptor CAR and a type I interferon targeting tumor cell markers. The cell can selectively kill tumor cells significantly.

Description

An immunotherapy method combining chimeric antigen receptor and type I interferon and its application

Technical field

The invention belongs to the field of biotechnology. Specifically, the present invention relates to an immunotherapy method combining chimeric antigen receptor and type I interferon and its application.

Background technique

Malignant tumors have become one of the main public health problems that seriously threaten people's health, and increasingly become the main risk factor for death of residents. The incidence of tumors in my country has continued to rise for many years. Because of its high mortality rate, more than one-fifth of deaths are caused by malignant tumors, and it has become the leading cause of death.

Traditional treatment methods include chemotherapy, radiotherapy and targeted therapy, which mainly target tumor cells. The effect only depends on the direct killing of tumor cells by drugs. However, these treatment methods are only effective in the short term, and most tumors can develop drug resistance. , So the treatment benefit is very limited.

As a new type of treatment, tumor immunotherapy mainly uses immune cells as the research object. It enhances the body's anti-tumor immune response by regulating the tumor microenvironment, innate immunity, and adaptive immunity, and has the potential for long-term benefits.

Programmed cell death-1 (PD-1)/programmed cell death-ligand1 (PDL1) inhibitors in immune checkpoint inhibitors have achieved success in tumor treatment A huge success, it can block tumor escape, activate its own immune system, and improve the body's anti-tumor immunity, thereby achieving the purpose of inhibiting and killing tumor cells, providing a new direction for cancer immunotherapy, but it may be due to tumor infiltration Low infiltration of sexual lymphocytes, difficulty in immune activation, and relative lack of PD-L1 expression in cancer tissues. Whether it is single treatment or combination treatment, the effect in clinical trials is not optimistic. Objective response (OR) rate Low. Therefore, it is still an unmet need to develop or upgrade new immunotherapies to enhance the therapeutic effect of solid tumors.

Although CAR-T manually replenishes "ammunition", although it has shown great relief in hematoma, it still faces exhaustion, which is manifested by proliferation and continuous decline. There are three main difficulties affecting the expansion of CAR-T to solid tumor treatment. First, the special vasculature and matrix barrier of solid tumors prevent T cell infiltration. The abnormal blood vessels promoted by tumors make it difficult for immune cells to infiltrate into tumor tissues, which may affect the formation of anti-tumor "base"-TLS, so it is impossible to obtain signals from innate immune cells such as DC and B cells. Second, the immunosuppressive tumor microenvironment. In the hypoxic and acidic solid tumor microenvironment, not only immunosuppressive factors such as TGF-β, but also a variety of immunosuppressive signals such as PD-L1 are expressed on the surface of tumor cells. In addition, there are immunosuppressive regulatory cells such as Treg and MDSC in the tumor microenvironment. After CART enters the tumor microenvironment, it is regulated by a variety of immunosuppressive signals to inhibit its activity. Third, the expression of solid tumor targets is more complicated. Most of the targets belong to tumor-associated antigens, that is, the target is also expressed in other normal tissues. Controlling the off-target toxicity of CART is also very important. Although there are some fourth-generation CARTs that regulate the deficiencies of ordinary CARTs through cytokines, they have not fundamentally improved the microenvironment of angiogenesis and immunosuppression, and their functions are still not perfect.

Therefore, there is an urgent need in the art to develop a new type of chimeric antigen receptor T cells that can directly act on tumor cells, inhibit and kill tumor cells, and can also regulate innate immune responses, thereby promoting antigen presentation and killing cell functions.

Summary of the invention

The purpose of the present invention is to provide a new type of chimeric antigen receptor T cells, which can directly act on tumor cells to inhibit and kill tumor cells, and can also regulate the innate immune response, thereby promoting antigen presentation and killing cell functions.

The first aspect of the present invention provides an engineered immune cell that expresses a chimeric antigen receptor CAR targeting tumor cell markers and type I interferon, the tumor cell marker Selected from the following group: PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or a combination thereof.

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

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

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

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

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

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

L-S-H-TM-C-CD3ζ (I)

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

L is no or signal peptide sequence;

S is an antigen binding domain targeting tumor cell markers, which are selected from the following group: PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or a combination thereof;

H is no or hinge area;

TM is the transmembrane domain;

C is a costimulatory signal molecule;

CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

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

In another preferred example, the structure of the single-chain variable region sequence of the antibody targeting tumor cell marker is as shown in formula A1 or A2:

V _H1 -V _L1 (A1); or

V _L1 -V _H1 (A2);

Wherein, V _L1 is the light chain variable region _{of the anti-tumor cell marker antibody; V H1} is the heavy chain variable region of the anti-tumor cell marker antibody; "-" is the connecting peptide (or flexible linker) or peptide bond.

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

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

In another preferred embodiment, the amino acid sequence of the flexible linker is shown in SEQ ID NO.: 15.

In another preferred example, _{the amino acid sequence of V L1} is shown in SEQ ID NO.:1, positions 22-128, and _{the amino acid sequence of V H1} is shown in SEQ ID NO.:1, positions 144-258.

In another preferred example, _{the amino acid sequence of V L1} is shown in SEQ ID NO.: 49, positions 22-134, and _{the amino acid sequence of V H1} is shown in SEQ ID NO.: 49, positions 150-264.

In another preferred example, _{the amino acid sequence of V L1} is shown in SEQ ID NO.: 56 at positions 22 to 131, and _{the amino acid sequence of V H1} is shown in SEQ ID NO.: 56 at positions 146 to 272.

In another preferred example, _{the amino acid sequence of V L1} is as shown in SEQ ID NO.: 2 or 51 or 58.

In another preferred example, _{the amino acid sequence of V H1} is as shown in SEQ ID NO.: 3 or 53 or 60.

In another preferred example, the sequence of the single-chain variable region of the antibody targeting tumor cell markers is murine, human, human and murine chimeric, or fully humanized single-chain antibody variable. District fragments.

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

In another preferred embodiment, the L is a signal peptide derived from CD8a.

In another preferred embodiment, the nucleotide sequence of L is shown in SEQ ID NO.: 12.

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

In another preferred example, the H is a hinge region of a protein selected from the group consisting of CD8, Ig (immunoglobulin) hinge, or a combination thereof.

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

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

In another preferred example, the TM is a transmembrane region of a protein selected from the group consisting of CD8a, CD8, CD28, CD33, CD37, CD8α, CD5, CD16, ICOS, CD9, CD22, CD134, CD137, CD154, CD19, CD45, CD4, CD3ε, or a combination thereof.

In another preferred embodiment, the TM is the transmembrane region derived from CD8a.

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

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

In another preferred embodiment, the C is a costimulatory signal molecule derived from 4-1BB.

In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO.:7.

In another preferred embodiment, the amino acid sequence of the CD3ζ is shown in SEQ ID NO.: 8.

In another preferred embodiment, the amino acid sequence of the CAR is shown in SEQ ID NO.: 1 or 49 or 56.

In another preferred example, the type I interferon is selected from the following group: IFNα1, IFNα2 (including IFNα2a, IFNα2b, IFNα2c), IFNα4, IFNα5, IFNα6, IFNα7, IFNα8, IFNα10, IFNα13, IFNα14, IFNα17, IFNα21, IFNβ, IFNε, IFNκ, IFNω, IFNδ, IFNτ, or a combination thereof, preferably IFNα2b.

In another preferred embodiment, the amino acid sequence of the type I interferon is shown in any one of SEQ ID NO.: 9, 36-48.

The second aspect of the present invention provides a method for preparing the engineered immune cells according to the first aspect of the present invention, including the following steps:

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

(B) The immune cells are modified so that the immune cells express the chimeric antigen receptor CAR and type I interferon targeted to tumor cell markers, thereby obtaining the engineering described in the first aspect of the present invention Of immune cells.

In another preferred example, in step (A), it further includes isolating and/or activating the immune cells to be modified.

In another preferred example, in step (B), it includes (B1) introducing the first expression cassette expressing the CAR targeting tumor cell marker into the immune cell; and (B2) expressing type I interference The second expression cassette of the protein is introduced into the immune cells; wherein the step (B1) can be performed before, after, at the same time, or alternately after the step (B2).

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

In another preferred example, when the immune cells to be modified in step (A) already express the CAR, step (B1) can be omitted.

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

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

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

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

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

In another preferred example, the vector is a viral vector or a transposon.

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 a combination thereof.

In another preferred example, the vector is a lentiviral vector or a transposon.

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

The third aspect of the present invention provides a preparation containing 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 formulation is a liquid formulation.

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

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

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

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

In another preferred example, the tumor includes highly expressing tumor cell markers (such as PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D Body, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138) tumors.

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

In another preferred example, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or a combination thereof.

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

In another preferred embodiment, the solid tumor is selected from the group consisting of prostate cancer, liver cancer, head and neck cancer, melanoma, non-Hodgkin’s lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma , Thyroid Cancer, Kidney Cancer, Mesothelioma, Osteosarcoma, Cholangiocarcinoma, Ovarian Cancer, Gastric Cancer, Bladder Cancer, Meningioma, Pancreatic Cancer, Multiple Squamous Cell Tumor, Esophageal Cancer, Lung Small Cell Carcinoma, Colorectal Cancer, Breast cancer, medulloblastoma, breast cancer, or a combination thereof.

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

(1) A first nucleic acid sequence, said first nucleic acid sequence containing a first expression cassette for expressing a chimeric antigen receptor CAR targeting tumor cell markers, the tumor cell markers being selected from the following group: PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or a combination thereof ;with

(2) A second nucleic acid sequence containing a second expression cassette for expressing type I interferon.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The eighth aspect of the present invention provides a fusion protein comprising a chimeric antigen receptor CAR targeting tumor cell markers and type I interferon, wherein the tumor cell marker is selected from the group consisting of PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or a combination thereof .

In another preferred embodiment, the CAR and the type I interferon are connected by a connecting peptide.

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

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

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

In another preferred example, the structure of the fusion protein is shown in the following formula III:

L-S-H-TM-C-CD3ζ-(Z3-P)m(I)

Where

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

L is no or signal peptide sequence;

H is no or hinge area;

TM is the transmembrane domain;

C is a costimulatory signal molecule;

CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ;

Z3 is a connecting peptide;

P is type I interferon;

m is 1, 2, 3, or 4.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO.: 10 or 54 or 61.

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

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

(a) A polynucleotide encoding the fusion protein shown in SEQ ID NO.: 10 or 54 or 61;

(b) A polynucleotide whose sequence is shown in SEQ ID NO.: 11 or 55 or 62;

(c) A polynucleotide whose nucleotide sequence is more than 75% (preferably more than 80%) homologous to the sequence shown in (b);

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

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

In another preferred embodiment, the polynucleotide sequence is as shown in SEQ ID NO.: 11 or 55 or 62.

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

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

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

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

In another preferred embodiment, the vector is selected from the group consisting of a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a transposon, or a combination thereof.

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

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

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

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

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

Description of the drawings

Figure 1 shows the structural schematic diagram of the expression elements contained in the first nucleic acid sequence and the second nucleic acid sequence in Example 1. In the figure, A is the structural schematic diagram of the first nucleic acid sequence targeting the PSMA target, and B is the structural schematic diagram of the first nucleic acid sequence targeting the PSMA target. A schematic diagram of the structure of the connection between the first nucleic acid and the second nucleic acid of the target; C is a schematic diagram of the structure of the first nucleic acid sequence targeting the GPC3 target, and D is the schematic diagram of the structure of the connection between the first nucleic acid and the second nucleic acid targeting the GPC3 target ; E is a schematic structural diagram of the first nucleic acid sequence targeting the BCMA target, and F is a schematic structural diagram of the first nucleic acid and the second nucleic acid targeting the BCMA target.

Figure 2 shows the flow chart of the virus titer detection of the control viruses PSMA-CAR and IFNα2b-CAR.

Figure 3 shows the flow chart of the positive detection of PSMA-CART and IFNα2b-CART CAR prepared with lentivirus.

Figure 4A is a flow diagram of positive detection of PB-PSMA-CART and PB-IFNα2b-PSMA-CART CAR prepared by transposon electroporation; Fig. 4B is PB-GPC3-CART and PB-IFNα2b- prepared by transposon electroporation The flow chart of GPC3-CART CAR positive detection; Figure 4C is the flow chart of the positive detection of PB-BCMA-CART and PB-IFNα2b-BCMA-CART CAR prepared by transposon electroporation; Figure 4D is the flow diagram of positive detection of PB-BCMA-CART and PB-IFNα2b-BCMA-CART CAR prepared by transposon electroporation PB-PSMA-CART and PB-IFNα2b-PSMA-CART IFNa2b ELISA test results.

Figure 5A shows the results of killing PC3-PSMA tumor cells by PB-PSMA-CART and PB-IFNα2b-PSMA-CART prepared by transposon electroporation. IFNα2b-GPC3-CART kills huh7 tumor cells. Figure 5C is the result of PB-BCMA-CART and PB-IFNα2b-BCMA-CART prepared by transposon electroporation to kill mm1s tumor cells.

Figure 6 is a graph showing the comparison of proliferation of PB-PSMA-CART and PB-IFNα2b-PSMA-CART prepared by transposon electrotransformation.

Figure 7 shows the flow cytometric comparison between PB-PSMA-CART and PB-IFNα2b-PSMA-CART prepared by transposon electroporation.

Figure 8A: PB-PSMA-CART and PB-IFNα2b-PSMA-CART cell supernatant prepared by transposon electrotransduction to stimulate human PBMC QPCR results; Figure 8B: PB-PSMA-CART and PB-IFNα2b prepared by transposon electrotransduction -Flow cytometric results of TRAIL expression after stimulation of human PBMC by PSMA-CART cell supernatant. Figure 8C is a graph of the killing effect of traditional CART and IFNα2b-CART cells prepared by electrotransduction with transposon incubation with PBMC and huh7 tumor cells.

Figure 9 The QPCR results (left) and the results of the migration experiment (right) after stimulating the tumor cells DU145 with the supernatant of PSMA-CART and IFNα2b-CART cells prepared by transposon electroporation.

Detailed ways

After extensive and in-depth research, the inventors unexpectedly discovered for the first time that they contained targeted tumor cell markers (such as PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1) ), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138) chimeric antigen receptor CAR and type I interferon engineered immune cells can mobilize endogenous immunity to exert a stronger anti-tumor effect Function, more effective and selective killing of tumor cells, such as tumor cells with high expression of PSMA. On this basis, the inventor completed the present invention.

Term Description

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

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

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

As used herein, "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding antigen, a transmembrane domain derived from a different polypeptide from the extracellular domain, and at least one cell Inner domain. "Chimeric antigen receptor (CAR)" is also called "chimeric receptor", "T-body" or "chimeric immune receptor (CIR)". The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a certain antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known as a domain that transmits signals to activate or inhibit biological processes in a cell.

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

As used herein, the terms "administration" and "treatment" refer to the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs, or biological fluids. "Administration" and "treatment" can refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells. "Administration" and "treatment" also mean treatment by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to humans, animals or research subjects, refers to treatment, preventive or preventive measures, research and diagnosis; including anti-human LAG-3 antibodies and humans or animals, subjects, cells, tissues , Physiological compartment or physiological fluid contact.

As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including any one CAR of the present invention and a composition thereof, to a patient who has one or more disease symptoms, and the therapeutic agent is known to These symptoms have a therapeutic effect. Generally, the patient is administered in an amount (therapeutically effective amount) of a therapeutic agent that is effective to alleviate one or more disease symptoms.

As used herein, the term "optional" or "optionally" means that the event or situation described later can occur but does not have to occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may but not necessarily have, and may be 1, 2, or 3.

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

PSMA

PSMA (prostrate specific membrane antigen, prostate specific membrane antigen) is highly expressed in prostate cancer and is an ideal target for immunotherapy of solid tumors. CAR-T cells targeting PSMA are extremely specific in tumor immunotherapy. Tropism and less limitation of major histocompatibility complexes. Junghans R et al. (2016) and Zuccolotto G (2014) have demonstrated the effectiveness and safety of CART cells in preclinical studies and clinical trials, but their application is still There are limitations, so further research and exploration are needed.

GPC3

Glypican 3 (GPC3) is highly expressed in hepatocellular carcinoma, gastric cancer and other cancers. It is a meaningful diagnostic, therapeutic and prognostic biomarker for liver cancer, and it has been used in the second generation /Research report on the treatment of hepatocellular carcinoma with CAR-T cells targeted by GPC3 of the third generation.

GD2

Gangliosides, such as GD2, are not only highly expressed in neuroblastoma, but also in a variety of solid tumors, including osteosarcoma, retinoblastoma, some soft tissue sarcomas, brain tumors and other tumors. A clinical trial (NCT02765243) was carried out to evaluate the safety and effectiveness of GD2CART.

HER2

The amplification or overexpression of human epidermal growth factor receptor HER2 occurs in approximately 15-30% of breast cancers and 10-30% of gastric/gastroesophageal cancers, and serves as a prognostic and predictive biomarker. HER2 overexpression is also seen in other cancers, such as ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer. HER2 is a fully validated target for breast cancer and sarcoma. Targeting HER2 with CAR T cells is very effective in the treatment of brain metastatic breast cancer and sarcoma in animal models, and relevant clinical trials have been carried out (NCT03696030, NCT00902044) .

MSLN

Mesothelin (MSLN) is a tumor differentiation antigen, which is usually highly expressed in human cancers, including malignant mesothelioma, pancreatic cancer, ovarian cancer and lung adenocarcinoma. Studies have reported that CART with this as a target can inhibit cancer growth.

CEA

Carcinoembryonic antigen (CEA) is widely expressed in cancers such as gastric cancer, lung cancer, pancreatic cancer, breast cancer and colorectal cancer. It has been developed and confirmed that anti-CEA CAR modified T cells play a role in solid tumors (NCT02349724).

EGFRvIII

Epidermal growth factor receptor EGFRvIII is the most common mutant of EGFR, and it is highly expressed in tumors such as malignant glioma, glioblastoma, brain cancer, and glioma. Its expression promotes tumorigenesis and is associated with poor prognosis. It is not expressed in normal tissues and is an attractive target for immunotherapy. In in vitro experiments, CART against EGFRvIII has a good tumor-killing ability, and there have been related clinical experimental studies (NCT01454596).

Claudin18.2(CLDN18.2)

The gastric-specific membrane protein Claudin 18.2 (CLDN18.2) is considered to be a potential therapeutic target for gastric cancer and other cancer types. CLDN18.2-specific CAR T cells may be the most promising for gastric cancer and other potential CLDN18.2-positive tumors Treatment strategy (NCT03874897).

MUC-1

MUC-1 is a glycosylated transmembrane protein. In normal cells, it is expressed on the apical surface of epithelial cells. Cancer cells express 100 times more MUC1 protein than normal cells. Studies have shown that MUC1-related antibody production and cellular immune responses have a positive impact on the prognosis of cancer patients. There have been clinical trials on the safety and effectiveness of MUC-1CART in the treatment of intrahepatic cholangiocarcinoma (NCT03633773).

NKG2D ligand

NKG2D ligand is not only expressed in most human tumor cells, including solid tumors (ovarian cancer, bladder cancer, breast cancer, lung cancer, liver cancer, colon cancer, kidney cancer, prostate cancer, melanoma, Ewing sarcoma, glioma) And neuroblastoma), and various leukemias (AML, CML, CLL), lymphoma and multiple myeloma. It is also expressed on immunosuppressive cells in the tumor microenvironment. Therefore, it provides an attractive target for cancer treatment.

CD19

CD19 is the most widely used hematological malignant tumor target in CAR-T cell therapy. It is used in acute B-cell lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL) and Clinical trials of diffuse large B-cell lymphoma (DLBCL) and other hematological malignancies have achieved good therapeutic effects. In addition to CD19, BCMA, CD20, CD22, CD30, IL3RA, CD38, CD138 and other molecules are also research targets for hematological malignancies, especially CD19-negative hematological tumors.

BCMA

BCMA (B cell maturation antigen), also called TNFRSF17 (TNF receptor superfamily member 17, TNF ligand superfamily member 17), is mainly expressed in plasma cells and mature B lymphocytes, and is basically undetectable in other normal human cells . BCMA is the most selectively expressed receptor on multiple myeloma cell lines, and its expression gradually increases with the differentiation of B cells, and it also gradually increases in the disease process of multiple myeloma. Immunotherapy targeting BCMA has significant effects in preclinical and clinical studies, especially CAR-T technology, which can specifically recognize tumor antigens in a non-major histocompatibility complex-dependent manner and exert a powerful anti-tumor immune effect .

Type I Interferon

Interferon is a glycoprotein, which is mainly produced by a variety of cells derived from hematopoietic and matrix. According to its structural characteristics, receptors, cell source and biological activity, it can be divided into three types, type I, II and III. Viruses, inhibiting cell proliferation, regulating immunity and anti-tumor effects all have important regulatory effects.

Among them, type I interferon has the potential to regulate the immune microenvironment. IFNα can enhance the activation of TH1 cells by inhibiting the proliferation and activity of Treg cells.

Type I interferons include seven types: IFNα, IFNβ, IFNε, IFNκ, IFNω, IFNδ and IFNτ. In humans and other animals due to selective splicing and other reasons, IFNα has multiple subtypes, and these subtypes have homology. , But the function is different. Its receptor is a heterodimeric transmembrane IFNα receptor (IFNAR). All type I IFNs can activate receptor-related JAK1 and TYK2 kinases and downstream signal transducers and activators of transcription (STAT ) Transcription factors. Thereby, an IFN stimulating gene factor 3 (ISGF3) complex composed of STAT1, STAT2 and IFN regulatory factor 9 (IRF9) is formed. The combination of ISGF3 complex and IFN stimulated response element (ISRE) induces the expression of IFN stimulating gene (ISG), It can regulate hundreds of genes downstream. Although the main signaling of type I IFN is through STAT1 and STAT2, the IFNα and IFNβ pathways can activate STAT3, STAT4, and Stat5, so they have a very wide range of effects.

In a preferred embodiment, Type I interferon includes, but is not limited to: IFNα1, IFNα2 (including IFNα2a, IFNα2b, IFNα2c), IFNα4, IFNα5, IFNα6, IFNα7, IFNα8, IFNα10, IFNα13, IFNα14, IFNα17, IFNα21 IFNβ, IFNε, IFNκ, IFNω, IFNδ, IFNτ, or a combination thereof, preferably IFNα2b.

The present invention finds for the first time that in the presence of type I interferons, on the one hand, they can directly act on tumor cells to inhibit and kill tumor cells; on the other hand, they can regulate the innate immune response, thereby promoting antigen presentation and killing cell functions; one On the one hand, they activate the adaptive immune system, thereby promoting the development of high-affinity antigen-specific T and B cell responses and immune memory; on the one hand, they not only inhibit tumor angiogenesis but also promote immune cell migration and infiltration. On the one hand, they can act on Treg and MDSC to regulate the immunosuppressive tumor microenvironment.

In a preferred embodiment, the amino acid sequence of Type I interferon is shown in any one of SEQ ID NO.: 9, 36-48.

Antigen binding domain

In the present invention, the antigen binding domain of the chimeric antigen receptor CAR specifically binds to tumor cell markers (such as PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138).

Hinge region and transmembrane region

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

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

Intracellular domain

The intracellular domain or another intracellular signaling domain of the CAR of the present invention is responsible for the activation of at least one normal effector function of the immune cell in which the CAR has been placed. The term "effector function" refers to the exclusive function of the cell. For example, the effector function of T cells may include cytolytic activity or auxiliary activity including cytokine secretion. Therefore, the term "intracellular signal transduction domain" refers to the part of the protein that transduces effector function signals and directs the cell to perform a specific function. Although the entire intracellular signaling domain can generally be used, in many cases, the entire chain need not be used. In terms of using truncated portions of intracellular signaling domains, such truncated portions can be used to replace the complete chain as long as it transduces effector function signals. The term "intracellular signaling domain" generally refers to any truncated portion of an intracellular signaling domain that is sufficient to transduce effector function signals.

Preferable examples of the intracellular signal transduction domain used in the CAR of the present invention include the cytoplasmic sequence of T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction after antigen receptor binding, and these sequences Any derivative or variant of and any synthetic sequence with the same functional capabilities.

In a preferred embodiment, the cytoplasmic domain of the CAR may be designed to include the CD3ζ signaling domain itself, or may be combined with any other desired cytoplasmic domains (one or more) useful in the content of the CAR of the present invention. A) joint. For example, the cytoplasmic domain of CAR may include a CD3ζ chain portion and a costimulatory signal transduction region. The costimulatory signal transduction region refers to a part of the CAR that includes the intracellular domain of costimulatory molecules. Co-stimulatory molecules are cell surface molecules required for effective response of lymphocytes to antigens, not antigen receptors or their ligands. Preferably, it includes 4-1BB (CD137) and the like.

The cytoplasmic signal transduction sequences in the cytoplasmic signal transduction portion of the CAR of the present invention can be connected to each other randomly or in a prescribed order. Optionally, short oligopeptide or polypeptide linkers, preferably between 2 and 10 amino acids in length, can form the link. The glycine-serine doublet provides a particularly suitable linker.

In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to include the signaling domain of 4-1BB (costimulatory molecule) and the signaling domain of CD3ζ.

Chimeric Antigen Receptor (CAR)

Chimeric antigen receptors (CARs) are composed of extracellular antigen recognition regions, usually scFv (single-chain variable fragment), transmembrane regions and intracellular co-stimulatory signal regions. The design of CARs has gone through the following process: The first generation CAR has only one intracellular signal component CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , And cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical effects. The second-generation CARs introduce a costimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function has been greatly improved, which further strengthens the persistence of CAR-T cells and the effect of tumor cells. The lethality. On the basis of the second-generation CARs, some new immunostimulatory molecules such as CD27 and CD134 are connected in series to develop into the third- and fourth-generation CARs.

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

CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. Such a high response rate could not be achieved by any previous treatment method. It has triggered an upsurge of clinical research in various countries around the world.

Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes target-specific binding elements (also called antigen binding domains). The intracellular domain includes a costimulatory signal transduction region and/or a zeta chain part. The costimulatory signal transduction region refers to a part of the intracellular domain that includes costimulatory molecules. Co-stimulatory molecules are cell surface molecules required for effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

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

When the CAR of the present invention is expressed in T cells, it can perform antigen recognition based on the antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, resulting in tumor cells not growing, being promoted to die or being affected in other ways, and causing the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused with an intracellular domain from one or more of the costimulatory molecule and/or zeta chain. Preferably, the antigen binding domain is fused with the intracellular domain combined with the 4-1BB signaling domain and/or the CD3ζ signaling domain.

As used herein, "antigen-binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments that have antigen-binding activity. The Fv antibody contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1/6 of that of a complete antibody. The single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred mode of the present invention, the scFv includes markers that specifically recognize tumors with high expression tumor cells (such as PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1) ), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138) antibodies, preferably single-chain antibodies.

In a preferred embodiment, the antigen-binding portion of the CAR of the present invention targets tumor cell markers. In a preferred embodiment, the antigen binding portion of the CAR of the present invention is a scFV targeting PSMA.

In a preferred embodiment, the structure of the scFv is as shown in formula A1 or A2:

V _H1 -V _L1 (A1); or

V _L1 -V _H1 (A2);

In a preferred embodiment, _{the amino acid sequence of V L1} is shown at positions 22-128 of SEQ ID NO.:1, and _{the amino acid sequence of V H1} is shown at positions 144-258 of SEQ ID NO.:1.

In a preferred embodiment, _{the amino acid sequence of V L1} is shown in SEQ ID NO.:2.

In a preferred embodiment, _{the amino acid sequence of V H1} is shown in SEQ ID NO.:3.

In a preferred embodiment, the antigen binding portion of the CAR of the present invention is a scFV targeting GPC3.

V _H1 -V _L1 (A1); or

V _L1 -V _H1 (A2);

In a preferred embodiment, _{the amino acid sequence of V L1} is shown in SEQ ID NO.: 49, positions 22-134, and _{the amino acid sequence of V H1} is shown in SEQ ID NO.: 49, positions 150-264.

In a preferred embodiment, _{the amino acid sequence of V L1} is shown in SEQ ID NO.:51.

In a preferred embodiment, _{the amino acid sequence of V H1} is shown in SEQ ID NO.:53.

In a preferred embodiment, the antigen binding portion of the CAR of the present invention is a scFV targeting BCMA.

V _H1 -V _L1 (A1); or

V _L1 -V _H1 (A2);

In a preferred embodiment, _{the amino acid sequence of V L1} is shown in SEQ ID NO.: 56 at positions 22 to 131, and _{the amino acid sequence of V H1} is shown in SEQ ID NO.: 56 at positions 146 to 272.

In a preferred embodiment, _{the amino acid sequence of V L1} is shown in SEQ ID NO.:58.

In a preferred embodiment, _{the amino acid sequence of V H1} is shown in SEQ ID NO.:60.

In a preferred embodiment, the scFV comprises a variant form which has ≥80%, ≥85%, ≥90%, ≥95%, ≥98% or ≥99% homology with its wild-type scFV sequence sex.

In the present invention, the scFV of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the scFV of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.

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

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

In the present invention, the intracellular domain in the CAR of the present invention includes the transmembrane region of CD8a, the costimulatory factor of 4-1BB, and the signal transduction domain of CD3ζ.

In a preferred embodiment of the present invention, the amino acid sequence of the CAR is shown in any one of SEQ ID NO.: 1, 10, 49, 54, 56 or 61.

In a preferred embodiment of the present invention, the nucleotide sequence of the CAR is shown in any one of SEQ ID NO.: 11, 55 or 62.

Among them, in SEQ ID NO.: 10, positions 1-21 are signal peptides; positions 22-258 are antigen-binding domains targeting tumor cell markers (such as PSMA-targeted antibody single-chain variable region sequences) ; 259-303 is the hinge region; 304-327 are transmembrane regions (such as the transmembrane region of CD8a); 328-370 are costimulatory components (such as 4-1BB); 371-481 are CD3ζ , Positions 482-500 are connecting peptides (such as self-cleaving proteins), and positions 501-689 are type I interferons (such as IFNα2b).

In SEQ ID NO.: 54, positions 1-21 are signal peptides; positions 22-264 are antigen-binding domains targeting tumor cell markers (such as the single-chain variable region sequence of an antibody targeting GPC3); 265-309 are the hinge region; 310-333 are transmembrane regions (such as the transmembrane region of CD8a); 334-375 are costimulatory components (such as 4-1BB); 376-487 are CD3ζ, and 488 Position -506 is for connecting peptide (such as self-cleaving protein), position 507-694 is for type I interferon (such as IFNα2b).

In SEQ ID NO.: 61, positions 1-21 are signal peptides; positions 22-272 are antigen-binding domains targeting tumor cell markers (such as antibody single-chain variable region sequences targeting BCMA); 273-317 are the hinge region; positions 318-341 are transmembrane regions (such as the transmembrane region of CD8a); positions 342-383 are costimulatory elements (such as 4-1BB); positions 384-495 are CD3ζ, and positions 496 Position -514 is a connecting peptide (such as self-cleaving protein), and positions 515-702 are type I interferons (such as IFNα2b).

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 of the present invention. The CAR-T cell of the present invention can target tumor surface antigens. (Such as PSMA), used to treat tumors with high or positive PSMA expression, especially solid tumors.

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) In view of the fact that many tumor cells express the same tumor antigen, they are targeted at a certain type of 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 body Cells reduce the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

In the present invention, the CAR of the present invention includes (i) an extracellular domain, which includes an antigen targeting tumor cell surface antigen; (ii) a transmembrane domain; (iii) a costimulatory factor; and (iv) a signal of CD3ζ Conduction domain; and; (v) connecting peptide (such as self-cleaving protein); (vi) type I interferon.

Chimeric antigen receptor macrophages (CAR-M cells)

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

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

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

Chimeric antigen receptor NK cells (CAR-NK cells)

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

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

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

Foreign T cell antigen receptor

As used herein, the foreign T cell antigen receptor (TCR) refers to the cloning of the α chain and β chain of TCR from tumor-reactive T cells through gene transfer technology, and the use of lentivirus or Retroviruses are vectors, which are transferred exogenously into TCR in T cells.

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

Carrier

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

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

To summarize briefly, the expression cassette or nucleic acid sequence of the present invention is usually operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. A typical cloning vector contains transcription and translation terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

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

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

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

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

Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Generally, these are located in the 30-110 bp region upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible in order to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before the activity begins to decrease. 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 immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter , Myosin promoter, heme promoter and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered part of the invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

In order to evaluate the expression of the CAR polypeptide or part thereof, the expression vector introduced into the cell may also contain either or both of the selectable marker gene or the reporter gene, so as to facilitate the search for the cell population to be transfected or infected by the viral vector. To identify and select expressing cells. In other aspects, the selectable marker can be carried on a single piece of DNA and used in the co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences so that they can be expressed in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

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

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

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

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

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

Where a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. Consider using 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 lipids. Lipid-associated nucleic acids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, and attached via linking molecules associated with both liposomes and oligonucleotides To liposomes, trapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles or Complexed with micelles, or otherwise associated with lipids. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in the solution. For example, they may exist in a bilayer structure, as micelles or have a "collapsed" structure. They can also simply be dispersed in the solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in such compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

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

Compared with the traditional retroviral system with limited loading capacity, complicated preparation process, and random insertion risk, the transposon system has a relatively simple preparation process, integrates foreign genes into the genome through transposase, and has the advantages of high loading capacity.

The earliest used mammalian transposon system is the "Sleeping-Beauty" transposon, but the "Sleeping-Beauty" transposon has defects such as excessive inhibitory effect and small carrying fragments (about 5kb), making it difficult to The application of genetic modification is restricted. The piggyBac (PB) transposon derived from lepidopteran insects is currently the most active transposon in mammals. The host range is extremely wide, from single-celled organisms to mammals, capable of carrying large foreign DNA fragments. When the transposable fragment is within 14kb, the transposition efficiency will not decrease significantly. PB transposon mainly adopts the "cut-paste" mechanism for transposition. After the transposition fragment is excised, it will not leave a footprint at the original site, and the genome can be accurately repaired after excision. In the application of reversible transgene Has an important role. In addition, PB transposase has high plasticity. By fusing with other functional proteins or changing the functional region of the transposase, it can not only change the activity and mode of action of the transposase, but also improve the targeting of foreign gene transposition. In recent years, the integration efficiency of PB in mammalian cells has been further improved through codon optimization, site-directed mutations of amino acids at specific sites, and the introduction of corresponding nuclear localization tags, making it widely used in cell therapy and gene therapy.

The lentiviral system has an upper limit on the sequence length when expressing long-segment sequences and cannot complete the packaging of large-segment expression vectors. However, the transposon system can insert long fragments within 14k at most, and the risk of random insertion is less than that of lentivirus, which has a wider range of applications. Scope and safety of use.

preparation

The present invention provides an engineered immune 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/Kg body weight, more preferably 1×10 ⁴ -1×10 ⁷ cells/Kg body weight.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine ; Antioxidant; Chelating agent such as EDTA or glutathione; Adjuvant (for example, aluminum hydroxide); and Preservative. The formulations of the invention are preferably formulated for intravenous administration.

Therapeutic application

The present invention includes therapeutic applications with cells (e.g., T cells) transduced with lentiviral vectors (LV) encoding the expression cassettes of the present invention. The transduced T cells can target tumor cell markers (such as PSMA, etc.) proteins to coordinately activate T cells and cause cellular immune responses, thereby selectively killing tumor cells, such as tumor cells with high expression of PSMA.

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

In one embodiment, the present invention includes a type of cell therapy in which the patient's autologous T cells (or heterologous donors) 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 disease is extremely low, and antigens are recognized by T cells in a non-MHC-restricted manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the present invention can undergo stable T cell expansion in vivo and last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, CAR-T cells that are tumor cell markers (such as PSMA, etc.) cause a specific immune response against cells expressing tumor cell markers (such as PSMA, etc.).

Although the data disclosed herein specifically discloses lentiviruses including antigen-binding domains, hinges and transmembrane regions that target tumor cell surface antigens, and 4-1BB and CD3ζ signaling domains, P2A, type I interferons (such as IFNα2b) Vector, but the present invention should be interpreted as including any number of changes in each part of the construct.

Treatable cancers include tumors that have not been vascularized or have not been substantially vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma) or solid tumors. The types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies such as sarcoma, carcinoma, and melanoma. It also includes adult tumors/cancers and childhood tumors/cancers.

Hematological cancer is cancer of the blood or bone marrow. Examples of hematological (or hematogenic) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic type , Monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non- Hodgkin's lymphoma (painless and high-grade form), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

A solid tumor is an abnormal mass of tissue that does not usually contain a cyst or fluid area. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that formed them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and cancer include prostate cancer, liver cancer, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

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

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

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

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

The present invention provides a method for treating tumors, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cell of the present invention.

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

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

When referring to "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by the physician, who considers the patient (subject ) Individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. May generally indicated: including those described herein, the pharmaceutical compositions of T cells may be ¹⁰⁴ to ¹⁰⁹ doses cells / kg body weight, preferably ¹⁰⁵ to ¹⁰⁶ cells / kg body weight doses (including all integers within that range Value) application. The T cell composition can also be administered multiple times at these doses. The cells can be administered by using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.

The administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intracutaneously, intratumorally, intranodal, intraspinal, intramuscular, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by i.v. injection. The composition of 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., previous , At the same time or after) administration to the patient, the treatment modality includes but not limited to treatment with the following agents: the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In a further embodiment, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies Or other immunotherapeutics. In a further embodiment, the cell composition of the present invention is administered to bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (for example, before, at the same time, or after). patient. For example, in one embodiment, the subject may undergo the standard treatment of high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

The dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to the practice accepted in the art. ^{Generally, 1×10 6} to 1×10 ¹⁰ modified T cells of the present invention (such as CAR-T cells of the present invention) can be injected into each treatment or course of treatment by, for example, intravenous infusion, Apply to the patient.

Fusion protein

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

As used herein, the term "fusion protein" also includes variant forms of the sequence of SEQ ID NO.: 10 or 54 or 61 having the above-mentioned activity. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletion, insertion and/or substitution, and addition or addition at the C-terminus and/or N-terminus One or several (usually within 3, preferably within 2, more preferably within 1) amino acid is missing. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

The present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion protein. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogues of the present invention can be (i) one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fusion A polypeptide formed from this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence, or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.

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

Table 1

最初的残基Initial residues	代表性的取代Representative substitution	优选的取代Preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Lys；ArgGln; His; Lys; Arg	GlnGln
Asp(D)Asp(D)	GluGlu	GluGlu
Cys(C)Cys(C)	SerSer	SerSer
Gln(Q)Gln(Q)	AsnAsn	AsnAsn
Glu(E)Glu(E)	AspAsp	AspAsp
Gly(G)Gly(G)	Pro；AlaPro; Ala	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe	LeuLeu

Leu(L)Leu(L)	Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr	LeuLeu
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	SerSer	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; Ala	LeuLeu

The present invention also provides analogs of the fusion protein of the present invention. The difference between these analogs and the polypeptide shown in SEQ ID NO.: 10 or 54 or 61 may be the difference in the amino acid sequence, the difference in the modification form that does not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

Modified forms (usually without changing the primary structure) include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or to optimize their solubility.

In one embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.: 10 or 54 or 61.

Coding sequence

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

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA can be a coding strand or a non-coding strand. The sequence of the coding region encoding the mature polypeptide may be the same as the sequence encoding the polypeptide shown in SEQ ID NO.: 10 or 54 or 61 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a polypeptide shown in SEQ ID NO.: 10 or 54 or 61, but differs in the sequence of the corresponding coding region.

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

The present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or polypeptide coding sequence of the present invention. The aforementioned polynucleotides, vectors or host cells may be isolated.

As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not separated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances that coexist in the natural state.

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

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

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

In one embodiment of the present invention, the polynucleotide sequence encoding the fusion protein is as shown in SEQ ID NO.: 11 or 55 or 62.

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

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.

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

The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or protein coding sequence of the present invention, and a method for expressing the fusion protein of the present invention on the NK cell by recombinant technology.

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

Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

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

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

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

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

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

The protein in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other properties can be used to separate and purify the protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The main advantages of the present invention include:

1. The present invention finds for the first time that it contains targeted tumor cell markers (such as PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138) chimeric antigen receptor CAR and type I interferon engineered immune cells can mobilize endogenous immunity to exert a stronger anti-tumor effect, and more effectively and selectively kill tumors Cells, such as tumor cells with high expression of PSMA, tumor cells with high expression of GPC3, and tumor cells with high expression of BCMA.

2. The present invention found for the first time that type I interferon can directly act on tumor cells, inhibiting and killing tumor cells;

3. The present invention found for the first time that type I interferon can regulate the innate immune response, thereby promoting antigen presentation and the killing function of CAR engineered immune cells;

4. The present invention finds for the first time that the adaptive immune system is activated, thereby promoting the development of high-affinity antigen-specific T and B cell responses and immune memory, and improving the persistence of CAR engineered immune cells;

5. The present invention finds for the first time that type I interferon not only inhibits tumor angiogenesis but also promotes the migration and infiltration of CAR engineered immune cells.

6. The present invention finds for the first time that type I interferon can act on Treg and MDSC, thereby releasing the tumor microenvironment that inhibits CAR engineered immune cells.

7. The present invention utilizes the transposon system to mediate the efficient integration of the chimeric antigen receptor and type I interferon into the host cell genome, and can obtain positive rates of stable expression of IFNα2b-CART at different targets, and the IFNα2b-CART can be known by ELISA verification. CART can express up to about 30 times IFNα2b than traditional CART.

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

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

Example 1

PSMA, GPC3, and BCMA were used as targets to construct plasmid vectors containing two expression cassettes expressing chimeric antigen receptor and IFNα2b. Refer to Figure 1 for the structure and positional relationship of each element on the expression cassette.

Specific steps are as follows:

(1) When the target is PSMA: GenScript Synthesizes the first expression cassette expressing the chimeric antigen receptor targeting prostate specific membrane antigen. The first expression cassette (PSMA-CAR) includes: CD8α signal peptide , PSMA single-chain antibody heavy chain variable region, Linker1, PSMA single-chain antibody light chain, CD8 hinge region, CD8α transmembrane domain, intracellular costimulatory element of 4-1BB and intracellular domain of CD3ζ (Figure 1, A) Connect the above sequences in sequence, and introduce the Kozak sequence and the corresponding restriction site at the forefront. Using XbaI and SalI double enzyme digestion, the first expression cassette was transferred to the lentiviral shuttle plasmid (obtained from Shanghai Bangyao Biotechnology Co., Ltd.), and the chimeric antigen receptor expression vector pELPS-PSMA-BBz transfer vector (as Control plasmid). Using the pELPS-PSMA-BBz transfer vector plasmid as the initial plasmid, a second expression cassette expressing IFNα2b was added. The first expression cassette and the second expression cassette were connected by P2A, and the second expression cassette (IFNα2b) was IFNα2b (Figure 1, B). The resulting plasmid was named pELPS-PSMA-BBz-2A-IFNα2b transfer vector plasmid.

(2) When the target is GPC3: The first expression cassette expressing chimeric antigen receptor targeting hepatocyte-specific membrane antigen is synthesized by GenScript, and the first expression cassette (GPC3-CAR) includes: CD8α signal Peptides, GPC3 single-chain antibody heavy chain variable region, Linker1, GPC3 single-chain antibody light chain, CD8 hinge region, CD8α transmembrane domain, intracellular costimulatory element of 4-1BB and intracellular domain of CD3ζ (Figure 1 , C), connect the above sequences in sequence, and introduce the Kozak sequence and the corresponding restriction site at the forefront. Using XbaI and SalI double enzyme digestion, the first expression cassette was transferred to the lentiviral shuttle plasmid (obtained from Shanghai Bangyao Biotechnology Co., Ltd.), and the chimeric antigen receptor expression vector pELPS-PSMA-BBz transfer vector (as Control plasmid). Using the pELPS-GPC3-BBz transfer vector plasmid as the initial plasmid, a second expression cassette expressing IFNα2b was added, the first expression cassette and the second expression cassette were connected by P2A, and the second expression cassette (IFNα2b) was IFNα2b (Figure 1, D). The resulting plasmid was named pELPS-GPC3-BBz-2A-IFNα2b transfer vector plasmid.

(3) When the target is BCMA: The first expression cassette that expresses the chimeric antigen receptor targeting the specific membrane antigen of multiple myeloma is synthesized by GenScript. The first expression cassette (BCMA-CAR) includes: CD8α signal peptide, BCMA single-chain antibody heavy chain variable region, Linker1, BCMA single-chain antibody light chain, CD8 hinge region, CD8α transmembrane domain, intracellular costimulatory element of 4-1BB and intracellular domain of CD3ζ ( Figure 1, E), connect the above sequences in sequence, and introduce the Kozak sequence and the corresponding restriction site at the forefront. Using XbaI and SalI double enzyme digestion, the first expression cassette was transferred to the lentiviral shuttle plasmid (obtained from Shanghai Bangyao Biotechnology Co., Ltd.), and the chimeric antigen receptor expression vector pELPS-BCMA-BBz transfer vector (as Control plasmid). Using the pELPS-BCMA-BBz transfer vector plasmid as the initial plasmid, a second expression cassette expressing IFNα2b was added, the first expression cassette and the second expression cassette were connected by P2A, and the second expression cassette (IFNα2b) was IFNα2b (Figure 1, F). The resulting plasmid was named pELPS-BCMA-BBz-2A-IFNα2b transfer vector plasmid. Wherein, the sequence of each element of the above-mentioned first expression cassette is as follows:

The base sequence of CD8α signal peptide (CD8αLeader) is shown in SEQ ID NO.: 12:

The amino acid sequence of CD8α signal peptide (CD8αLeader) is shown in SEQ ID NO.: 4: MALPVTALLLPLALLLHAARP;

The amino acid sequence of PSMA-CAR without type I interferon is shown in SEQ ID NO.:1:

The base sequence of the PSMA single-chain antibody light chain variable region (PSMA-ScFv VL) is shown in SEQ ID NO.: 13:

The amino acid sequence of the PSMA single-chain antibody light chain variable region (PSMA-ScFv VL) is shown in SEQ ID NO.: 2:

The base sequence of the Linker of PSMA-ScFv VL and PSMA-ScFv VH is shown in SEQ ID NO.: 14:

The amino acid sequence of the Linker of PSMA-ScFv VL and PSMA-ScFv VH is shown in SEQ ID NO.: 15:

The base sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO.: 16:

The amino acid sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO.: 3:

The base sequence of CD8 hinge region (CD8hinge) is shown in SEQ ID NO.: 17:

The amino acid sequence of the CD8 hinge region (CD8hinge) is shown in SEQ ID NO.: 5:

The base sequence of CD8α transmembrane domain (CD8a-TM) is shown in SEQ ID NO.: 18:

The amino acid sequence of CD8α transmembrane domain (CD8a-TM) is shown in SEQ ID NO.: 6:

The base sequence of the intracellular costimulatory element of 4-1BB is shown in SEQ ID NO.: 19:

The amino acid sequence of the intracellular costimulatory element of 4-1BB is shown in SEQ ID NO.: 7:

The base sequence of the intracellular domain of CD3ζ is shown in SEQ ID NO.: 20:

The amino acid sequence of the intracellular domain of CD3ζ is shown in SEQ ID NO.: 8:

The base sequence of P2A is shown in SEQ ID NO.: 21:

The base sequence of type I interferon is as follows:

IFNα:

IFNα2a:

IFNα2b:

IFNα4:

IFNα5:

IFNα6:

IFNα7:

IFNα8:

IFNα10:

IFNα13:

IFNα14:

IFNα17:

IFNα21:

IFNβ:

The amino acid sequence of type I interferon is shown below:

IFNα:

IFNα2a:

IFNα2b:

IFNα4:

IFNα5:

IFNα6:

IFNα7:

IFNα8:

IFNα10:

IFNα13:

IFNα14:

IFNα17:

IFNα21:

IFNβ:

The amino acid sequence of PSMA-CAR containing type I interferon (IFNα2b) is shown in SEQ ID NO.: 10:

The nucleotide sequence of the PSMA-CAR containing type I interferon (IFNα2b) is shown in SEQ ID NO.: 11:

Among them, the first expression cassette PSMA-ScFv VL-Linker-PSMA-ScFv VL (Figure 1-A) expressing the chimeric antigen receptor targeting prostate-specific membrane antigen can be replaced with the following expressions targeting GPC3 and BCMA Corresponding components, other components can remain unchanged.

The PB-GPC3-CAR plasmid (Figure 1C) and PB-IFNα2b-GPC3-CAR (Figure 1D) plasmids were synthesized with reference to the method in Example 3 below.

The amino acid sequence of GPC3-CAR without type I interferon is shown in SEQ ID NO.: 49:

The base sequence of the GPC3 single-chain antibody light chain variable region (GPC3-ScFv VL) is shown in SEQ ID NO.: 50:

The amino acid sequence of the GPC3 single-chain antibody light chain variable region (GPC3-ScFv VL) is shown in SEQ ID NO.: 51:

The Linker of GPC3-ScFv VL and GPC3-ScFv VH is the base sequence of SEQ ID NO.: 14 above.

The amino acid sequence of the Linker of GPC3-ScFv VL and GPC3-ScFv VH is shown in SEQ ID NO.: 15 above.

The base sequence of the GPC3 single-chain antibody heavy chain variable region (GPC3-ScFv VH) is shown in SEQ ID NO.: 52:

The amino acid sequence of the GPC3 single-chain antibody heavy chain variable region (GPC3-ScFv VH) is shown in SEQ ID NO.: 53:

The amino acid sequence of GPC3-CAR containing type I interferon (IFNα2b) is shown in SEQ ID NO.: 54:

The nucleotide sequence of GPC3-CAR containing type I interferon (IFNα2b) is shown in SEQ ID NO.: 55:

Refer to the following Example 3 method to synthesize PB-BCMA-CAR plasmid (Figure 1E) and PB-IFNα2b-BCMA-CAR plasmid (Figure 1F)

The amino acid sequence of BCMA-CAR without type I interferon is shown in SEQ ID NO.: 56:

The base sequence of the BCMA single-chain antibody light chain variable region (BCMA-ScFv VL) is shown in SEQ ID NO.: 57:

The amino acid sequence of the BCMA single-chain antibody light chain variable region (BCMA-ScFv VL) is shown in SEQ ID NO.: 58:

The Linker of BCMA-ScFv VL and BCMA-ScFv VH is the base sequence of SEQ ID NO.: 14 above.

The amino acid sequence of the Linker of BCMA-ScFv VLBCMA-ScFv VH is shown in SEQ ID NO.: 15 above.

The base sequence of the BCMA single-chain antibody heavy chain variable region (BCMA-ScFv VH) is shown in SEQ ID NO.: 59:

The amino acid sequence of the BCMA single-chain antibody heavy chain variable region (BCMA-ScFv VH) is shown in SEQ ID NO.: 60:

The amino acid sequence of BCMA-CAR containing type I interferon (IFNα2b) is shown in SEQ ID NO.: 61:

The nucleotide sequence of BCMA-CAR containing type I interferon (IFNα2b) is shown in SEQ ID NO.: 62:

Example 2

(1) Construction of virus expressing chimeric antigen receptor and IFNα2b

The method is as follows: Use Escherichia coli to amplify the pELPS-PSMA-BBz and pELPS-PSMA-BBz-2A-IFNα2b plasmids and the lentivirus packaging auxiliary plasmids pMD2.G and psPAX2. After extracting the plasmids, perform agarose gel electrophoresis and sequencing for identification The correctness of the plasmid. The 293T with the highest generation number in good condition was selected as the lentivirus packaging cell, and the above three plasmids were transfected into the 293T cell with the transfection reagent PEI. The transfection was completed in a 10cm culture dish with a total system of 10mL. The cell transfection mixture for each dish should be prepared as a 1mL system with serum-free DMEM, so that the psPAX2 plasmid: pMD2.G plasmid: pELPS-PSMA-BBz-2A-IFNα2b plasmid: PEI=5μg:3μg:5μg:50μL, mix the transfection mixture at room temperature, let it stand for 20 minutes, slowly add it to the 293T that has 9mL of medium with a cell density of 70-80%, and replace with fresh medium (DMEM) after 6-8h +10%FBS+1%P/S). The culture supernatant was harvested at 48h and 72h respectively, and the virus expressing chimeric antigen receptor and IFNα2b was obtained after ultrafiltration and ultraisolation concentration. The resulting virus was named IFNα2b-PSMA-CAR virus. Use the pELPS-PSMA-BBz transfer vector plasmid (compared to the pELPS-PSMA-BBz-2A-IFNα2b plasmid, the PSMA-CAR plasmid lacks the second expression cassette expressing IFNα2b) as a control, and refer to the above methods for transfection and treatment. Named PSMA-CAR virus.

(2) Virus titer detection

Methods as below:

Select a good 293T to detect the virus titer, inoculate 500μL of cells with a density of 4*10^5/mL in a 24-well plate, after the cells adhere to the wall, add different gradient volumes of concentrated virus solution, culture for 48h and then digest the cells , Use CAR to recognize the bound biotinylated PSMA protein and wash it after incubating with the cells at 4°C for 50min, and then stain it with APC-streptavidin SA that can bind to biotin for 30min at 4°C, and wash after staining. Install the tube and use the flow cytometer to detect the CAR positive rate, select the virus volume with the appropriate positive rate to calculate the virus titer, the titer calculation formula: titer (TU/mL) = (2*10^5*CAR positive rate)/virus volume .

Titer detection follows the above-mentioned titer detection method. After the cells are attached to the plate, two volume gradients of 2μL and 5μL are set for the control viruses PSMA-CAR and IFNα2b-CAR viruses, respectively. To avoid false positives caused by non-specific staining, it is necessary to set CTRL carries out the CAR-positive gate, and the CAR-positive cells fall into the APC-positive gate, and the ratio value shown is the CAR-positive rate. According to the flow cytometry results shown in Figure 2, 2μL of PSMA-CAR concentrated virus infection can reach 60.4% positive rate of 200,000 293T, 5μL corresponds to 81.3%; 2μL of IFNα2b-CAR virus infection can reach 27.4% of 200,000 293T The positive rate, 5μL corresponds to a positive rate of 43.9%. Because the positive rate is too high to reflect the true titer of the virus, a volume of 2μL is used to calculate the titer. The titer of the control virus PSMA-CAR can reach 6.04×10^7TU/ mL, and the IFNα2b-PSMA-CAR virus titer is 1.8×10^7TU/mL.

Example 3

(1) Use lentiviral vectors to construct T cells expressing chimeric antigen receptors and IFNα2b

Methods as below:

Use lymphatic separation fluid to separate PBMC from human blood, and then use CD4 and CD8 magnetic bead sorting to separate T cells. After 48 hours of activation by the CD3/CD28 complex, the packaged PSMA-CAR and IFNα2b-PSMA- CAR virus was infected by centrifugation at MOI=10 for 2h, and after 24h, it was replaced with fresh medium (XVIVO+10%FBS+IL-2). The two CART cells were named PSMA-CART cells and IFNα2b-PSMA-CART cells, respectively.

The CAR expression levels of the above two CARTs were detected according to the similar method of titer detection 48h after infection. Among them, the positive rate of IFNα2b-PSMA-CART was 49.3%, and the positive rate of PSMA-CART was 89.5%.

(2) Using the transposon system to construct T cells expressing chimeric antigen receptors and IFNα2b

The lentiviral vector was replaced with a transposon system, and electroporation technology was used for delivery. The IFNα2b-CART with stable positive rate was successfully prepared, and the expression detection of IFNα2b was completed:

The above-mentioned first expression cassette was synthesized by GenScript and cloned into the vector PB513B-1 (SBI) vector

The PiggyBac transposon vector containing PSMA CAR was constructed and named PB-PSMA-CAR vector. The first expression cassette and the second expression cassette (the first expression cassette and the second expression cassette) in Example 1 were synthesized by GenScript Connected by P2A) and cloned into PB513B-1 (SBI) vector to synthesize PB-IFNα2b-PSMA-CAR vector. Prepare 1M T cells with good growth status after 48 hours of activation, and use the Lonza 2b-Nucleofector instrument (according to the instrument operating instructions) to separately transfer the above transposon plasmids PB-PSMA-CAR or PB-IFNα2b-CAR, PB220PA-1 ( Provide the expression plasmid of PB transposase, purchased from System Bioscience) The plasmid is transfected into the cell nucleus and _{cultured in a 37°C, 5% CO 2} incubator. At 48 hours after transfection, use the method in Example 2 above to detect CAR positive Rate.

The PB-BCMA-CAR vector and the PB-IFNα2b-BCMA-CAR vector, the PB-GPC3-CAR vector and the PB-IFNα2b-GPC3-CAR vector were synthesized using the above-mentioned transposon system and method respectively, and the same method was used to prepare PB-BCMA -CART, PB-IFNα2b-BCMA-CART, PB-GPC3-CART, PB-IFNα2b-GPC3-CART, the positive rate of CAR was detected by the method of Example 2. The results are shown in Figure 4A, the positive rate of PB-IFNα2b-PSMA-CART is 20.9%, the positive rate of PB-PSMA-CART is 20.81%, Figure 4B shows that the positive rate of PB-GPC3-CART CAR is 23.86%, and the positive rate of PB-PSMA-CART is 23.86%. The positive rate of IFNα2b-GPC3-CART was 19.44%. Figure 4C shows that the positive rate of PB-BCMA-CART was 19.64%, and the positive rate of PB-IFNα2b-BCMA-CART was 24.84%. Therefore, IFNα2b-CART with stable CAR expression can be obtained by using PiggyBac transposon and electroporation method.

For IFNα2b-CAR, it is not only necessary to detect the expression of its CAR, but also to verify whether it has the ability to express IFNα2b, so collect the above two CART cells under the same culture system conditions (the positive rate is adjusted to be the same, the density is 1M/mL) for 48h The supernatant of ELISA was used to verify the expression of IFNα2b.

The results are shown in Figure 4D, PB-IFNα2b-PSMA-CART can express higher IFNα2b than traditional PB-PSMA-CART, and the average is as high as about 30 times.

Both the IFNα2b-CART and the traditional CART in the following examples (ie, Examples 4-10) are prepared using the transposon system of this example. Among them, CART (PB-PSMA-CART, PB-GPC3-CART, PB-BCMA-CART) expressing only the first expression cassette mentioned above is collectively referred to as conventional CART, and CART co-expressing the second expression cassette (PB-IFNα2b -PSMA-CART, PB-IFNα2b-GPC3-CART, PB-IFNα2b-BCMA-CART) are collectively referred to as IFNα2b-CART.

Wherein, the transposon system in the present invention adopts the PiggyBac ^™ Transposon Vector System from SBI Company, the transposon PB513B-1 is purchased from SBI Company, and the transposase PB220PA-1 is purchased from System Bioscience Company. The base sequence of PB transposase (PB220PA-1) is shown in SEQ ID NO.: 63:

The amino acid sequence of PB transposase (PB220PA-1) is shown in SEQ ID NO.: 64:

Example 4

IFNα2b enhances the killing effect of CART on tumor cells

PB-IFNα2b-PSMA-CART and PB-PSMA-CART, which have the same adjustment of CAR positive rate, are used as effector cells, and PC3-PSMA (human prostate cancer cell line, using lentivirus to stably express PSMA and luciferase) as target cells, first Add the same amount of target cells (20,000) to the low-adsorption well plate, and add the corresponding number of CART effector cells according to the effective target ratio (effector cell: target cell) 1:1, 0.5:1, 0.25:1, and do different things at the same time The gradient has only the wells of target cells (0.5, 1, 1.5, 2, 2.5, 3, 50,000) as the standard curve. Since the target cells can express luciferase, after 12 hours of co-incubation (cc:co-culture), after adding the substrate, the absorbance is linearly related to the number of target cells. You can make a curve and calculate the number of remaining target cells. Calculate the killing efficiency Lysis(%)=(starting target cell number-remaining target cell number)/starting target cell number, taking killing efficiency Lysis(%) as the ordinate, and different effective target ratios (E:T) as Abscissa.

The results are shown in Figure 5A. As the effective-to-target ratio increases, the killing effect of CART gradually increases. When the effective-to-target ratio is 1:1, the average killing efficiency of PB-IFNα2b-PSMA-CART is 84%. The killing efficiency of PSMA-CART is 73%, indicating that CART cells overexpressing IFNα2b can enhance their ability to kill tumor cells. Figure 5B shows that PB-IFNα2b-GPC3-CART also has stronger tumor killing ability than PB-GPC3-CART. In terms of hematoma, PB-IFNα2b-BCMA-CART has stronger killing mm1s than PB-BCMA-CART. The capacity of multiple myeloma cell lines (Figure 5C).

Example 5

IFNα2b did not affect the proliferation of CART

In order to verify the ability of IFNα2b to promote the proliferation of T cells, 200,000 IFNα2b-CART and PSMA-CART with the same positive rate were cultured in the same culture system, and the absolute count was followed by a counter to track the two different CARTs within two weeks. Proliferation.

The results are shown in Figure 6, PB-IFNα2b-PSMA-CART and PB-PSMA-CART have the same proliferation ability, indicating that IFNα2b secreted CART does not affect the expansion ability of CART cells.

Example 6

IFNα2b-CART has a high proportion of Tn (memory T cell) phenotype

CART cells 72 hours after electroporation were used for staining with CD4, CD8, CD45RA, CCR7 flow cytometry antibodies, and flow cytometry software was used to analyze the expression of memory marker genes CD45RA and CCR7 in CD4 and CD8 T cells, respectively. The results are shown in Figure 7. Among CD4 T cells, CCR7 ⁺ and CD45RA ⁺ double positive cells (Tn:

The ratio of T cells) in PB-IFNα2b-PSMA-CART is 29.5%, and the ratio in PB-PSMA-CART cells is 13.0%; in CD8 T cells,

The proportion of T cells in PB-IFNα2b-PSMA-CART is 37.9%, and the proportion in PB-PSMA-CART cells is 13.5%. IFNα2b-CART has more memory T cell subtypes than PSMA-CART.

Example 7

IFNα2b-CART activates PBMC cells and innate immunity

Collect the above two CART cells (traditional CART: PB-PSMA-CART and IFNα2b-CART: PB-IFNα2b-PSMA-CART) under the same culture system conditions (the positive rate is adjusted to be the same, the density is 1M/mL) for 48h After verifying the expression of IFNα2b by ELISA. The supernatant was used to stimulate PBMC cells (3M/mL) for 24 hours and then RNA was extracted for QPCR. The results are shown in Figure 8A: the supernatant from IFNα2b-CART can effectively activate human PBMC to secrete chemokines (CXCL10, CXCL11, CCL2, CXCL9) that are beneficial to T cells and reduce IL10, CSF2, IL1β cytokines, and at the same time enhance Cytokines (IL15, IL18) that promote the proliferation of T cells and other immune cells.

At the same time, the IFNα2b-CART supernatant stimulated the expression of NK cells and mononuclear cells (mono) in PBMC to up-regulate TRAI1, indicating that IFNα2b-CART can more strongly activate innate immune cells to exert tumor-killing functions (Figure 8B).

Because type I interferon can enhance NK up-regulation of TRAIL by regulating innate immune cells, and mediating the killing effect of NK on tumor cells by binding to TRAIL receptors on the surface of tumor cells, the supernatant collected from the above-mentioned traditional CART and IFNα2b-CART cells is combined with After homologous PBMC were incubated in a medium containing GM-CSF and IL 2 cytokines for 24 hours, the co-incubated PBMC and huh7 cells were co-incubated at a ratio of 1:1. The four groups of experiments were set up as follows: A: huh7: Only huh7 cells; B: huh7+PBMC: huh7 and PBMC are cultured at a ratio of 1:1; C: huh7+PBMC (conventional CART supernatant stimulation 24h): huh7 and PBMC are cultured at a ratio of 1:1:1; D: huh7+ PBMC (IFNα2b-CART supernatant stimulation for 24h): huh7 and PBMC were cultured at a ratio of 1:1; 24h later, the death of huh7 was observed under a microscope. As shown in Figure 8C, almost all huh7 cells in group D died, but the huh7 cells in group C still existed More adherent huh7 tumor cells indicate that IFNα2b-CART can enhance the killing of liver cancer cells by regulating and activating immune cells in PBMC.

Example 8

IFNα2b-CART supernatant activates tumor cells to up-regulate the expression of chemokines and promote T cell migration.

Take the culture supernatant of PB-PSMA-CART and PB-IFNα2b-PSMA-CART under the same culture conditions for 48 hours, count the tumor cells DU145 cells and plate them, use the collected cell supernatant to stimulate for 24 hours, extract RNA, and screen by QPCR Detect chemokines.

Results Figure 9 shows that IFNα2b-CART supernatant (DU145+IFNα2b-PSMA-CART) can activate DU145 cells to up-regulate the expression of CCL8 (left). Therefore, a migration experiment was designed for verification: the lower chamber plating (24-well plate) 20 Ten thousand DU145 cells were stimulated with PB-IFNα2b-PSMA-CART and PB-PSMA-CART supernatant for 24 hours, and 400,000 CFSE-labeled HT (human T cells) were added to the upper chamber. After 4 hours of migration, use counting magnetic beads for absolute Count the cells that migrated to the lower chamber. The results are shown in Figure 9 (right), IFNα2b-CART can significantly promote the migration of HT to the lower ventricle.

Example 9

IFNα2b-CART inhibits tumor angiogenesis

Through animal experiments, combined with immunofluorescence technology, the results show that IFNα2b-CART can significantly inhibit tumor angiogenesis.

Example 10

(1) IFNα2b-CART mobilizes endogenous immunity to exert a stronger anti-tumor effect

(2) IFNα2b-CART promotes tumor regression in vivo

The PC3-PSMA cell line (labeled with Luciferase) was injected subcutaneously into the mouse, and the volume of the tumor was tracked using vernier calipers and mouse in vivo imaging technology. When it reached a certain size (200mm ³ ), grouping was set for treatment.

The results show that IFNα2b-CART has a better ability to regress tumors in vivo than PSMA-CART.

Sequence information table:

The amino acid sequence of CD8α signal peptide (CD8αLeader) is shown in SEQ ID NO.: 4:

IFNα2b:

The base sequence of CD8 hinge region (CD8hinge) is shown in SEQ ID NO.: 17:

The base sequence of P2A is shown in SEQ ID NO.: 21:

The base sequence of type I interferon is as follows:

IFNα:

IFNα2a:

IFNα2b:

IFNα4:

IFNα5:

IFNα6:

IFNα7:

IFNα8:

IFNα10:

IFNα13:

IFNα14:

IFNα17:

IFNα21:

IFNβ:

The amino acid sequence of type I interferon is shown below:

IFNα:

IFNα2a:

IFNα4:

IFNα5:

IFNα6:

IFNα7:

IFNα8:

IFNα10:

IFNα13:

IFNα14:

IFNα17:

IFNα21:

IFNβ:

The base sequence of PB transposase (PB220PA-1) is shown in SEQ ID NO.: 63:

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

Claims

An engineered immune cell, characterized in that the engineered immune cell expresses a chimeric antigen receptor CAR and type I interferon targeting tumor cell markers, and the tumor cell markers are selected from the following group: PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or Its combination.
A method for preparing the engineered immune cells according to claim 1, characterized in that it comprises the following steps:

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

(B) The immune cells are modified so that the immune cells express the chimeric antigen receptor CAR and type I interferon targeted to tumor cell markers, thereby obtaining the engineered Immune Cells.
A preparation, characterized in that the preparation contains the engineered immune cell according to claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.
A use of the engineered immune cells according to claim 1, characterized in that they are used to prepare drugs or preparations for selectively killing tumors.
A kit for selectively killing tumors, characterized in that the kit contains a container, and inside the container:

(1) A first nucleic acid sequence, said first nucleic acid sequence containing a first expression cassette for expressing a chimeric antigen receptor CAR targeting tumor cell markers, the tumor cell markers being selected from the following group: PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or a combination thereof ;with

(2) A second nucleic acid sequence containing a second expression cassette for expressing type I interferon.
A method for selectively killing tumors, which is characterized in that it comprises:

A safe and effective amount of the engineered immune cell according to claim 1 or the preparation according to claim 3 is administered to a subject in need of treatment.
A fusion protein, characterized in that the fusion protein comprises a chimeric antigen receptor CAR targeting tumor cell markers and type I interferon, wherein the tumor cell marker is selected from the group consisting of PSMA, GPC3, GD2 , HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or a combination thereof.
The fusion protein of claim 7, wherein the structure of the fusion protein is shown in the following formula III:

L-S-H-TM-C-CD3ζ-(Z3-P)m (I)

Where

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

L is no or signal peptide sequence;

S is an antigen binding domain targeting tumor cell markers, which are selected from the following group: PSMA, GPC3, GD2, HER2, Mesothelin (MSLN), CEA, EGFR/EGFRvIII, Claudin 18.2, Mucin 1 (MUC1), NKG2D ligand, CD19, CD20, BCMA, CD22, CD30, IL3RA, CD38, CD138, or a combination thereof;

H is no or hinge area;

TM is the transmembrane domain;

C is a costimulatory signal molecule;

CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ;

Z3 is a connecting peptide;

P is type I interferon;

m is 1, 2, 3, or 4.
A polynucleotide characterized in that it encodes the fusion protein of claim 7.
A vector, characterized in that the vector comprises the polynucleotide of claim 9.