Artificial receptor with exfoliated structure

Abstract

The present invention provides artificial receptors comprising a ligand binding site, a transmembrane domain having an exfoliated structure, and a signal transduction domain into an immune cell, and nucleic acids encoding the artificial receptors.

Description

Technical Field

[0001] The present invention relates to an artificial receptor with a shedding structure. More specifically, it relates to an artificial receptor comprising a ligand binding site, a transmembrane domain with a shedding structure, and a signal transduction domain into immune cells. In addition, it also relates to a design method for a receptor regulated by tropocytosis or a nucleic acid encoding the receptor. Background Art

[0002] If continuous input strong stimulation, the phenomenon of decreased sensitivity is well known. The decreased sensitivity caused by the continuous signal of this receptor is considered to be biologically significant, and known fall-off is one of the mechanisms that can be expected to prevent decreased sensitivity. Falling off is the mechanism that the extracellular domain of receptor is cut by metalloproteinase. Even if only studied, it is also known to cause falling off (non-patent literature 1) in more than half of the tyrosine kinase receptors. It is believed that after receptor and ligand reaction cause the phosphorylation of intracellular domain, the reaction may be temporarily reset by falling off, and the signal is regulated, but for falling off, signal regulation is carried out with what kind of mechanism, and it is not clear that the details.

[0003] The T cell receptor (T cell receptor; TCR) used for cancer immunotherapy, which is a target for T cell recognition, is also a type of tyrosine kinase receptor. As one of the signal controls, it is known that if TCR is stimulated, the cell surface expression of the TCR-CD3 complex decreases. In addition, in recent years, the artificial chimeric receptor (Chimeric antigen receptor; CAR), which is considered to have epoch-making therapeutic effects in immune cell therapy, is a structure that recognizes antigens and transduces TCR signals to T cells by binding antibodies to auxiliary receptors such as CD3 intracellular domains, CD28, and 4-1BB, but CD3 and co-stimulatory molecules are both tyrosine kinase receptors.

[0004] It is known that if CAR cell therapy is continued, the therapeutic effect is weakened. It is known that cell bite is one of the mechanisms related to the weakening effect of such CAR cell therapy or recurrence after treatment (non-patent literature 2). Cell bite is such a mechanism that, if the target cell reacts with the T cell expressing CAR, the membrane exchange is caused in the form of biting the cell membrane at the reaction site, and the T cell seizes the target antigen. Therefore, in CAR cell therapy, the antigen expression of cancer cells is reduced, causing immune escape and becoming the cause of recurrence. In addition, it is also reported that since the T cells that have captured the cancer antigen express the captured cancer antigen on their own cell membrane, the suppression of the immune response is caused by becoming the target of other CAR-T cells. It is known that cell bite is discovered as a membrane exchange phenomenon caused by an immune response, and it is a physiological phenomenon preserved in protists such as amoeba cells. However, the detailed mechanism of membrane exchange is not clear, and it is not known to suppress cell bite in CAR-T cell therapy to solve the problem.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Merilahti JAM and Elenius K, Oncogene 38(2):151-163 (2019),

[0008] Non-patent document 2: Hamieh M et al., Nature. 568(7750): 112-116 (2019). Summary of the invention

[0009] Problems to be solved by the invention

[0010] Therefore, an object of the present invention is to provide a method for inhibiting intercellular cytotoxicity for establishing an immunotherapy for diseases such as cancer that is less likely to cause recurrence and more likely to maintain therapeutic effects.

[0011] Means of solving problems

[0012] In order to solve the above problems, the inventors of the present invention focus on the signal control of CAR. First, by changing the transmembrane domain of CAR to a structure that is shed (also referred to as a "shed structure"), the structure of CAR is closer to the physiological tyrosine kinase receptor, and the effect on immune cell therapy is verified. CAR with a shed structure lacks the site for recognizing the antigen presented on the surface of cancer cells due to shedding, and it is believed that the recognition ability of cancer cells is reduced. In fact, when CAR-expressing cells and cancer cells are reacted in a short time of 3 hours, CAR with a shed structure shows a slight decrease in reactivity compared to CAR without the structure. However, surprisingly, if CAR-expressing cells are reacted with cancer cells for a long time, CAR with a shed structure is more reactive than usual CAR, and the anti-tumor effect is also confirmed to be enhanced in an in vivo model using tumor-transplanted mice. Based on such insights, further verification was carried out, and the results clearly showed that CAR with a shed structure prevents the cell bite of the target antigen in cancer cells by shedding. It was found that maintaining long-term reactivity by inhibiting cell bite is one of the reasons for the improvement of the anti-tumor effect in CAR with a shed structure.

[0013] The inventors further focused on the intracellular domain of CAR and proposed the following hypothesis, that is, by adjusting the size of such a domain, whether cytotoxicity can be further controlled, and whether the regulation of cytotoxicity does not necessarily require a signal transduction domain. Based on such a hypothesis, a study was conducted, and the results confirmed the following situation, that is, the signal transduction domain is not necessary for the occurrence of cytotoxicity, the cytotoxicity intensity depends on the size of the intracellular domain and varies, and the signal transduction domain causes stronger cytotoxicity than the non-signaling domain when the size is the same. In addition, it has also been confirmed that the cytotoxicity intensity can be further suppressed by combining the addition of the shedding structure and the change of the number or order of the intracellular signal transduction domain. It can be said that based on these insights, a technology for adjusting the cytotoxicity level of CAR has been successfully established. Based on these insights, the inventors further repeatedly studied and completed the present invention.

[0014] That is, the present invention is as follows.

[0015] [1] An artificial receptor comprising a ligand binding site, a transmembrane domain with a shedding structure, and a signal transduction domain into immune cells.

[0016] [2] The artificial receptor according to [1], which is selected from a chimeric antigen receptor, a modified T cell receptor, a modified Fc receptor and a modified tyrosine kinase receptor.

[0017] [3] The artificial receptor according to [1] or [2], wherein the immune cell is selected from T cells, natural killer cells and macrophages.

[0018] [4] The artificial receptor according to [3], wherein the T cell is a cytotoxic T cell.

[0019] [5] The artificial receptor according to any one of [1] to [4], wherein the shedding structure is derived from a protein selected from the group consisting of notch protein, amyloid precursor protein and CD28.

[0020] [6-1] The artificial receptor according to any one of [1] to [5], wherein the signal transduction domain into immune cells comprises a signal transduction domain derived from at least one selected from the group consisting of CD3 ζ chain, CD28, and 4-1BB.

[0021] [6-2] The artificial receptor according to any one of [1] to [6-1], wherein the ligand binding site is a cancer antigen binding site.

[0022] [7-1] The artificial receptor according to any one of [1] to [6-2], wherein the transmembrane domain having a shedding structure comprises:

[0023] (1) the amino acid sequence represented by SEQ ID NO: 26, or

[0024] (2) An amino acid sequence obtained by substituting, deleting, inserting and / or adding one or more amino acids to the amino acid sequence of (1).

[0025] [7-2] The artificial receptor according to any one of [1] to [7-1], comprising an extracellular hinge domain consisting of a sequence of 29 or less amino acids.

[0026] [8-1] The artificial receptor according to any one of [1] to [7-2], which does not have a cleavage site for γ-secretase in the transmembrane domain.

[0027] [8-2] The artificial receptor according to any one of [1] to [8-1], which does not have a cleavage site for γ-secretase in the intracellular domain.

[0028] [9-1] The artificial receptor according to any one of [1] to [8-2], which has two or more identical signal transduction domains.

[0029] [9-2] The artificial receptor according to [9-1], wherein the signal transduction domain is the signal transduction domain of CD28.

[0030] [10-1] A nucleic acid encoding the artificial receptor according to any one of [1] to [9-2].

[0031] [10-2] A vector comprising the nucleic acid according to [10-1].

[0032] [11-1] An immune cell comprising the artificial receptor according to any one of [1] to [9-2], the nucleic acid according to [10-1], or the vector according to [10-2].

[0033] [11-2] The immune cell according to [11-1], wherein the immune cell is selected from T cells, natural killer cells and macrophages.

[0034] [12-1] A drug comprising the immune cell according to [11-1] or [11-2].

[0035] [12-2] The drug according to [12-1], which is used for the treatment or prevention of cancer.

[0036]

[13] A method for designing a cytotoxic receptor or a nucleic acid encoding the receptor, comprising:

[0037] (1) a step of selecting a receptor comprising a ligand binding site, a transmembrane domain, and a signal transduction domain into immune cells, and

[0038] (2A) a step of regulating the sensitivity of the transmembrane domain selected in step (1) to shedding, and / or

[0039] (2B) A step of adding, inserting and / or deleting one or more amino acid residues and / or substituting one or more amino acid residues in the signal transduction domain of the receptor prepared in step (1).

[0040]

[14] A method for producing a cytotoxic receptor or a nucleic acid encoding the receptor, comprising:

[0041] (1) a step of preparing a receptor comprising a ligand binding site, a transmembrane domain, and a signal transduction domain into immune cells, and

[0042] (2A) a step of regulating the sensitivity of the transmembrane domain prepared in step (1) to shedding, and / or

[0043] (2B) A step of adding, inserting and / or deleting one or more amino acid residues and / or substituting one or more amino acid residues in the signal transduction domain of the receptor prepared in step (1).

[0044] [15-1] The method according to

[13] or

[14] , wherein the receptor selected or prepared in step (1) is selected from a chimeric antigen receptor, a T cell receptor, an Fc receptor and a tyrosine kinase receptor.

[0045] [15-2] The method according to any one of

[13] to [15-1], wherein the immune cell is selected from T cells, natural killer cells and macrophages.

[0046]

[16] A method for treating or preventing cancer in a mammal, characterized in that an effective amount of the immune cells according to [11-1] or [11-2] or the drug according to [12-1] or [12-2] is administered to the mammal.

[0047]

[17] The immune cell according to [11-1] or [11-2] or the drug according to [12-1] or [12-2], which is used for the treatment or prevention of cancer.

[0048]

[18] Use of the immune cell according to [11-1] or [11-2] or the drug according to [12-1] or [12-2] for the manufacture of a therapeutic or preventive drug for cancer.

[0049] Effects of the Invention

[0050] By utilizing the present invention, the cytophagy of immune cells can be prevented, receptors such as CAR or TCR can be made to maintain reactivity for a long time, and the effect of existing T cell therapy can be greatly increased. In addition, by changing the transmembrane domain or intracellular domain of the receptor, cytophagy can also be controlled. In addition, the insights of the present invention can also be useful in clarifying the partial mechanism of the common physiological phenomenon of the role of cytophagy in vivo or the reason why tyrosine kinase receptors become structures that are shedding (shedding). BRIEF DESCRIPTION OF THE DRAWINGS

[0051] [ Figure 1 ] CAR with a shedding structure (cleavable-CAR, Cleavable-CAR) tends to maintain reactivity for a long time and is more effective even in vivo. a: Structure of cleavable-CAR construct and control (normal-CAR) construct. The transmembrane domain (TM) of Notch1 protein was used as a shedding structure (also called "cleavable structure"), and a CAR with a general CD8 TM (normal-CAR) was used as a control. b: Various CAR constructs were introduced into NFAT-Luc jurkat (Promega) at 4×10 5After 1:1 co-culture of NALM6 cells for 3 hours (3h), fluorescein was added and the luminescence intensity was measured. After 3h, 28bbz (a CAR constructed by connecting the ScFv (CD19 ScFv) for CD19 (recognizing CD19) with the signal transduction domain of CD28, the signal transduction domain of 4-1BB and the signal transduction domain of CD3ζ chain) and 28z (a CAR constructed by connecting the signal transduction domain of CD19 ScFv with the signal transduction domain of CD28 and the signal transduction domain of CD3ζ chain) showed the same degree of reactivity, and bbz (a CAR constructed by connecting the signal transduction domain of CD19 ScFv with the signal transduction domain of 4-1BB and the signal transduction domain of CD3ζ chain) showed a slight decrease in reactivity. c: In the same experimental system as b, co-culture was continued for 4 days, and the luminescence intensity was measured. It was confirmed that after long-term culture, regardless of the intracellular domain (ICD), the reactivity of the cleavable-CAR was higher than that of the normal CAR. d, e, f: 5×10 5 NALM6 cells were intravenously administered at 2×10 on days 4 and 10 5 Survival curves of peripheral blood CD8 cells after various CARs were introduced. d is a comparison of 28bbz and 28bbz-cleavable, e is a comparison of 28z and 28z-cleavable, and f is a comparison of bbz and bbz-cleavable. No significant difference was found in bbz, but a significant survival extension was clearly found in 28z and 28bbz-cleavable-CAR.

[0052] [ Figure 2] Cleavable-CAR inhibits cytotoxicity. ab: Cytotoxicity assay of NALM6 by PBMCs introduced with various CARs. The effector / target ratio was changed, CAR-T and NALM6 were co-cultured, and the analysis was performed according to the protocol of the cytotoxicity assay kit (TECHNO SUZUTA). No significant differences were found. c: NALM6 cells and jurkata cells introduced with CAR were co-cultured at a 1:1 ratio overnight, and the CD19 expression level of NALM6 was analyzed by flow cytometry. It was confirmed that in cleavable-CAR, the reduction of CD19 (cytotoxicity) was significantly inhibited compared with normal-CAR. d, e: A Hibit-tag (Promega) was added to the front end of the CAR of Jurkat cells into which 28bbz-transfected normal-CAR or cleavable-CAR was introduced, and after co-culture with NALM6, the cells were fixed, stained with mouse anti-Hibit mAb (Promega), AlexaFluor647 goat anti-mouse IgG (Invitrogen), and CD19-FITC antibody, and imaged and analyzed using image-Stream (MKII). (d) and a schematic diagram (e) of NALM6. In normal-CAR, a decrease in CD19-FITC and CAR uptake into cells were found, but in cleavable-CAR, CD19 was not reduced, confirming that CAR was present on the cell membrane surface.

[0053] [ Figure 3]Cleavage of the extracellular domain is important for inhibiting cytotoxicity and maintaining signals for a long time. a: Results of FACS analysis of CD19 expression in NALM6 after co-culturing Jurkat cells expressing cleavable-CAR (28bbz) or normal-CAR with NALM6 cells. If the ADAM inhibitor GI254023X, which induces cleavage of the extracellular domain, is added at various concentrations, there is no change at all in normal-CAR, but in co-culture with cleavable-CAR, a concentration-dependent decrease in CD19 expression was confirmed. It was confirmed that even in cleavable-CAR, cytotoxicity can be caused by inhibiting ADAM. b: Cytotoxicity levels when ADAM or γ-secretase inhibitors are added to the co-culture medium of Jurkat expressing cleavable-CAR (28bbz) and NALM6. Although either inhibition is likely to cause cytotoxicity, ADAM has a stronger tendency. c: After cells expressing cleavable-CAR (28bbz) were co-cultured with NALM6 at a 1:1 ratio for 4 days under the addition of various inhibitors, luciferin was added to NFAT jurkat for luminescence detection. In the case of ADAM inhibitors, a decrease in the signal was found, but in the case of γ-secretase inhibitors, no decrease was found. Although there was no significant difference, there is also the possibility of signal enhancement. d: Cytokinesis of cleavable-CAR using TM of amyloid precursor protein (APP). It was confirmed that as long as it is a shedding structure, it is not limited to Notch and inhibits cytokinesis. e: It shows that if a mutation that enhances the shedding of the extracellular domain of APP (Swe mutation (APP695 K595N / M596L APP double mutant)) is used, cytokinesis is further inhibited. It was confirmed that the degree of shedding controls the level of cytokinesis. f: After co-culturing a cell line expressing CD19, Raji and a normal or cleavable 28bbz CAR, the results of analyzing the expression of CD19 in Raji cells. In the case of cleavable-CAR, inhibition of cytotoxicity was confirmed even in Raji cells.

[0054] [ Figure 4] The level of cell bites may vary due to the size of the intracellular domain of the receptor. In order to investigate what factors are important to the degree of cell bites, first, jurkat expressing CAR without CD3ζ (only bb (CAR constructed by connecting the signal transduction domain of CD19 ScFv to 4-1BB) or 28bb (CAR constructed by connecting the signal transduction domain of CD19 ScFv to the signal transduction domain of CD28 and the signal transduction domain of 4-1BB)) was co-cultured with NALM6 to analyze the CD19 expression of NALM6. Surprisingly, cell bites were found even without CD3ζ. In addition, the intensity of cell bites was 28bb>28. Rather than an increase in signals, it is better to think that the length or weight of the intracellular domain is important for cell bites. bbGFP and 28bbGFP with GFP added to the C-terminus were made, and co-culture was performed in the same way. The results confirmed that cell bites were significantly enhanced compared with bb and 28bb.

[0055] [ Figure 5 ]A CAR in which a portion of the EGFP protein is added to the intracellular domain is made under CD19ScFv-CD8TM, and the average CD19 expression level of NALM6 after co-culture with NALM6 is measured. Since EGFP obviously does not have a signal transduction function, it is shown that even without a signal transduction domain, it can cause cell bites. In addition, compared with a CAR that adds a portion of EGFP, about 1 / 3 of the total length (EGFP267) or about 2 / 3 of the total length (EGFP510), it is confirmed that cell bites are enhanced by length, and cell bites are easily caused by the size of the intracellular domain. In addition, if the same length of the signal-free domain (EGFP267) and the tyrosine kinase domain (28bb) are compared, it is clear that the tyrosine kinase domain causes stronger cell bites, thereby suggesting that the signal transduction domain can further enhance the traction force even if it is the same size.

[0056] [ Figure 6 ] If the signaling domains of the same size are arranged near the membrane where the cleavage site exists, as in 2828 (the signaling domains of CD28 are connected in series), the cytotoxicity can be more strongly inhibited. It is believed that the stronger inhibition of cytotoxicity is due to the increase in the traction force during signal transduction. In this way, it is known that in a CAR with a shedding structure, if the configuration or number of the intracellular domains is changed, the cytotoxicity level can be controlled.

[0057] [ Figure 7 In the cleavable sequence from mouse notch1, by tandemly connecting the CD28 intracellular signaling domain (cleavable-28bbz), it was found that the anti-tumor activity in vivo was significantly enhanced compared with the sequence with only one CD28 intracellular signaling domain (cleavable-2828z).

[0058] [ Figure 8 ]NALM6 CD19 expression results after co-culturing NALM6 cells with Jurkat cells expressing CAR. If 19-cleavable 2828z (cleavable-2828z with CD19 ScFv) and 19-cleavable mutant 2828z (a sequence that is not cleaved by γ-secretase by introducing a deletion mutation (the region of amino acid residues 329 to 335 of sequence number 28) into 19-cleavable 2828z) are compared, the result is that the mutant further inhibits cell cleavage. It is believed that the effect of inhibiting cell cleavage is due to the cleavage of the extracellular domain.

[0059] [ Fig. 9 ] It was found that in the same cell-binding detection system, even in a CAR using the TM of human CD28 and its upper 12 amino acids (amino acid residues 141 to 152 of CD28) at the TM site, cell-binding was inhibited by connecting the signaling domain in series. It is speculated that CD28 is also a shedding protein.

[0060] [ Fig.10 ]Jurkat cells express TRBV12 as a T cell receptor. NFAT-Jurkat cells were co-cultured with various CAR-T cells integrated with ScFv for TRBV12 (aTRBV12 ScFv), and the TCR signal intensity on the NFAT-jurkat side was detected. A tendency for signal reduction was confirmed using CAR with hCleavable-2828z CAR. This suggests that by forming a cleavable sequence in the receptor, it may lead to escape from immune cells, etc. UT refers to a control sample co-cultured with cells that do not express CAR.

[0061] [ Fig.11 ] The results of the measurement of the tandem effect of a cleavable-CAR and a signaling domain using ScFV (Her2 ScFV) for Her2. FACS analysis was performed on the Her2 expression on the skOV3 side after skOV3, a tumor strain expressing Her2, was co-cultured with various Her2-CARs overnight. It shows that even in Her2-CAR, if the number of signaling domains is increased when hCleavable is used, cell necrosis can be inhibited. The skOV3 on the left side of the figure represents the control Her2 expression level when co-cultured with cells without CAR.

[0062] [ Fig.12] Results of a study on the length of human Notch1 protein. The structure of Notch1 is shown in the figure. A CAR (respectively 19hL31447 2828z, 19hL314482828z, 19hL31530 2828z, 19hL31560 2828z) was prepared with the amino acid residues at positions 1447, 1488, 1530 or 1560 of human Notch1 (NCBI accession number: NP_060087.3) as the first amino acid residue of the transmembrane domain with a shedding structure, and the cells obtained by introducing it into NFAT-Jurkat were compared with the cells obtained by co-culture with control cells or NALM6 to confirm the reactivity. The CAR at position 1447, which completely contains LNR, shows the same strong reactivity as hCleavable CAR. In addition, if LNR is deleted, the reactivity is reduced, but a significant reaction is found compared to the control. Therefore, it is considered that the reactivity can be controlled by changing the length of the Notch1 sequence used. The control (cont) indicates the luminescence intensity without co-culture. DETAILED DESCRIPTION

[0063] 1. Artificial receptors containing transmembrane domains with shedding structures

[0064] The present invention provides an artificial receptor comprising a transmembrane domain having a shedding structure (also referred to as a "transmembrane region" or "transmembrane site"). More specifically, an artificial receptor comprising a ligand binding site, a transmembrane domain having a shedding structure, and a signal transduction domain into immune cells is provided (hereinafter sometimes referred to as the "receptor of the present invention"). In addition, the receptor of the present invention generally comprises an extracellular hinge domain that connects the ligand binding site and the transmembrane domain. By having a shedding structure, the receptor of the present invention can inhibit cell erosion with cells expressing a ligand recognized by the receptor, compared to receptors that do not have the structure. Cell erosion is mainly a phenomenon in which a cell produced in immune cells gnaws off proteins located on other cells together with the membrane.

[0065] In this specification, "receptor" means a construct comprising a ligand binding site, a transmembrane domain and an intracellular domain, and "artificial receptor" means a receptor other than a natural receptor, in other words, a receptor that does not exist in a mammalian organism unless exogenously introduced. As a specific receptor, for example, chimeric antigen receptors, T cell receptors, Fc receptors, tyrosine kinase receptors, etc. can be listed, but are not limited thereto. Receptors have receptors that function as monomers in vivo (at least with ligand binding ability) (for example: CAR, etc.) or receptors that function as multimers of dimers or above (for example: TCR, etc.), and the receptors in this specification can be each constituent element of a receptor that functions as a multimer, generally referring to a form that functions in vivo. For example, a TCR receptor generally refers to a dimer composed of an α chain β chain or a dimer composed of a γ chain δ chain, or a complex formed by further binding CD3 on these dimers.

[0066] The ligand binding site of the receptor is not particularly limited as long as it constitutes a part of the extracellular domain and has the ability to bind to the ligand. For example, the antigen binding site of an antibody, a single-chain variable region fragment (scFv) formed by connecting the heavy chain variable region (VH) and the light chain variable region (VL) constituting the antigen binding site with a linker peptide, the antigen binding site of a T cell receptor (TCR), the ligand binding site of a tyrosine kinase receptor other than TCR, the immunoglobulin Fc binding site of an Fc receptor, etc. The above-mentioned ligand binding site, for example, preferably does not show the ability to bind to the above-mentioned artificial receptor or the cell expressing the above-mentioned artificial receptor. That is, the above-mentioned artificial receptor preferably does not contain a ligand of the above-mentioned ligand binding site inside. The above-mentioned artificial receptor may have one or more of the above-mentioned ligand binding sites.

[0067] The ligand is usually a cell surface antigen of a cancer cell, but is not limited thereto. Examples of the ligand include BCMA, B7-H3, B7-H6, CD7, CD10, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD34, CD38, CD41, CD44, CD56, CD70, CD74, CD97, CD123, CD133, CD138, CD171, CD248, CAIX, CEA, c-Met, CS1 (CD319), CSPG4, CLDN6, CLD18A2, CYP1B1, DNAM-1, GD2, GD3, GM2, GFRα4, GPC3, GPR20, GPRC5D, globoH, Gp100, GPR20, GPRC5D, EGFR, EGFR variants, EpCAM, EGP2, EGP40, FAP, FITC, HER2, HER3, and HPV. E6, HPV E7, hTERT, IgGκ chain, IL-11Ra, IL-13Ra2, KIT, Lewis A, Lewis Y, Legumain, LMP1, LMP2, Ly6k, LICAM, MAD-CT-1, MAD-CT-2, MAGE-A1, MUC1, MUC16, NA-17, NY-BR-1, NY-ESO-1, O-acetyl-GD2, h5T4, PANX3, PDGRFb, PLAC1, polysialic acid, PSCA, PSMA, RAGE1, ROR1, sLe, SSEA-4, TARP, TAG-72, TEM7R, Tn antigen, TRAIL receptor, TRP2, TSHR, α-fetoprotein, mesothelin, folate receptor α (FRα), folate receptor β (FRβ), FBP, UPK2, VEGF-R2, WT-1, TCR (e.g., TRBV12, etc.), etc.

[0068] The transmembrane domain of a receptor means a protein domain that penetrates the cell membrane. In this specification, a transmembrane domain refers to a region that penetrates the cell membrane, but a region including a portion of the extracellular domain or a portion of the intracellular domain is sometimes referred to as a transmembrane domain. The transmembrane domain usually takes an α-helical topology, but it can also be a β-barrel or other structural domain. In addition, a "transmembrane domain with a shedding structure" means a transmembrane domain with a region that is cut by a cell-endogenous shedding enzyme (sheddase) in the extracellular domain region (in other words, near the membrane of the extracellular domain) or in the transmembrane domain. The transmembrane domain with a shedding structure usually includes at least a portion of the extracellular hinge domain and the transmembrane domain. The shedding enzyme is a membrane-bound protein that has the ability to cut a portion of the transmembrane protein with a shedding structure. In the case where the receptor of the present invention has an extracellular hinge domain (hereinafter sometimes referred to as a "hinge domain"), the region cut by the shedding enzyme may be present in the hinge domain, may be present in the transmembrane domain, or may be present in a position other than them. In addition, the extracellular hinge domain refers to the region between the ligand binding site and the transmembrane domain.

[0069] The shedding structure is a structure found in the transmembrane domain of many tyrosine kinase receptors. As shown in the following examples, it is shown that no matter what the type of shedding structure is, as long as it is a structure cut by the shedding enzyme in the body, it can inhibit cell phagocytosis. It is inferred that this is because the adhesion of the receptor to its ligand is inhibited by shedding, thereby inhibiting cell phagocytosis. Therefore, the shedding structure used in the present invention can be any structure as long as it can be cut by the shedding enzyme in the body. As proteins having a shedding structure, for example, notch proteins (specifically, notch1 protein, notch2 protein, notch3 protein, notch4 protein) (SEQ ID NOs. 2 and 1 represent the amino acid sequence of the transmembrane domain having a shedding structure of mouse notch1 and the base sequence encoding the domain, respectively. In addition, SEQ ID NOs. 28 and 27 represent the amino acid sequence of the transmembrane domain having a shedding structure of human notch1 and the base sequence encoding the domain, respectively. In addition, for the amino acid sequence represented by SEQ ID NO. 28, SEQ ID NOs. 33 to 36 represent amino acid sequences in which a part of the amino acid sequence is deleted.), amyloid precursor protein (SEQ ID NOs. 4 and 3 represent the amino acid sequence of the transmembrane domain having a shedding structure and the base sequence encoding the domain, respectively), CD28 (SEQ ID NOs. 22 and 21 represent the amino acid sequence of the transmembrane domain having a shedding structure and the base sequence encoding the domain, respectively), ALCAM (activated leukocyte cell adhesion molecule, activated leukocyte cell adhesion molecule The transmembrane domains derived from these proteins may be wild-type transmembrane domains (e.g., transmembrane domains comprising a sequence represented by any one of SEQ ID NOs: 2, 4, 22, 28, 33 to 36), or mutant transmembrane domains comprising an amino acid sequence that is highly similar or identical to the wild-type amino acid sequence and that can be cleaved by a sheddase. The receptor of the present invention can be designed and produced, for example, by replacing the transmembrane domain of a receptor that does not have a shedding structure (for example, the transmembrane domain of CD8 (the amino acid sequence and the base sequence encoding the domain are represented by SEQ ID NOs. 6 and 5, respectively) with a transmembrane domain having a shedding structure, or by replacing the extracellular domain side of the transmembrane domain or its vicinity (for example, within the hinge domain) with a corresponding region of the transmembrane domain having a shedding structure.In addition, the receptor of the present invention can also be designed and produced by replacing the transmembrane domain of a receptor having a shedding structure with a transmembrane domain having a different shedding structure with higher sensitivity to shedding, introducing a mutation with higher sensitivity to shedding (e.g., Swe mutation of APP, etc.) into the transmembrane domain, etc. The amino acid sequence of the transmembrane domain from the Swe mutant of APP and the base sequence encoding the domain are represented by SEQ ID NOs: 8 and 7, respectively.

[0070] Most of the shedding enzymes that have the ability to cut the above-mentioned shedding structure belong to the family of ADAM (a disintegrin and metalloproteinase) or BACE (beta-site amyloid precursor protein cleaving enzyme). As the shedding enzymes belonging to the ADAM family, for example, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAMDEC1 can be listed, and as the shedding enzymes belonging to the BACE family, for example, BACE1 can be listed.

[0071] The transmembrane domain of the wild-type notch protein has an S2 cleavage site cut by ADAM and an S3 cleavage site and an S4 cleavage site cut by γ-secretase. The intracellular domain of the notch protein is released into the cytoplasm by cleavage of the S3 and S4 cleavage sites by γ-secretase, and then migrates to the nucleus. From the viewpoint of inhibiting cell chewing, in the receptor of the present invention, since the release of the intracellular domain into the cytoplasm cannot be said to be necessary, the receptor of the present invention may be a receptor having a release function of the intracellular domain into the cytoplasm, or a receptor without a release function. In the case of notch protein, the release function of such an intracellular domain can introduce a mutation into the S3 cleavage site of the transmembrane domain to inhibit the cleavage of γ-secretase, and can further introduce a mutation into the S4 cleavage site. Therefore, the receptor of the present invention may be a receptor without a cleavage site of the transmembrane domain γ-secretase, and may also be a receptor without a cleavage site of the γ-secretase in the intracellular domain. In one embodiment, the transmembrane domain of the wild-type notch protein is removed from the transmembrane domain of the receptor of the present invention.

[0072] In the present specification, an amino acid sequence with high similarity or identity to a specific amino acid sequence means an amino acid sequence with 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity or identity to the specific amino acid sequence, or an amino acid sequence in which one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids are substituted, deleted, inserted, and / or added.

[0073] The intracellular domain of a receptor is a domain present in the cell when the receptor is fixed to the cell membrane by a transmembrane domain, whereas the domain exposed outside the cell is called an extracellular domain. The intracellular domain usually includes a signal transduction domain. As shown in the following examples, since cytokinesis is also shown when a receptor without a signal transduction domain is used, the intracellular domain may not include a signal transduction domain.

[0074] The signal transduction domain is not particularly limited as long as it can perform signal transduction into immune cells, and examples thereof include those selected from MHC class I molecules, TNF receptors, immunoglobulin-like proteins, cytokine receptors, integrins, activated NK cell receptors, Toll-like receptors, B7-H3, BAFFR, BTLA, BY55CD160), CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD4, CD5, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD22, CD27, CD28, CD29, CD 30, CD40, CD49a, CD49D, CD49f, D66d, CD69, CD79a, CD79b, CD84, CD96 (Touch), CD103, 4-1BB (CD137), CDS, CEACAM1, CRTAM, CNAM1 (CD226), DAP10, Fc receptor β chain, Fc receptor γ chain, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS (CD278), IL2Rβ, IL 2Rγ, IL7Rα, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, Ly9(CD229), LAT, L FA-1(CD11a / CD18), LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX40, PAG / Cbp, PSGL1, SEL PLG (CD162), SEMA4D (CD100), SLAM (SLAMF1, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, TNFR2, TRANCE / RANKL, VLA1 and VLA-6, one or more intracellular regions of proteins (these examples are sometimes referred to as examples of typical signal transduction domains). Alternatively, a mutant signal transduction domain comprising an amino acid sequence with high similarity or identity to the natural amino acid sequence of the above-mentioned signal transduction domain can also be used. In one embodiment of the present invention, the signal transduction domain comprises a signal transduction domain of at least one protein selected from CD3ζ chain, CD28 and 4-1BB, preferably a signal transduction domain of two proteins (for example: the signal transduction domain of CD28 and CD3ζ chain), and more preferably the signal transduction domain of all proteins.Sequence numbers 10 and 9 represent the amino acid sequence of the signal transduction domain of the CD3ζ chain and the base sequence encoding the domain, respectively, sequence numbers 12 and 11 represent the amino acid sequence of the signal transduction domain of CD28 and the base sequence encoding the domain, respectively, and sequence numbers 14 and 13 represent the amino acid sequence of the signal transduction domain of 4-1BB and the base sequence encoding the domain, respectively.

[0075] T cells are activated by intracellular signals via TCR (primary activation), and then the above-mentioned activation is enhanced by intracellular signals via costimulatory molecules (secondary activation). Therefore, in the case where the above-mentioned immune cells are T cells, the above-mentioned signal transduction domain is preferably used in combination to induce the above-mentioned primary activation signal transduction domain and the signal transduction domain induced by the above-mentioned secondary activation. The signal transduction domain induced by the above-mentioned primary activation can list the signal transduction domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), as a specific example, CD3γ, CD3δ, CD3ε, CD3ζ, Fc receptor β chain, Fc receptor γ chain, CD5, CD22, CD66d, CD79a, CD79b, etc., preferably CD3ζ. As the signal transduction domain inducing the above-mentioned secondary activation, for example, the signal transduction domain other than the signal transduction domain listed as the signal transduction domain inducing the above-mentioned primary activation in the example of a typical signal transduction domain can be listed. When the signal transduction domain that induces one activation and the signal transduction domain that induces the second activation are used in combination, the signal transduction domain comprises one or more signal transduction domains that induce one activation and one or more signal transduction domains that induce the second activation, and in a preferred embodiment, comprises one signal transduction domain that induces one activation and multiple signal transduction domains that induce the second activation. In any one of the signal transduction domain that induces one activation and the signal transduction domain that induces the second activation, a mutant signal transduction domain may also be used.

[0076] In addition, as shown in the following examples, a CAR having an intracellular domain formed by connecting two identical (same) signal transduction domains in series (tandem) shows higher anti-tumor activity than a CAR having one signal transduction domain. Therefore, the receptor of the present invention may have more than two (e.g., 2, 3, 4, 5 or more) identical signal transduction domains (e.g., CD28 signal transduction domain or 4-1BB). In the case where the receptor of the present invention has more than two intracellular domains, each intracellular domain may be directly connected to each other or connected via a linker such as a linker peptide.

[0077] In this specification, "similarity" means the ratio (%) of identical amino acids and similar amino acids to all overlapping amino acid residues in the optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can consider introducing gaps into one or both sequences for optimal alignment). "Similar amino acids" means amino acids that are similar in physicochemical properties, for example, aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met) and the like are listed as amino acids classified as the same group. It is expected that substitution of such similar amino acids will not change the expression type of the protein (i.e., conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various references (e.g., see Bowie et al., Science, 247: 1306-1310 (1990)). The similarity or identity of the amino acid sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10, allowed interval, matrix = BLOSUM62, filter = OFF).

[0078] Signal transduction domain is a domain with a signal transduction function into a cell, particularly an immune cell. "Immune cell" used in the present invention is not particularly limited as long as it is a cell (so-called immune effector cell) that has the ability to damage target cells such as cancer cells through a certain mechanism of action, for example, T cells responsible for cellular immunity in acquired immunity or NK cells, monocytes, macrophages, dendritic cells, B cells, etc. responsible for natural immunity. Among them, T cells, natural killer cells, macrophages are preferred, and T cells are particularly preferred. In this specification, "T cell" means CD3 positive cells, for example, cytotoxic T cells (CTL) as CD8 positive cells can be listed, helper T cells, regulatory T cells, effector T cells as CD4 positive cells, most of the cells are CD8 and CD4 negative γδT cells, etc., preferably cytotoxic T cells and helper T cells.

[0079] In this specification, "modified receptors" such as modified T cell receptors, modified Fc receptors, and modified tyrosine kinase receptors refer to receptors in which one or more amino acids constituting natural receptors or known artificial receptors are deleted, replaced, inserted, and / or added. The site of modification is not particularly limited, and the modification is usually performed in the transmembrane domain or its vicinity (e.g., in the hinge domain).

[0080] In this specification, "chimeric antigen receptor (CAR)" means an artificially constructed hybrid protein comprising an antigen binding domain (e.g., scFv) of an antibody connected to a T cell signaling domain. As a feature of CAR, the ability to convert the specificity and reactivity of immune cells to a selected target in a non-MHC restricted manner using the antigen binding properties of monoclonal antibodies can be cited. Non-MHC restricted antigen recognition gives CAR-expressing immune cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, if CAR is expressed in T cells, CAR has the advantage of not dimerizing with endogenous TCR α and β chains.

[0081] The CAR of the present invention comprises an antigen binding domain, a transmembrane domain and a signal transduction domain of an antibody that can specifically recognize a surface antigen of a target cell (e.g., a cell surface antigen of a cancer cell, etc.). The CAR of the present invention may also comprise an extracellular hinge domain.

[0082] In the case where the receptor or CAR of the present invention has an extracellular hinge domain, the extracellular hinge domain is, for example, composed of 1 to 300 amino acids, 5 to 100 amino acids, and 10 to 70 amino acids. The extracellular hinge domain may also be composed of 6 to 29 amino acids. As the extracellular hinge domain possessed by CAR, for example, hinge domains from CD3, CD8, CD28, KIR2DS2 or IgG4, IgD or other immunoglobulins can be listed. In addition, it may also be a hinge domain from a protein identical to the transmembrane domain. Alternatively, a mutant hinge domain containing an amino acid sequence with high similarity or identity to the natural amino acid sequence of the above-mentioned hinge domain may also be used. In one embodiment of the present invention, the hinge domain is a hinge domain of IgG4. Sequence numbers 18 and 17 represent the amino acid sequence of the hinge domain of IgG4 and the base sequence encoding the domain, respectively.

[0083] In addition, the effect of inhibiting cell chewing can also be found in CARs that connect a part of the hinge domain of CD28 to the transmembrane domain. Therefore, as a hinge domain, the hinge domain of CD28 and a part thereof can be listed. The amino acid sequence of the hinge domain of CD28 and the base sequence encoding the domain are represented by sequence numbers 24 and 23, respectively. As a part of the hinge domain of CD28, for example, a hinge domain comprising a part (sequence number 26) consisting of amino acid residues at positions 19 to 30 in the amino acid sequence of sequence number 24 can be listed, or a mutant hinge domain comprising an amino acid sequence with high similarity or identity to the amino acid sequence of sequence number 26. A part of the hinge domain of these CD28 and its mutant are usually composed of less than 29 (for example: 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12) amino acids. In addition, it is preferred that the extracellular hinge domain and the transmembrane domain are derived from the same protein (for example, both are derived from CD28). Thus, in one embodiment of the present invention, the receptor of the present invention contains a sequence comprising the amino acid sequence of SEQ ID NO: 24 and the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence with high similarity or identity to the sequence. In addition, in another embodiment, the hinge domain of the receptor of the present invention does not include a hinge domain consisting of the amino acid sequence of SEQ ID NO: 24 and / or a region consisting of an amino acid sequence that is 80% or more identical to the sequence.

[0084] In this specification, "T cell receptor (TCR)" means a receptor composed of a dimer of TCR chains (α chain, β chain, γ chain, δ chain), which recognizes antigens or the antigen-HLA (human leukocyte antigen) (MHC, major histocompatibility complex) complex, thereby transducing stimulation signals to T cells. TCR is usually composed of a dimer of an α chain and a β chain or a γ chain and a δ chain. Various TCR chains are composed of a variable region and a constant region, and there are 3 complementary determining regions (CDR1, CDR2, CDR3) in the variable region.

[0085] The constant region of the TCR chain used in the present invention is preferably modified in the constant region of the natural TCR chain. As such modification, for example, the dimer expression efficiency generated by the disulfide bond between the TCR chains is enhanced by replacing the specific amino acid residues in the constant region of the natural TCR chain with cysteine ​​residues, but it is not limited thereto.

[0086] Fc receptors are receptors that have the ability to bind to the Fc region of antibodies. Examples of Fc receptors include Fcα receptors (e.g., FcαRI, etc.) that are specific receptors for IgA, Fcγ receptors (e.g., CD64 (FCGRI), CD32 (FCGRII), CD16 (FCGRIII), etc.) that are receptors for the Fc portion of IgG, Fcε receptors that bind to IgE (e.g., FcεRI, FcεRII, etc.), neonatal Fc receptors that bind to maternal IgG, Fc receptor-like proteins, and Fcα / μ receptors that bind to IgM and IgA. Among them, Fcγ receptors are preferred.

[0087] Tyrosine kinase receptors are also called receptor tyrosine kinases. They have tyrosine kinase activity and are cell surface receptors with high affinity for many polypeptide growth factors, cytokines, and hormones. Although TCR is also a tyrosine kinase receptor, in this specification, unless otherwise specified, tyrosine kinase receptors refer to tyrosine kinase receptors other than TCR. As a family of tyrosine kinase receptors, for example, the EGF receptor family (for example: ErbB1, ErbB2, ErbB3, ErbB4, etc.), the insulin receptor family (for example: IR-A, IR-B, etc.), the PDGF receptor family (for example: PDGFR-αα, PDGFR-αβ, PDGFR-ββ, etc.), the VEGF receptor family (for example: VEGFR-1, VEGFR-2, VEGFR-3, etc.), the FGF receptor family (for example: FGFR1, FGFR2, FGFR3, FGFR4, FGFRL1, FGFR6, etc.), the CCK family, the GF receptor family , HGF receptor family (e.g., MET, etc.), Eph receptor family (e.g., EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, etc.), AXL family, TIE family, RYK family, DDR family, RET family (e.g., RET51, RET43, RET9, etc.), ROS family, LTK family, ROR family, MuSK family, LMR family, families other than the above, etc. Tyrosine kinase receptors may belong to any of these families. In addition, as tyrosine kinase receptors, CD3 and costimulatory molecules (e.g., CD28, 4-1BB, CD357, CD40, ICOS, OX40, etc.) may also be listed.

[0088] The source of the receptor used in the present invention is not particularly limited, and is preferably derived from mammals (eg, humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, dogs, cats, monkeys, etc.), with human receptors being more preferred.

[0089] The receptor of the present invention can be made by genetic engineering using the following nucleic acid or vector of the present invention. For example, in the case where the receptor is a monomer such as CAR, the nucleic acid encoding the receptor can be introduced into the cell, the receptor is expressed in the cell, and the receptor is separated to make it. In addition, in the case where the receptor is a dimer such as TCR, for example, a nucleic acid encoding one of the polypeptides constituting the receptor of the present invention and a nucleic acid encoding another polypeptide can be introduced into the cell, each polypeptide is expressed to form a dimer, and the dimer is separated by a method known to itself to make it. It is the same in the case where the receptor is a multimer of more than a trimer.

[0090] 2. Nucleic acid encoding the receptor of the present invention and a vector containing the nucleic acid

[0091] The present invention provides a nucleic acid encoding the receptor of the present invention (hereinafter sometimes referred to as "the nucleic acid of the present invention"). When the receptor is a dimer, as the nucleic acid of the present invention, a nucleic acid encoding one of the polypeptides constituting the receptor of the present invention and a nucleic acid encoding another polypeptide may be contained in different molecules, or a nucleic acid encoding two polypeptides may be contained in a single molecule. The same is true when the receptor is a multimer of trimers or higher.

[0092] The nucleic acid of the present invention can be DNA or RNA, or can also be a DNA / RNA chimera, preferably DNA. In addition, the nucleic acid can be double-stranded or single-stranded. In the case of double-stranded, it can be a double-stranded DNA, double-stranded RNA or a hybrid of DNA:RNA. In the case where the nucleic acid is RNA, for the sequence of RNA, the T in the base sequence is replaced and read as U. In addition, the nucleic acid of the present invention can contain natural nucleotides, modified nucleotides, nucleotide analogs or mixtures thereof as long as the polypeptide can be expressed in vitro or in cells.

[0093] Nucleic acid of the present invention can be made by known methods, for example, can be based on the known DNA sequence information of receptor, synthetic oligo DNA primers to cover the desired part of the sequence, using the total RNA or mRNA fraction prepared by the cell with the sequence as a template, amplified by RT-PCR method, thus cloned. Alternatively, the partially overlapping oligo DNA short chains synthesized can be connected by chemically synthesized DNA chains or by utilizing PCR method (overlapping PCR method) or Gibson Assembly method, thus constructing a DNA encoding its full length or a part. In addition, mutations can also be appropriately introduced in the sequence.

[0094] The nucleic acid of the present invention can be incorporated into an expression vector. Therefore, the present invention provides an expression vector comprising the nucleic acid of the present invention (hereinafter sometimes referred to as "the vector of the present invention").

[0095] Examples of promoters used in the vector of the present invention include ubiquitin promoter, EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. Among them, ubiquitin promoter, EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, and SRα promoter are preferred.

[0096] In addition to the above-mentioned promoter, the vector of the present invention may further contain transcription and translation regulatory sequences, ribosome binding sites, enhancers, replication origins, poly A signals, selectable marker genes, etc. as desired. Examples of selectable marker genes include dihydrofolate reductase genes, neomycin resistance genes, and puromycin resistance genes.

[0097] In the case where the receptor is a dimer, an expression vector comprising a nucleic acid encoding one of the polypeptides constituting the receptor of the present invention and a nucleic acid encoding another polypeptide can be introduced into the target cell to form a heterodimer of the two polypeptides in the cell or on the cell surface. In this case, the nucleic acid encoding one of the polypeptides constituting the receptor of the present invention and the nucleic acid encoding another polypeptide can be integrated into separate expression vectors or into one expression vector. In the case of being integrated into one expression vector, the two nucleic acids are preferably integrated via a sequence capable of multicistronic expression. By using a sequence capable of multicistronic expression, it is possible to more effectively express multiple genes integrated into one expression vector. As sequences capable of polycistronic expression, for example, 2A sequences (e.g., 2A sequences (F2A) derived from foot-and-mouth disease virus (FMDV), 2A sequences (E2A) derived from equine rhinitis A virus (ERAV), 2A sequences (P2A) derived from porcine teschovirus (PTV-1), 2A sequences (T2A) derived from tricholoma virus (TaV)) (PLoS ONE, 3: e2532, 2008, Stem Cells 25, 1707, 2007, etc.), internal ribosome entry site (IRES) (U.S. Patent No. 4,937,190), etc. are listed. From the viewpoint of uniform expression level, 2A sequences are preferred. In addition, among 2A sequences, P2A sequences and T2A sequences are preferred. The same is true when the receptor is a multimer of trimers or higher.

[0098] As expression vectors that can be used in the present invention, viral vectors, plasmid vectors, etc. can be listed. As viral vectors, retroviral vectors (including lentiviral vectors or pseudotype vectors), adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, Sendai virus, episomal vectors, etc. can be listed. In addition, transposon expression systems (e.g., PiggyBac systems) can also be used. As plasmid vectors, animal cell expression plasmids (e.g., pal-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo), etc. can be listed.

[0099] 3. Immune cells having the receptor of the present invention and drugs containing the same

[0100] The present invention provides immune cells having a nucleic acid or vector of the present invention (hereinafter sometimes referred to as "immune cells of the present invention") and drugs containing immune cells of the present invention (hereinafter sometimes referred to as "drugs of the present invention"). The immune cells of the present invention preferably express the receptors of the present invention on the cell surface. The immune cells of the present invention may express one receptor of the present invention, or may express two or more receptors of the present invention (e.g., a combination of CAR and TCR, etc.).

[0101] The immune cells or drugs of the present invention can be administered to mammals (e.g., humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cattle, dogs, cats, monkeys, etc.). Therefore, a method for treating or preventing cancer in a mammal is also provided, characterized in that the immune cells or drugs of the present invention are administered to the mammal. In addition, unless otherwise specified, the therapeutic or preventive drug for cancer also includes drugs that can treat and prevent the disease. The same is true for the therapeutic or preventive method for cancer.

[0102] In this specification, "therapeutic drugs" include not only drugs for the purpose of radical treatment of cancer, but also include, for example, drugs for the purpose of inhibiting the progression of cancer, drugs for the purpose of alleviating symptoms (for example, improving mild symptoms (minimal manifestations, MM) that do not affect life and work) or alleviating sequelae. For example, since cancer is a disease that progresses over a long period of time (usually in years), the progression of symptoms can be prevented by starting treatment at an early stage. In addition, in this specification, "preventive drugs" include not only drugs for the purpose of reducing the risk of cancer in subjects who have not developed cancer, but also drugs for the purpose of reducing the risk of cancer recurrence in subjects who have already developed cancer. The same is true for methods for treating or preventing cancer.

[0103] As immune cells having the nucleic acid or vector of the present invention, the same cells as those listed in the above "1. An artificial receptor comprising a transmembrane domain having a shedding structure" can be listed, preferably T cells, more preferably cytotoxic T cells. Immune cells expressing the receptor of the present invention can be obtained by introducing the nucleic acid or vector of the present invention into immune cells collected from an organism (e.g., peripheral blood T cells) or into immune cells differentiated and induced from precursor cells or pluripotent stem cells of the cells. Alternatively, the nucleic acid or vector of the present invention can be introduced into precursor cells or pluripotent stem cells of target immune cells, and differentiated into target immune cells by a differentiation induction method known per se.

[0104] Immune cells can be collected from peripheral blood, bone marrow, and umbilical cord blood of humans or non-human mammals. When immune cells expressing the receptor of the present invention are used for the treatment or prevention of cancer, the immune cells are preferably collected from the subject or a donor with the same HLA type as the subject.

[0105] Precursor cells of the above-mentioned immune cells include, for example, hematopoietic stem cells, lymphoid stem cells, multipotent precursor cells that have lost the ability to self-replicate (multipotent progenitor cells (multipotent progenitor: MMP), myeloid-lymphoid common precursor cells (MLP), myeloid precursor cells (MP), granulocyte-monocyte precursor cells (GMP), macrophage-dendritic cell precursor cells (MDP), dendritic cell precursor cells (DCP), pre-T cells (for example: DN1 cells, DN2 cells, DN3 cells, DN4 cells, DP cells, etc.), etc.

[0106] Examples of the pluripotent stem cells include embryonic stem cells (ES cells), artificial pluripotent stem cells (induced pluripotent stem cells: iPS cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), etc. When the pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be cells produced by destroying the embryo or cells produced without destroying the embryo, preferably cells produced without destroying the embryo.

[0107] In this specification, "pluripotent stem cells" refer to embryonic stem cells (ES cells) and cells that potentially have the same pluripotent differentiation as embryonic stem cells, that is, the ability to differentiate into various tissues of an organism (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotent differentiation as ES cells include "artificial pluripotent stem cells (iPS cells or iPSCs)".

[0108] ES cells are stem cells with pluripotency and self-replication-based proliferation ability established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans or mice. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and since then, ES cell lines have also been established in primates such as humans and monkeys (JA Tomson et al. (1998), Science 282: 1145-1147; JA Tomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Tomson et al. (1996), Biol. Reprod., 55: 254-259; JA Tomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165). ES cells can be established by removing the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere of an embryo in the cleavage stage before the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or using an embryo that has stopped producing (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

[0109] As the ES cell strain used in the present invention, any human ES cell strain may be used, and for example, various human ES cell strains established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Developmental Medicine, Cellartis, etc. can be used. Specifically, for example, as human ES cell strains, CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc. sold by ESI Bio, H1 strains, H9 strains, etc. sold by WiCell Research, KhES-1 strains, KhES-2 strains, KhES-3 strains, KhES-4 strains, KhES-5 strains, SSES1 strains, SSES2 strains, SSES3 strains, etc. sold by RIKEN can be cited.

[0110] iPS cells are cells obtained by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells and reprogramming them. Currently, there are many types of iPS cells, including human iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into human fibroblasts (Takahashi K, Yamanaka S. et al., Cell, (2007) 131: 861-872.), Nanog-iPSCs established by screening with Nanog expression as an index after the introduction of the above four factors (Okita, K., Ichisaka, T. and Yamanaka, S. (2007). Nature 448, 313-317.), iPSCs prepared by a method that does not include c-Myc (Nakagawa M, Yamanaka S. et al., Nature Biotechnology, (2008) 26, 101-106), and iPSCs established by introducing six factors by a virus-free method (Okita K et al., Nat. Methods 2011May;8(5):409-12, Okita K et al., Stem Cells.31(3):458-66.) etc. In addition, artificial pluripotent stem cells established by introducing four factors, namely OCT3 / 4, SOX2, NANOG and LIN28, prepared by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920.), artificial pluripotent stem cells prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), artificial pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Application Laid-Open No. 2008-307007), etc. can also be used.

[0111] In addition, all published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. Artificial pluripotent stem cells known in the art and described in, for example, Japanese Patent Application Publication No. 7,795-797) or patents (e.g., Japanese Patent Application Publication No. 2008-307007, Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0112] As artificial pluripotent stem cell strains, various iPSC strains established by NIH, RIKEN, Kyoto University, etc. can be used. For example, as long as they are human iPSC strains, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc. of RIKEN, 253G1 strain, 253G4 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, FfI-01s04 strain, etc. of Kyoto University can be listed, and 1231A3 strain is preferred.

[0113] In this specification, "hematopoietic precursor cell" means a pluripotent stem cell (multipotent stem cell) that can differentiate into blood cell lineage cells. In humans, it is mainly present in the bone marrow and differentiates into leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages), erythrocytes, platelets, mast cells, dendritic cells. In addition, in the present invention, "hematopoietic precursor cell" means CD34 positive cells, preferably CD34 / CD43 double positive (DP) cells. The source of the hematopoietic precursor cell used in the present invention is not limited, for example, it can be a hematopoietic precursor cell obtained by differentiating induced pluripotent stem cells using the following method, or it can be a hematopoietic precursor cell separated from a living body tissue by a known method.

[0114] As a method for differentiating pluripotent stem cells into immune cells such as T cells, for example, a method including (1) a step of differentiating pluripotent stem cells into hematopoietic progenitor cells introduced with the nucleic acid or vector of the present invention and (2) a step of differentiating the hematopoietic progenitor cells into immune cells can be cited. The above step (1) can be, for example, a method of culturing pluripotent stem cells in a culture medium induced into hematopoietic progenitor cells as described in International Publication No. 2013 / 075222, International Publication No. 2016 / 076415, and Liu S. et al., Cytotherapy, 17 (2015); 344-358, etc. In addition, for example, in the case where the immune cells are T cells, the above step (2) can be cited as a method including (2-1) a step of inducing CD4CD8 double positive T cells from hematopoietic progenitor cells and (2-2) a step of inducing CD8 positive T cells from CD4CD8 double positive T cells as described in International Publication No. 2016 / 076415, etc.

[0115] The culture medium for inducing hematopoietic progenitor cells is not particularly limited, and the culture medium for culturing animal cells can be prepared as a basal culture medium. As a basal culture medium, for example, Iscove's Modified Dulbecco's Medium (IMDM) culture medium, Medium 199 culture medium, Eagle's Minimum Essential Medium (EMEM) culture medium, αMEM culture medium, Dulbecco's modified Eagle's Medium (DMEM) culture medium, Ham's F12 culture medium, RPMI 1640 culture medium, Fischer's culture medium, Neurobasal culture medium (Life Technologies), mixed culture medium thereof, etc. can be listed. Serum can be contained in the culture medium, or it can be used in a serum-free form. As needed, the basal culture medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, etc.

[0116] The culture medium for inducing differentiation into CD4CD8 double positive T cells used in step (2-1) is not particularly limited, and the culture medium for culturing animal cells can be prepared as a basal culture medium. The basal culture medium can include the same basal culture medium as the basal culture medium used in the above step (1). The culture medium may contain serum, or it may be used in a serum-free form. As needed, the basal culture medium may contain, for example, vitamin C, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, etc.

[0117] As the basal medium and medium additive used in step (2-2), the same substances as the basal medium and medium additive described in step (1) can be listed. The above-mentioned medium may contain an adrenocortical hormone agent. As an adrenocortical hormone agent, for example, glucocorticoids and their derivatives can be listed, and as the glucocorticoid, for example, cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, beclomethasone propionate, etc. can be listed. Among them, dexamethasone is preferred.

[0118] The method for introducing the nucleic acid or vector of the present invention into cells is not particularly limited, and known methods can be used. In the case of introducing nucleic acids or plasmid vectors, for example, calcium phosphate coprecipitation, PEG method, electroporation, microinjection, lipofection, etc. can be used. For example, the methods described in Cell Engineering Special Volume 8 New Cell Engineering Experimental Program, 263-267 (1995) (published by Xiujunsha); Virology, 52, 456 (1973); Folia Pharmacol. Jpn., Vol. 119 (No. 6), 345-351 (2002), etc. can be used. In the case of using viral vectors, the nucleic acid of the present invention can be introduced into appropriate packaging cells (e.g., Plat-E cells) or complementary cell strains (e.g., 293 cells), the viral vector produced in the culture supernatant can be recovered, and the vector can be transfected into cells by an appropriate method suitable for each viral vector, thereby introducing it into the cell. For example, specific methods using retroviral vectors as vectors are disclosed in International Publication No. 2007 / 69666; Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007), etc. In addition, specific methods using lentivirus as vectors are disclosed in Zufferey R. et al., Nat Biotechnol, 15 (9): 871-895 (1997), etc. In particular, when retroviral vectors are used, by using CH-296 (manufactured by Takara Bio Co., Ltd.), which is a recombinant fibronectin fragment, efficient gene introduction into various cells becomes possible. Alternatively, the nucleic acid or vector of the present invention can also be introduced into the genome of the cell by genome editing (e.g., CRISPR / Cas system, TALEN, ZFN, etc.).

[0119] The nucleic acid of the present invention can also be directly introduced into cells in the form of RNA to express the receptor of the present invention in cells. As the method for introducing RNA, known methods can be used, for example, lipofection or electroporation can be preferably used.

[0120] The expression of the receptor of the present invention can be detected or measured by immunological methods, for example, using an antibody that recognizes a portion of the receptor of the present invention (e.g., a constant region of a TCR chain, etc.). Examples of immunological methods include antibody arrays, flow cytometry analysis, radioisotope immunoassay (RIA method), ELISA, protein blotting, immunohistostaining, enzyme immunoassay (EIA method), fluorescent immunoassay (FIA), and immunochromatography.

[0121] The present invention also provides a method for producing a cell comprising the step of introducing the nucleic acid or vector of the present invention into a cell. The cell and the method for introducing the nucleic acid or vector of the present invention are as described above. The above-mentioned cell preferably expresses the receptor of the present invention. The expression of the receptor of the present invention can be detected or measured by the above-mentioned method. In addition, in another embodiment, an immune cell obtained by the above-mentioned production method is also provided.

[0122] As the target of the drug of the present invention, as long as it is a cancer containing cancer cells expressing cancer antigens that the receptor of the present invention targets in the cancer tissue, all cancers can be the object. As such cancers, for example, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer (e.g., melanoma, Merkel cell carcinoma), breast cancer, prostate cancer, bladder cancer, vaginal cancer, neck cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, non-small cell lung cancer, tracheal cancer, bronchial cancer, colon cancer, rectal cancer, small intestine cancer, large intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, fallopian tube cancer, nasal cancer, Pharyngeal cancer and other cancers; cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue and hematopoietic tissue; sarcomas such as chondrosarcoma, Ewing's sarcoma, rhabdomyosarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, soft tissue sarcoma, etc.; blastomas such as hepatoblastoma, medulloblastoma, Wilms' tumor, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, etc.; germ cell tumors; lymphomas; leukemia, acute myeloid leukemia, multiple myeloma, etc.

[0123] As components other than immune cells that may be included in the drug of the present invention, for example, pharmaceutically acceptable additives may be listed, and more specifically, physiological saline, buffered physiological saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonic agents, fillers, lubricants, etc. may be listed.

[0124] Examples of methods for administering the immune cells or drugs of the present invention include injection into a tumor, into a vein, into an artery, into a muscle, subcutaneously, or into the peritoneum.

[0125] The amount of the immune cells of the present invention contained in the drug of the present invention can be appropriately adjusted according to the type of immune cells, purpose, dosage form, and target therapeutic effect, taking into account, for example, the type, location, severity of cancer, age, weight, and condition of the subject to be treated. For example, the drug of the present invention can be administered in a single administration, usually 1×10 4 ~1×10 10 1×10 5 ~1×10 9 , more preferably 5×106 ~5×10 8 The immune cells of the present invention are formulated into a single formulation.

[0126] The administration interval of the immune cells or drugs of the present invention is not particularly limited and can be appropriately adjusted while taking into account the amount of the immune cells of the present invention administered each time, for example, it can be administered independently 4 times, 3 times, 2 times or 1 time a day, every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, once a month or twice.

[0127] The immune cells or drugs of the present invention can be used in combination with known cancer therapeutic drugs. Examples of cancer therapeutic drugs include alkylating drugs such as cyclophosphamide, bendamustine, ifosfamide, and dacarbazine; metabolic antagonists such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine; molecular targeted drugs such as rituximab, cetuximab, and trastuzumab; kinase inhibitors such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib; proteasome inhibitors such as bortezomib; calcineurin inhibitors such as cyclosporine and tacrolimus; anticancer antibiotics such as idarubicin, doxorubicin, and mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum preparations such as cisplatin, oxaliplatin, and carboplatin; hormone therapy drugs such as tamoxifen and bicalutamide; immunosuppressants such as interferon, nivolumab, and pembrolizumab, and the like.

[0128] 4. Method for designing a receptor whose cytotoxicity is regulated or method for producing the receptor

[0129] As shown in the following examples, it is shown that the cytophagy of the receptor can be regulated by the presence or absence of a shedding structure or the shedding intensity of the receptor, the size of the intracellular domain and / or the presence or absence of a signal transduction domain. More specifically, as shown in the following examples, it was found that by introducing the shedding structure into a transmembrane domain that does not have a shedding structure, cytophagy is inhibited, and by further introducing a mutation that is more likely to produce shedding, the inhibitory effect of cytophagy is enhanced. In addition, it was found that the occurrence of cytophagy does not require the receptor to have a signal domain, the intensity of cytophagy varies depending on the size of the intracellular domain, and when the size is the same, the signal transduction domain causes cytophagy more strongly than the non-signaling domain.

[0130] Therefore, in another embodiment, the present invention provides a method for designing a cytotoxic receptor or a nucleic acid encoding the receptor (hereinafter sometimes referred to as "the design method of the present invention"), which comprises:

[0131] (1) a step of selecting a receptor comprising a ligand binding site, a transmembrane domain, and a signal transduction domain into immune cells, and

[0132] (2A) a step of regulating the sensitivity of the transmembrane domain selected in step (1) to shedding, and / or

[0133] (2B) A step of adding, inserting and / or deleting one or more amino acid residues and / or substituting one or more amino acid residues in the signal transduction domain of the receptor prepared in step (1).

[0134] In addition, a method for producing a cytotoxic receptor or a nucleic acid encoding the receptor (hereinafter sometimes referred to as "the production method of the present invention") is provided, which comprises:

[0135] (I) a step of preparing a receptor comprising a ligand binding site, a transmembrane domain, and a signal transduction domain into immune cells, and

[0136] (IIA) a step of regulating the sensitivity of the transmembrane domain prepared in step (I) to shedding, and / or

[0137] (IIB) A step of adding, inserting and / or deleting one or more amino acid residues, and / or replacing one or more amino acid residues in the signal transduction domain of the receptor prepared in step (I). Hereinafter, the term "the method of the present invention" may be used as a general term for both the design method of the present invention and the preparation method of the present invention.

[0138] In the present specification, "the cytokine activity is regulated" means that when a receptor (i.e., a designed or manufactured receptor) (hereinafter sometimes referred to as "the receptor after the implementation of the present invention") after implementing steps (2A), (2B), (IIA) and / or (IIB) of the method of the present invention is introduced into an immune cell, the intensity of cytokine activity changes (i.e., decreases or increases), or a change in the intensity of cytokine activity is expected, compared to the case where the receptor prepared in step (1) or (I) (hereinafter sometimes referred to as "the target receptor") is introduced into an immune cell.

[0139] The receptor prepared in step (1) or (I) of the method of the present invention is not particularly limited, and examples thereof include known CAR, TCR, Fc receptor, tyrosine kinase receptor, etc. The specific examples or structures of these receptors can all be cited in the above-mentioned "1. Artificial receptor comprising a transmembrane domain having a shedding structure".

[0140] In the case where the transmembrane domain of the target receptor does not have a shedding structure, step (2A) or (IIA) of the method of the present invention can be performed by replacing the transmembrane domain with a transmembrane domain having a shedding structure, or replacing the extracellular domain side of the transmembrane domain or its vicinity (e.g., in the hinge domain) with a corresponding region of a transmembrane domain having a shedding structure, etc. Alternatively, shedding can also be induced by adding a structure that can impart shedding ability to the transmembrane domain, such as the hinge domain of CD28, a portion thereof, or a mutant thereof. In addition, in the case where the transmembrane domain of the target receptor is shedding, it can be performed by replacing it with a transmembrane domain that does not have a shedding structure, deleting the hinge domain or replacing it with another hinge domain, replacing it with a transmembrane domain that has a shedding structure with a different sensitivity to shedding, and introducing a mutation with a different sensitivity to shedding (e.g., Swe mutation of APP, etc.) into the transmembrane domain, etc. In addition, it is reported that when about half of the negatively charged amino acid residues are present in the sequence near the extracellular domain side of the sequence cleaved by the shedding enzyme, or when there is a sugar chain modification, shedding can be inhibited (Iwagishi R et al., J Biol Chem. 295 (35): 12343-12352 (2020)). Therefore, for the sequence near the extracellular domain side of the sequence cleaved by the shedding enzyme, the sensitivity to shedding can also be adjusted by adjusting the charge or the degree of sugar modification.

[0141] Since the step (2B) or (IIB) of the method of the present invention maintains the signal transduction function even after such a change when adding, inserting and / or deleting amino acid residues in the signal transduction domain of the target receptor, amino acid addition is usually performed to make it a signal transduction domain containing an amino acid sequence with high similarity or identity to the amino acid sequence of the signal transduction domain before the change. In addition, in the following examples, it is shown that in a receptor containing a transmembrane structure with a shedding structure, cell mitosis is further inhibited by increasing the number of signal transduction domains. Therefore, the step (2B) or (IIB) of the method of the present invention can be a step of changing the order or number of intracellular domains of the target receptor. In the case of changing the number of intracellular domains, it is preferred to connect the same intracellular domains in series. In addition, in the following examples, even if EGFP (sequence number 20) with a length of about 240 amino acids is added to the signal transduction domain, cell mitosis is found to be enhanced. Therefore, when the signal transduction domain of the target receptor is replaced with a non-signaling domain, or when a non-signaling domain is further added to the signal transduction domain, the upper limit of the size of the replaced or added non-signaling domain is not particularly limited, and examples of the upper limit include 500 amino acids long, 400 amino acids long, 300 amino acids long, 250 amino acids long, 200 amino acids long, 150 amino acids long, 100 amino acids long, and the like.

[0142] In steps (2A) and (2B) of the design method of the present invention, it is not necessary to actually make the receptor, but it is sufficient to image it in the brain. However, the imaged receptor is usually concretized on a program (e.g., graphic design tools, office software, etc.) running on an electronic computer or on paper. Therefore, designing a receptor that anticipates changes in the intensity of cell chewing and organizing the behavior of the receptor in the form of a table is also equivalent to the implementation of the design method of the present invention. In addition, in steps (IIA) and (IIB) of the preparation method of the present invention, it is also not necessary to actually make the receptor, but it is sufficient to make a nucleic acid encoding the receptor designed in a manner that expresses a receptor that changes the shedding structure and / or changes the signal transduction domain in the cell.

[0143] For the target receptor, by implementing steps (2A), (2B), (IIA) and / or (IIB) of the method of the present invention, in theory, when introduced into cells, a receptor whose cytotoxicity is regulated compared to the target receptor is designed or manufactured. However, in practice, it can be confirmed that the cytotoxicity is regulated in the receptor after the implementation of the present invention, and the cytotoxicity intensity in the receptor after the implementation of the present invention can also be evaluated. The cytotoxicity intensity can be evaluated by the following method, for example, as described in the following examples, but is not limited thereto. Immune cells (1×10 5The target cells are co-cultured with the target cells in a 48-well plate at 37°C overnight at a ratio of 1:1, and the expression level of the ligand is measured by flow cytometry using an antibody against the ligand targeting the receptor. The expression levels of the ligands in each cell are compared, and if there is a difference, it can be evaluated that the cytotoxicity is regulated.

[0144] By introducing the receptor designed or produced by the method of the present invention or the nucleic acid encoding the receptor into a cell (e.g., cancer cell) that causes cytotoxicity with an immune cell or the cell, the cytotoxicity can also be controlled. For example, by replacing the nucleic acid encoding the receptor designed or produced by the method of the present invention with at least one (preferably all) of the nucleic acids encoding the target receptor inherent in the cell by genome editing, or inserting it into the genome of the cell, the cytotoxicity can also be controlled by replacing the inherent target receptor or acting competitively.

[0145] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0146] Example

[0147] <Materials and Methods>

[0148] Vector construction and virus production

[0149] Using the clinically used FCM63 CD19-specific scFv (sequence numbers 15 and 16), the transmembrane domain of CD8 (sequence numbers 5 and 6) is used as a normal-TM, the transmembrane domain of Notch1 (sequence numbers 1 and 2) or APP protein (a part of the extracellular domain of each protein) (sequence numbers 3 and 4 and sequence numbers 7 and 8) is used as a cleavable-TM, and the intracellular domain of CD28 (sequence numbers 11 and 12), the intracellular domain of 4-1BB (sequence numbers 13 and 14), the intracellular domain of CD3ζ chain (sequence numbers 9 and 10) or the intracellular domain composed of these intracellular domains is used as an intracellular domain, and a CAR vector is constructed by a general molecular biology method. FCM63 CD19-specific scFv is connected to the transmembrane domain of CD8 via the hinge domain of IgG4 (sequence numbers 17 and 18). On the other hand, FCM63 CD19-specific scFv and cleavable-TM are not connected via the hinge domain of IgG4. Using 293T cells, packaging vectors, and envelope vectors, viral fluid for gene introduction is produced. For other CARs, the same is made. Sequence numbers 21 and 22 represent the sequence of the transmembrane domain of CD28 containing a hinge domain (sequence numbers 25 and 26) consisting of 12 amino acid residues, sequence numbers 27 and 28 represent the sequence of hCleavable (a transmembrane domain containing a part of the extracellular domain of human Nothc1), sequence numbers 29 and 30 represent the sequence of aTRBV12ScFv, and sequence numbers 31 and 32 represent the sequence of Her2ScFV. In addition, sequence numbers 33 to 36 represent hCleavable of 19hL31447 2828z, 19hL31448 2828z, 19hL31530 2828z, and 19hL31560 2828z, respectively.

[0150] Detection of NFAT-Jurkat-based CAR signaling

[0151] Various CAR constructs were introduced into NFAT-Luc Jurkat cells (Promega J1621) using lentivirus to produce CAR-NFAT-Luc Jurkat cells. These cells were incubated with a cell line expressing the CD19 target (NALM6, Raji) at 5×10 4 After short-term (3 h) and long-term (4 days) co-culture, the recommended amount of luciferin (VivoGlo Promega) was added to each well and Nivo TM (PerkinElmer Japan) The activity intensity of NFAT signal was detected by luminescence.

[0152] T cell stimulation and gene transfer

[0153] Peripheral blood from healthy individuals was obtained according to an experimental plan approved by the Ethics Committee of Kyoto University. Peripheral blood T cells (PBMC) were isolated by using Leucosep TM (Greiner) centrifugal concentration gradient method for separation. Add the recommended amount of Dynabeads human T-activator CD3 / CD28 (VERITAS) to the separated PBMCs, and culture and stimulate with medium added with IL15 and IL-7 in CD15% FBS+a-MEM medium. Add lentivirus on the second and third days of stimulation to introduce various CAR genes. Remove Dynabeads on the third day of stimulation, and then add culture medium to proliferate CAR-PBMCs.

[0154] Mouse tumor models

[0155] NSG mice (Jackson) aged 8-12 weeks were used according to an experimental plan approved by the Animal Experiment Committee of Kyoto University based on animal ethics. On day 0, 5 × 10 5 NALM-6 cells were used as a mouse tumor model. 2×10 5 CAR-T cells as a therapeutic model.

[0156] Flow cytometry-based cytotoxicity assay

[0157] 1×10 5 CAR-T cells or CAR-jurkat cells were co-cultured with NALM-6 cells at a ratio of 1:1 in a 48-well plate at 37°C overnight. The cells were recovered with FACS buffer, and various antibodies were added. After 30 minutes of staining, the cells were measured with FACSAria (BD). The data were analyzed using Flowjo software. In the metalloproteinase inhibitor experiment, GI254023X and DAPT (Sigma) were added to the co-culture at various concentrations and the experiment was performed.

[0158] Cytotoxicity assay

[0159] CAR-T cells were mixed with target cells at various effector / target ratios, and cytotoxicity was measured with Nivo (PerkinElmer) using the N-SPC kit (TECHNO SUZUTA) as recommended.

[0160] Imaging analysis

[0161] NALM6 cells were co-cultured with Jurkat cells into which CARs with Hibit-tagged proteins added to the front end of various CARs (19-28bbz or cleavable-19-28bbz) were introduced overnight, and then fixed and cell membrane permeabilized using Biolegend's intracellular staining kit, and intracellular staining was performed according to the Biolegend intracellular flow cytometry staining protocol. After staining with mouse anti-Hibit mAb (Promega), AlexaFluor647 goat anti-mouse IgG (Invitrogen), and CD19-FITC antibody, imaging analysis was performed using Image-Stream (MKII).

[0162] Statistical analysis

[0163] In the in vitro experiments using cultured cells, all groups were tested multiple times with n=3 to confirm reproducibility. Data analysis was performed using Excel, and the comparison of the mean values ​​of each group was performed using a T test for significant differences, which are recorded as *(p<0.05), **(p<0.01), and ***(p<0.001). In the in vivo experiments, 5 mice were used per group, and the survival rate curve was prepared using Prism, and the difference in survival period was tested using the log-rank test.

[0164] Example 1: Verification of the effect of shedding on CAR effect

[0165] The study was initiated based on the assumption that the reactivity of CAR may change if a CAR with a structure that is shed (cleavable-CAR) is compared with a conventional CAR with a CD8 transmembrane domain that is not shed (normal-CAR). CARs were prepared in which the transmembrane domain was changed to a transmembrane domain derived from the Notch protein, which is widely known as a shed structure, and a CAR that maintains the reactivity of CAR ( Figure 1 -a). In order to detect the reactivity with target cells in the short term among CARs with various intracellular domains, NFAT-Jurkat cells were expressed with various CAR genes and co-cultured with NALM6 cells for 3 hours, and luciferase expression was detected. The results showed that the cleavable-CAR showed the same to slightly lower reactivity than the conventional CAR ( Figure 1 -b). If there is a detached structure, it can be expected to cause the signal to turn on / off, so if it is viewed in the long term, it is believed that the signal intensity is easy to maintain. After 4 days of co-culture, the reactivity was detected. The results showed that regardless of the structure, the signal intensity of the cleavable-CAR was significantly stronger ( Figure 1-c). Long-term signal enhancement is expected to improve the long-term therapeutic effect in vivo. In order to verify the actual therapeutic effect, an experimental system in which PBMCs introduced with CAR were intravenously administered to NSG mice with a blood cancer model of NALM6 cells was used for in vivo verification. The results showed that CARs with intracellular domains of 28bbz and 28z can significantly prolong survival. Bbz also tends to prolong survival ( Figure 1 -def). In addition, among 28bbz CARs, even in the validation using a subcutaneous tumor model, cleavable-CAR showed a higher effect than conventional CAR, indicating prolonged survival ( Figure 1 -g).

[0166] Example 2: Verification of the efficient mechanism of CAR caused by shedding

[0167] To investigate the mechanism by which cleavable-CARs can exert a higher effect, we first examined CD8 + T cell cytotoxicity, no significant difference was found between normal-CAR and cleavable-CAR ( Figure 2 -ab). In recent years, it has been reported that the degree of cytotoxicity caused by CAR varies with CAR reactivity, so various CARs were used to verify the degree to which CD19 molecules expressed by NALM6 cells were removed by cytotoxicity when co-cultured with NALM6 cells. The results clearly showed that no matter which intracellular domain of CAR was present, as long as it was made into a cleavable-CAR, it could significantly inhibit the reduction of CD19 expression in target cells ( Figure 2 -c). In the 28bbz CAR with the largest difference in cytotoxicity between normal-CAR and cleavable-CAR, a CAR with Tag protein added to the front end of the CAR was produced, and the extracellular domain of the CAR after reacting with the target was detected by image. The results showed that the normal-CAR was integrated into some target cells at the site of immune synapse formation, but the cleavable-CAR was attached to the target cells in a manner that covered the periphery ( Figure 2 -d, e). In addition, in normal-CAR, when co-cultured with target cells, it is observed that the cells are pulled and crushed, but in cleavable-CAR, repeated contact and separation, like pecking, are observed. It can be said that the above results show that the dynamics of CAR-T cells change depending on whether the receptor has a shedding structure.

[0168] Example 3: Verification of the mechanism of inhibiting cell phagocytosis

[0169] In order to verify how cleavable-CAR inhibits cytokinesis, an inhibition experiment on metalloproteinases that induce shedding was performed. First, ADAM inhibitors that cause cleavage of the extracellular domain were added, and the results confirmed that even in cleavable-CAR, the inhibition of cytokinesis was relieved in a concentration-dependent manner ( Figure 3 -a). Although γ-secretase inhibitors also easily cause cytotoxicity when inhibiting the cleavage of the intracellular domain, the degree is milder than that of ADAM inhibitors. In addition, in the long-term stimulation experiment of NFAT-jurkat, if ADAM inhibitors are added, the signal is significantly reduced, but no change is found in γ-secretase inhibition ( Figure 3 -bc). Next, in order to verify whether other shedding structures also cause inhibition of cytokinesis, CARs using the transmembrane domain of amyloid precursor protein (APP) as shedding structures were produced, and inhibition of cytokinesis was also observed in them. If APP-TM is used, which is known to cause familial Alzheimer's disease, the cleavage of the extracellular domain of APP is increased by mutations (Swe mutations), it is shown that cytokinesis is further inhibited, indicating that the ease of cleavage of the CAR extracellular domain by metalloproteinases largely determines the level of inhibition of cytokinesis.

[0170] Example 4: Verification of the correlation between cytokinesis and intracellular signals

[0171] It was verified what kind of signal determines the intensity of cytotoxicity. First, in order to verify whether cytotoxicity in normal-CAR requires TCR signals via CD3ζ, a construct without CD3ζ was used to detect cytotoxicity. The results made it clear that although the level of cytotoxicity became mild, cytotoxicity would be caused even without a CD3ζ signal. Although no significant difference was found in the intensity of the NFAT signal, the construct with 28bb as the signal transduction domain showed a stronger induction of cytotoxicity compared to only the 4-1BB signal transduction domain or the CD28 signal transduction domain. Therefore, based on the assumption that the size of the intracellular domain is more important than the signal intensity, the level of cytotoxicity was verified by attaching a structure of GFP, which should be obviously unrelated to signal transduction, to the N-terminus of CAR. As a result, it was found that cytotoxicity was more strongly induced due to the addition of the GFP domain ( Figure 4 -a). Next, in order to verify whether cytotoxicity requires a tyrosine kinase domain, a receptor was prepared in which a portion of EGFP (SEQ ID NOs. 19 and 20) was modified to increase the length of the transmembrane domain. The results showed that cytotoxicity was induced even without a tyrosine kinase domain, and the extent of cytotoxicity was dependent on the length of the intracellular domain ( Figure 5 In addition, when the non-signaling domain (EGFP267) and the tyrosine kinase domain (28bb) of the same length are compared, it is clear that the tyrosine kinase domain strongly induces cytotoxicity ( Figure 5In addition, it was found that if the signaling domain of the same size is arranged near the membrane where the cleavage site exists, as in 2828, cell mitosis can be more strongly inhibited ( Figure 6 ). In addition, these CARs were validated using a subcutaneous tumor model, and the results showed that CARs with tandem insertion of the signaling domain prolonged the survival of mice (exerting higher anti-tumor activity) ( Figure 7 ).

[0172] Example 5: Verification of the correlation between cell mitosis and release of intracellular domains

[0173] The transmembrane domain of the wild-type notch protein has an S3 cleavage site and an S4 cleavage site that are cleaved by γ-secretase. The S3 and S4 cleavage sites are cleaved by γ-secretase, and the intracellular domain of the notch protein is released into the cytoplasm. Therefore, the effect of inhibiting cell nibbling was verified by using a cleavable-CAR (19 cleavable mutation 2828z) that loses γ-secretase cleavage due to partial disappearance mutations of the S3 and S4 cleavage sites. After co-culturing NALM6 cells with Jurkat cells expressing CAR, NALM6 CD19 expression was measured, and the results showed that the 19 cleavable mutation 2828z significantly inhibited the reduction in CD19 expression in NALM6 cells compared with the CAR (19 cleavable 2828z) with the transmembrane domain of wild-type notch (i.e., cell nibbling was further inhibited) ( Figure 8 ). It is believed that the effect of inhibiting the cleavage of cleavable-CAR is mainly due to the cleavage of the extracellular domain caused by the shedding enzyme, and does not necessarily require the release of the intracellular domain caused by γ-secretase.

[0174] Example 6: Validation of cytokines in CARs using different transmembrane domains

[0175] The same cell-binding detection system as in Example 5 was used to verify the inhibitory effect of cell-binding when the transmembrane (TM) site was changed to the TM of CD28. Specifically, in a CAR using the TM of human CD28 and 12 amino acids of its extracellular domain as a hinge, the signaling domain was connected in series, and the results showed that cell-binding was further inhibited ( Fig. 9 ). It is expected that CD28 is also a shedding protein, and the cleavage site of its extracellular domain exists within 12 amino acids starting from TM.

[0176] Example 7: Validation of CARs using different ligand binding sites

[0177] Instead of CD19 ScFv, CAR with aTRBV12 ScFv or Her2 ScFv was used for verification. NFAT-Jurkat cells were co-cultured with various CAR-T cells integrated with aTRBV12ScFv, and the TCR signal intensity on the NFAT-juakat side was detected. It was confirmed that the CAR using hCleavable-2828z CAR had a tendency to reduce the signal ( Fig.10 ). In addition, FACS analysis was performed on the Her2 expression on the skOV3 side after co-culturing skOV3, a tumor strain expressing Her2, with various Her2-CARs overnight. This indicates that even in Her2-CAR, cytotoxicity is inhibited in CARs with hCleavable, and even in the 4-1BB signaling domain, cytotoxicity can be further inhibited by increasing the number of signaling domains when using a transmembrane site containing a shedding structure ( Fig.11 ).

[0178] Example 8: Study on the effects of Notch1 protein length

[0179] The effect on the reactivity of CAR when shortening the length of the transmembrane domain with the shedding structure of human Notch1 was studied. CARs with different lengths of the extracellular hinge domain were made (19hL31447 2828z, 19hL31448 2828z, 19hL31530 2828z, 19hL31560 2828z, respectively), and the cells obtained by introducing them into NFAT-Jurkat were compared with the cells obtained by co-culture with the control or NALM6 to confirm the reactivity. The CAR at position 1447 that completely contains LNR showed the same strong reactivity as hCleavable CAR. In addition, if LNR is deleted, the reactivity is reduced, but a significant response is found compared to the control ( Fig.12 ). Therefore, it is believed that the reactivity can be controlled by changing the length of the Notch1 usage sequence.

[0180] Industrial Applicability

[0181] By utilizing the present invention, cell nibbling in immune cells can be prevented, and the responsiveness of CAR preparations such as Kymriah (Novartis Pharma) and YESCARTA (Gilead Sciences) or receptors such as TCR used as gene therapy preparations in medical clinics can be maintained for a long time, which can greatly improve the effect of existing T cell therapy. In addition, since it is not considered to be obviously harmful in principle, it can be applied to clinical practice at an early stage. In addition, by changing the transmembrane domain or intracellular domain of the receptor, it is also possible to control cell nibbling. By promoting the research on the cell nibbling control technology of the present invention, it may be related to the improvement of xenotransplantation technology, the improvement of immune cell function, and the development of control technology for signal transduction via various cleavable proteins in the future.

[0182] This application is based on Japanese Patent Application No. 2022-118674 (filing date: July 26, 2022) filed in Japan, the contents of which are all incorporated herein by reference.

Claims

1. An artificial receptor comprising a ligand binding site, a transmembrane domain with a shedding structure, and a signal transduction domain into immune cells.

2. The artificial receptor of claim 1, which is selected from the group consisting of a chimeric antigen receptor, a modified T cell receptor, a modified Fc receptor, and a modified tyrosine kinase receptor.

3. The artificial receptor of claim 1 or 2, wherein the immune cell is selected from the group consisting of a T cell, a natural killer cell, and a macrophage.

4. The artificial receptor of claim 3, wherein the T cell is a cytotoxic T cell.

5. The artificial receptor according to any one of claims 1 to 4, wherein the shedding structure is derived from a protein selected from the group consisting of notch protein, amyloid precursor protein and CD28. 6 . The artificial receptor according to claim 1 , wherein the signal transduction domain into immune cells comprises a signal transduction domain derived from at least one selected from the group consisting of CD3 zeta chain, CD28, and 4-1BB.

7. The artificial receptor according to any one of claims 1 to 6, wherein the transmembrane domain having a shedding structure comprises: (1) the amino acid sequence represented by SEQ ID NO: 26, or (2) An amino acid sequence obtained by substituting, deleting, inserting and / or adding one or more amino acids to the amino acid sequence of (1). 8 . The artificial receptor according to claim 1 , wherein the transmembrane domain does not have a cleavage site for gamma-secretase. 9 . The artificial receptor according to claim 1 , which has two or more identical signal transduction domains.

10. A nucleic acid encoding the artificial receptor according to any one of claims 1 to 9. 11 . An immune cell comprising the artificial receptor according to any one of claims 1 to 9 or the nucleic acid according to claim 10 .

12. A medicine comprising the immune cell according to claim 11.

13. A method for designing a cytotoxic receptor or a nucleic acid encoding the receptor, comprising: (1) a step of selecting a receptor comprising a ligand binding site, a transmembrane domain, and a signal transduction domain into immune cells, and (2A) a step of regulating the sensitivity of the transmembrane domain selected in step (1) to shedding, and / or (2B) A step of adding, inserting and / or deleting one or more amino acid residues and / or substituting one or more amino acid residues in the signal transduction domain of the receptor prepared in step (1).

14. A method for producing a cytotoxic receptor or a nucleic acid encoding the receptor, comprising: (1) a step of preparing a receptor comprising a ligand binding site, a transmembrane domain, and a signal transduction domain into immune cells, and (2A) a step of regulating the sensitivity of the transmembrane domain prepared in step (1) to shedding, and / or (2B) A step of adding, inserting and / or deleting one or more amino acid residues and / or substituting one or more amino acid residues in the signal transduction domain of the receptor prepared in step (1).

15. The method according to claim 13 or 14, wherein the receptor selected or prepared in step (1) is selected from the group consisting of a chimeric antigen receptor, a T cell receptor, an Fc receptor and a tyrosine kinase receptor.

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