Immunocompetent cell, therapeutic agent, dna, and vector

Abstract

The present invention addresses the problem of providing a novel tumor treatment technology targeting meflin.　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.

Description

Immunocompetent cells, therapeutic agents, DNA, and vectors

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

[0002] Meflin (immunoglobulin superfamily containing leucine-rich repeat, ISLR) is a protein 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 exhibits an antitumor effect against sarcoma (Patent Document 1).

[0004] International Publication No. 2021 / 157601

[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 aims to provide a novel tumor treatment technique that targets meflin.

[0007] The present inventors have discovered that the above-mentioned 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, wherein 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, and 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.

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

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

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

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

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

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

[0015] Fig. 1 shows an outline of the constructs designed in the Examples. Fig. 2 shows the results of flow cytometry in the Examples. Fig. 3 shows the results of a cytotoxicity assay in the Examples. Fig. 4 shows the results of a real-time cytotoxicity assay in the Examples.

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

[0017] <Immunocompetent 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 on their surface a specific antibody (chimeric antigen receptor) that recognizes a specific antigen expressed by tumor cells. It is known that when CAR-T cells are administered to a patient, the CAR-T cells bind to the tumor cells via the specific antibody and antigen, 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 immunocompetent cells. The immunocompetent cells of the present invention having such a configuration bind to meflin-positive tumor cells and exert a tumor therapeutic effect by their cytotoxicity.

[0020] "Meflin" is a protein also known as a leucine-rich repeat-containing immunoglobulin superfamily (ISLR). Human meflin may have the amino acid sequence registered under GenBank accession number 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 number BAA85970.1.

[0021] The anti-meflin single-chain antibody of the present invention has a common structure with the antibody described in WO 2021 / 157601 (see "Clone 27-7" in the same publication). WO 2021 / 157601 shows that this antibody is specific to meflin and that the antitumor effect of the antibody is enhanced by using this antibody in combination with a cytotoxic agent. On the other hand, it is known that when an antibody expressed in CAR-T cells has high antigen specificity, 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 antibody is applied to CAR-T cells, an antitumor effect equal to or greater than that achieved when used in combination with a cytotoxic agent is achieved, leading to the completion of the present invention.

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

[0023] (1) Immunocompetent Cells "Immunocompetent cells" in the present invention include cells with cytotoxic function, such as T cells (T lymphocytes), natural killer (NK) cells, macrophages, and polymorphonuclear leukocytes (PMNs). Examples of T cells (T lymphocytes) include cytotoxic T cells (killer T cells), helper T cells, and suppressor T cells. Of these, T cells are preferred from the viewpoint of facilitating stable antitumor effects. Note 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 treatment subject (human, non-human mammal, etc.). From this perspective, the immunocompetent cells are preferably derived from an organism of the same species as the treatment subject, and more preferably derived from the treatment subject 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 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 contains an extracellular domain (chimeric antigen receptor), a transmembrane domain, and an intracellular domain, in this order.

[0027] The anti-meflin single-chain antibody of the present invention satisfies the following requirements. Note that the following variable regions are all derived from anti-meflin antibodies that recognize 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] The term "single-chain antibody" as used herein refers to an antibody comprising a light chain variable region and a heavy chain variable region that constitute the antigen-binding site of the antibody. Typically, a flexible linker is present between the light chain variable region and the heavy chain variable region.

[0030] When an anti-meflin single-chain antibody contains a linker, its sequence is not particularly limited as long as it exhibits 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 nucleotide 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 sites other than the extracellular domain (anti-meflin single-chain antibody) in the chimeric antigen receptor include the following commonly used sites. The sequences of these domains can be determined in accordance with the method described in Leukemia, 2004, 18:676-684. - Transmembrane domain: hinge of CD8α - Intracellular domain: intracellular domain of 4-1BB and intracellular domain of CD3ζ

[0034] (3) Diseases to be Treated The immunocompetent cells of the present invention have an antitumor 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 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 decrease 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 antitumor effect against tumors containing meflin-positive cells. The present invention encompasses therapeutic agents for treating tumors (such as meflin-positive sarcomas), which contain the immunocompetent cells of the present invention.

[0038] The method of using the immunocompetent cells of the present invention may employ conditions similar to those for conventional therapies using CAR-T cells. For example, an antitumor effect can be achieved by administering the immunocompetent cells to a subject to be treated (preferably a subject from which the immunocompetent cells are derived) by injection or the like.

[0039] <Method for Producing Immunocompetent Cells> The immunocompetent cells of the present invention can be produced by the same method as the conventionally known method for producing CAR-T cells, except that a predetermined anti-meflin single-chain antibody is expressed.

[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 step, hybridoma cells producing an antibody against the meflin antigen are first prepared. Next, mRNA is extracted from the obtained hybridoma cells, and cDNAs (SEQ ID NOs: 6 and 8) encoding the light-chain variable region and heavy-chain variable region of the anti-meflin antibody, respectively, are obtained according to the method described in WO 2021 / 157601.

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

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

[0044] In a preferred embodiment of the present invention, the construct comprises, in this order: a nucleotide sequence encoding the signal peptide of CD8α (linked in the order of the light chain variable region and the heavy chain variable region); a nucleotide 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; and a nucleotide sequence encoding the intracellular domain of CD3ζ, wherein the light chain variable region of the anti-meflin single-chain antibody consists of the nucleotide sequence set forth in SEQ ID NO: 2; and 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 contained between the light chain variable region and the heavy chain variable region. A preferred sequence of the linker is SEQ ID NO: 3. The present invention also encompasses DNA having the above 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. Examples of the vector include viral (lentiviral, etc.) vectors. A marker gene (enhanced green fluorescent protein (EGFP) or the like) may be introduced into the vector for expression analysis or the like. The present invention also encompasses vectors having the above configuration.

[0046] (Step 3) Preparation of packaging cells In this step, packaging cells are prepared by transfecting the viral vector obtained in step 2 into packaging cells. This step allows the preparation of viral particle (virion)-producing cells that express anti-meflin single-chain antibody.

[0047] (Step 4) Transduction of T cells In this step, immunocompetent cells are infected with the virus particles released from the packaging cells obtained in step 3. Through 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 immunocompetent 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] Selection methods include the following: Confirmation of the presence or absence of expression of anti-meflin single-chain antibodies and the expression level thereof by flow cytometry. Confirmation of the killing ability of target cells by a cytotoxicity assay.

[0051] The present invention will be explained 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 by the following method. The method for preparing the immunocompetent cells was in accordance with the method described in Japanese Patent No. 5,328,018.

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

[0054] An anti-meflin single-chain antibody gene was designed from the obtained cDNA. 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 is linked in the order of VL, linker, and VH. 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 the VL is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 6. The nucleotide 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 the VH is shown in SEQ ID NO: 4, and the amino acid sequence is shown in SEQ ID NO: 8.

[0055]

[0056]

[0057] (2) Preparation of anti-meflin-CAR construct A construct containing, in this order, a CD8α signal peptide, an anti-meflin single-chain antibody (VL, linker, VH), a CD8α hinge, a CD8α transmembrane domain, a 4-1BB intracellular domain, and a CD3ζ intracellular domain was prepared 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 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] The designed anti-meflin-CAR construct is outlined in Figure 1.

[0060] (3) Preparation of Cell Lines. Jurkat, a human T lymphocyte cell line, was purchased from the American Type Culture Collection (ATCC). HS-Os-1, a cell line 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). 293T, a human fetal kidney epithelial cell line, was purchased from ATCC. Jurkat, HS-Os-1, and KYM-1 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin (P / S). 293T, a cell line used for lentivirus packaging, was maintained in DMEM containing 10% FBS and P / S. All cells were incubated at 37°C and 5% CO ２ was maintained.

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

[0062] (5) Transduction of T Cells and Obtainment of Immunocompetent Cells of the Present Invention Before transduction of Jurkat cells, a culture dish was coated with RetroNectin overnight at 4°C, blocked with 2% bovine serum albumin, and then washed with phosphate-buffered saline (PBS). Next, Jurkat cells were transduced with "Mock-CAR" or "Meflin-CAR" using the lentiviral particles obtained in (4) above, according to the RetroNectin operating instructions. After transduction, Jurkat cells were grown using a refresh culture medium, and ZsGreen-positive Jurkat cells were enriched by cell sorting according to the level of fluorescence. Through the above steps, "Mock-CAR-T cells," which are T cells transduced with "Mock-CAR," and "Meflin-CAR-T cells," which are T cells transduced with "Meflin-CAR," were obtained. These T cells correspond to the "immunocompetent cells" of 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 used as a control. "Anti-meflin-allophycocyanin (APC)" was used to detect meflin in HS-Os-1 cells and KYM-1 cells, respectively. Anti-meflin-APC was provided by Professor Atsushi Enomoto (Nagoya University).

[0064] All flow cytometry data were 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, in T cells into which "meflin-CAR" was introduced (Meflin-targeted CAR-T), "meflin-CAR"-specific expression was observed.

[0066] (7) Cytotoxicity Assay A lactate dehydrogenase (LDH) cytotoxicity assay was performed using osteosarcoma cells and rhabdomyosarcoma cells as target cells. Non-transduced cells (Jurkat cells) were used as a control. First, osteosarcoma cells (HS-Os-1) and rhabdomyosarcoma cells (KYM-1) were transduced at 1 × 10 ５ The cells were seeded in 96-well flat-bottom plates at 1 / well and incubated at 37°C in 5% CO in RPMI 1640 medium supplemented with 10% FBS. ２ Then, meflin-CAR-T cells were added as effector cells 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 centrifuged (250 g, 4 minutes) and incubated at 37°C, 5% CO ２ was cultured in.

[0067] After 48 hours of culture, the lactate dehydrogenase (LDH) level in the cell culture supernatant was measured using a lactate dehydrogenase cytotoxicity assay kit (Dojindo Laboratories, Inc.). Based on the measurement results, 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 in target cells - maximum LDH level in 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. Note that similar results to those shown in Figure 3 were also obtained for KYM-1.

[0069] (8) Real-time Cytotoxicity Assay The cytotoxicity of each CAR-T cell was evaluated in real time using an "xCELLigence" real-time cell analyzer (Agilent Technologies). First, osteosarcoma cells and rhabdomyosarcoma cells (1 × 10) in normal culture medium were cultured. ４ / well) were seeded into a 16-well plate and left to stand on a clean bench for 30 minutes. The plate was then placed in an 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 plate was then placed in an analyzer (37°C, 5% CO ２ ) and cell proliferation measurements were initiated using an iCELLigence RTCA (Agilent Technologies). Cell proliferation was measured continuously every 15 minutes for 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 with "meflin-CAR-T cells." Similar results to those shown in Figure 4 were also obtained for KYM-1.

Claims

1. An immunocompetent cell expressing an anti-meflin single-chain antibody on the cell surface, wherein 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, and 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.

2. The immunocompetent cell according to claim 1, wherein the anti-meflin single-chain antibody comprises, in this order, the light chain variable region, a linker, and the heavy chain variable region, and the linker consists of the amino acid sequence set forth in SEQ ID NO:

7.

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 immunocompetent cell according to any one of claims 1 to 3, and the tumor is a meflin-positive sarcoma.

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

4.

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

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