CN116102662A

CN116102662A - Fusion chimeric antigen receptor targeting CA9, recombinant expression vector and application thereof

Info

Publication number: CN116102662A
Application number: CN202211250823.0A
Authority: CN
Inventors: 张同存; 孙钒; 吕梦清; 崔烨挺
Original assignee: Wuhan Bio Raid Biotechnology Co ltd
Current assignee: Wuhan Bio Raid Biotechnology Co ltd
Priority date: 2022-10-10
Filing date: 2022-10-10
Publication date: 2023-05-12

Abstract

The invention provides a fusion chimeric antigen receptor targeting CA9, a recombinant expression vector and application thereof, and belongs to the technical field of genetic engineering. A fusion chimeric antigen receptor that targets cells expressing CA9 surface antigen and that secretes an IL-15SA fusion protein or IL-36 γ, CXCL11 and expresses CCR8 or CXCR6 on a membrane, comprising a CD8 a signal peptide, a scFv that targets CA9, a Strep II tag protein, a CD8 a hinge domain, a CD28 transmembrane domain and a CD28 intracellular signal domain, a 41-BB co-stimulatory domain, a CD3 zeta signaling domain, a domain that secretes an IL-15SA, IL-36 γ or CXCL11 fusion protein, and a domain that membrane expresses CCR8 or CXCR6. The fusion chimeric antigen receptor of the invention is expressed in T cells, enhances immune cell infiltration, recruits more immune cells, and plays a better role in inhibiting tumors.

Description

Fusion chimeric antigen receptor targeting CA9, recombinant expression vector and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a fusion chimeric antigen receptor targeting CA9, a recombinant expression vector and application thereof.

Background

Cellular immunotherapy is an emerging tumor treatment method that constructs an expression vector of a chimeric antigen receptor (chimeric antigen receptor, CAR) by molecular biology techniques, and introduces the expression vector into immune cells isolated from a human body, so that the CAR is expressed on the cell surface, and then returns the cell surface to the human body. The CAR-expressing immune cells are able to specifically recognize and kill target cells. However, CAR T cells frequently collide with walls in the course of treating solid tumors, and now still face a number of problems and challenges, and the novel CAR structure, the discovery of new targets, is the key of CAR-T treatment of solid tumors.

CA9 (Carbonic anhydrase) is a transmembrane protein, one of the only two tumor-associated carbonic anhydrase isozymes known. In the past, CA9 expression was found to be elevated in various cancers such as lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and adenocarcinoma, cholangiocarcinoma, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, pancreatic cancer, rectal cancer, sarcoma, thymus cancer, gastric cancer, endometrial cancer, and the like, in addition to the expression in normal tissues. Among the many tumor immune-related molecules, CA9 can be an ideal target for solid tumor immunotherapy, and therefore, in the treatment of the above tumor diseases, it is preferable to use a drug or vaccine that can target CA 9.

The IL-15SA fusion protein can prolong the half-life of IL15 in serum and increase the affinity of IL15 beta receptor to immune cells, recruit CD8+ T cells, CD4+ T cells, natural killer cells and mast cells, can specifically bind to IL-15 and form an immune synaptic structure with surrounding effector NK cells and IL-15 Rbeta-gamma chains on T cells, activate JAK/STAT and other channels of NK/T cells to play a role (Cheever MA, et al 2008; mortier E, et al 2006). Promoting proliferation of cd3+ T cells (predominantly cd8+ T cells) and NK1.1+ NK cells; enhancing the effect of cytokines on IgG or T cells in vivo on antibody-dependent cell-mediated cytotoxicity (Waldmann TA, et al 2020; akdis M, et al 2011); IL-15 also promotes the induction of CD8+ T cells with longer life and higher affinity, can kill tumor cells very effectively, and finally enhances the anti-tumor effect of immunotherapy (Xu H, et al 2021; qiaue Hu, et al 2018; martin A, et al 2008).

The IL1RL2/IL-36R receptor binds to and signals cytokines through it, thereby activating NF-kappa-B and MAPK signaling pathways in target cells. Part of the IL-36 signaling system is thought to be present in the epithelial barrier and involved in the local inflammatory response; similar to the IL-1 system with which the accessory receptor IL1RAP is shared. Tissue infiltration, cell maturation and cell proliferation are driven by action on keratinocytes, dendritic cells and indirectly on T cells, thus participating in the skin inflammatory response (Bachmann M, et al 2012). Inducing the expression of macrophages, T cells and neutrophil chemokines in cultured keratinocytes, such as CCL3, CCL4, CCL5, CCL2, CCL17, CCL22, CL20, CCL5, CCL2, CCL17, CCL22, CXCL8, CCL20 and CXCL1; but also stimulated its own expression, and the expression of the prototype skin pro-inflammatory parameters TNF-alpha, S100A 7/psoriasis and inducible NOS (Gresnigt M.S, et al 2013). May play a role in the pro-inflammatory response during specific neutrophil airway inflammation: activates mitogen-activated protein kinase and NF-kappa B in primary lung fibroblasts and stimulates the expression of IL-8 and CXCL3 and the Th17 chemokine CCL20 in lung fibroblasts. May be involved in an innate immune response to fungal pathogens such as Aspergillus fumigatus (Debets R, et al 2001; foster A.M, et al 2014). The IL-36-gamma gene engineering into the CAR can significantly improve proliferation and persistence. The IL-36 gamma expressing CAR-T cells were able to enhance MHC class II and CD86 expression on tumor mouse spleen macrophages and dendritic cells, suggesting that IL-36 plays a role in the maturation of antigen presenting cells. In the mouse model, the anti-tumor activity was shown to be very strong (Li X, et al 2020).

CXCL11 (C-X-C motif chemokine ligand 11, C-X-C motif chemokine 11, also known as interferon-induced T cell alpha chemokine (I-TAC)) is a small molecular weight cytokine in the CXC chemokine family (Huoman J, et al 2021). It is highly expressed in peripheral blood leukocytes, pancreas and liver, moderately expressed in thymus, spleen and lung, and poorly expressed in small intestine, placenta and prostate tissue (Moreno B, et al 2022). CXCL11 acts on target cells by interacting with the cell surface chemokine receptor CXCR 3. CXCL11 can chemotactic activated T cells (Rot A, et al 2010; liL, et al 2022).

CCR8 (C-C motif chemokine receptor 8) is one of the members of the chemokine receptor subfamily, belonging to the G protein-coupled receptor. Human CCL1 is the primary ligand for CCR8, with the other ligands CCL8, CCL16, and CCL18.CCR8 is specifically expressed on tumor-infiltrating regulatory T cells (tregs), and is expressed on both Th2 cells and tumor cells; however, it is not substantially expressed on peripheral blood tregs or normal tissues. CCR8 has a major role in the recruitment of Tregs and Th2 cells to inflammatory and tumor sites. A number of studies have found that highly expressed CCR8 is associated with a variety of cancers including colorectal, breast, gastric, metastatic brain, metastatic liver cancers, especially on Tregs cells isolated from breast cancer (Karin, et al 2018; das, suvendu, et al 2013).

CCLI binds CCR8 on cd4+ T cells, resulting in conversion of cd4+ T cells to Tregs, which are recruited; CCL1 binds CCR8 on Tregs, which recruit to inflammatory or tumor sites. The CCR8-CCL1 axis recruits not only CCR8+ Tregs to TME. CCR8-CCL1 may also induce up-regulation of FO x P3 and CD39, as well as IL-10, which are critical for Tregs inhibition, which will enhance the immunosuppressive activity of Tregs. It was found that CCR8 was significantly up-regulated in tumor-invasive Tregs of human breast cancer compared to Tregs in normal tissues, suggesting that CCR8 is a promising therapeutic target on Tregs in TME without eliciting systemic autoimmunity (Whiteside, sarah k., et al 2021; roose, helen, et al 2021; korbecki, jan et al 2020).

CXCR6 (C-X-C motif chemokine receptor 6), the encoded protein and its unique ligand chemokine ligand 16 (CCL 16) are part of a signaling pathway that regulates T lymphocyte migration to various peripheral tissues (liver, spleen red marrow, intestine, lung and skin) and promotes cell-cell migration. Interact with dendritic cells and fibroblast reticulocytes. CXCR6/CCL16 also controls the localization of resident memory T lymphocytes in different areas of the lung and maintains airway resident memory T lymphocytes (Angelo LS, et al 2022; dai Y, et al 2021).

Disclosure of Invention

The invention aims at providing a fusion chimeric antigen receptor targeting CA9, a recombinant expression vector and application thereof.

The present invention relates to a fusion protein comprising a Chimeric Antigen Receptor (CAR) targeting CA9 and a potentiating domain, having the effect of increasing the infiltration capacity of CAR-T cells and the ability to recruit immune cells.

The present invention relates to a fusion chimeric antigen receptor targeting cells expressing CA9 surface antigen, which can secrete IL-15SA fusion protein, CXCL11 or IL-36 gamma, and recruit other immune cells, such as T cells, NK cells, dendritic cells, macrophages, monocytes and the like.

The invention relates to a fusion chimeric antigen receptor of a target cell expressing CA9 surface antigen, which expresses CCR8 or CXCR6 and can enhance the infiltration of CAR-T cells into cancer tissues.

The invention relates to a fusion chimeric antigen receptor of a cell targeted to express CA9 surface antigen, which can secrete IL-15SA fusion protein, IL-36 gamma or CXCL11 and express CCR8 or CXCR6. Other immune cells, e.g., T cells, NK cells, dendritic cells, macrophages, monocytes, etc., are recruited and the CAR-T cells are promoted to infiltrate the cancer tissue.

The technical scheme of the invention is realized as follows:

as shown in fig. 1-4, the present invention provides a fusion chimeric antigen receptor comprising a CD8 a signal peptide, a scFv targeting CA9, a Strep II tag protein, a CD8 a hinge domain, a CD28 transmembrane domain and a CD28 intracellular signal domain, a 41-BB co-stimulatory domain, a CD3 zeta signaling domain, a domain that secretes an IL-15SA, IL-36 gamma or CXCL11 fusion protein, a domain that targets CA9 surface antigen cells and secretes an IL-15SA, IL-36 gamma or CXCL11 fusion protein, and a membrane that expresses CCR8 or CXCR6.

As a further improvement of the present invention, the amino acid sequence of the scFv targeting CA9 comprises the amino acid sequence as set forth in SEQ ID NO:1 to SEQ ID NO:24, a step of detecting the position of the base; the corresponding nucleotide sequence comprises SEQ ID NO:25 to SEQ ID NO:48.

as a further improvement of the present invention, the amino acid sequence of the CD8 a signal peptide is as shown in SEQ ID NO:49, the amino acid sequence of Strep II tag protein is shown in SEQ ID NO:51, the amino acid sequence of the CD8 a hinge domain is set forth in SEQ ID NO:53, the amino acid sequence of the CD28 transmembrane domain is shown in SEQ ID NO:55, the amino acid sequence of the CD28 intracellular signaling domain is set forth in SEQ ID NO:57, the amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:59, the amino acid sequence of the CD3 zeta signaling domain is shown in SEQ ID NO: shown at 61.

As a further improvement of the present invention, the nucleotide sequence of the CD8 a signal peptide is as shown in SEQ ID NO:50, the nucleotide sequence of the Strep II tag protein is shown as SEQ ID NO:52, the nucleotide sequence of the CD8 a hinge domain is set forth in SEQ ID NO:54, the nucleotide sequence of the CD28 transmembrane domain is set forth in SEQ ID NO:56, the nucleotide sequence of the CD28 intracellular signaling domain is set forth in SEQ ID NO:58, the nucleotide sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:60, and the nucleotide sequence of the CD3 zeta signaling domain is shown in SEQ ID NO: shown at 62.

As a further improvement of the invention, the amino acid sequence of the secreted IL-15SA fusion protein domain is shown in SEQ ID NO:63, the amino acid sequence of the secreted IL-36 gamma fusion protein domain is shown in SEQ ID NO:65, the amino acid sequence of the CXCL11 fusion protein secretion domain is set forth in SEQ ID NO: 67.

As a further improvement of the invention, the nucleotide sequence of the secreted IL-15SA fusion protein domain is shown in SEQ ID NO:64, the nucleotide sequence of the secreted IL-36 gamma fusion protein domain is shown in SEQ ID NO:66, and the nucleotide sequence of the CXCL11 fusion protein domain is shown in SEQ ID NO: shown at 68.

As a further improvement of the present invention, the amino acid sequence of the membrane expression CCR8 domain is set forth in SEQ ID NO:69, the amino acid sequence of the membrane-expressed CXCR6 domain is set forth in SEQ ID NO: shown at 71.

As a further improvement of the present invention, the nucleotide sequence of the membrane expression CCR8 domain is set forth in SEQ ID NO:70, the nucleotide sequence of the membrane-expressed CXCR6 domain is set forth in SEQ ID NO: 72.

Preferably, the cleavage signal peptides are T2A and P2A, and the amino acid sequences thereof are SEQ ID NOs: 73, and SEQ ID NO:75, the nucleotide sequences are SEQ ID NOs: 74, and SEQ ID NO:76.

preferably, the Linker between the light chain and the heavy chain of the CA9 scFv is (G4S) 3Linker, and the amino acid sequence of the Linker is SEQ ID NO:77, the nucleotide sequence of which is SEQ ID NO:78.

the amino acid sequences of the light and heavy chains of the CA9 scFv are shown in Table 1.

TABLE 1

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The invention further provides a recombinant expression vector for expressing the fusion chimeric antigen receptor, which comprises a fusion chimeric antigen receptor expression frame, wherein the fusion chimeric antigen receptor expression frame consists of a promoter capable of being expressed in T cells and a nucleic acid which is positioned downstream of the promoter and used for encoding the fusion chimeric antigen receptor.

As a further improvement of the present invention, the promoter is RSV promoter, CMV promoter, SV40 promoter, EF-1. Alpha. Promoter.

As a further improvement of the present invention, the recombinant expression vector is obtained by inserting the nucleic acid encoding the fusion chimeric antigen receptor 3' downstream of the EF-1. Alpha. Promoter in a lentiviral expression vector.

In another aspect, the present application provides a method of enhancing the killing ability of a tumor cell by a chimeric antigen receptor that targets CA9, wherein the method comprises the steps of: using the chimeric antigen receptor targeting CA9 to bind to a potentiating domain, wherein the potentiating domain comprises a protein or functional fragment selected from the group consisting of: IL-15SA, IL-36 gamma, CXCL11, CCR8 and CXCR6.

In certain embodiments, the C-terminus of the CA 9-targeting chimeric antigen receptor is directly or indirectly linked to the N-terminus of the potentiating domain.

The invention also provides a recombinant expression vector for expressing the fusion chimeric antigen receptor, which comprises a chimeric antigen receptor expression frame and a replication system, wherein the chimeric antigen receptor expression frame consists of a promoter capable of being expressed in T cells and a nucleic acid for encoding the chimeric antigen receptor positioned downstream of the promoter.

The invention further provides the application of the fusion chimeric antigen receptor or the recombinant expression vector in preparing products for treating tumors.

As a further improvement of the invention, the tumors include lung squamous carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous carcinoma and adenocarcinoma, cholangiocarcinoma, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, pancreatic carcinoma, rectal carcinoma, sarcoma, thymus carcinoma, gastric carcinoma, endometrial carcinoma. Provides a more efficient method with fewer side effects and adverse reactions.

In the invention, the corresponding relationship among the binding target, the single-chain antibody amino acid sequence, the single-chain antibody nucleotide sequence, the carrier and the cell name is shown in the following table 2:

TABLE 2

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The invention has the following beneficial effects:

the expression of CCR8 or CXCR6 on the surface of T cells can increase the infiltration of immune cells, thereby enhancing the killing effect of the immune cells on tumor cells. In this method of treatment, the role of CAR, IL-15SA fusion protein, IL-36 γ, CXCL11 and chemokines CCR8, CXCR6 is of paramount importance. Fusion chimeric antigen receptors include single chain antibody domains, transmembrane domains, intracellular signaling domains, IL-15SA fusion proteins, IL-36 gamma, CXCL11 secretion domains, chemokine CCR8, CXCR6 domains. Wherein, the single-chain antibody domain targets immune cells to target cells, the intracellular signal domain releases intracellular signals, the killing activity of the immune cells is started, the IL-15SA fusion protein, IL-36 gamma and CXCL11 recruit other immune cells, and CCR8 and CXCR6 promote the tumor infiltration of the CAR-T cells.

By expressing the fusion chimeric antigen receptor of the invention in T cells, it is possible to obtain CAR-T cells capable of efficiently and specifically targeting malignant cells expressing CA9 surface antigens, secreting IL-15SA fusion proteins or CXCL11, IL-36 γ and membrane expressing CCR8 or CXCR6. Can better activate T cells, enhance immune cell infiltration, and recruit more immune cells, such as NK cells, dendritic cells, macrophages and granulocytes. Thus providing a more efficient and less adverse effect and side effects for the treatment of some solid tumors expressing CA9 surface antigen, such as lung squamous carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous carcinoma and adenocarcinoma, cholangiocarcinoma, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, pancreatic carcinoma, rectal carcinoma, sarcoma, thymus carcinoma, gastric carcinoma, endometrioid carcinoma, etc.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a schematic representation of a DNA fragment encoding a CA9 chimeric antigen receptor CAR domain according to the examples;

FIG. 2 is a schematic representation of a DNA fragment encoding a CA9 fusion chimeric antigen receptor CAR domain and secreted IL-15SA, IL-36 gamma, CXCL11 fusion proteins according to an example;

FIG. 3 is a schematic representation of a DNA fragment encoding a CA9 fusion chimeric antigen receptor CAR domain and expressing CCR8, CXCR6 chemokine receptors in an example;

FIG. 4 is a schematic representation of DNA fragments encoding CA9 fusion chimeric antigen receptor CAR domains and combinations with different potentiating domains in examples;

FIG. 5 is the efficiency of lentivirus infection of T cells in example 2;

FIGS. 6 to 9 are graphs comparing killing effect of 24 CAR-T cells and control T cells, control virus modified T cells (GFP-T) and unmodified T cells (T) on tumor cells A549, SUN-245, IM95, ACHM from different types of cancers and highly expressing CA 9;

FIGS. 10-13 are graphs comparing killing effects of 24 CAR-T cells and control T cells, control virus modified T cells (GFP-T) and unmodified T cells (T) on tumor cells T98G, HT1197, MKN28, HL60 from different types of cancers and not expressing CA 9;

FIG. 14 is a comparison of the efficacy of 11 different fusion chimeric antigen receptor expression plasmids harboring a synergistic domain of example 5;

FIGS. 15 to 18 are graphs comparing killing effect of 11 different synergistic domain-bearing CAR-T cells, control virus-modified T cells (GFP-T) and unmodified T cells (T) of example 6 on tumor cells A549, SUN-245, IM95, ACHM from different types of cancers and highly expressing CA 9;

FIGS. 19-22 are graphs comparing killing effects of 11 different synergistic domain bearing CAR-T cells, control virus modified T cells (GFP-T) and unmodified T cells (T) of example 6 on tumor cells T98G, HT1197, MKN28, HL60 from different types of cancers and not expressing CA 9;

FIGS. 23 to 24 are schematic representations of the treatment of tumor-bearing mice with 11 types of CAR-T cells bearing a potentiating domain of example 7.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 construction of CA9 chimeric antigen receptor expression plasmid

The corresponding relation among the binding target of the CA9 chimeric antigen receptor, the amino acid sequence of the single-chain antibody, the nucleotide sequence of the single-chain antibody, the carrier and the cell name is shown in the table 1;

the 24 different nucleotide sequences encoding CA9 scF γ are shown in table 1.

The nucleotide sequence of the coded tag protein Strep II, the CD8 hinge region and the transmembrane domain CD28TM is synthesized artificially, and the nucleotide sequence is shown as SEQ ID NO: 52. SEQ ID NO:54 and SEQ ID NO: shown at 56.

The nucleotide sequences of the intracellular signal domains CD28 ICD, 4-1BB ICD and CD3 ζICD are artificially synthesized, and SEQ ID NO: 58. SEQ ID NO:60 and SEQ ID NO: shown at 62.

The DNA fragments are inserted into the downstream of EF-1 alpha promoter of lentiviral expression vector pTK-EF-1a according to the combination mode in the table to obtain 24 different CA9 fusion chimeric antigen receptor expression plasmids.

EXAMPLE 2 transfection of CA9 chimeric antigen receptor expression plasmid into T cells

1) Packaging preparation of lentiviruses

CA9 chimeric antigen receptor expression plasmid and helper plasmid pMDLg, pRSV, pMD2.G were transfected with PEI transfection reagent (Invitrogen) at a ratio of 12:10:6:5, see PEI transfection reagent Specification for specific procedures. After 48h of transfection, the virus-containing cell culture supernatant was aspirated into a centrifuge tube, centrifuged at 3000g for 30min at 4℃and transferred to a fresh centrifuge tube, and after filtration through a 0.22 μm filter 30000g was centrifuged for 3h, and 500. Mu.L CTS was used ^TM OpTmizer ^TM The T cell expansion SFM culture medium was resuspended, and the cells were stored at-80℃in aliquots of 100. Mu.L.

2) Preparation of T cells

10mL of fresh blood from healthy persons was taken, CD3+ T cells were isolated with lymphocyte isolates (Ficol) and MicroBeads, and activated with CD3/CD 28T cell activation, for details as described in the specification. With IL-2, 6mM L-glutamine CTS containing 300IU/mL ^TM OpTmizer ^TM T cell expansion SFM Medium (Gibco ^TM ) Culturing for 24h to obtain activated T cells.

3) Expanded culture of lentivirus-infected T cells and post-infection T cells

Lentiviral concentrate was removed from-80℃and T cells were infected at MOI=5, and polybrene (purchased from Sigma Co.) was added at a final concentration of 8. Mu.g/mL. The cell suspension was added to 1 well of a 24-well plate at 1000g,32℃and centrifuged for 1h. After 8h incubation in a 5% CO2 incubator at 37℃1000g, centrifugation was carried out again for 1h at 32 ℃. The culture supernatant was aspirated and fresh L-glutamine CTS containing 300IU/mL IL-2 and 6mM was added ^TM OpTmizer ^TM T cell expansion SFM Medium (Gibco ^TM ) Culturing was continued. Detecting cell density every 2-3 days, sucking 1mL of cell suspension when cell density reaches 2×106 cells/mL, adding into another well, adding 1mL of fresh L-glutamine CTS containing 300IU/mL IL-2 and 6mM into two wells respectively ^TM OpTmizer ^TM T cell expansion SFM Medium (Gibco ^TM ) The expansion culture was continued. This is repeated until the cells have been expanded to a sufficient amount.

As shown in fig. 5, CAR-T cells against CA9 were obtained after testing for efficient expression of the CA9 chimeric antigen receptor in T cells.

Example 3 specific killing Activity of CAR-T cells on CA 9-Positive tumor cells

26 96-well plates were prepared, and inoculated with 5X 10 plates, respectively ⁴ Cancer cells expressing CA9 (A549, SUN-245, IM95, ACHM) and cancer cells not expressing CA9 (T98G, HT1197, MKN28, HL 60) were cultured overnight, and 24 CAR-T cells, control virus modified T cells (GFP-T) and unmodified T cells (T) were added to the two cell culture wells at a ratio of effector cells (E) to target cells (T) =10:1, 5:1, respectively. After 2.5h incubation, specific killing activity of 24 CAR-T cells against cancer cells was detected, operating according to calcein method instructions. The results show that the CAR-T cell group has stronger killing effect on cancer cells highly expressing CA9 compared with GFP-T and T cell groups at the effective target ratio of 10:1 and 5:1, and the difference is significant (p < 0.05) (FIGS. 6-9); there was no significant difference in killing effect on cancer cells that did not express CA9 (p>0.05 (fig. 10-13). Therefore, 24 CAR-T cells in the embodiment all have specific killing activity on tumor cells which highly express CA 9.

In addition, the tumor killing results show that B1 CA9 scFv is the single chain antibody with the best effect in B1-B24 CAR-T.

Example 4 construction of a synergistic CAR-T cell Using B1 scFv as CA9 surface antigen binding Domain

Artificially synthesizing a nucleotide sequence of the secreted IL-15SA fusion protein or IL-36 gamma, CXCL11 as shown in SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO: shown at 68.

Artificially synthesizing a film to express a CCR8 fusion protein, wherein the nucleotide sequence of the CXCR6 structural domain is shown in SEQ ID NO:70, SEQ ID NO: 72.

The synergistic structural domain DNA fragment is inserted into the downstream of EF-1 alpha promoter of lentiviral expression vector pTK-EF-1 alpha according to the combination mode in the following table, and the C end of the synergistic structural domain is sequentially connected with a shearing signal peptide and B1 CAR from upstream to obtain 11 different CA9 fusion chimeric antigen receptor expression plasmids.

EXAMPLE 5 transfection of fusion chimeric antigen receptor expression plasmids containing the synergistic Domain into T cells

11 different fusion chimeric antigen receptor expression plasmids containing the synergy domain were transfected into T cells following the procedure in example 2 and CAR expression was detected using a flow cytometer (fig. 14).

Example 611 specific killing Activity of different synergistic Domain-bearing CAR-T cells against CA 9-Positive tumor cells

13 96-well plates were prepared and inoculated with 5X 10 plates, respectively ⁴ Cancer cells highly expressing CA9 (A549, SUN-245, IM95, ACHM) and cancer cells not expressing CA9 (T98G, HT1197, MKN28, HL 60) were cultured overnight, and 11 kinds of CAR-T cells with a synergistic domain, control virus-modified T cells (GFP-T) and unmodified T cells (T) were added to the two kinds of cell culture wells at a ratio of effector cells (E) to target cells (T) =10:1, 5:1, respectively. After 2.5h incubation, the specific killing activity of 11 CAR-T cells against cancer cells was tested, operating according to calcein method instructions. The results show that the CAR-T cell group with the synergistic domain has an effective target ratio of 10:1 and 5:1, has a stronger killing effect on cancer cells highly expressing CA9, and this difference is significant (p < 0.05) (fig. 15-18); there was no significant difference in killing effect on cancer cells that did not express CA9 (p>0.05 (fig. 19-22). Thus, 11 of the CAR-T cells with the potentiating domain in this example all have specific killing activity against tumor cells that highly express CA9, whereas the results indicate that CAR-T cells with the potentiating domain exhibit more surprising killing ability against cancer cells that highly express CA9 than the single CAR in example 3.

Example 7 treatment of tumor-bearing mice with synergistic Domain-bearing CAR-T cells

Taking 1×10 ⁷ Individual PBMC cells were resuspended in 0.2mL RPMI-1640 and tail vein injected into 4 week old female NOG-B2M (Taconic) mice. After 4 weeks, each was subcutaneously injected 5×10 ⁶ A549 cells (high expression CA 9) to tumor volume up to 1000mm ³ At this time, PBS (0.2 mL), unmodified T cells (T, 0.2mL, 3X 10) ⁶ ) Control virusModified T cells (GFP-T, 0.2mL, 3X 10) ⁶ ) B1 CAR-T cells (0.2 mL, 3X 10) ⁶ )、B1&IL-15SA CAR-T cells (0.2 mL, 3X 10) ⁶ )、B1&IL-36 gamma CAR-T cells (0.2 mL, 3X 10) ⁶ )、B1&CXCL11 CAR-T cells (0.2 mL, 3X 10) ⁶ )，B1&CCR8 CAR-T cells (0.2 mL, 3X 10) ⁶ )，B1&CXCR6 CAR-T(0.2mL，3×10 ⁶ ) Cells (0.2 mL, 3X 10) ⁶ )，B1&IL-15SA&CCR8 CAR-T cells (0.2 mL, 3X 10) ⁶ )，B1&IL-36γ&CCR8 CAR-T cells (0.2 mL, 3X 10) ⁶ )，B1&CXCL11&CCR8 CAR-T cells (0.2 mL, 3X 10) ⁶ )，B1&IL-15SA&CXCR6 CAR-T cells (0.2 mL, 3X 10) ⁶ )，B1&IL-36γ&CXCR6 CAR-T cells (0.2 mL, 3X 10) ⁶ )，B1&CXCL11&CXCR6 CAR-T cells (0.2 mL, 3X 10) ⁶ ) Tumor volume changes were monitored. The results show that secretion of IL-15SA or CXCL11 or IL-36 γ and CAR-T membrane expressing CCR8 or CXCR6 can achieve a stronger tumor killing function by recruiting immune cells and enhancing infiltration. Comparison of various combinations shows that: the decrease in tumor volume was more pronounced and durable with BRD CA9-1 CAR-T (IL-15 SA-CCR 8) cells, and this difference was significant (p < 0.05) (FIGS. 23-24).

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A fusion chimeric antigen receptor that targets cells expressing CA9 surface antigen and that secretes an IL-15SA fusion protein or IL-36 γ, CXCL11 and expresses CCR8 or CXCR6 on a membrane, comprising a CD8 a signal peptide, a scFv that targets CA9, a strep ii tag protein, a CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular signal domain, a 41-BB co-stimulatory domain, a CD3 zeta signaling domain, a domain that secretes an IL-15SA, IL-36 γ, or CXCL11 fusion protein, and a domain that membrane expresses CCR8 or CXCR6.

2. The fusion chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the scFv targeting CA9 comprises the amino acid sequence set forth in SEQ ID NO:1 to SEQ ID NO:24, a step of detecting the position of the base; the corresponding nucleotide sequence comprises SEQ ID NO:25 to SEQ ID NO:48.

3. the fusion chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the CD8 a signal peptide is set forth in SEQ ID NO:49, the amino acid sequence of Strep II tag protein is shown in SEQ ID NO:51, the amino acid sequence of the CD8 a hinge domain is set forth in SEQ ID NO:53, the amino acid sequence of the CD28 transmembrane domain is shown in SEQ ID NO:55, the amino acid sequence of the CD28 intracellular signaling domain is set forth in SEQ ID NO:57, the amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:59, the amino acid sequence of the CD3 zeta signaling domain is shown in SEQ ID NO: shown at 61.

4. The fusion chimeric antigen receptor according to claim 1, wherein the nucleotide sequence of the CD8 a signal peptide is set forth in SEQ ID NO:50, the nucleotide sequence of the Strep II tag protein is shown as SEQ ID NO:52, the nucleotide sequence of the CD8 a hinge domain is set forth in SEQ ID NO:54, the nucleotide sequence of the CD28 transmembrane domain is set forth in SEQ ID NO:56, the nucleotide sequence of the CD28 intracellular signaling domain is set forth in SEQ ID NO:58, the nucleotide sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:60, and the nucleotide sequence of the CD3 zeta signaling domain is shown in SEQ ID NO: shown at 62.

5. The fusion chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the secreted IL-15SA fusion protein domain is as set forth in SEQ ID NO:63, the amino acid sequence of the secreted IL-36 gamma fusion protein domain is shown in SEQ ID NO:65, the amino acid sequence of the CXCL11 fusion protein secretion domain is set forth in SEQ ID NO: 67.

6. The fusion chimeric antigen receptor according to claim 1, wherein the nucleotide sequence of the secreted IL-15SA fusion protein domain is set forth in SEQ ID NO:64, the nucleotide sequence of the secreted IL-36 gamma fusion protein domain is shown in SEQ ID NO:66, and the nucleotide sequence of the CXCL11 fusion protein domain is shown in SEQ ID NO: shown at 68.

7. The fusion chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the membrane-expressed CCR8 domain is set forth in SEQ ID NO:69, the amino acid sequence of the membrane-expressed CXCR6 domain is set forth in SEQ ID NO: shown at 71.

8. The fusion chimeric antigen receptor according to claim 1, wherein the membrane expresses the CCR8 domain with a nucleotide sequence set forth in SEQ ID NO:70, the nucleotide sequence of the membrane-expressed CXCR6 domain is set forth in SEQ ID NO: 72.

9. Recombinant expression vector for expressing the fusion chimeric antigen receptor according to any one of claims 1 to 8, characterized in that it comprises a fusion chimeric antigen receptor expression cassette consisting of a promoter expressible in T cells and a nucleic acid encoding said fusion chimeric antigen receptor downstream of said promoter.

10. The recombinant expression vector of claim 9, wherein the promoter is an RSV promoter, a CMV promoter, an SV40 promoter, an EF-1a promoter.

11. The recombinant expression vector of claim 9, wherein the recombinant expression vector is obtained by inserting the nucleic acid encoding the fusion chimeric antigen receptor 3' downstream of the EF-1a promoter in a lentiviral expression vector.

12. Use of the fusion chimeric antigen receptor according to any one of claims 1 to 8 or the recombinant expression vector according to any one of claims 9 to 11 for the preparation of a product for the treatment of tumors.

13. The use according to claim 12, wherein the tumour comprises lung squamous carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous carcinoma and adenocarcinoma, cholangiocarcinoma, glioblastoma multiforme, head and neck carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, pancreatic carcinoma, rectal carcinoma, sarcoma, thymus carcinoma, gastric carcinoma, endometrial carcinoma.