Immunocompetent cell, therapeutic agent, dna, and vector

Abstract

To provide a novel tumor treatment technology targeting meflin.SOLUTION: The present invention provides an immunocompetent cell in which an anti-meflin single-stranded antibody is expressed on the surface thereof, wherein a light-chain variable region in the anti-meflin single-stranded antibody comprises the amino acid sequence represented by SEQ ID NO: 6 and a heavy-chain variable region in the anti-meflin single-stranded antibody comprises the amino acid sequence represented by SEQ ID NO: 8.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to immunocompetent cells, therapeutic agents, DNA, and vectors. [Background technology]

[0002] Meflin (Immunoglobulin superfamily containing leucine-rich repeat, ISLR) is a protein that is specifically expressed in mesenchymal stem cells. Meflin is known to play important roles, for example, in maintaining the undifferentiated state of cells and in tissue repair in various organs.

[0003] It has also been reported that an antibody-drug conjugate of an anti-meflin antibody and a cytotoxic agent exerts an antitumor effect against sarcoma (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2021 / 157601 Summary of the Invention [Problem to be solved by the invention]

[0005] On the other hand, there is a need for more effective tumor treatments that target meflin.

[0006] The present invention has been made in view of the above circumstances, and has an object to provide a novel tumor treatment technique that targets meflin. [Means for solving the problem]

[0007] The present inventors have found that the above problems can be solved by introducing a specific anti-meflin single-chain antibody as a chimeric antigen receptor in CAR-T cell therapy, and have completed the present invention. More specifically, the present invention provides the following.

[0008] (1) An immunocompetent cell expressing an anti-meflin single-chain antibody on the cell surface, the light chain variable region of the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO:6; The heavy chain variable region of the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO: 8. Immune-competent cells.

[0009] (2) the anti-meflin single chain antibody comprises, in this order, the light chain variable region, a linker, and the heavy chain variable region; The linker consists of the amino acid sequence set forth in SEQ ID NO: 7. An immunocompetent cell described in (1).

[0010] (3) The immunocompetent cell according to (1), wherein the cell is a T cell.

[0011] (4) A therapeutic agent for treating a tumor, comprising: The therapeutic agent comprises the immunocompetent cell according to any one of (1) to (3), The tumor is a meflin-positive sarcoma. Therapeutic agent.

[0012] (5) DNA comprising: The DNA comprises a base sequence encoding a signal peptide of CD8α; A base sequence encoding an anti-meflin single-chain antibody; A nucleotide sequence encoding the hinge of CD8α; A nucleotide sequence encoding the transmembrane domain of CD8α; A nucleotide sequence encoding the intracellular domain of 4-1BB; A nucleotide sequence encoding the intracellular domain of CD3ζ; in this order, the light chain variable region of the anti-meflin single chain antibody comprises the nucleotide sequence set forth in SEQ ID NO: 2, The heavy chain variable region of the anti-meflin single chain antibody consists of the nucleotide sequence set forth in SEQ ID NO: 4. DNA.

[0013] (6) A vector incorporating the DNA described in (5). Effect of the Invention

[0014] According to the present invention, a novel tumor treatment technique that targets meflin is provided. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows an overview of the constructs designed in the Examples. [Diagram 2] FIG. 1 shows the results of flow cytometry in an example. [Diagram 3] FIG. 1 shows the results of a cytotoxicity assay in an example. [Figure 4] FIG. 1 shows the results of a real-time cytotoxicity assay in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited thereto.

[0017] <Immune-competent cells> The present invention is a technology related to CAR (chimeric antigen receptor)-T cell therapy.

[0018] "CAR-T cells" are T cells that express a specific antibody (chimeric antigen receptor) on their surface that recognizes a specific antigen expressed by tumor cells. It is known that by administering CAR-T cells to a patient, the CAR-T cells bind to tumor cells via specific antibodies and antigens, making it possible to kill the tumor.

[0019] The main technical feature of the present invention is that a specific anti-meflin single-chain antibody is expressed on the surface of an immunocompetent cell. The immunocompetent cells of the present invention having such a configuration bind to meflin-positive tumor cells and exert a tumor therapeutic effect due to their cytotoxic function.

[0020] "Meflin" is a protein also known as the immunoglobulin superfamily containing leucine-rich repeat (ISLR). Human meflin may have the amino acid sequence registered under GenBank Accession No. BAA85970.1. Meflin may be a meflin (e.g., human meflin) having an amino acid sequence corresponding to the amino acid sequence registered under GenBank Accession No. BAA85970.1.

[0021] The anti-meflin single-chain antibody of the present invention has a common configuration with the antibody described in WO 2021 / 157601 (see "Clone 27-7" in the same publication). WO 2021 / 157601 shows that the antibody is specific to meflin and that the antitumor effect of the antibody is enhanced by combining the antibody with a cytotoxic agent. On the other hand, it is known that if the antibodies expressed in CAR-T cells have high specificity for the antigen, the therapeutic effect may actually be reduced (Park, S. et al. Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity. Sci. Rep. 7, 14366 (2017)). On the other hand, the present inventors unexpectedly found that when the above-mentioned antibody is applied to CAR-T cells, it exerts an antitumor effect equal to or greater than that obtained when used in combination with a cytotoxic agent, and thus completed the present invention.

[0022] The immunocompetent cells of the present invention are described in detail below.

[0023] (1)Immune-competent cells In the present invention, the "immunocompetent cells" include cells having cytotoxic function such as T cells (T lymphocytes), natural killer (NK) cells, macrophages, polymorphonuclear leukocytes (PMN), etc. Examples of T cells (T lymphocytes) include cytotoxic T cells (killer T cells), helper T cells, and suppressor T cells. Among these, T cells are preferred from the viewpoint of ease of obtaining a stable antitumor effect. It should be noted that B lymphocytes (humoral antibody-producing cells) are not included in the immunocompetent cells of the present invention.

[0024] The immunocompetent cells of the present invention exert a therapeutic effect when administered into the body of a subject to be treated (humans, non-human mammals, etc.). From this viewpoint, the immunocompetent cells are preferably derived from an organism of the same species as the subject of treatment, and more preferably derived from the subject of treatment itself.

[0025] The method for isolating immunocompetent cells from an organism is not particularly limited, and known methods such as density separation can be used.

[0026] (2) Chimeric antigen receptor In the present invention, the chimeric antigen receptor expressed in the immunocompetent cells may have a structure similar to that of a chimeric antigen receptor employed in the design of conventional CAR-T cells, except that it contains an anti-meflin single-chain antibody having a predetermined variable region. That is, in a representative example, the chimeric antigen receptor comprises an extracellular domain (chimeric antigen receptor), a transmembrane domain, and an intracellular domain, in that order.

[0027] The anti-meflin single-chain antibody of the present invention satisfies the following requirements: All of the following variable regions are derived from an anti-meflin antibody that recognizes human meflin protein. The light chain variable region of the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO:6. The heavy chain variable region of the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO:8.

[0028] The base sequence corresponding to the amino acid sequence set forth in SEQ ID NO:6 is SEQ ID NO:2. The base sequence corresponding to the amino acid sequence set forth in SEQ ID NO:8 is SEQ ID NO:4.

[0029] In the present invention, a "single chain antibody" is an antibody that comprises a light chain variable region and a heavy chain variable region that constitute the antigen-binding site of the antibody. Usually, a flexible linker is present between the light chain variable region and the heavy chain variable region.

[0030] When the anti-meflin single-chain antibody contains a linker, the sequence thereof is not particularly limited as long as it can provide the desired flexibility. In a preferred embodiment of the present invention, the linker of the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO:7. The base sequence corresponding to the amino acid sequence set forth in SEQ ID NO:7 is SEQ ID NO:3.

[0031] In a preferred embodiment of the present invention, the anti-meflin single chain antibody comprises the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8 consecutively in this order.

[0032] In a preferred embodiment of the present invention, the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO:5.

[0033] The site other than the extracellular domain (anti-meflin single-chain antibody) in the chimeric antigen receptor may be the following, which are commonly used. The sequences of these domains may be set according to the method described in Leukemia, 2004, 18:676-684. · Transmembrane domain: hinge of CD8α Intracellular domain: the intracellular domain of 4-1BB and the intracellular domain of CD3ζ

[0034] (3) Disease to be treated The immunocompetent cells of the present invention have an anti-tumor effect against tumors containing meflin-positive cells.

[0035] Tumors that can be treated with the immunocompetent cells of the present invention include sarcomas (osteosarcoma, chondrosarcoma, liposarcoma, rhabdomyosarcoma, undifferentiated pleomorphic sarcoma, desmoid tumor, meningioma, etc.).

[0036] Whether or not the immunocompetent cells of the present invention have the desired therapeutic effect can be evaluated, for example, by co-culturing the immunocompetent cells with target cells (meflin-positive tumor cells) and confirming the reduction in the number of target cells over time by flow cytometry, cytotoxicity assay, or the like.

[0037] (4) Uses of immunocompetent cells As described above, the immunocompetent cells of the present invention have an anti-tumor effect against tumors containing meflin-positive cells. The present invention includes a therapeutic agent for treating tumors (such as meflin-positive sarcomas) comprising the immunocompetent cells of the present invention.

[0038] The method of using the immunocompetent cells of the present invention may adopt the same conditions as those of conventional therapies using CAR-T cells. For example, the immunocompetent cells may be administered to a subject (preferably a subject from which the immunocompetent cells are derived) by injection or the like to exert an antitumor effect.

[0039] <Method of producing immunocompetent cells> The immunocompetent cells of the present invention can be produced by methods similar to those used for producing conventional CAR-T cells, except that they express a specific anti-meflin single-chain antibody.

[0040] The method for producing immunocompetent cells of the present invention includes, for example, the following steps.

[0041] (Step 1) Preparation of anti-meflin single-chain antibody In this process, first, hybridoma cells that produce antibodies against the meflin antigen are prepared. Next, mRNA is extracted from the obtained hybridoma cells, and cDNAs (sequence numbers 6 and 8) encoding the light chain variable region and heavy chain variable region of the anti-meflin antibody, respectively, are obtained in accordance with the method described in WO2021 / 157601.

[0042] (Step 2) Preparation of anti-meflin-CAR construct In this step, the cDNA obtained in step 1 is amplified to produce a construct containing an antibody cDNA.

[0043] An example of a construct according to a preferred embodiment of the present invention is shown in FIG.

[0044] In a preferred embodiment of the present invention, the construct comprises: A base sequence encoding the signal peptide of CD8α (linked in the order of the light chain variable region and the heavy chain variable region), A base sequence encoding an anti-meflin single-chain antibody; A nucleotide sequence encoding the hinge of CD8α; A nucleotide sequence encoding the transmembrane domain of CD8α; A nucleotide sequence encoding the intracellular domain of 4-1BB; A nucleotide sequence encoding the intracellular domain of CD3ζ; in this order, The light chain variable region of the anti-meflin single chain antibody comprises the nucleotide sequence set forth in SEQ ID NO:2, The heavy chain variable region of the anti-meflin single-chain antibody consists of the nucleotide sequence set forth in SEQ ID NO:4. In a more preferred embodiment, a linker may be included between the light chain variable region and the heavy chain variable region. A preferred sequence for the linker is SEQ ID NO:3. The present invention also includes DNA having the above-mentioned configuration.

[0045] Next, an expression cassette containing the obtained construct is inserted into a vector to prepare a vector that expresses an anti-meflin single-chain antibody, such as a viral (lentiviral or other) vector. A marker gene (such as EGFP (Enhanced green fluorescent protein)) may be introduced into the vector for expression analysis or the like. The present invention also includes a vector having the above-mentioned configuration.

[0046] (Step 3) Preparation of packaging cells In this step, the viral vector obtained in step 2 is transfected into packaging cells to produce packaging cells. By this process, cells producing virus particles (virions) expressing anti-meflin single-chain antibody can be prepared.

[0047] (Step 4) Transduction of T cells In this step, the virus particles released from the packaging cells obtained in step 3 are used to infect immunocompetent cells. By this step, the immunocompetent cells of the present invention can be obtained.

[0048] The method for infecting immunocompetent cells is not particularly limited, but examples include mixed culture of virus particle-producing cells and immunocompetent cells.

[0049] (Step 5) Selection of immune cells If necessary, the immunocompetent cells obtained in step 4 may be selected by any method to determine whether or not they are to be used for therapy.

[0050] The selection methods include the following: Confirmation of the presence or absence of expression of anti-meflin single-chain antibody and the expression level, etc. by flow cytometry. - Confirmation of the killing ability of target cells by cytotoxicity assay. EXAMPLES

[0051] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0052] Immunocompetent cells were prepared and their cytotoxicity was evaluated according to the following method, which was in accordance with the method described in Japanese Patent No. 5328018.

[0053] (1) Preparation of anti-meflin single-chain antibody mRNA was extracted from anti-meflin antibody-producing hybridoma cells that recognize human meflin protein. The anti-meflin antibody-producing hybridoma cells were provided by Professor Atsushi Enomoto (Nagoya University). From the obtained mRNA, cDNAs encoding the light chain variable region and heavy chain variable region of the anti-meflin antibody were obtained in accordance with the method described in WO2021 / 157601.

[0054] From the obtained cDNA, an anti-meflin single-chain antibody gene was designed. The anti-meflin single-chain antibody (scFv) used in this test consists of a light chain variable region (VL), a linker, and a heavy chain variable region (VH), and the VL, linker, and VH are linked in this order. The nucleotide sequence of the scFv is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:5. The nucleotide sequence of VL is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:6. The base sequence of the linker is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:7. The nucleotide sequence of VH is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:8.

[0055] [Table 1]

[0056] [Table 2]

[0057] (2) Preparation of anti-meflin-CAR construct A construct was prepared containing, in this order, a signal peptide of CD8α, an anti-meflin single-chain antibody (VL, linker, VH), a hinge of CD8α, a transmembrane domain of CD8α, an intracellular domain of 4-1BB, and an intracellular domain of CD3ζ, according to the method described in Leukemia, 2004, 18:676-684. The obtained construct is hereinafter referred to as "meflin-chimeric antigen receptor (CAR)". The expression cassette was subcloned into the pLVSIN-EF1a-IRES-ZsGreen lentiviral plasmid (Takara Bio Inc.).

[0058] In addition, a construct was obtained in the same manner as above, except that the anti-meflin single-chain antibody was not introduced. The obtained construct is hereinafter referred to as "Mock-chimeric antigen receptor (CAR)".

[0059] An overview of the designed anti-meflin-CAR construct is shown in Figure 1.

[0060] (3) Preparation of cell lines Human T lymphocyte cells, "Jurkat," were purchased from ATCC (American Type Culture Collection). "HS-Os-1," derived from osteoblastic osteosarcoma, and "KYM-1," a human rhabdomyosarcoma cell line derived from a cervical tumor, were provided by Professor Atsushi Enomoto (Nagoya University). The 293T cell line, a human fetal kidney epithelial cell line, was purchased from ATCC. "Jurkat," "HS-Os-1," and "KYM-1" were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin (P / S). The 293T cell line, a cell line for lentivirus packaging, was maintained in DMEM containing 10% FBS and P / S. All cells were maintained at 37°C and 5% CO2.

[0061] (4) Lentivirus packaging Using "TransIT (registered trademark)-Lenti Transfection Reagent" (Mirus Bio, Inc.), 293T cells were transfected with "Mock-CAR" or "Meflin-CAR" in pLVSIN-EF 1α-IR ES-ZsGreen lentiviral plasmid to obtain packaging cells. 48 hours after transfection, lentiviral particles were harvested and filtered through a 0.45 μm hydrophilic PVDF membrane (Millipore). The collected lentiviral particles were stored at -80°C until the next step.

[0062] (5) Transduction of T cells and obtaining the immunocompetent cells of the present invention Prior to transduction of Jurkat cells, culture dishes were coated with retronectin overnight at 4°C, blocked with 2% bovine serum albumin, and then washed with phosphate-buffered saline (PBS). Next, according to the RetroNectin operating instructions, Jurkat cells were transduced with "Mock-CAR" or "Meflin-CAR" using the lentiviral particles obtained in (4) above. After transduction, Jurkat cells were expanded using refresh culture medium, and ZsGreen-positive Jurkat cells were enriched by cell sorting according to the degree of fluorescence. Through the above steps, "Mock-CAR-T cells", which are T cells introduced with "Mock-CAR", and "Meflin-CAR-T cells", which are T cells introduced with "Meflin-CAR", were obtained. These T cells correspond to the "immunocompetent cells" according to the present invention.

[0063] (6) Flow cytometry The expression of "Mock-CAR" and "Meflin-CAR" on the surface of T cells (Jurkat cells) was stained with "Biotin-SA-goat anti-rat IgG" (Jackson 112-065-167). Non-transduced cells (Jurkat cells) were set up as a control. To detect meflin in HS-Os-1 and KYM-1 cells, we used anti-meflin-allophycocyanin (APC), which was kindly provided by Professor Atsushi Enomoto (Nagoya University).

[0064] All flow cytometry data was analyzed using a BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences) and BD Accuri™ C6 Plus Software.

[0065] The results are shown in Figure 2. As shown in Figure 2, Meflin-CAR-specific expression was observed in T cells introduced with Meflin-CAR (Meflin-targeted CAR-T).

[0066] (7) Cytotoxicity assay Lactate dehydrogenase (LDH) cytotoxicity assays were performed using osteosarcoma and rhabdomyosarcoma cells as target cells. Non-transduced cells (Jurkat cells) were set up as a control. First, osteosarcoma cells (HS-Os-1) and rhabdomyosarcoma cells (KYM-1) were cultured at 1 × 10 5 The cells were seeded at 1 / well into a 96-well flat-bottom plate and allowed to attach for 24 hours at 37° C., 5% CO 2 in RPMI 1640 medium supplemented with 10% FBS. Next, "meflin-CAR-T cells" as effector cells were added to the plate at a predetermined E:T ratio (20:1, 10:1, 5:1, 2.5:1, or 1:1), and the plate was then centrifuged (250g, 4 minutes) and cultured at 37°C, 5% CO2.

[0067] After 48 hours of culture, lactate dehydrogenase (LDH) levels in the cell culture supernatant were measured using a lactate dehydrogenase cytotoxicity assay kit (Dojindo Laboratories, Ltd.). Based on the measurement results, the cytotoxicity was calculated using the following formula: The test was repeated three times, and the results were expressed as the mean ± standard deviation. Cytotoxicity (%) = (total number of cells tested - number of effector cells - number of target cells) / (maximum LDH level of target cells - maximum LDH level of target cells) x 100

[0068] The results for HS-Os-1 are shown in Figure 3. As shown in Figure 3, cytotoxicity of "meflin-CAR-T cells" to HS-Os-1 was observed. In addition, results similar to those shown in FIG. 3 were obtained for KYM-1.

[0069] (8) Real-time cytotoxicity assay The cytotoxicity of each CAR-T cell was evaluated in real time using the "xCELLigence" real-time cell analysis device (Agilent Technologies). First, osteosarcoma cells and rhabdomyosarcoma cells (1 × 10 4 / well) were seeded onto a 16-well plate and allowed to stand on a clean bench for 30 minutes. The plate was then placed in the analyzer overnight. After confirming the adhesion of each cell, each CAR-T cell was added to the plate at a predetermined E:T ratio (1:1 or 2:1) and left to stand on a clean bench for 30 minutes. The plates were then placed into the analyzer (37° C., 5% CO 2 ) and measurement of cell proliferation was initiated using the iCELLigence RTCA (Agilent Technologies). Cell proliferation was measured continuously every 15 min over a period of 5 days.

[0070] The results for HS-Os-1 are shown in Figure 4. As shown in Figure 4, a reduction in HS-Os-1 cells was observed by "meflin-CAR-T cells." In addition, results similar to those shown in FIG. 4 were obtained for KYM-1.

Claims

1. An immunocompetent cell expressing an anti-meflin single chain antibody on the cell surface, the light chain variable region of the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO:6; The heavy chain variable region of the anti-meflin single chain antibody consists of the amino acid sequence set forth in SEQ ID NO:

8. Immune-competent cells.

2. the anti-meflin single chain antibody comprises, in this order, the light chain variable region, a linker, and the heavy chain variable region; The linker consists of the amino acid sequence set forth in SEQ ID NO:

7. The immunocompetent cell described in claim 1.

3. The immunocompetent cell according to claim 1 , wherein the cell is a T cell.

4. A therapeutic agent for treating a tumor, comprising: The therapeutic agent comprises the immunocompetent cell according to any one of claims 1 to 3, The tumor is a meflin-positive sarcoma. Therapeutic agent.

5. DNA, The DNA comprises a base sequence encoding a signal peptide of CD8α; A base sequence encoding an anti-meflin single-chain antibody; A nucleotide sequence encoding the hinge of CD8α; A nucleotide sequence encoding the transmembrane domain of CD8α; A base sequence encoding the intracellular domain of 4-1BB; A base sequence encoding the intracellular domain of CD3ζ; in this order, the light chain variable region of the anti-meflin single chain antibody comprises the nucleotide sequence set forth in SEQ ID NO: 2, The heavy chain variable region of the anti-meflin single chain antibody consists of the nucleotide sequence set forth in SEQ ID NO:

4. DNA.

6. A vector incorporating the DNA according to claim 5.

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