KR20240035662A - CAR-T cell targeting CT83 and use thereof - Google Patents

Abstract

The present invention relates to CAR-T cells targeting CT83, which is located on the cell surface and acts as a cancer-specific antigen. In the present invention, CAR is a region that can specifically bind to CT83 - hinge region - transmembrane region - The CAR-T of the present invention has a structure of an intracellular co-stimulatory domain and a signaling domain, and can be used for the treatment of CT83-overexpressing carcinoma.

Description

CAR-T cell targeting CT83 and use thereof {CAR-T cell targeting CT83 and use thereof}

The present invention relates to CAR-T cells targeting CT83 and their anticancer use.

CT (Cancer/Testis) antigen is known to be a tumor-related antigen that is expressed limitedly in testicular germ cells and is not expressed in normal tissues, but is abnormally overexpressed in certain types of carcinoma. As tumor-related antigens, the majority of CT antigens are located in the nucleus and/or cytoplasm, which limits their use as targets for targeted therapy or immunotherapy. A recent bioinformatics study identified 22 CT antigens located on the cell surface and characterized by upregulation in hematologic or solid malignancies.

CT antigen 83 (CT83), also known as Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1), is a single-pass type II membrane protein encoded by the CT83 or Cxorf61 gene located on chromosome Xq23. CT83 was reported to be overexpressed in 81.6% of stomach cancer tissues, 75% of triple-negative breast cancer tissues, 32.6-40% of non-small cell lung cancer tissues, 62.5% of colorectal cancer tissues, and 90.2% of nasopharyngeal cancer tissues. Additionally, it has been shown that CT83 can be recognized by cytotoxic T lymphocytes (CTL) and T cells expressing the engineered T cell receptor (TCR).

Meanwhile, thanks to recent advances in cell engineering, chimeric antigen receptor (CAR)-T cells are emerging. CAR-T cells are T lymphocytes that express a chimeric antigen receptor consisting of the constant portion of the TCR fused with an immunoglobulin variable fragment. Target recognition is not limited by HLA and can therefore target all types of tumor markers.

The immunogenic characteristics and relative cancer-specific expression of CT83 make it an attractive target for cancer immunotherapy. However, no targeting has yet been attempted as CAR-T cell therapy or antibody-based therapy. The present inventors developed CT83-specific CAR-T cells (CT83.CAR-T) and confirmed their anti-tumor activity to complete the present invention.

da Cunha JP, Galante PA, de Souza JE, de Souza RF, Carvalho PM, Ohara DT, et al. Bioinformatics construction of the human cell surfaceome. Proc Natl Acad Sci U S A 2009;106:16752-7.

The present inventors intend to produce CAR-T cells targeting CT83 and provide anticancer uses thereof.

Accordingly, the purpose of the present invention is to provide a CAR encoding nucleic acid molecule capable of binding to CT83, an expression vector containing the nucleic acid molecule, and a T-cell transformed with the expression vector to express the CAR.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

In order to solve the above problems, the present invention provides a nucleic acid molecule encoding CAR targeting CT83.

In the present invention, the CAR may include a CT83 (Cancer/Testis Antigen 83) binding domain, a transmembrane domain, and an intracellular co-stimulatory domain and signaling domain.

In one embodiment of the present invention, the CT83 binding domain may be a single chain variable fragment (scFv) of a monoclonal antibody specific for CT83, and the scFv specific for CT83 includes the light chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 5. A heavy chain variable region may be included.

In another embodiment of the present invention, the CT83-specific scFv may have a light chain variable region and a heavy chain variable region connected by a linker, and the linker may include or consist of the amino acid sequence of SEQ ID NO: 4.

As another embodiment of the present invention, the present inventors used a transmembrane domain consisting of the amino acid sequence of SEQ ID NO: 7 in a specific experiment, but the transmembrane domain includes an extracellular binding portion and an intracellular co-stimulatory domain and/or a signaling domain. It is not limited as long as it is an amino acid sequence that performs the function of fusing, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and It may be derived from one or more selected from the group consisting of CD154 and may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

In another embodiment of the present invention, the CT83 binding domain and the transmembrane domain may be connected by a hinge region. In a specific experiment, the present inventors used the hinge region consisting of the amino acid sequence of SEQ ID NO: 6, but provided a physical distance between the CT83 binding domain and the cell to prevent the binding of the CT83 binding domain to CT83 and intracellular signal transmission through this binding. There are no restrictions as long as it is done smoothly.

In another embodiment of the invention, the intracellular costimulatory domains include OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). It may be a functional signaling domain obtained from one or more proteins selected from the group consisting of, and preferably may be derived from CD28, and the costimulatory domain derived from CD28 includes or consists of the amino acid sequence of SEQ ID NO: 8. It may be.

As another embodiment of the present invention, the intracellular signaling domain may be derived from CD3 zeta, and the signaling domain derived from CD3 zeta may include or consist of the amino acid sequence of SEQ ID NO: 9.

As another embodiment of the present invention, the nucleic acid molecule may further include a sequence encoding a signal peptide for movement of CAR expressed within the cell to the cell surface, and the present inventors have identified SEQ ID NO: 2 as the signal peptide. A leader region consisting of the amino acid sequence was used, but is not limited as long as it is a signal peptide that induces the movement of proteins expressed within the cell to the cell membrane.

Additionally, the present invention provides an expression vector containing the nucleic acid molecule for intracellular expression of the CAR.

In one embodiment of the present invention, a lentiviral vector was used as the vector in the specific experiment of the present invention, but the vector is not limited thereto, and the vector is operable by a suitable control sequence capable of expressing the CAR in human T cells. Linked nucleic acid molecules, which may be viral vectors or non-viral vectors. Non-limiting examples of non-viral vectors include DNA, RNA, and plasmids, and non-limiting examples of viral vectors include lentivirus, adenovirus, and retrovirus.

In addition, the present invention provides T cells that are transformed with the expression vector and express the CAR, and the T cells may be isolated from humans for use as a cell therapy agent. At this time, the human may be the subject for which the cell therapy product will be used, or it may be a human of the same species other than the subject.

Additionally, the present invention provides a pharmaceutical composition for treating cancer comprising T cells expressing the CAR as an active ingredient.

In one embodiment of the present invention, the cancer is not limited as long as it is a carcinoma that overexpresses CT83, and the CT83 overexpressing carcinoma includes breast cancer, hepatocellular cancer, stomach cancer, lung cancer, testicular cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, Examples include esophageal cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, kidney cancer, bladder cancer, urothelial cancer, sarcoma, prostate cancer, thyroid cancer, thymic cancer, malignant melanoma, nasopharyngeal cancer, malignant mesothelioma, and colorectal cancer, and the above breast cancer is triple negative breast cancer. Includes, and the lung cancer includes non-small cell lung cancer.

The present invention relates to CAR-T cells targeting CT83, which is located on the cell surface and acts as a cancer-specific antigen. The present invention provides effective treatment of cancer by effectively killing CT83-overexpressing cancer cells and inducing T cell activation. You can.

Figure 1 is a schematic diagram of the CT83.CAR-T and Mock-T structures.
Figure 2 shows the results of flow cytometry analysis of transduced T-cells.
Figure 3 shows the results confirming the antitumor efficacy of CT83.CAR-T in vitro. Specifically, Figures 3A to 3B are graphs showing the survival rates of KATO-III and H358 cells treated with T cells transduced with CT83.CAR-T or Mock-T, Figure 3C is a fluorescence micrograph, and Figure 3D is a This is the result of measuring the concentrations of IFN-γ and TNFα released from CT83.CAR-T and Mock-T co-cultured with target cells (KATO-III and H358) at an E:T ratio of 20:1.
Figure 4 shows the results confirming that CT83.CAR-T activation and proliferation are increased by CT83 antigen stimulation. Specifically, Figure 4a shows the results of flow cytometry analysis of CFSE-labeled CT83.CAR-T after 5 days of co-culture, and Figure 4b shows the results of counting the number of CT83.CAR-T expressing CD107a after 12 hours of co-culture. , Figure 4c shows the results of counting cells expressing CD25 (Mock-T and CT83.CAR-T), and Figure 4d shows the results of counting cells expressing CD69.

[Experimental materials and methods]

1. Design and production of recombinant lentiviral vectors

The lentiviral vector plasmid encoding CT83.CAR consists of a single-chain variable fragment of a monoclonal antibody specific for CT83 and the intracellular costimulatory and signaling domains of the CD28 and CD3ζ chains. In order to identify T cells transformed by the plasmid vector, the plasmid was designed to additionally contain a green fluorescent protein gene, and the CT83.CAR coding region and the green fluorescent protein gene were linked to the T2A (Thesa asigna virus 2A) gene. It was designed.

2. Construction of recombinant lentivirus

에서 90분 동안 75,000 x g에서 초원심분리하여 농축하고 -80

에서 보관하였다. Lentivirus encoding CT83.CAR or CT83 was produced by the method described below. Lentiviral vector plasmids, pMDLg/pRRE, pRSV-Rev, and pMD2.G pMD2.G packing plasmids (Addgene) were mixed in HEK-293T cells at a ratio of 2:1:1 and incubated with PEI (polyethyleneimine) (Sigma-Aldrich, St Louis). , MO) was used for transformation. After 48 and 72 hours of transformation, the supernatant containing the virus was obtained and filtered through a 0.45um PES filter. Then, the virus

Concentrate by ultracentrifugation at 75,000 xg for 90 minutes at -80

It was stored in .

3. Generation of CT83.CAR T-cells

에서 4시간 동안 50 uL 농축된 CT83.CAR-인코딩 렌티바이러스 또는 GFP-인코딩 렌티바이러스로 형질전환하였다. 렌티바이러스 형질도입은 24시간 후에 반복하였다. 2주 동안 세포 배양 후 PE anti-human CD3 (BioLegend), PerCP anti-human CD4 (BioLegend), and APC anti-human CD8a (BioLegend)로 염색하였다. CytoFLEX(Beckmann)를 사용하여 유세포 분석을 수행하였다.Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of healthy donors by density gradient centrifugation using Ficoll-Paque (GE Healthcare) and cultured in TexMACS medium (Miltenyi Biotec, Auburn, USA). Activation of PBMC was induced using T Cell TransAct® (Miltenyi Biotec, Auburn, USA) and 20 IU/mL IL-2 (Human IL-2 IS premium grade, Miltenyi Biotec, Auburn, USA) for 3 days. Activated T-cells were then incubated for 37 days in the presence of 10 ug/mL protamine sulfate (Sigma-Aldrich) and 50 IU/mL IL-2.

were transfected with 50 uL concentrated CT83.CAR-encoding lentivirus or GFP-encoding lentivirus for 4 hours. Lentiviral transduction was repeated 24 hours later. After culturing the cells for 2 weeks, they were stained with PE anti-human CD3 (BioLegend), PerCP anti-human CD4 (BioLegend), and APC anti-human CD8a (BioLegend). Flow cytometry was performed using CytoFLEX (Beckmann).

3. Target cell line

KATO-III (stomach cancer), H358 (lung cancer), and MDA-MB-231 (breast cancer) cell lines were transformed to express luciferase and cultured in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS (fetal bovine serum). It was cultured in . HEK-293T cells were transformed using a lentiviral vector to express CT83 antigen and luciferase.

4. ELISA (Enzyme-Linked Immunosorbent Assay)

After co-culturing CT83.CAR-T and Mock-T (2.5 × 10 ⁵ ) with target cells at a 20:1 ratio overnight, supernatants were collected and assayed for IFN using an ELISA kit (DuoSet® R&D system) according to the manufacturer's instructions. ELISA was performed to measure the concentrations of -γ, IL-2, and TNF-α.

5. Bioluminiscent Luciferase Reporter

KATO-III (gastric cancer), H358 (lung cancer), MDA-MB-231 (breast cancer), and recombinant lentivirus-transfected HEK-293T cell lines were co-cultured with CT83.CAR-T or Mock-T for 72 h. Transduced T cells and target cells (1 x 10 ⁴ ) were co-cultured in 96-well plates at a ratio of 2.5:1, 5:1, 10:1, or 20:1. Luciferase expression from target cells was analyzed using the One-Glo Luciferase Assay System (Promega) and a luminometer (Wallac Victor 2 Multi-label Counter, Perkin Elmer, Waltham, MA) according to the manufacturer's instructions. The luciferase activity of target cells not exposed to T cells was set to 100% cell viability.

6. T cell proliferation assay

CT83.CAR-T and Mock-T were labeled in PBS containing 5 uM Cell Trace Far Red (Invitrogen) at 37°C for 20 minutes. T cells were washed, and 1 x 10 ⁵ cells were co-cultured with equal amounts of target KATO-III (gastric cancer), H358 (lung cancer), and MDA-MB-231 (breast cancer) cells. On day 1, low dose IL-2 (10 IU/mL) was added to the medium. Labeled cells were harvested and flow cytometry was performed on day 5 to detect dye dilution for each cell division.

7. Flow cytometry for T cell activation

CT83.CAR-T and Mock-T (2.5 x 10 ⁵ ) were co-cultured with target cells at a 10:1 ratio in 96-well plates for 16 hours. To measure the percentage of CD107a ⁺ , CD25 ⁺ or CD69 ⁺ , T cells were analyzed by flow cytometry after staining with fluorescently labeled antibodies.

[Experiment result]

1. Generation of CT83.CAR-T-cells

A second-generation CAR consisting of a chimeric anti-human CT83 antibody, a single-chain variable fragment (scFv) derived from the CD3ζ intracellular domain and the CD28 costimulatory domain was constructed (Figure 1). T cells transfected with only ZsGreen1 fluorescent protein (Mock-T) were used as a control (Figure 1). For efficient expression of CAR molecules on the cell surface of transduced cells, the leader sequence of the CD8α signal peptide (MALPVTALLLLPLALLLHAARP) was inserted, and the scFv sequence was linked to the transmembrane domain using CD8 as a hinge to allow better flexibility. .

In the present invention, the amino acid sequence of the region constituting CT83 and the CAR targeting it is shown in Table 1 below.

sequence number item amino acid sequence One CT83 MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST 2 Leader MALPVTALLLPLALLLHAARP 3 Light chain variable region GAIGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTFLYWFLQRPGQSPQLLIYRMSNNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK 4 Linker GGGGSGGGGSGGGGS 5 Heavy chain variable region EVQLQQSGPELVKPGASVKMSCKTSGYTSTEYTMHWVKQSHGQSLEWIGVIYPNNGDTSYNQKFRGKATLTVDKSSNTAYMELRSLTSEDSAVYYCARLGWQGFANWGQGTLVTVSA 6 Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY 7 Transmembrane domain IWAPLAGTCGVLLLSLVITLYC 8 CD28 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY 9 CD3z RSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

PBMCs isolated from healthy donors were activated with TransAct and recombinant IL-2 (20 IU/mL), and activated T cells were transduced with CT83.CAR-encoding lentivirus or GFP-encoding lentivirus on day 3 of activation. . After transduction, T cells were proliferated under IL-2 (50 IU/mL) conditions. Flow cytometry was performed 48 hours after transduction to confirm CAR expression through detection of GFP-positive T cells (Figure 2). T cells expressed significant levels of CT83.CAR.

2. Confirmation of CT83 expression in various cancer cells

Flow cytometry was performed to confirm the expression of CT83 on the surface of several cancer cell lines, including KATO-III, H358, and MDA-MB-231 cell lines. Furthermore, RT-PCR was performed to confirm the expression of CT83 in the cancer cell line. The above results suggest that CT83 can be used as a new target for CAR-T therapy.

3. Confirmation of cytotoxicity of CT83.CAR-T in cancer cells

To confirm the cytotoxicity of transduced T cells, the genes of three target cell lines (KATO-III, H358, and MDA-MB-231) were modified to express mCherry and luciferase. Cell viability was determined using a luciferase reporter system and a luminometer. CT83.CAR-T and Mock-T were mixed and cultured with target cancer cell lines at E:T (effector-target) ratios of 20:1, 10:1, 5:1, and 2.5:1. As a result, it was confirmed that CT83.CAR-T exhibited more potent cytotoxicity than Mock-T after 72 hours of co-culture (FIGS. 3A to 3C).

The cytokine secretion profile of CT83.CAR-T in response to target cancer cells was further investigated. After the co-culture, the culture supernatant was obtained and ELISA was performed to quantify the concentration of cytokines (IFN-γ, IL-2, and TNF-α). As a result, it was confirmed that CT83.CAR-T produced much more functional cytokines than Mock-T (Figure 3d).

To evaluate the proliferative ability of CT83.CAR-T, CT83.CAR-T cells were labeled with CFSE (5-(and 6)-carboxyfluorescein diacetate succinimidyl ester) and co-cultured with target cells for 5 days. It was confirmed that co-culture with target cells expressing CT83 induced proliferation of CT83.CAR-T (Figure 4a).

CD107a (LAMP-1), a degranulation marker located mainly inside the granules of T cells, is detected by T cells releasing perforin and granzyme to activate target cytolysis. A significant increase in CD107a was confirmed after co-culture with target cells and CT83.CAR-T (Figure 4b). Furthermore, it was confirmed that the levels of CD25 (Figure 4c) and CD69 (Figure 4d) on the surface of CT83.CAR-T cells exposed to target cells increased.

From the above results, it can be seen that when CT83.CAR-T encounters target cells expressing CT83, it produces cytotoxicity-related cytokines and is activated, showing strong cytotoxicity.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising:
The CAR is a nucleic acid molecule comprising a CT83 (Cancer/Testis Antigen 83) binding domain, a transmembrane domain, and an intracellular co-stimulatory domain and signaling domain. According to paragraph 1,
A nucleic acid molecule wherein the CT83 binding domain is a single chain variable fragment (scFv) of a monoclonal antibody specific for CT83. According to paragraph 2,
The CT83-specific scFv is a nucleic acid molecule comprising a light chain variable region of SEQ ID NO: 3 and a heavy chain variable region of SEQ ID NO: 5. According to paragraph 3,
The CT83-specific scFv is a nucleic acid molecule in which a light chain variable region and a heavy chain variable region are connected by a linker of SEQ ID NO: 4. According to paragraph 1,
A nucleic acid molecule wherein the transmembrane domain includes the amino acid sequence of SEQ ID NO: 7. According to paragraph 1,
The CT83 binding domain and the transmembrane domain are connected by a hinge region, and the hinge region includes the amino acid sequence of SEQ ID NO: 6. According to paragraph 1,
The intracellular costimulatory domain is one or more selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137) A nucleic acid molecule derived from a protein. According to paragraph 1,
A nucleic acid molecule, wherein the intracellular signaling domain is derived from CD3 zeta. In clause 7,
A nucleic acid molecule, wherein the intracellular co-stimulatory domain is derived from CD28 and includes the amino acid sequence of SEQ ID NO: 8. According to clause 8,
A nucleic acid molecule in which the signaling domain derived from CD3 zeta includes the amino acid sequence of SEQ ID NO: 9. An expression vector comprising the nucleic acid molecule of any one of claims 1 to 10. T cells transformed with the expression vector of claim 11. A pharmaceutical composition for treating cancer comprising the transformed T cells of claim 12 as an active ingredient,
A pharmaceutical composition, wherein the cancer is a CT83 overexpressing carcinoma. According to clause 13,
The CT83-overexpressing carcinomas include breast cancer, hepatocellular cancer, stomach cancer, lung cancer, testicular cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, esophagus cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, kidney cancer, bladder cancer, urothelial cancer, sarcoma, and prostate cancer. , a pharmaceutical composition, characterized in that it is one or more types of cancer selected from the group consisting of thyroid cancer, thymic cancer, malignant melanoma, nasopharyngeal cancer, malignant mesothelioma, and colorectal cancer.