Nucleic acid molecule encoding CAR, vector comprising same, CAR, immune cell comprising CAR, pharmaceutical composition comprising same, and method for preparing immune cell comprising CAR

Abstract

The present invention relates to nucleic acid molecules having a nucleotide sequence encoding a chimeric antigen receptor (CAR). The invention relates to a vector comprising the nucleic acid molecule. The present invention relates to CARs. The present invention relates to immune cells comprising CARs. The present invention relates to a pharmaceutical composition comprising an immune cell comprising a CAR. The present invention relates to a method for preparing an immune cell comprising a CAR.

Description

Technical Field

[0001] The present invention relates to a nucleic acid molecule having a nucleotide sequence encoding a chimeric antigen receptor (hereinafter, also referred to as "CAR"). The present invention relates to a vector comprising the nucleic acid molecule. The present invention relates to CAR. The present invention relates to an immune cell comprising CAR. The present invention relates to a pharmaceutical composition for treating a malignant tumor comprising an immune cell comprising CAR. The present invention relates to a method for preparing an immune cell comprising CAR. Background Art

[0002] CAR refers to a receptor protein produced by genetically fusing an extracellular domain containing an antigen binding region, a transmembrane domain, and an intracellular domain that transmits activation signals of immune cells. For example, as described in Patent Document 1, the antigen binding region of CAR uses a single-chain antibody that recognizes antigens expressed in tumor cells. In recent years, cancer immunotherapy, which introduces genes encoding CAR into immune cells and transplants immune cells expressing CAR on the cell surface into patients to treat cancer, has attracted attention. Immune cells expressing CAR are activated when they recognize antigens of tumor cells in vivo, and express cell-killing molecules or cytokines such as Fas ligand, perforin, and granzyme, exerting anti-tumor effects. On the other hand, it is known that the production of cytokines by immune cells expressing CAR may cause cytokine release syndrome (CRS).

[0003] Prior art literature Patent Literature Patent Document 1: U.S. Patent No. 7,741,465 Summary of the invention Problems to be solved by the invention The object of the present invention is to provide a CAR that can inhibit the production of cytokines by immune cells when expressed in immune cells. In addition, the object of the present invention is to provide a nucleic acid molecule encoding such a CAR and a vector of its nucleic acid molecule. Furthermore, the object of the present invention is to provide an immune cell comprising such a CAR, a pharmaceutical composition comprising the immune cell, and a method for preparing the immune cell.

[0004] Means for solving problems The present inventors have discovered that by making the prescribed amino acid residues of the framework region 3 (FR3) of the light chain variable region of the antigen binding region possessed by CAR into basic amino acid residues, the production of cytokines by immune cells containing CAR can be suppressed, thereby completing the present invention. Therefore, the present inventions described in the following [1] to

[21] are provided.

[0005] [1] A nucleic acid molecule having a nucleotide sequence encoding a CAR, wherein the nucleic acid molecule comprises a segment encoding an extracellular domain, a segment encoding a transmembrane domain, and a segment encoding an intracellular domain, the segment encoding the extracellular domain comprises a nucleotide sequence encoding an antigen binding region comprising a light chain variable region and a heavy chain variable region, and in the nucleotide sequence encoding FR3 of the light chain variable region defined by the Kabat method, at least 3 codons are codons encoding basic amino acid residues.

[0006] [2] The nucleic acid molecule according to [1] above, wherein the at least three codons include at least three selected from the group consisting of a codon encoding the amino acid residue at position 60 of the light chain variable region, a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, a codon encoding the amino acid residue at position 70, a codon encoding the amino acid residue at position 72, a codon encoding the amino acid residue at position 74, a codon encoding the amino acid residue at position 76, a codon encoding the amino acid residue at position 77, a codon encoding the amino acid residue at position 79, and a codon encoding the amino acid residue at position 81.

[0007] [3] The nucleic acid molecule according to [1] or [2] above, wherein three or more and five or less codons selected from the group consisting of a codon encoding the 60th amino acid residue of the light chain variable region defined by the Kabat method, a codon encoding the 63rd amino acid residue, a codon encoding the 65th amino acid residue, a codon encoding the 67th amino acid residue, a codon encoding the 70th amino acid residue, a codon encoding the 72nd amino acid residue, a codon encoding the 74th amino acid residue, a codon encoding the 76th amino acid residue, a codon encoding the 77th amino acid residue, a codon encoding the 79th amino acid residue and a codon encoding the 81st amino acid residue are codons encoding basic amino acid residues.

[0008] [4] A nucleic acid molecule according to any one of [1] to [3] above, wherein the antigen-binding region comprises a single-chain antibody, and the single-chain antibody is a single-chain antibody that binds to a complex of a peptide from MAGE-A4 and HLA-A2, a complex of a peptide from PRAME and HLA-A24, CD19, BCMA or CEA.

[0009] [5] A nucleic acid molecule according to any one of [1] to [4] above, wherein the transmembrane domain comprises a transmembrane region of any protein selected from the group consisting of an α chain of a T cell receptor, a β chain of a T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS and GITR.

[0010] [6] A nucleic acid molecule according to any one of [1] to [5] above, wherein the intracellular domain comprises a signal transduction domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ and FcRβ.

[0011] [7] The nucleic acid molecule according to the above-mentioned [6], wherein the segment encoding the intracellular domain further comprises a nucleotide sequence encoding a co-stimulatory domain, and the co-stimulatory domain is a co-stimulatory domain of at least one protein selected from the group consisting of 4-1BB, CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 and ICOS.

[0012] [8] The nucleic acid molecule according to any one of [1] to [7] above, further comprising a segment encoding a hinge domain between the nucleotide sequence encoding the antigen-binding region and the segment encoding the transmembrane domain.

[0013] [9] The nucleic acid molecule according to any one of [1] to [8] above, wherein the nucleic acid molecule is DNA or RNA.

[0014]

[10] A vector comprising the nucleic acid molecule according to any one of [1] to [9] above.

[0015]

[11] A CAR comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain includes an antigen binding region comprising a light chain variable region and a heavy chain variable region, and in FR3 of the light chain variable region defined by the Kabat method, at least 3 amino acid residues are basic amino acid residues.

[0016]

[12] The CAR according to

[11] above, wherein the at least three amino acid residues include at least three selected from the group consisting of the amino acid residue at position 60, the amino acid residue at position 63, the amino acid residue at position 65, the amino acid residue at position 67, the amino acid residue at position 70, the amino acid residue at position 72, the amino acid residue at position 74, the amino acid residue at position 76, the amino acid residue at position 77, the amino acid residue at position 79 and the amino acid residue at position 81 of the light chain variable region.

[0017]

[13] The CAR according to

[11] or

[12] above, wherein three or more and five or less amino acid residues selected from the group consisting of amino acid residue at position 60, amino acid residue at position 63, amino acid residue at position 65, amino acid residue at position 67, amino acid residue at position 70, amino acid residue at position 72, amino acid residue at position 74, amino acid residue at position 76, amino acid residue at position 77, amino acid residue at position 79 and amino acid residue at position 81 of the light chain variable region defined by the Kabat method are basic amino acid residues.

[0018]

[14] A CAR according to any one of

[11] to

[13] above, wherein the antigen binding region comprises a single-chain antibody that binds to a complex of a peptide from MAGE-A4 and HLA-A2, a complex of a peptide from PRAME and HLA-A24, CD19, BCMA or CEA.

[0019]

[15] A CAR according to any one of

[11] to

[14] above, wherein the transmembrane domain comprises a transmembrane region of any protein selected from the group consisting of an α chain of a T cell receptor, a β chain of a T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS and GITR.

[0020]

[16] The CAR according to any one of

[11] to

[15] above, wherein the intracellular domain comprises a signal transduction domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ and FcRβ.

[0021]

[17] The CAR according to the above-mentioned

[16] , wherein the intracellular domain further comprises a co-stimulatory domain, and the co-stimulatory domain is a co-stimulatory domain of at least one protein selected from the group consisting of 4-1BB, CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 and ICOS.

[0022]

[18] The CAR according to any one of

[11] to

[17] above, further comprising a hinge domain between the antigen binding region and the transmembrane region.

[0023]

[19] An immune cell comprising the chimeric antigen receptor according to any one of

[11] to

[18] above.

[0024]

[20] A pharmaceutical composition for treating malignant tumors, comprising the immune cells described in

[19] above.

[0025]

[21] A method for preparing immune cells, comprising: introducing the nucleic acid molecule described in any one of [1] to [9] or the vector described in

[10] into immune cells, so that the immune cells express a chimeric antigen receptor.

[0026] Effects of the Invention According to the present invention, immune cells containing CAR that suppresses cytokine production can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A : is a diagram schematically showing an example of a nucleic acid molecule according to the present embodiment. The figure also shows an enlarged view of a segment (VL) encoding a light chain variable region in a nucleic acid molecule. In the figure, A shows a segment encoding an extracellular domain, B shows a segment encoding a transmembrane domain, and C shows a segment encoding an intracellular domain. D shows a segment encoding an antigen binding region contained in the extracellular domain. Among these segments, VH shows a segment encoding a heavy chain variable region, L shows a segment encoding a linker, HD shows a segment encoding a hinge domain, TMD shows a segment encoding a transmembrane domain, co-STD shows a segment encoding a co-stimulatory domain, and SD shows a segment encoding a signal transduction domain. In the enlarged view of VL, the dotted line with * shows a codon encoding a basic amino acid residue. Figure 1A In the region encoding FR3, there are three codons encoding basic amino acid residues, but the present invention is not limited thereto.

[0028] Figure 1B: is a diagram schematically showing an example of a CAR of the present embodiment. In the figure, VH shows a heavy chain variable region, VL shows a light chain variable region, HD shows a hinge domain, TMD shows a transmembrane domain, co-STD shows a co-stimulatory domain, and SD shows a signal transduction domain. The curve connecting VH and VL shows a linker. In VL, the dotted line with * shows a basic amino acid residue. Figure 1B In the embodiment, the number of basic amino acid residues in FR3 is 3, but the present invention is not limited thereto.

[0029] Figure 2A This is a diagram schematically showing a nucleic acid molecule encoding the CAR of Example 1-3.

[0030] Figure 2B This is a diagram schematically showing a nucleic acid molecule encoding the CAR of Example 4.

[0031] Figure 3 This graph shows the tumor area (average value) of each mouse after PBS or CAR-T cells were administered to mice transplanted with tumor cells. DETAILED DESCRIPTION

[0032] 1. Nucleic acid molecules The nucleic acid molecule of the present embodiment has a nucleotide sequence encoding CAR. In this specification, "nucleotide sequence" is synonymous with "base sequence" and "nucleic acid sequence". A nucleotide sequence refers to a one-dimensional arrangement (sequence) of nucleotides in a nucleic acid molecule. Therefore, a nucleic acid molecule encoding a polypeptide has a nucleotide sequence encoding the polypeptide. In this specification, the expression "having a nucleotide sequence" refers to both being composed of the nucleotide sequence and containing the nucleotide sequence. In this specification, the term "polypeptide" includes protein molecules, partial regions in protein molecules, and fragments of protein molecules. In this specification, a portion of a nucleic acid molecule is also referred to as a "segment", and a portion of a protein molecule is referred to as a "domain".

[0033] like Figure 1A As illustrated, the nucleic acid molecule of the present embodiment includes, from the 5' side, a segment encoding an extracellular domain, a segment encoding a transmembrane domain, and a segment encoding an intracellular domain. The segment encoding the extracellular domain includes a nucleotide sequence encoding an antigen binding region comprising a light chain variable region and a heavy chain variable region. In the present specification, "an antigen binding region comprising a light chain variable region and a heavy chain variable region" refers to a domain that comprises at least one light chain variable region and at least one heavy chain variable region and is capable of binding to a specified antigen via them. As an antigen binding region, for example, a single-chain antibody can be cited. A single-chain antibody is also called scFv, in which the light chain variable region and the heavy chain variable region are connected by a peptide linker, and is a partial region of the CAR construct of the present embodiment. In Figure 1AIn the nucleic acid molecule, the segment encoding the antigen binding region includes a nucleotide sequence encoding a single-chain antibody composed of VH, L and VL. The details of each segment of the nucleic acid molecule of this embodiment and the CAR encoded by the nucleic acid molecule are described later.

[0034] like Figure 1A As shown, the nucleic acid molecule of the present embodiment has the following characteristics: in the nucleotide sequence encoding the FR3 of the light chain variable region (hereinafter, also referred to as "light chain FR3"), at least 3 codons are codons encoding basic amino acid residues. That is, the CAR encoded by the nucleic acid molecule of the present embodiment has an antigen binding region in which at least 3 amino acid residues of the light chain FR3 are basic amino acid residues. Hereinafter, the antigen binding region will also be referred to as the "modified antigen binding region". In addition, the CAR having the modified antigen binding region will also be referred to as the "modified CAR" below. The nucleic acid molecule of the present embodiment can be said to be a nucleic acid molecule encoding the modified CAR. In this specification, "codon" refers to 3 consecutive nucleotides of DNA or RNA.

[0035] The nucleic acid molecule encoding the altered antigen binding region can be obtained by changing the codons described later on the nucleic acid molecule encoding the original antigen binding region. In this specification, "original antigen binding region" refers to the antigen binding region before the change, which is an antigen binding region in which the number of basic amino acid residues in the light chain FR3 is 2 or less. That is, in the nucleotide sequence encoding the original antigen binding region, the number of codons encoding basic amino acid residues in the nucleotide sequence encoding the light chain FR3 is 2 or less. Hereinafter, the CAR having the original antigen binding region will also be referred to as "CAR before the change".

[0036] The framework region (FR) refers to the region other than the complementary determining region (CDR) present in the light chain variable region and the heavy chain variable region of an antibody. FR acts as a scaffold connecting the three CDRs and contributes to the structural stability of the CDRs. Therefore, the amino acid sequence of FR is highly conserved between antibodies of the same species. The variable regions of the heavy chain and light chain each have three CDRs, CDR1, CDR2, and CDR3, and four FRs, FR1, FR2, FR3, and FR4. They are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminal side of the variable region. Hereinafter, the CDR of the heavy chain will also be referred to as "HCDR" and the CDR of the light chain will be referred to as "LCDR".

[0037] In the technical field, a method for numbering the amino acid residues of CDRs (hereinafter also referred to as "numbering method") for defining the boundaries and lengths of CDRs is known. When the amino acid residues of CDRs are numbered by the numbering method, the amino acid residues of FRs are also numbered. The number assigned to the amino acid residue by the numbering method indicates the position of the amino acid residue in the amino acid sequence of the light chain or heavy chain. Examples of the numbering method include the Kabat method (Kabat EA et al., Sequences of Proteins of Immunological Interest., NIH publication No. 91-3242), the Chothia method (Chothia C. and Lesk AM., Canonical Structures for the Hypervariable Regions of Immunoglobulins., J Mol Biol., vol. 196, p. 901-917, 1987), the IMGT method (Lefranc MP. et al., Developmental and Comparative Immunology 29 (2005) 185-203), the Honerger method (Honegger A. et al., Yet Another Numbering Scheme for Immunoglobulin Variable Domains: An Automatic Modeling and Analysis Tool., J Mol Biol., vol. 309, p. 657-670, 2001), the ABM method, and the Contact method.

[0038] About antigen binding region, the boundary and length of CDR and FR in each variable region of light chain and heavy chain can be defined by arbitrary numbering method.In this specification, the boundary and length of CDR and FR are defined by Kabat method.For example, in the case where the antigen binding region comprises single-chain antibody or is constituted by it, according to Kabat method, the light chain FR1 of single-chain antibody is defined as the region consisting of amino acid residues at positions 1-23 of light chain variable region.The light chain FR2 of single-chain antibody is defined as the region consisting of amino acid residues at positions 35-49 of light chain variable region.The light chain FR3 of single-chain antibody is defined as the region consisting of amino acid residues at positions 57-88 of light chain variable region.The light chain FR4 of single-chain antibody is defined as the region consisting of amino acid residues at positions 98-109 of light chain variable region.In this specification, in the case of recording the position of amino acid residues in the light chain variable region of antigen binding region, as long as there is no special explanation, the position of the amino acid residue represents the position defined by Kabat method.

[0039] The nucleic acid molecule of the present embodiment can be obtained by sequentially connecting a nucleic acid molecule encoding an extracellular domain, a nucleic acid molecule encoding a transmembrane domain, and a nucleic acid molecule encoding an intracellular domain from the 5' side. The connection of these nucleic acid molecules can be carried out by known gene recombination techniques and other molecular biology techniques. The nucleic acid molecule encoding the extracellular domain can be composed of a nucleic acid molecule encoding a modified antigen binding region. Preferably, the nucleic acid molecule encoding the extracellular domain is obtained by connecting a nucleic acid molecule encoding a modified antigen binding region with a nucleic acid molecule encoding a hinge domain using the above-mentioned technology.

[0040] Hereinafter, in the nucleotide sequence of the light chain FR3 encoding the original antigen binding region, more than 3 codons encoding the amino acid residues of the non-basic amino acid residues are made into codons encoding the basic amino acid residues, also referred to as "changing codons" or "changing codons". The nucleic acid molecule encoding the changed antigen binding region can be obtained by changing the codons of the nucleic acid molecule encoding the original antigen binding region. The amino acid residues of the non-basic amino acid residues refer to neutral amino acid residues and / or acidic amino acid residues, preferably neutral amino acid residues. The nucleotide sequence encoding the changed antigen binding region and the nucleotide sequence encoding the original antigen binding region are preferably the same except for the position of the changed codons.

[0041] By changing the codons in this way, at least three codons in the nucleotide sequence encoding the light chain FR3 of the original antigen binding region become codons encoding basic amino acid residues, and a nucleic acid molecule encoding the altered antigen binding region can be obtained. The codon change can be performed by replacing or inserting codons in the nucleic acid molecule encoding the original antigen binding region.

[0042] Basic amino acid residues refer to lysine residues, arginine residues and histidine residues. Among them, arginine residues and lysine residues are preferred. Neutral amino acid residues refer to alanine residues, asparagine residues, cysteine ​​residues, glycine residues, glutamine residues, isoleucine residues, leucine residues, methionine residues, phenylalanine residues, proline residues, serine residues, threonine residues, tryptophan residues, tyrosine residues and valine residues. Acidic amino acid residues refer to aspartic acid residues and glutamic acid residues.

[0043] In the nucleotide sequence encoding light chain FR3, at least 3 codons introduced by the change of codons may all be codons encoding arginine residues, or may all be codons encoding lysine residues. Alternatively, in the nucleotide sequence encoding light chain FR3, at least 3 codons introduced by the change of codons may be part of which are codons encoding arginine residues, and the rest are codons encoding lysine residues.

[0044] The nucleic acid molecule of the present embodiment can be DNA or RNA. In the nucleotide sequence encoding light chain FR3, the type of the codon introduced by the change of codon is not particularly limited as long as it encodes basic amino acid residues. When the nucleic acid molecule of the present embodiment is DNA, as the codon encoding basic amino acid residues, AGA, AGG, CGA, CGC, CGG and CGT encoding arginine residues, AAA and AAG encoding lysine residues, CAC and CAT encoding histidine residues can be enumerated. When the nucleic acid molecule of the present embodiment is RNA, as the codon encoding basic amino acid residues, AGA, AGG, CGA, CGC, CGG and CGU encoding arginine residues, AAA and AAG encoding lysine residues, CAC and CAU encoding histidine residues can be enumerated.

[0045] In the nucleic acid molecule of the present embodiment, the number of codons encoding basic amino acid residues in the nucleotide sequence encoding light chain FR3 is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. The number of codons encoding basic amino acid residues in the nucleotide sequence encoding light chain FR3 is preferably 3 or more and 6 or less, more preferably 3 or more and 5 or less.

[0046] In the light chain FR3 of the altered antigen-binding region, at least 3 basic amino acid residues from the change of the codon are preferably located at the position of the amino acid residues of the vernier region residues and non-exposed residues removed from the amino acid sequence of the light chain FR3." Vernier region residues" refers to the amino acid residues that contribute to the structural stability of CDR in the amino acid sequence of FR. "Non-exposed residues" refers to the amino acid residues that are folded inside the molecule and not exposed on the surface. It is expected that even if the non-exposed residues are changed, the effect of the change is small or absent. For example, the amino acid residues after removing the vernier region residues and non-exposed residues from the amino acid sequence of the light chain FR3 are the 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th, 80th and 81st amino acid residues of the light chain variable region.

[0047] In the nucleotide sequence encoding the altered antigen-binding region, it is preferred that at least three codons selected from the group consisting of the codon encoding the amino acid residue at position 60 of the light chain variable region, the codon encoding the amino acid residue at position 63, the codon encoding the amino acid residue at position 65, the codon encoding the amino acid residue at position 67, the codon encoding the amino acid residue at position 70, the codon encoding the amino acid residue at position 72, the codon encoding the amino acid residue at position 74, the codon encoding the amino acid residue at position 76, the codon encoding the amino acid residue at position 77, the codon encoding the amino acid residue at position 79, and the codon encoding the amino acid residue at position 81 are codons encoding basic amino acid residues. More preferably, more than 3 and less than 6 codons selected from the above group are codons encoding basic amino acid residues. Particularly preferably, more than 3 and less than 5 codons selected from the above group are codons encoding basic amino acid residues. For example, each codon described in any one of the following 1) to 21) is a codon encoding a basic amino acid residue.

[0048] 1) A codon encoding the 60th amino acid residue, a codon encoding the 63rd amino acid residue, and a codon encoding the 65th amino acid residue in the light chain variable region; 2) a codon encoding the amino acid residue at position 60, a codon encoding the amino acid residue at position 63, and a codon encoding the amino acid residue at position 76 of the light chain variable region; 3) a codon encoding the amino acid residue at position 60, a codon encoding the amino acid residue at position 74, and a codon encoding the amino acid residue at position 76 of the light chain variable region; 4) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, and a codon encoding the amino acid residue at position 67 of the light chain variable region; 5) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, and a codon encoding the amino acid residue at position 70 of the light chain variable region; 6) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 7) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 67, and a codon encoding the amino acid residue at position 70 of the light chain variable region; 8) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 67, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 9) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 70, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 10) a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, and a codon encoding the amino acid residue at position 70 of the light chain variable region; 11) a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 12) a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 70, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 13) a codon encoding the amino acid residue at position 67, a codon encoding the amino acid residue at position 70, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 14) a codon encoding the 70th amino acid residue, a codon encoding the 72nd amino acid residue, and a codon encoding the 74th amino acid residue of the light chain variable region; 15) a codon encoding the amino acid residue at position 77, a codon encoding the amino acid residue at position 79, and a codon encoding the amino acid residue at position 81 of the light chain variable region; 16) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, and a codon encoding the amino acid residue at position 70 of the light chain variable region; 17) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 18) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 70, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 19) a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 67, a codon encoding the amino acid residue at position 70, and a codon encoding the amino acid residue at position 72 of the light chain variable region; 20) a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, a codon encoding the amino acid residue at position 70, and a codon encoding the amino acid residue at position 72 of the light chain variable region; and 21) A codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, a codon encoding the amino acid residue at position 70, and a codon encoding the amino acid residue at position 72 of the light chain variable region.

[0049] In the nucleotide sequence encoding the altered antigen-binding region, the codons encoding the amino acid residues at at least three positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region and the codons encoding the amino acid residues at at least one position selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region are preferably codons encoding basic amino acid residues.

[0050] In the nucleotide sequence encoding the altered light chain FR3 of the antigen-binding region, when 3 or more and 6 or less codons are codons encoding basic amino acid residues, the 3 or more and 6 or less codons preferably include: codons encoding amino acid residues at 3, 4 or 5 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region, and codons encoding amino acid residues at 1, 2 or 3 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region.

[0051] In the nucleotide sequence encoding the altered light chain FR3 of the antigen-binding region, when 3 or more and 5 or less codons are codons encoding basic amino acid residues, the 3 or more and 5 or less codons preferably include: codons encoding amino acid residues at 3 or 4 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region, and codons encoding amino acid residues at 1 or 2 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region.

[0052] In the nucleotide sequence encoding the altered antigen-binding region, the codons encoding the amino acid residues at at least 3 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region and the codons encoding the amino acid residues at at least 1 position selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region are preferably codons encoding basic amino acid residues.

[0053] In the nucleotide sequence encoding the altered light chain FR3 of the antigen-binding region, when 3 or more and 6 or less codons are codons encoding basic amino acid residues, the 3 or more and 6 or less codons preferably include: codons encoding amino acid residues at 3, 4 or 5 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region, and codons encoding 1, 2 or 3 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region.

[0054] In the nucleotide sequence encoding the altered light chain FR3 of the antigen-binding region, when 3 or more and 5 or less codons are codons encoding basic amino acid residues, the 3 or more and 5 or less codons preferably include: codons encoding amino acid residues at 3 or 4 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region, and codons encoding amino acid residues at 1 or 2 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region.

[0055] In the nucleotide sequence encoding the altered antigen-binding region, when a codon other than the codons encoding the amino acid residues at positions 60, 63, 65, 67, 70, 72, 74, 76, 77, 79 and 81 of the light chain variable region is changed, the altered codon is preferably a codon encoding an amino acid residue at a position selected from the group consisting of positions 57, 58, 59, 61 and 62.

[0056] CDR participates in the affinity of the antigen-binding region for the antigen, so in the nucleic acid molecule of the present embodiment, the nucleotide sequence encoding the CDR of the antigen-binding region is preferably not changed. That is, it is preferred that the amino acid sequence of the CDR of the altered antigen-binding region and the nucleotide sequence encoding it are the same as the amino acid sequence of the CDR of the original antigen-binding region and the nucleotide sequence encoding it.

[0057] Hereinafter, immune cells containing CAR before the change will also be referred to as "immune cells before the change", and immune cells containing CAR after the change will be referred to as "immune cells after the change". When the amino acid sequence of the changed CAR is the same as the amino acid sequence of the CAR before the change, except that at least 3 amino acid residues of the light chain FR3 are basic amino acid residues, the production of cytokines and the cell killing effect of the changed immune cells can be compared with the immune cells before the change. In the changed immune cells, the production of cytokines is suppressed compared with the immune cells before the change. The types of cytokines are not particularly limited, and for example, IFNγ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, etc. can be cited. In this specification, "the production of cytokines is suppressed" means that tumor cells and immune cells are mixed at a prescribed ratio, and at a time point after a prescribed period of time from the mixing, the amount of cytokines released by the changed immune cells is less than the amount released by the immune cells before the change. For example, the amount of cytokines released by the modified immune cells is 93% or less, 92% or less, 91% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the amount released by the immune cells before the modification. The amount of cytokines released by immune cells containing CAR can be investigated, for example, by measuring the cytokines in the culture supernatant by a known method such as enzyme-linked immunosorbent assay (ELISA) after co-culturing the immune cells and tumor cells. Specifically, the method of Example 1 described later is exemplified.

[0058] Preferably, in the changed immune cells, the cell killing effect on tumor cells is maintained compared with the immune cells before the change, and it is further preferred that the cell killing effect is improved. In this specification, "cell killing effect maintenance" means that tumor cells and immune cells are mixed at a specified ratio, and at a time point after a specified period of time from the mixing, the cell killing effect of the immune cells before the change and the immune cells after the change are substantially the same. In a preferred embodiment, "cell killing effect maintenance" means that the cell killing effect of the immune cells after the change is more than 95% and less than 105% of the cell killing effect of the immune cells before the change. Even if the cell killing effect of the immune cells after the change is lower than that of the immune cells before the change, as long as the amount of cytokines is greatly reduced, it can be considered that a better therapeutic effect is obtained. In the case of administering such immune cells to patients, if the amount of immune cells applied is increased, the cell killing effect on tumor cells is improved, and cytokines can still be at a low level. That is, as long as the cytokines can be maintained at a low level, the amount of immune cells applied can be appropriately adjusted in order to maintain or improve the cell killing effect. "Cell killing effect" can be evaluated in vitro. For example, the cell killing effect can be evaluated by indicators such as the number of tumor cells that survive and the survival rate when immune cells containing CAR and tumor cells are co-cultured. Specifically, the method of Example 1 described later is exemplified. In addition, in the case of solid cancer as the object, it is also possible to administer immune cells containing CAR after transplanting tumor cells into non-human animals such as mice, and measure the size of the tumor to evaluate. Specifically, the method of Example 5 described later can be exemplified. In the case of blood cancer as the object, it is also possible to administer immune cells containing CAR after transplanting tumor cells into non-human animals such as mice, and evaluate by measuring the number of tumor cells in the blood by a microscope or flow cytometer.

[0059] As mentioned above, the nucleic acid molecules encoding the changed antigen binding region can be obtained from the nucleic acid molecules encoding the original antigen binding region by known gene recombination techniques and other molecular biology techniques. First, based on the nucleotide sequence of the nucleic acid molecules encoding the original antigen binding region, a primer set for changing the codon is made. For example, when the change of the codon is replaced by the codon, a primer set designed in a manner that at least 3 codons are replaced by codons encoding basic amino acid residues in the nucleotide sequence encoding light chain FR3 is made. Then, by using the PCR method of the primer set, the nucleic acid molecules encoding the original antigen binding region are amplified as a template, thereby the nucleic acid molecules encoding the antigen binding region in which at least 3 amino acid residues of the light chain FR3 are replaced by basic amino acid residues can be obtained. Alternatively, when the change of the codon is inserted into the codon, a primer set designed in a manner that at least 3 positions in the nucleotide sequence encoding the light chain FR3 are inserted into the codons encoding basic amino acid residues is made. Then, by using the PCR method of the primer set, the nucleic acid molecules encoding the original antigen binding region are amplified as a template, thereby the nucleic acid molecules encoding the antigen binding region in which at least 3 positions of the light chain FR3 are inserted with basic amino acid residues can be obtained.

[0060] As mentioned above, the nucleic acid molecule encoding the altered antigen binding domain is obtained based on the nucleic acid molecule encoding the original antigen binding domain, so it is preferred to obtain the nucleic acid molecule that the original antigen binding domain contains the nucleotide sequence encoding it. For example, in the case of being able to obtain the E. coli clone of the plasmid DNA encoding the original antigen binding domain, by extracting the plasmid DNA from the E. coli clone, the nucleic acid molecule containing the nucleotide sequence encoding the original antigen binding domain can be obtained. In addition, in order to make the above-mentioned primer set, the nucleotide sequence encoding the original antigen binding domain is preferably known or can be confirmed. In the case where the antigen binding domain contains a single-chain antibody or is composed of it, the nucleotide sequence encoding the single-chain antibody can be investigated from the known databases such as PDB, GeneBank, abYsis, IMGT. In the case of having a nucleic acid molecule containing the nucleotide sequence encoding the original antigen binding domain, by sequencing the nucleic acid molecule, the nucleotide sequence encoding the original antigen binding domain can be investigated.

[0061] Preferably, the altered antigen binding region and the original antigen binding region bind to at least an antigen expressed in tumor cells. The antigen may be an antigen also expressed in normal cells, or an antigen specifically expressed in tumor cells. The antigen includes not only a full-length antigen, but also a fragment of the antigen, and a complex of a fragment of the antigen and an MHC (major histocompatibility complex) protein. As a fragment of an antigen, for example, a part of the antigen presented by an antigen presenting cell or a tumor cell itself, a synthetic peptide consisting of a part of the amino acid sequence of the antigen, etc. can be cited. MHC protein is called HLA (human leukocyte antigen) in humans, and HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HAL-DP, etc. can be cited. For example, in the case where the antigen is a protein expressed in tumor cells, the altered antigen binding region and the original antigen binding region can bind to a complex of a fragment of the antigen and an MHC protein.

[0062] As the type of antigen recognized by the antigen binding region, it is preferred that the antigen is present on the surface of the tumor cell and the antigen is present in the tumor cell. As such an antigen, for example, CD19, CD20, CD30, CD44, CD133, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, PRAME, NY-ESO-1, SSX2, GP100, MART-1, HER2, carcinoembryonic antigen (CEA), MUC-1, CA125, Glypican (GPC3), B cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), ganglioside GM2, etc. can be cited. In addition, the antigen recognized by the antigen binding region can also be a complex of a fragment of the antigen and an MHC protein. The complex is formed by antigen fragmentation in the tumor cell, and its fragment is combined with the MHC of the tumor cell. In addition, the formed complex is presented to the surface of the tumor cell. Examples of such complexes include complexes of peptides derived from MAGE-A4 and HLA-A2 (hereinafter also referred to as "MAGE-A4 / HLA-A2 complexes"), complexes of peptides derived from PRAME and HLA-A24 (hereinafter also referred to as "PRAME / HLA-A24 complexes"), and the like. Examples of HLA-A2 that forms a complex with a peptide derived from MAGE-A4 include HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, and HLA-A*02:11. Among them, HLA-A*02:01 is preferred. Examples of HLA-A24 that forms a complex with a peptide derived from PRAME include HLA-A*24:02, HLA-A*24:03, and the like. Among them, HLA-A*24:02 is preferred. The peptides derived from MAGE-A4 and PRAME also form complexes with HLA that are different from the above, depending on the sequence of the fragmented peptides. The peptides derived from MAGE-A4 and PRAME are oligopeptides composed of a portion of each amino acid sequence of MAGE-A4 and PRAME (e.g., 8 to 20 amino acids). As peptides derived from MAGE-A4, for example, oligopeptides composed of the amino acid sequence of GVYDGREHTV (sequence number 1) can be cited. As peptides derived from PRAME, for example, oligopeptides composed of the amino acid sequence of LYVDSLFFL (sequence number 2) can be cited.

[0063] The nucleic acid molecule encoding the original antigen binding region itself can be obtained by known gene recombination technology and other molecular biology techniques. In the case where the antigen binding region contains a single-chain antibody or is composed thereof, for example, by using a phage display method of an antibody phage library, it is possible to separate nucleic acid molecules encoding single-chain antibodies that bind to the antigens of tumor cells. Alternatively, a hybridoma that produces an antibody that binds to the antigens of tumor cells can be prepared, and RNA extracted from the hybridoma is used to prepare a nucleic acid molecule encoding a single-chain antibody by reverse transcription reaction and PCR. Hybridomas can be prepared by known methods such as the method described in Kohler G. and Milstein C., Nature, vol. 256, p. 495-497, 1975.

[0064] In the segment encoding the extracellular domain (hereinafter, also referred to as the "extracellular segment"), the nucleotide sequence encoding the antigen binding region includes a nucleotide sequence encoding a light chain variable region and a nucleotide sequence encoding a heavy chain variable region. The nucleotide sequence encoding the antigen binding region may include a portion or all of the nucleotide sequence encoding the constant region. The constant region may be a constant region of any one of the heavy chain and the light chain, preferably a light chain constant region. In the nucleotide sequence encoding the antigen binding region, the order of the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region is not particularly limited. For example, the nucleotide sequence encoding the antigen binding region may include a nucleotide sequence encoding the light chain variable region and a nucleotide sequence encoding the heavy chain variable region in sequence from the 5' side. Alternatively, the nucleotide sequence encoding the antigen binding region may also include a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region in sequence from the 5' side. In the case of including a portion or all of the nucleotide sequence encoding the light chain constant region, it is preferred that the nucleotide sequence is contiguous with the nucleotide sequence encoding the light chain variable region. In the case of including a portion or all of the nucleotide sequence encoding the heavy chain constant region, it is preferred that the nucleotide sequence is contiguous with the nucleotide sequence encoding the heavy chain variable region.

[0065] The segment encoding the antigen binding region comprises a nucleotide sequence encoding a single-chain antibody that binds to any of the above-mentioned tumor cell antigens or is composed of its nucleotide sequence. Preferably, the segment encoding the antigen binding region comprises a nucleotide sequence encoding a complex of a peptide from MAGE-A4 and HLA-A2, a complex of a peptide from PRAME and HLA-A24, a single-chain antibody that binds to CD19 or CEA or is composed of its nucleotide sequence.

[0066] Preferably, the nucleotide sequence encoding the single-chain antibody of the complex of the peptide derived from MAGE-A4 and HLA-A2 comprises the nucleotide sequence shown in sequence numbers 153 to 158. Sequence number 153 shows the nucleotide sequence encoding HCDR1, sequence number 154 shows the nucleotide sequence encoding HCDR2, sequence number 155 shows the nucleotide sequence encoding HCDR3, sequence number 156 shows the nucleotide sequence encoding LCDR1, sequence number 157 shows the nucleotide sequence encoding LCDR2, and sequence number 158 shows the nucleotide sequence encoding LCDR3. A specific example of the nucleotide sequence encoding the single-chain antibody comprising sequence numbers 153 to 158 is shown in sequence number 159. Based on this sequence number 159, the nucleotide sequence encoding the single-chain antibody after the FR3 is changed is shown in sequence numbers 160 to 169.

[0067] Preferably, the nucleotide sequence encoding the single-chain antibody that binds to the complex of the peptide derived from PRAME and HLA-A24 comprises the nucleotide sequence shown in sequence numbers 170 to 175. Sequence number 170 is a nucleotide sequence encoding HCDR1, sequence number 171 is a nucleotide sequence encoding HCDR2, sequence number 172 is a nucleotide sequence encoding HCDR3, sequence number 173 is a nucleotide sequence encoding LCDR1, sequence number 174 is a nucleotide sequence encoding LCDR2, and sequence number 175 is a nucleotide sequence encoding LCDR3. A specific example of a nucleotide sequence encoding a single-chain antibody comprising sequence numbers 170 to 175 is shown in sequence number 176. Based on this sequence number 176, the nucleotide sequence encoding the single-chain antibody after changing FR3 is shown in sequence numbers 177 to 179.

[0068] Preferably, the nucleotide sequence encoding the single-chain antibody binding to CD19 includes the nucleotide sequence shown in sequence numbers 180-185. Sequence number 180 is a nucleotide sequence encoding HCDR1, sequence number 181 is a nucleotide sequence encoding HCDR2, sequence number 182 is a nucleotide sequence encoding HCDR3, sequence number 183 is a nucleotide sequence encoding LCDR1, sequence number 184 is a nucleotide sequence encoding LCDR2, and sequence number 185 is a nucleotide sequence encoding LCDR3. A specific example of a nucleotide sequence encoding a single-chain antibody comprising sequence numbers 180-185 is shown in sequence number 186. Based on this sequence number 186, the nucleotide sequence encoding the single-chain antibody after changing FR3 is shown in sequence numbers 187-205.

[0069] The nucleotide sequence encoding the single-chain antibody preferably includes a nucleotide sequence encoding a peptide linker between the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region. In a single-chain antibody, the peptide linker is a portion connected to the light chain variable region and the heavy chain variable region. The amino acid sequence of the peptide linker is not particularly limited, and a sequence of 15-20 amino acids in length containing a repeat of an amino acid sequence consisting of glycine residues and serine residues (e.g., GGGGS: sequence number 206) is generally used. For example, the nucleotide sequence encoding the single-chain antibody may include, from the 5' side, a nucleotide sequence encoding a light chain variable region, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding a heavy chain variable region. Alternatively, the nucleotide sequence encoding the single-chain antibody may also include, from the 5' side, a nucleotide sequence encoding a heavy chain variable region, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding a light chain variable region.

[0070] In CAR, the transmembrane domain is a site for fixing CAR on the cell membrane of immune cells. The transmembrane domain of CAR comes from a transmembrane protein. For example, in the nucleic acid molecule of the present embodiment, a segment having a nucleotide sequence encoding a transmembrane domain (hereinafter, also referred to as a "transmembrane segment") may include a nucleotide sequence encoding the full length of a transmembrane protein. Alternatively, the transmembrane segment may also include a nucleotide sequence encoding a portion of a transmembrane protein as long as it maintains its function as a transmembrane domain. A portion of a transmembrane protein preferably includes all or part of the transmembrane region of the protein. Transmembrane proteins are not particularly limited, and examples thereof include the α chain of T cell receptors, the β chain of T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB (also referred to as CD137), ICOS (Inducible T-cell co-stimulator) and GITR (Glucocorticoid-induced TNF receptor). Preferably, the transmembrane segment comprises a nucleotide sequence encoding a transmembrane region of any protein selected from the group of these transmembrane proteins. Among them, a nucleotide sequence encoding a transmembrane region of CD8α or CD28 is particularly preferred. A specific example of a nucleotide sequence encoding a transmembrane region of CD28 is shown in SEQ ID NO: 207.

[0071] The nucleic acid molecule of the present embodiment preferably includes a segment encoding a hinge domain between the nucleotide sequence encoding the antigen binding region and the transmembrane segment (hereinafter also referred to as a "hinge segment"). In CAR, the hinge domain can give the extracellular domain a length for the antigen binding region to approach the antigen and flexibility for avoiding steric obstacles. The hinge domain is also referred to as a spacer. The hinge domain of CAR can be derived from, for example, a transmembrane protein or IgG. Specifically, in the nucleic acid molecule of the present embodiment, the hinge segment may include all of the nucleotide sequences encoding the extracellular region of the transmembrane protein or all of the constant region of IgG. Alternatively, as long as the hinge segment maintains the function as a hinge domain, it may also include a nucleotide sequence encoding a part of the extracellular region of the transmembrane protein or a part of the constant region of IgG. Hereinafter, the part of the extracellular region of the membrane protein that can be used as a hinge domain is also referred to as a "hinge region". Preferably, the hinge segment includes a nucleotide sequence encoding any one of the regions selected from the group consisting of the light chain constant region of IgG, the CD8α hinge region, and the CD28 hinge region. IgG is preferably IgG4. A specific example of a nucleotide sequence encoding an IgG light chain constant region is shown in SEQ ID NO: 208. A specific example of a nucleotide sequence encoding a CD28 hinge domain is shown in SEQ ID NO: 209.

[0072] In CAR, the intracellular domain includes a signal transduction domain. The signal transduction domain of CAR is a site for inducing signal transduction that activates immune cells expressing the CAR when the antigen binding region binds to the antigen. In the nucleic acid molecule of the present embodiment, the segment encoding the intracellular domain (hereinafter, also referred to as the "intracellular segment") may include a nucleotide sequence encoding a signal transduction domain. The signal transduction domain of CAR may be derived from a membrane protein having an intracellular region such as a cell membrane receptor or a transmembrane protein. A signal transduction domain may exist in the intracellular region of the membrane protein. That is, the intracellular domain of CAR may include a signal transduction domain of a membrane protein. Therefore, the intracellular segment may include a nucleotide sequence encoding the full length of the membrane protein. Alternatively, the intracellular segment may also include a nucleotide sequence encoding a portion of the above-mentioned membrane protein as long as it maintains its function as a signal transduction domain. A portion of the membrane protein preferably includes all or part of the signal transduction domain of the protein. The membrane protein is not particularly limited, and examples thereof include CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ, and FcRβ. Preferably, the intracellular segment comprises a nucleotide sequence encoding a signal transduction domain of at least one protein selected from the group of these membrane proteins. Among them, CD3ζ is preferred. A specific example of a nucleotide sequence encoding a signal transduction domain of CD3ζ is shown in SEQ ID NO: 210.

[0073] The intracellular segment preferably has a nucleotide sequence encoding a costimulatory domain in addition to the nucleotide sequence encoding the above-mentioned signal transduction domain. It is known that the signal from the costimulatory domain is transmitted to immune cells (especially T cells) together with the signal from the above-mentioned signal transduction domain, thereby improving the proliferation ability, cytotoxic activity, survival rate, etc. of the immune cells. The costimulatory domain of CAR can be derived from a membrane protein having an intracellular region such as a cell membrane receptor or a transmembrane protein. A costimulatory domain may exist in the intracellular region of the membrane protein. That is, the intracellular domain of CAR may include a costimulatory domain of a membrane protein. Therefore, the intracellular segment may include a nucleotide sequence encoding the full length of the membrane protein. Alternatively, the intracellular segment may also include a nucleotide sequence encoding a portion of the above-mentioned membrane protein as long as it maintains the function as a costimulatory domain. A portion of the membrane protein preferably includes all or part of the costimulatory domain of the protein. Membrane proteins are not particularly limited, and examples thereof include 4-1BB (also referred to as CD137), CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, and ICOS. Preferably, the intracellular segment comprises a nucleotide sequence encoding a co-stimulatory domain of at least one protein selected from the group of these membrane proteins. Among them, at least one selected from the group consisting of 4-1BB, CD28, and GITR is preferred. A specific example of a nucleotide sequence encoding a co-stimulatory domain of CD28 is shown in sequence number 211. A specific example of a nucleotide sequence encoding a co-stimulatory domain of GITR is shown in sequence number 212.

[0074] In the intracellular domain segment, the order of the nucleotide sequence encoding the signal transduction domain and the nucleotide sequence encoding the costimulatory domain is not particularly limited. For example, the nucleotide sequence encoding the intracellular domain may include the nucleotide sequence encoding the signal transduction domain and the nucleotide sequence encoding the costimulatory domain in sequence from the 5' side. Alternatively, the nucleotide sequence encoding the intracellular domain may also include the nucleotide sequence encoding the costimulatory domain and the nucleotide sequence encoding the signal transduction domain in sequence from the 5' side.

[0075] The nucleic acid molecule of the present embodiment may include various nucleotide sequences in addition to the nucleotide sequence encoding CAR as needed. As such nucleotide sequences, for example, leader sequences, recognition sequences for restriction enzymes, nucleotide sequences encoding peptide tags, stop codons, etc. can be cited. The peptide tag can be appropriately selected from well-known peptide tags such as histidine tags, glutathione-S-transferase (GST) tags, and FLAG (registered trademark) tags.

[0076] Another embodiment of the present invention relates to a vector comprising the nucleic acid molecule of 1. above. Specifically, the vector of this embodiment can be in the form of integrating the nucleic acid molecule of this embodiment into a known vector. The type of vector is not particularly limited, and examples thereof include plasmid vectors, viral vectors, etc. The vector can be linear or circular. The type of plasmid vector is not particularly limited, and examples thereof include expression vectors, vectors for making viral vectors, transposon vectors, cloning vectors, etc. An expression vector is a vector that can express a protein encoded by a nucleotide sequence of a nucleic acid molecule integrated into the vector in an appropriate host cell such as a mammalian cell, an insect cell, a yeast, or an Escherichia coli. A transposon vector is a vector that can integrate a nucleic acid molecule integrated into a transposon vector into the genome of a host cell by being introduced into an appropriate host together with an expression vector in which a gene encoding a transposase is integrated.

[0077] The type of viral vector is not particularly limited, and examples thereof include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, Epstein-Barr virus (EBV) vectors, etc. The viral vector is preferably replication-deficient so that the virus does not replicate itself in infected cells.

[0078] The vector may contain appropriate control sequences as required, such as promoter sequences, operator sequences, enhancer sequences, nucleotide sequences encoding drug resistance markers, and multiple cloning sites.

[0079] As described above, the nucleic acid molecule of the present embodiment can be any one of DNA and RNA. Compared with RNA, DNA is a stable substance, and various DNA vectors are commercially available. Therefore, the nucleic acid molecule of the present embodiment as DNA is advantageous in terms of easy storage and handling. It is known that protein-encoding RNA can express the protein without being affected by the transcriptional regulation process when introduced into cells. Therefore, the nucleic acid molecule of the present embodiment as RNA is advantageous in terms of being able to express rapid CAR in immune cells.

[0080] 2.CAR Another embodiment of the present invention relates to CAR. The CAR of this embodiment is a protein molecule encoded by a nucleotide sequence possessed by the nucleic acid molecule described in 1. above. The CAR of this embodiment is a protein produced by a genetic engineering method that includes an extracellular domain, a transmembrane domain, and an intracellular domain in sequence from the N-terminal side. The extracellular domain includes an antigen binding region comprising a light chain variable region and a heavy chain variable region. The CAR of this embodiment is characterized in that at least 3 amino acid residues in the light chain FR3 of the antigen binding region are basic amino acid residues. That is, the CAR of this embodiment comprises an antigen binding region in which at least 3 amino acid residues of the light chain FR3 are basic amino acid residues. The antigen binding region is the same as the above-mentioned modified antigen binding region. Hereinafter, the antigen binding region possessed by the CAR of this embodiment is also referred to as a “modified antigen binding region”. The CAR of this embodiment can also be said to be a mutant of the CAR having the original antigen binding region. The CAR of this embodiment is preferably expressed in immune cells and fixed to the cell membrane of immune cells. In immune cells expressing the CAR, the production of cytokines is suppressed compared to immune cells expressing the CAR containing the original antigen binding region. Furthermore, it is preferred that the cytotoxic effect of the immune cells on tumor cells be maintained, and it is further preferred that the cytotoxic effect be enhanced.

[0081] In the CAR of the present embodiment, at least 3 basic amino acid residues of the light chain FR3 are derived from the above-mentioned codon changes. In the light chain FR3 of the altered antigen-binding region, at least 3 basic amino acid residues derived from the codon changes may all be arginine residues, or may all be lysine residues. Alternatively, at least 3 basic amino acid residues derived from the codon changes may be part of them as arginine residues, and the rest as lysine residues.

[0082] The amino acid residue before at least 3 amino acid residues in the light chain FR3 become basic amino acid residues is a neutral amino acid residue or an acidic amino acid residue, preferably a neutral amino acid residue. That is, at least 3 basic amino acid residues in the light chain FR3 of the altered antigen-binding region are residues changed by at least 3 residues selected from the neutral amino acid residue and / or acidic amino acid residue in the light chain FR3 of the original antigen-binding region.

[0083] In the altered light chain FR3 of the antigen-binding region, the number of at least three basic amino acid residues derived from the change of codons is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. The number of at least three basic amino acid residues derived from the change of codons in the altered light chain FR3 of the antigen-binding region is preferably 3 or more and 6 or less, more preferably 3 or more and 5 or less. As described above, the at least three basic amino acid residues in the light chain FR3 of the altered antigen-binding region are preferably located at the position of the amino acid residue selected from the group consisting of 57, 58, 59, 60, 61, 62, 63, 65, 67, 70, 72, 74, 76, 77, 79, 80 and 81 of the light chain variable region.

[0084] In the altered antigen-binding region, at least three amino acid residues selected from the group consisting of amino acid residues at positions 60, 63, 65, 67, 70, 72, 74, 76, 77, 79 and 81 of the light chain variable region are preferably basic amino acid residues derived from codon changes. More preferably, more than three and less than six amino acid residues selected from the above group are basic amino acid residues. Particularly preferably, more than three and less than five amino acid residues selected from the above group are basic amino acid residues. For example, the amino acid residues described in any one of 1) to 21) below are basic amino acid residues derived from codon changes.

[0085] 1) amino acid residues 60, 63, and 65 in the light chain variable region; 2) amino acid residues 60, 63, and 76 in the light chain variable region; 3) amino acid residues 60, 74, and 76 of the light chain variable region; 4) amino acid residues 63, 65, and 67 of the light chain variable region; 5) amino acid residues 63, 65, and 70 of the light chain variable region; 6) amino acid residues 63, 65, and 72 of the light chain variable region; 7) amino acid residues 63, 67 and 70 of the light chain variable region; 8) amino acid residues 63, 67 and 72 of the light chain variable region; 9) amino acid residues 63, 70 and 72 of the light chain variable region; 10) amino acid residues 65, 67 and 70 of the light chain variable region; 11) amino acid residues 65, 67 and 72 of the light chain variable region; 12) amino acid residues 65, 70 and 72 of the light chain variable region; 13) amino acid residues 67, 70 and 72 of the light chain variable region; 14) amino acid residues 70, 72, and 74 of the light chain variable region; 15) amino acid residues 77, 79 and 81 of the light chain variable region; 16) amino acid residues 63, 65, 67 and 70 of the light chain variable region; 17) amino acid residues 63, 65, 67 and 72 of the light chain variable region; 18) amino acid residues 63, 65, 70 and 72 of the light chain variable region; 19) amino acid residues 63, 67, 70 and 72 of the light chain variable region; 20) amino acid residues 65, 67, 70 and 72 of the light chain variable region; and 21) Amino acid residues 63, 65, 67, 70 and 72 in the light chain variable region.

[0086] In the altered antigen-binding region, the amino acid residues at at least three positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region and the amino acid residue at at least one position selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region are preferably basic amino acid residues.

[0087] When more than 3 and less than 6 amino acid residues in the altered light chain FR3 of the antigen-binding region are basic amino acid residues, the more than 3 and less than 6 amino acid residues preferably include: amino acid residues at 3, 4 or 5 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region, and amino acid residues at 1, 2 or 3 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region.

[0088] When 3 or more and 5 or less amino acid residues in the altered light chain FR3 of the antigen-binding region are basic amino acid residues, the 3 or more and 5 or less amino acid residues preferably include: amino acid residues at 3 or 4 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region, and amino acid residues at 1 or 2 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region.

[0089] In the altered antigen-binding region, the amino acid residues at at least three positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region and the amino acid residue at at least one position selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region are preferably basic amino acid residues.

[0090] When more than 3 and less than 6 amino acid residues in the altered light chain FR3 of the antigen-binding region are basic amino acid residues, the more than 3 and less than 6 amino acid residues preferably include: amino acid residues at 3, 4 or 5 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region, and amino acid residues at 1, 2 or 3 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region.

[0091] When more than 3 and less than 5 amino acid residues in the altered light chain FR3 of the antigen-binding region are basic amino acid residues, the more than 3 and less than 5 amino acid residues preferably include: amino acid residues at 3 or 4 positions selected from the group consisting of positions 60, 74, 76, 77, 79 and 81 of the light chain variable region, and amino acid residues at 1 or 2 positions selected from the group consisting of positions 63, 65, 67, 70 and 72 of the light chain variable region.

[0092] In the altered antigen-binding region, when an amino acid residue other than amino acid residues at positions 60, 63, 65, 67, 70, 72, 74, 76, 77, 79 and 81 of the light chain variable region is altered, the altered amino acid residue is preferably an amino acid residue at a position selected from the group consisting of positions 57, 58, 59, 61 and 62.

[0093] In the CAR of the present embodiment, the extracellular domain includes an antigen binding region comprising a light chain variable region and a heavy chain variable region. As an antigen binding region, for example, a single-chain antibody can be cited. The antigen binding region may include a portion or all of the constant region of the antibody. The constant region may be any constant region of the heavy chain and the light chain, preferably a light chain constant region. In the antigen binding region, the order of the light chain variable region and the heavy chain variable region is not particularly limited. For example, the antigen binding region may include a light chain variable region and a heavy chain variable region in sequence from the N-terminal side. Alternatively, the antigen binding region may also include a heavy chain variable region and a light chain variable region in sequence from the N-terminal side. In the antigen binding region, part or all of the light chain constant region is preferably included after the light chain variable region. In the antigen binding region, part or all of the heavy chain constant region is preferably included after the heavy chain variable region.

[0094] In the CAR of this embodiment, the antigen binding region may include a single-chain antibody that binds to any one of the antigens present on the surface of the above-mentioned tumor cells and the antigens present in the tumor cells, or is composed of a single-chain antibody thereof. Preferably, the antigen binding region includes a single-chain antibody that binds to a complex of a peptide from MAGE-A4 and HLA-A2, a complex of a peptide from PRAME and HLA-A24, CD19 or CEA, or is composed of the single-chain antibody.

[0095] Preferably, the amino acid sequence of the single-chain antibody that binds to the complex of the peptide derived from MAGE-A4 and HLA-A2 comprises the amino acid sequence shown in sequence numbers 213 to 218. Sequence number 213 is the amino acid sequence of HCDR1, sequence number 214 is the amino acid sequence of HCDR2, sequence number 215 is the amino acid sequence of HCDR3, sequence number 216 is the amino acid sequence of LCDR1, sequence number 217 is the amino acid sequence of LCDR2, and sequence number 218 is the amino acid sequence of LCDR3. A specific example of the amino acid sequence of the single-chain antibody comprising sequence numbers 213 to 218 is shown in sequence number 219. The amino acid sequence of the single-chain antibody in which FR3 is modified based on sequence number 219 is shown, for example, in sequence numbers 220 to 229.

[0096] Preferably, the amino acid sequence of the single-chain antibody that binds to the complex of the peptide derived from PRAME and HLA-A24 comprises the amino acid sequence shown in sequence numbers 230 to 235. Sequence number 230 is the amino acid sequence of HCDR1, sequence number 231 is the amino acid sequence of HCDR2, sequence number 232 is the amino acid sequence of HCDR3, sequence number 233 is the amino acid sequence of LCDR1, sequence number 234 is the amino acid sequence of LCDR2, and sequence number 235 is the amino acid sequence of LCDR3. A specific example of the amino acid sequence of the single-chain antibody comprising sequence numbers 230 to 235 is shown in sequence number 236. The amino acid sequence of the single-chain antibody with FR3 modified based on sequence number 236 is shown, for example, in sequence numbers 237 to 239.

[0097] Preferably, the amino acid sequence of the single-chain antibody that binds to CD19 comprises the amino acid sequence shown in sequence numbers 240-245. Sequence number 240 is the amino acid sequence of HCDR1, sequence number 241 is the amino acid sequence of HCDR2, sequence number 242 is the amino acid sequence of HCDR3, sequence number 243 is the amino acid sequence of LCDR1, sequence number 244 is the amino acid sequence of LCDR2, and sequence number 245 is the amino acid sequence of LCDR3. A specific example of the amino acid sequence of the single-chain antibody comprising sequence numbers 240-245 is shown in sequence number 246. The amino acid sequence of the single-chain antibody with FR3 modified based on sequence number 246 is shown, for example, in sequence numbers 247-265.

[0098] Preferably, the single-chain antibody comprises a peptide linker between the light chain variable region and the heavy chain variable region. The peptide linker is as described above. For example, the single-chain antibody can sequentially comprise the light chain variable region, the peptide linker and the heavy chain variable region from the N-terminal side. Alternatively, the single-chain antibody can also sequentially comprise the heavy chain variable region, the peptide linker and the light chain variable region from the N-terminal side.

[0099] In the CAR of this embodiment, the transmembrane domain may include the full length of the transmembrane protein. Alternatively, as long as its function is maintained, the transmembrane domain may also include a portion of the transmembrane protein. A portion of the transmembrane protein preferably includes all or part of the transmembrane region of the protein. As transmembrane proteins that can be used in the transmembrane domain, the proteins exemplified in 1. above can be cited. Preferably, the transmembrane domain is, for example, a transmembrane region of any protein selected from the group consisting of the α chain of the T cell receptor, the β chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB (also referred to as CD137), ICOS and GITR. Among them, the transmembrane region of CD8α or CD28 is preferred. A specific example of the amino acid sequence of the transmembrane region of CD28 is shown in sequence number 266.

[0100] The CAR of this embodiment preferably includes a hinge domain between the antigen binding region and the transmembrane domain. The hinge domain may include the entire extracellular region of a transmembrane protein or the entire constant region of IgG. Alternatively, as long as its function is maintained, the hinge domain may also include a portion of the extracellular region of a transmembrane protein or a portion of the constant region of IgG. As proteins that can be used in the hinge domain, the proteins exemplified in 1. above can be cited. Preferably, the hinge domain is, for example, any one region selected from the group consisting of the light chain constant region of IgG, the hinge domain of CD8α, and the hinge domain of CD28. IgG is preferably IgG4. Specific examples of the amino acid sequences of the IgG light chain constant regions are shown in sequence number 267. Specific examples of the amino acid sequences of the CD28 hinge domain are shown in sequence number 268.

[0101] In the CAR of the present embodiment, the intracellular domain may include the full length of a membrane protein having an intracellular region. Alternatively, as long as the function as a signal transduction domain is maintained, the intracellular domain may also include a portion of the above-mentioned membrane protein. The signal transduction domain may be present in the intracellular region of a membrane protein. Preferably, a portion of a membrane protein having an intracellular region includes all or part of the signal transduction domain of the protein. As membrane proteins that can be used in the intracellular domain, the proteins exemplified in 1. above can be cited. Preferably, the intracellular domain, for example, includes a signal transduction domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ and FcRβ. Among them, the signal transduction domain of CD3ζ is preferred. A specific example of the amino acid sequence of the signal transduction domain of CD3ζ is shown in sequence number 269.

[0102] In the CAR of the present embodiment, the intracellular domain preferably has a costimulatory domain in addition to the above-mentioned signal transduction domain. The intracellular domain may include the full length of a membrane protein having an intracellular region. Alternatively, as long as the function as a costimulatory domain is maintained, the intracellular domain may also include a portion of the above-mentioned membrane protein. The costimulatory domain may be present in the intracellular region of each of the above-mentioned proteins. Preferably, a portion of a membrane protein having an intracellular region includes all or part of the costimulatory domain of the protein. As membrane proteins that can be used in the costimulatory domain, the proteins exemplified in 1. above can be cited. Preferably, the costimulatory domain, for example, includes a costimulatory domain of at least one protein selected from the group consisting of 4-1BB (also referred to as CD137), CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 and ICOS. Among them, the costimulatory domains of 4-1BB, CD28 and GITR are preferred. A specific example of the amino acid sequence of the costimulatory domain of CD28 is shown in SEQ ID NO: 270. A specific example of the amino acid sequence of the costimulatory domain of GITR is shown in SEQ ID NO: 271.

[0103] In the intracellular domain, the order of the signal transduction domain and the costimulatory domain is not particularly limited. For example, the intracellular domain may include the signal transduction domain and the costimulatory domain in sequence from the N-terminal side. Alternatively, the intracellular domain may also include the costimulatory domain and the signal transduction domain in sequence from the N-terminal side.

[0104] The CAR of this embodiment may contain additional oligopeptides or polypeptides as needed. As such oligopeptides and polypeptides, signal peptides, peptide tags, etc. can be cited. The peptide tag can be appropriately selected from well-known peptide tags such as histidine tags, GST tags, FLAG (registered trademark) tags, etc.

[0105] The CAR of the present embodiment can be generated by a protein expression system using the nucleic acid molecule of the present embodiment. The protein expression system can be an expression system using a host cell or a cell-free protein synthesis system. In the generation of the expression system using a host cell, for example, by introducing the nucleic acid molecule of the present embodiment integrated into an expression vector suitable for the host cell to be used into the host cell, the CAR of the present embodiment can be expressed. As a cell-free protein synthesis system, for example, a synthesis system from wheat germ, a synthesis system from Escherichia coli, a recombinant cell-free protein synthesis system, etc. can be cited. In the case of generating CAR in a host cell, the host cell can be dissolved with a solution containing an appropriate solubilizing agent to free CAR in the solution. In a cell-free protein synthesis system, the synthesized CAR is contained in the reaction solution. CAR free in the liquid can be recovered by known methods such as column chromatography. For example, in the case where the generated CAR has a histidine tag or a GST tag as a peptide tag, it can be recovered by affinity chromatography using a carrier containing Ni-NTA (aminotriacetic acid that forms a chelate with nickel ions) or glutathione. If necessary, the recovered CAR can be purified by a known method such as gel filtration and dialysis.

[0106] 3. CAR-containing immune cells Another embodiment of the present invention relates to an immune cell comprising a CAR. The immune cell comprising a CAR of this embodiment is an immune cell that expresses the CAR of 2. above by introducing the nucleic acid molecule of 1. above into the immune cell. The details of CAR and nucleic acid molecules are as described above. As immune cells before the introduction of the nucleic acid molecule of 1. above, for example, immune cells collected from mammals including humans, immune cells modulated from stem cells, and immune cells established as cell lines can be cited. Specifically, immune cells obtained by blood collection of leukocyte components, immune cells separated from blood, etc. can be exemplified.

[0107] As immune cells separated or isolated from biological samples, for example, peripheral blood mononuclear cells (PBMC), T cells and natural killer (NK) cells can be cited. Among them, T cells and NK cells are preferred. T cells include CD8 positive T cells, CD4 positive T cells, cytotoxic T cells, helper T cells, regulatory T cells and tumor infiltrating lymphocytes. Preferred T cells are CD8 positive T cells and cytotoxic T cells. As cell strains of immune cells, cell strains derived from lymphocytes are preferred, for example, Jurkat cells, MOLT-4 cells, U-937 cells, etc. can be cited. Hereinafter, T cells expressing CAR will also be referred to as "CAR-T cells".

[0108] The method for introducing the nucleic acid molecule of 1. above into immune cells is not particularly limited and can be appropriately selected from known gene introduction methods. Examples of gene introduction methods include liposome transfection, electroporation, calcium phosphate method, gene introduction based on cationic polymers, gene introduction based on viral vectors, gene introduction based on transposons, and the like. In liposome transfection and cationic polymer-based gene introduction, commercially available transfection reagents such as FuGENE (registered trademark) and JetPEI (registered trademark) can be used.

[0109] The immune cells containing the CAR of the present embodiment can be cultured in the same manner as the immune cells before the nucleic acid molecules of the present embodiment are introduced. The culture medium, serum, additives, etc. can be appropriately determined according to the type of immune cells used. As a culture medium, for example, MEM, DMEM, RPMI-1640, etc. can be mentioned. In the case where the immune cells are lymphocytes, for example, commercially available lymphocyte culture media such as GT-T502, GT-T503, and GT-T551 (Takara Bio Co., Ltd.) can be used. As serum, for example, fetal bovine serum (FBS), human AB type serum, etc. can be mentioned. As additives, for example, L-glutamine, insulin, IL-2, etc. can be mentioned. As culture conditions for immune cells, for example, conditions under a 5% CO2 atmosphere at 37°C can be mentioned.

[0110] In the immune cells containing CAR of the present embodiment, CAR is expressed as a transmembrane protein. When the extracellular domain of the CAR binds to the antigen of the tumor cell, the immune cell is activated by the signal from the intracellular domain of the CAR. In addition, the activated immune cells containing CAR of the present embodiment release cytotoxic proteins (such as perforin, granzyme, etc.) and anti-tumor cytokines (such as tumor necrosis factor (TNF)-α, lymphokines, etc.), and induce the expression of cell surface molecules such as Fas ligand that cause cell death, and exert a cell killing effect. Preferably, the immune cells containing CAR of the present embodiment maintain the cell killing effect compared with the immune cells containing CAR having the original antigen binding region, and more preferably the cell killing effect is improved. The cell killing effect based on the immune cells containing CAR can be investigated by known methods such as cytotoxicity determination. Specifically, the method of Example 3 described later can be exemplified. In addition, as in Example 4 described later, the immune cells containing CAR can also be administered to non-human animals transplanted with tumor cells, and the size of the tumor can be measured to evaluate the cell killing effect. On the other hand, the immune cells containing CAR of the present embodiment have a reduced release of cytokines (e.g., IFNγ, granulocyte-macrophage colony stimulating factor (GM-CSF), etc.) that are the cause of CRS compared to immune cells containing CAR having the original antigen binding region. The release of cytokines by immune cells containing CAR can be investigated by known methods such as enzyme-linked immunosorbent assay (ELISA). Specifically, the method of Example 5 described later can be exemplified.

[0111] 4. Preparation method of CAR immune cells Another embodiment of the present invention relates to a method for preparing immune cells containing CAR (hereinafter, also referred to as "the preparation method of this embodiment"). In the preparation method of this embodiment, the nucleic acid molecule of 1. above is introduced into an immune cell, and the immune cell is made to express the CAR of 2. above, thereby preparing an immune cell containing the CAR of 3. above. The details of CAR and nucleic acid molecules are as described above. In addition, the details of the immune cells before the introduction of the nucleic acid molecules of 1. above are as described above. PBMC, T cells and NK cells are preferred. When the immune cells containing CAR obtained by the preparation method of this embodiment are transplanted into a living body, it is preferred to use immune cells separated or isolated from the living body sample collected from the living body itself or from other living bodies of the same species as the living body.

[0112] The method for introducing the nucleic acid molecule of 1. above into immune cells is as described above. Preferably, after the nucleic acid molecule is introduced into the immune cell, the immune cell is cultured for a prescribed period. The method for culturing immune cells is as described above. The prescribed period may be a period during which CAR is expressed at least in the immune cell. The period until CAR is expressed is determined according to the method for introducing the nucleic acid molecule, for example, more than 3 hours and less than 72 hours, preferably more than 6 hours and less than 48 hours. In the case where the method for introducing the nucleic acid molecule is a method for stably expressing CAR in immune cells, the prescribed period may be a period sufficient to proliferate immune cells expressing CAR. Such a period is not particularly limited, for example, more than 48 hours and less than 20 days, preferably more than 72 hours and less than 14 days.

[0113] By introducing the nucleic acid molecule of 1. above into the immune cell, the CAR of 2. above is expressed in the immune cell. If necessary, it can be confirmed that the immune cell expresses CAR. The expression of CAR in the immune cell can be confirmed by a known protein detection method such as ELISA, flow cytometry, immunoprecipitation, polyacrylamide gel electrophoresis, and Western blotting.

[0114] 5. Pharmaceutical compositions Another embodiment of the present invention relates to a pharmaceutical composition for treating malignant tumors comprising immune cells containing CAR (hereinafter also referred to as "the pharmaceutical composition of this embodiment"). In the pharmaceutical composition of this embodiment, as an active ingredient, an immune cell containing the CAR of 3. above is included. The details of CAR and the immune cells containing it are as described above. The pharmaceutical composition of this embodiment may also contain a pharmaceutically acceptable additive. As such additives, aqueous media, D-glucose, dextran, serum albumin, dimethyl sulfoxide (DMSO), etc. for stably preserving immune cells can be cited. As the above-mentioned aqueous medium, for example, physiological saline, phosphate buffered saline (PBS), composite electrolyte solution, etc. can be cited. The pharmaceutical composition of this embodiment is preferably administered to the patient non-orally. That is, the pharmaceutical composition is preferably in a form suitable for non-oral administration, such as injection, infusion, etc.

[0115] The malignant tumor that is the object of treatment of the pharmaceutical composition of this embodiment is a tumor containing tumor cells with antigens recognized by CAR. Malignant tumors can be blood cancers or solid cancers. For example, acute leukemia (acute myeloid leukemia, B-cell acute lymphocytic leukemia, acute monocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, etc.), chronic leukemia (chronic myeloid leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia, etc.), lymphoma (diffuse large cell B cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma, lymphoplasmacytic lymphoma, marginal zone lymphoma, etc.), multiple myeloma, melanoma, breast cancer, prostate cancer, bladder cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, osteosarcoma, neuroblastoma, etc.

[0116] 6. Treatment Methods Another embodiment of the present invention relates to a method for treating a malignant tumor using an immune cell comprising the CAR of 3. above (hereinafter also referred to as "the method of treating a malignant tumor of the present embodiment"). It includes a step of administering the above-mentioned pharmaceutical composition to a patient with a malignant tumor. The malignant tumor to be treated is as described above.

[0117] In the administration step, the immune cells are preferably administered to the patient non-orally. Examples of non-oral administration include intravenous, intra-arterial, intramuscular, intraperitoneal, and subcutaneous administration. Among them, intravenous administration is preferred.

[0118] The administration of the pharmaceutical composition of this embodiment can be appropriately determined according to the type of cancer, the condition of the patient, age, weight, etc. The administration amount is, for example, 1×10 immune cells containing CAR per time for an adult weighing 50 kg or more. 4 cells and above 1×10 10 cells or less, preferably 1×10 5 cells and above 1×10 9 cells or less, more preferably 1×10 6 cells and above 5×10 8 cells or less. The number of administrations may be only one administration or multiple administrations. After administration, the immune cells containing CAR survive and proliferate in the patient's body, and the number of administrations is usually one. However, if it is determined that the survival and proliferation of immune cells are insufficient, multiple administrations may be performed. The administration interval may be, for example, 1-4 times a day, every week, every 10-30 days, every month, every 3-6 months, every year, etc.

[0119] The pharmaceutical composition of this embodiment can be used in combination with other anticancer agents. The administration of other anticancer agents and the administration of the pharmaceutical composition of 5. above can be on the same day or on another day. As other anticancer agents, alkylating drugs such as cyclophosphamide, metabolic antagonists such as pentostatin, molecular targeted drugs such as rituximab, kinase inhibitors such as imatinib, proteasome inhibitors such as bortezomib, calcineurin inhibitors such as cyclosporin, anticancer antibiotics such as idarubicin, plant alkaloids such as irinotecan, platinum preparations such as cisplatin, hormone therapeutic agents such as tamoxifen, immunosuppressive drugs such as nivolumab and pembrolizumab can be cited, but they are not limited to these.

[0120] As treatment before administration of the pharmaceutical composition of this embodiment, lymphocyte removal chemotherapy can be performed to reduce the number of leukocytes in the patient. For example, fludarabine, cyclophosphamide, bendamustine, etc. are used in lymphocyte removal chemotherapy.

[0121] 7. Methods to reduce cytokine production by immune cells Another embodiment of the present invention relates to a method for reducing the production of cytokines by immune cells containing CAR (hereinafter, also referred to as "the reduction method of this embodiment"). In the reduction method of this embodiment, it includes: a process of making the nucleic acid molecule of 1. above; and a process of introducing the nucleic acid molecule of 1. above into immune cells so that the immune cells express the CAR of 2. above. The obtained immune cells containing CAR express CAR containing a modified antigen binding region. Compared with immune cells expressing CAR containing the original antigen binding region, the production of cytokines of immune cells expressing the CAR is reduced. On the other hand, compared with immune cells expressing CAR containing the original antigen binding region, the cell killing effect of immune cells expressing CAR containing the modified antigen binding region is maintained or improved. The reduction method of this embodiment can help to further reduce the side effects caused by cytokines in the treatment method using immune cells expressing CAR. The details of the nucleic acid molecule and CAR are as described above. The method of introducing nucleic acid molecules into immune cells and the method of measuring cytokines produced by immune cells containing CAR are as described above.

[0122] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

[0123] Example Example 1: Preparation of CAR-T cells binding to MAGE-A4 / HLA-A2 complex and confirmation of its effect (1) (1) Obtaining a nucleic acid molecule encoding CAR serving as a template Similar to the example of US2020 / 0276237 (which is incorporated herein by reference), two plasmid DNAs for viral vector preparation containing genes encoding CARs having single-chain antibodies that bind to the MAGE-A4 / HLA-A2 complex were obtained. The complex recognized by the single-chain antibody is a complex of MAGE-A4 and HLA-A*02:01. Hereinafter, the CAR encoded by the gene in one plasmid DNA is referred to as "MAGE-A4-zG", and the CAR encoded by the gene in another plasmid DNA is referred to as "MAGE-A4-zG-s1". The amino acid sequence of the peptide from MAGE-A4 in the above-mentioned MAGE-A4 / HLA-A2 complex is GVYDGREHTV (sequence number 1). In order to prepare nucleic acid molecules encoding mutants of MAGE-A4-zG and MAGE-A4-zG-s1 by PCR, the above-mentioned plasmid DNAs are used as templates.

[0124] Reference Figure 2A , the gene encoding MAGE-A4-zG is connected from the 5' side in sequence: a leader sequence (Leader), a nucleotide sequence encoding a heavy chain variable region (VH), a nucleotide sequence encoding a linker (L), a nucleotide sequence encoding a light chain variable region (VL), a nucleotide sequence encoding a light chain constant region (CL), a nucleotide sequence encoding a transmembrane region (CD28TM) of CD28, a nucleotide sequence encoding CD3ζ, and a nucleotide sequence encoding an intracellular region (GITRICD) of GITR. VH, L, VL, and CL constitute a segment having a nucleotide sequence encoding an extracellular domain of CAR. VH, L, and VL constitute a segment having a nucleotide sequence encoding a single-chain antibody that specifically binds to the MAGE-A4 / HLA-A2 complex. CD3ζ and GITRICD constitute a segment having a nucleotide sequence encoding an intracellular domain of CAR. CL is a segment having a nucleotide sequence encoding the hinge domain of CAR.

[0125] The gene encoding MAGE-A4-zG-s1 has the same structure as the gene encoding MAGE-A4-zG, except that a remaining nucleotide sequence is added to the 3' end (3' side of GITRICD) of the gene encoding MAGE-A4-zG. It was confirmed that the search results of the remaining nucleotide sequence in a known database did not match the nucleotide sequence encoding any protein. In addition, the results of computer analysis predicted that the amino acid sequence corresponding to the remaining nucleotide sequence formed a straight-chain polypeptide without any secondary structure.

[0126] MAGE-A4-zG and MAGE-A4-zG-s1 are CARs that contain a single-chain antibody consisting of VH, VL, and a linker connecting them, and CL as an intracellular domain, CD28TM as an extracellular domain, and CD3ζ and GITRICD as transmembrane domains.

[0127] (2) Obtaining Nucleic Acid Molecules Encoding Mutants of MAGE-A4-zG and MAGE-A4-zG-s1 [Reagents] QIAprep Spin Miniprep Kit (QIAGEN) PrimeSTAR (registered trademark) Max Premix (Takara Bio Co., Ltd.) Ligation high ver.2 (Toyobo Co., Ltd.) T4 Polynucleotide Kinase (Toyobo Co., Ltd.) Dpn I (Toyobo Co., Ltd.) Competent high DH5α (Toyobo Co., Ltd.) (2.1) Primer design and PCR Based on the nucleotide sequence of sequence number 3, a primer set for obtaining a polynucleotide encoding CAR was designed, wherein the CAR comprises a single-chain antibody in which the amino acid residues shown in the following a) or b) in MAGE-A4-zG are substituted with arginine residues. In addition, based on the nucleotide sequence of sequence number 9, a primer set for obtaining a polynucleotide encoding CAR was designed, wherein the CAR comprises a single-chain antibody in which the amino acid residues shown in the following a) in MAGE-A4-zG-s1 are substituted with arginine residues.

[0128] a) amino acid residues 63, 65, 67 and 70 of VL as defined by Kabat; and b) Amino acid residues 63, 65, 67 and 72 of VL defined by Kabat Each of the above plasmid DNAs containing genes encoding MAGE-A4-zG and MAGE-A4-zG-s1, respectively, was used as a template DNA. A PCR reaction solution containing the template DNA, a primer set (sequence numbers 77 and 78) and PrimeSTAR (registered trademark) Max Premix was prepared, and a PCR reaction was performed. As a result, a PCR product with a restriction enzyme recognition site (hereinafter referred to as a restriction site) added to the 5' end and the 3' end was obtained. DpnI was added to the obtained PCR product to fragment the template plasmid DNA. Using the PCR product treated with DpnI as a template, a PCR reaction solution containing a mutagenic primer set and PrimeSTAR (registered trademark) Max Premix was prepared, and a PCR reaction was performed. The obtained PCR product, a plasmid DNA for viral vector production (empty vector) treated with restriction enzymes, Ligation high ver.2 and T4 polynucleotide kinase were mixed, and incubated at 16°C for 1 hour to perform a ligation reaction.

[0129] Hereinafter, the CAR of the single-chain antibody in which the amino acid residues shown in a) and b) above in MAGE-A4-zG are replaced with arginine residues will be referred to as "MAGE-A4-zG-m1" and "MAGE-A4-zG-m2", respectively. Hereinafter, the CAR of the single-chain antibody in which the amino acid residue shown in a) above in MAGE-A4-zG-s1 is an arginine residue will be referred to as "MAGE-A4-zG-m1-s1". Table 1 shows the correspondence between the sequence numbers of each mutant and the primer set used for its production. In the table, "F" represents the sequence number of the nucleotide sequence of the forward primer, and "R" represents the sequence number of the nucleotide sequence of the reverse primer. The description of the table is the same for the table of the following examples.

[0130] [Table 1]

[0131] (2.2) Transformation, plasmid DNA extraction and sequencing The solution after the ligation reaction and DH5α were used to obtain the transformant of E. coli by the heat shock method. The single colony on the agar medium was cultured in LB liquid medium containing ampicillin. The plasmid DNA was extracted from the obtained E. coli using the QIAprep Spin Miniprep kit. Each plasmid DNA obtained was sequenced. The sequencing results confirmed that the nucleic acid molecules encoding MAGE-A4-zG and MAGE-A4-zG-s1 mutants were obtained.

[0132] (3) Production of MAGE-A4-zG, MAGE-A4-zG-s1, and T cells expressing their mutants (3.1) Modulation of human lymphocytes PBMCs were isolated from blood provided by healthy donors using Ficoll-Paque (trademark) PLUS (GE Healthcare). The obtained PBMCs were used as human lymphocytes. In addition, the collection and analysis of human peripheral blood and other samples used in this study were carried out in accordance with the Declaration of Helsinki, all in accordance with the protocol approved by the Research Ethics Committee of the School of Medicine of Mie University, and were implemented based on the written consent of the subjects themselves. The collected samples were encrypted so that they could not be identified by the subjects themselves and stored in refrigerators and liquid nitrogen tanks with anti-theft disposal. The personal information of the subjects was anonymized, and strict attention and disposal were implemented in a way that the privacy of the individuals and the results of the genetic analysis were not leaked to the outside.

[0133] (3.2) Isolation and culture of PBMC Anti-CD3 antibody OKT-3 (eBioscience) and RetroNectin (registered trademark) (Takara Bio Co., Ltd.) were added to ACD-A solution (Terumo Corporation) at final concentrations of 5 μg / mL and 25 μg / mL, respectively. 400 μL of the obtained liquid was added to each well of a 12-well plate (Nunc) and allowed to stand overnight at 4°C. The liquid in each well was removed, and the plates were washed three times with PBS to prepare a PBMC culture plate. AB serum (300 μL, Veritastk) was added to the lymphocyte culture medium GT-T503 (50 mL, Takara Bio Co., Ltd.), and human IL-2 (NIPRO) was further added to a final concentration of 300 IU / mL to prepare the PBMC culture medium. The PBMCs obtained in the above (3.1) were cultured at 2.5×10 5 ~3.0×10 5 cells / mL in the prepared culture medium. Add 2 mL of the cell suspension to each well of the PBMC culture plate and culture at 37°C in a CO2 incubator for 3 days. Replace 1 mL of the culture medium on the 3rd day and culture for another day.

[0134] (3.3) Using retrovirus to introduce CAR genes into immune cells (i) Production of retrovirus using packaging cells plat-A The plasmid DNA for producing each viral vector containing MAGE-A4-zG, MAGE-A4-zG-s1 and genes encoding these mutants obtained in (2) above was introduced into packaging cells plat-A using FuGENE (registered trademark) (Promega Corporation) and cultured for 2 days. Then, the culture supernatant was recovered to obtain a liquid containing the viral vector (hereinafter also referred to as "virus liquid").

[0135] (ii) Preparation of retrovirus diluent and washing solution To a mixture of PBS (40 mL) and ACD-A solution (2.4 mL), 25% Albuminar (registered trademark) (CSL Behring Co., Ltd.) was added to a final concentration of 2.5% to prepare a retrovirus diluent. Separately, 25% Albuminar (registered trademark) was added to PBS (39 mL) to a final concentration of 1.5% to prepare a retrovirus washing solution.

[0136] (iii) Preparation of plates for retroviral infection RetroNectin (registered trademark) (Takara Bio Co., Ltd.) was diluted with retrovirus diluent to a final concentration of 20 μg / mL. Add 250 μL of RetroNectin (registered trademark) diluent to each well of a 24-well plate and let stand overnight at 4°C. Remove the liquid in each well and wash twice with retrovirus washing solution. Add the virus solution prepared in (3.1) above to each well, centrifuge at 2000×g, 2 hours, and 32°C, and apply the retrovirus to each well. Remove the virus solution in each well, wash twice with retrovirus washing solution, and prepare a plate for retrovirus infection.

[0137] (iv) Production of CAR-T cells using retroviral infection The PBMCs cultured in the above (3.2) were recovered and 1.3×10 5 cells / mL in PBMC culture medium. 1.5 mL of the cell suspension was added to each well of the retroviral infection plate and centrifuged at 1000×g, 10 minutes, and 32°C. The plate was then placed in a CO2 incubator and the cells were cultured at 37°C. Thus, T cells (CAR-T cells) expressing various CARs including MAGE-A4-zG, MAGE-A4-zG-s1, and their mutants were obtained. CAR-T cells were used for various assays 12 days after being isolated and counted from PBMC.

[0138] (4) Confirmation of the cell killing effect and cytokine secretion of CAR-T cells (4.1) Cytotoxicity assay The various CAR-T cells prepared in (3) above were used as effector cells, and their cytotoxic effects were measured using the N-SPC (registered trademark) non-RI cytotoxicity assay kit (Techno Suzuta Co., Ltd.). As target cells, human melanoma cell lines SK-MEL-37 or NW-MEL-38 were used. These target cells were A2-positive MAGE-A4-positive tumor cells.

[0139] The measurement principle of the above-mentioned measurement kit is as follows. When the BM-HT Reagent included in the above-mentioned measurement kit is added to the target cells, the BM-HT Reagent is hydrolyzed by intracellular esterase to generate HT chelate in the target cells. If the target cells are killed by effector cells, the HT chelate leaks into the culture supernatant. When the Eu Solution included in the above-mentioned measurement kit is added to the culture supernatant containing the HT chelate, a Eu / HT complex is formed. When the complex is excited by a laser, time-resolved fluorescence is generated. The leakage of the HT chelate depends on the cytotoxic activity of the effector cells, so the cell killing effect can be quantitatively measured by measuring the time-resolved fluorescence.

[0140] The target cells were cultured at 1×10 4 cells / mL in the culture medium, and BM-HT Reagent was added. Effector cells (CAR-T cells) and target cells were mixed at a ratio of 3:1 or 1:1 in terms of cell number, added to each well of a 96-well plate, and co-cultured at 37°C for 2 hours. For comparison, two wells (control wells) with target cells added without effector cells were prepared and cultured in the same manner. At 1.5 hours from the start of culture, the Detergent attached to the above kit was added to one of the control wells, and the fluorescence detected from the control well was taken as the maximum fluorescence. In addition, the other control well was untreated, and the fluorescence detected from the control well was taken as the minimum fluorescence. EuSolution (120 μL) was added to the culture supernatant (12 μL) of the co-culture, and after standing at room temperature for 15 minutes, the time-resolved fluorescence was measured.

[0141] (4.2) Determination of IFNγ (i) Sample preparation As effector cells, CAR-T cells containing MAGE-A4-zG-s1 and CAR-T cells containing MAGE-A4-zG-m1-s1 prepared in (3) above were used. For comparison, control cells prepared in (3) above were also used. As target cells, SK-MEL-37 and NW-MEL-38, A2-positive MAGE-A4-positive tumor cells, were used. The effector cells and target cells were 1×10 5 The cells were suspended in a culture medium at 10 cells / mL and added to each well of a 96-well plate, and cultured at 37°C for 12 hours. The culture supernatant was recovered and used as a sample.

[0142] (ii) Preparation of reagents In the measurement of IFNγ in each sample, invitrogen (registered trademark) Human IFN gamma Uncoated ELISA with Plates (Thermo Fisher Scientific) was used. 10× Coating Buffer was diluted 10 times with purified water to prepare Coating Buffer. Capture antibody (anti-human IFNγ antibody) (48 μL) was added to Coating Buffer (12 mL) to prepare a dilution solution of capture antibody. 100 μL of the dilution solution of capture antibody was added to each well of a 96-well flat-bottom plate Costar (registered trademark) 9018 (Corning). The plate was left to stand overnight at 4°C. The solution in the wells was removed, and the wells were washed five times with 0.05% PBS-T (PBS, 0.05% Tween (trademark)-20). 5× Assay Diluent was diluted 5 times with purified water to prepare Assay Diluent. 200 μL of Assay Diluent was added to each well and blocked at room temperature for 1 hour. Remove the Assay Diluent in the wells and wash the wells 5 times with 0.05% PBS-T to obtain a plate coated with capture antibodies. Dissolve recombinant human IFN-γ in Assay Diluent to prepare a solution with a final concentration of 1000 pg / mL. Dilute the solution 2-fold in 7 stages to prepare a standard. Add the detection antibody (biotin-labeled anti-human IFNγ antibody) (48 μL) to the coating buffer (12 mL) to prepare a dilution solution of the detection antibody. Add the enzyme (streptavidin-HRP) (48 μL) to the coating buffer (12 mL) to prepare a dilution solution of the enzyme.

[0143] (iii) Determination of IFNγ The samples and standards were added to the wells of the plate and incubated at room temperature for 2 hours. The wells were washed 5 times with 0.05% PBS-T. 100 μL of the detection antibody dilution was added to each well. The plate was incubated at room temperature for 1 hour. The solution in the wells was removed and the wells were washed 5 times with 0.05% PBS-T. 100 μL of the enzyme dilution was added to each well. The plate was incubated at room temperature for 30 minutes. The solution in the wells was removed and the wells were washed 7 times with 0.05% PBS-T. 100 μL of TMB substrate solution was added to each well. The plate was incubated at room temperature in the dark for 15 minutes. 50 μL of 0.18M H2SO4 was added to each well to stop the reaction, and the absorbance at a wavelength of 450 nm was immediately measured using a microplate reader Model 680 (Bio-Rad). A standard curve was drawn based on the measurement results of the standard. The absorbance of each sample was substituted into the standard curve to obtain the concentration of IFNγ in each sample. The obtained values ​​were used as the IFNγ secretion amounts of each effector cell and control cell.

[0144] (2) Results Table 2 shows the results of cytotoxic activity and IFNγ production when SK-MEL-37 was used as a target cell and T cells containing MAGE-A4-zG or its mutants were used as effector cells. In addition, Table 3 shows the results of cytotoxic activity and IFNγ production when SK-MEL-37 was used as a target cell and T cells containing MAGE-A4-zG-s1 or its mutants were used as effector cells. In the table, "CAR type" is a symbol annotated by the present inventors and others to each CAR, and contains information related to the mutation of CAR. "WT" indicates an unaltered CAR (hereinafter, WT CAR is also referred to as "wild type"). The symbol "R" in "CAR type" indicates a CAR in which the amino acid residue shown in the "mutation position" in the table is substituted with an arginine residue. "NA" in the "Sequence Number" column shows the sequence number of the nucleotide sequence encoding CAR, and "AA" shows the sequence number of the amino acid sequence of CAR. The numerical value of "cytotoxic activity" is shown as a ratio when the ratio of target cells killed by CAR-T cells whose CAR type is WT is set to 1.00. "3:1" and "1:1" indicate the ratio of the number of effector cells to target cells (hereinafter, this ratio is also referred to as "cell mixing ratio"). The numerical value of "IFNγ production" is shown as the ratio when the IFNγ production amount of CAR-T cells whose CAR type is WT is set to 1.00. The description of the table is the same for the table of the following examples.

[0145] As shown in Table 2, the cytotoxic activity of each mutant was as follows, assuming that the cytotoxic activity of T cells containing MAGE-A4-zG was 1.00.

[0146] The cytotoxic activity of T cells containing MAGE-A4-zG-m1 was 1.59 when the cell mixing ratio was 3:1, and 1.14 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-m2 was 1.41 in the case of a cell mixing ratio of 3:1 and was 1.60 in the case of a cell mixing ratio of 1:1.

[0147] Regarding cytotoxic activity, T cells containing MAGE-A4-zG-m1 or MAGE-A4-zG-m2 were higher than T cells containing MAGE-A4-zG.

[0148] On the other hand, the IFNγ production amount of each mutant was as follows, assuming that the IFNγ production amount of T cells containing MAGE-A4-zG was 1.00.

[0149] The IFNγ production of T cells containing MAGE-A4-zG-m1 was 0.67, The IFNγ production level of T cells containing MAGE-A4-zG-m2 was 0.47.

[0150] The IFNγ production amount of T cells containing MAGE-A4-zG-m1 or MAGE-A4-zG-m2 was lower than that of T cells containing MAGE-A4-zG.

[0151] As shown in Table 3, the cytotoxic activity of T cells containing MAGE-A4-zG-m1-s1 was 2.34 in the case of a cell mixing ratio of 3:1, and 2.25 in the case of a cell mixing ratio of 1:1, when the cytotoxic activity of T cells containing MAGE-A4-zG-s1 was set to 1.00. On the other hand, the IFNγ production amount of T cells containing MAGE-A4-zG-m1-s1 was 0.66, when the IFNγ production amount of T cells containing MAGE-A4-zG-s1 was set to 1.00. The cytotoxic activity of T cells containing MAGE-A4-zG-m1-s1 was higher than that of T cells containing MAGE-A4-zG-s1, but the IFNγ production amount was lower than that of T cells containing MAGE-A4-zG-s1.

[0152] [Table 2]

[0153] [Table 3]

[0154] Table 4 shows the cytotoxic activity and IFNγ production when NW-MEL-38 was used as the target cell and T cells containing MAGE-A4-zG-s1 or MAGE-A4-zG-m1-s1 were used as the effector cells. As shown in Table 4, the cytotoxic activity of T cells containing MAGE-A4-zG-s1 was 1.35 when the cell mixing ratio was 3:1, and 1.65 when the cell mixing ratio was 1:1, when NW-MEL-38 was used as the target cell and T cells containing MAGE-A4-zG-s1 or MAGE-A4-zG-m1-s1 were used as the effector cells. As shown in Table 4, the cytotoxic activity of T cells containing MAGE-A4-zG-s1 was 1.00, and the cytotoxic activity of T cells containing MAGE-A4-zG-m1-s1 was 1.35 when the cell mixing ratio was 3:1, and 1.65 when the cell mixing ratio was 1:1. Regarding the cytotoxic activity, T cells containing MAGE-A4-zG-m1-s1 were higher than T cells containing MAGE-A4-zG-s1. On the other hand, the IFNγ production of T cells containing MAGE-A4-zG-m1-s1 was 0.40 when the IFNγ production of T cells containing MAGE-A4-zG-s1 was 1.00. T cells containing MAGE-A4-zG-m1-s1 were lower than T cells containing MAGE-A4-zG-s1.

[0155] [Table 4]

[0156] It is suggested that the immune cells of this embodiment are CAR-T cells that suppress the production of cytokines. Furthermore, the cell killing effect of the CAR-T cells is improved.

[0157] Example 2: Preparation of CAR-T cells binding to the MAGE-A4 / HLA-A2 complex and confirmation of its effect (2) (1) Obtaining nucleic acid molecules encoding the MAGE-A4-zG mutant Using the nucleic acid molecule encoding MAGE-A4-zG prepared in Example 1 as a template DNA, a nucleic acid molecule encoding a mutant different from that in Example 1 was prepared by PCR. Specifically, the method is as follows.

[0158] (1.1) Primer design, PCR and ligation reactions Based on the nucleotide sequence of sequence number 3, a primer set for obtaining a polynucleotide encoding CAR was designed, wherein the CAR comprises a single-chain antibody in which the amino acid residues shown in the following a), c), e), f) or h) in MAGE-A4-zG are replaced by arginine residues. In addition, based on the nucleotide sequence of sequence number 3, a primer set for obtaining a polynucleotide encoding CAR was designed, wherein the amino acid residues shown in the following b), d) or g) in MAGE-A4-zG are replaced by lysine residues. Using these primer sets and template DNA, PCR and ligation reactions were performed in the same manner as in Example 1.

[0159] a) amino acid residues 63, 65 and 72 of VL as defined by Kabat; b) amino acid residues 63, 67 and 70 of VL as defined by Kabat; c) amino acid residues at positions 63, 70 and 72 of VL as defined by Kabat; d) amino acid residues at positions 65, 67 and 70 of VL as defined by Kabat; e) amino acid residues at positions 65, 70 and 72 of VL as defined by Kabat; f) amino acid residues 67, 70 and 72 of VL as defined by Kabat; g) amino acid residues at positions 63, 65, 67, 70 and 72 of VL as defined by Kabat; and h) Amino acid residues 60, 74 and 76 of VL defined by Kabat (1.2) Transformation, plasmid DNA extraction and sequencing The solution after the ligation reaction and DH5α were used to obtain E. coli transformants in the same manner as in Example 1. Then, plasmid DNA was extracted from E. coli cultured in a liquid medium and sequenced in the same manner as in Example 1. The sequencing results confirmed that a nucleic acid molecule encoding a mutant of MAGE-A4-zG was obtained.

[0160] Hereinafter, CARs having a single-chain antibody in which the amino acid residues shown in a), c), e), f) or h) in MAGE-A4-zG are substituted with arginine residues are referred to as "MAGE-A4-zG-m5", "MAGE-A4-zG-m7", "MAGE-A4-zG-m9", "MAGE-A4-zG-m10" and "MAGE-A4-zG-m12", respectively. In addition, CARs having a single-chain antibody in which the amino acid residues shown in b), d) or g) are substituted with lysine residues are referred to as "MAGE-A4-zG-Km6", "MAGE-A4-zG-Km8" and "MAGE-A4-zG-Km11", respectively.

[0161] The correspondence between each mutant and the sequence number of the primer set used for its preparation is shown in Tables 5 and 6. In Table 6, "1st" represents the primer set used to replace the 74th and 76th amino acid residues of VL, and "2nd" represents the primer set used to replace the 60th amino acid residue of VL.

[0162] [Table 5]

[0163] [Table 6]

[0164] (2) Preparation of CAR-T cells expressing MAGE-A4-zG and its mutants In the same manner as in Example 1, a gene encoding MAGE-A4-zG and its mutants was introduced into PBMC separated and cultured from the blood of a healthy donor using a retrovirus to obtain each CAR-T cell.

[0165] (3) Confirmation of the cell killing effect and cytokine secretion of CAR-T cells The cytotoxic activity and IFNγ production of each CAR-T cell were measured in the same manner as in Example 1. SK-MEL-37 was used as the target cell. The determination of MAGE-A4-zG-m5, MAGE-A4-zG-Km6, MAGE-A4-zG-m7, MAGE-A4-zG-Km8, MAGE-A4-zG-m9 and MAGE-A4-zG-m10 was carried out on different dates from the determination of MAGE-A4-zG-Km11 ​​and MAGE-A4-zG-m12. The results are shown in Tables 7 and 8. The symbol "K" included in the "Type of CAR" in the table indicates a CAR in which the amino acid residue shown in the "Variation Position" in the table is substituted with a lysine residue. This is also the same for the tables in the examples below.

[0166] [Table 7]

[0167] [Table 8]

[0168] As shown in Table 7, the cytotoxic activity of each mutant was as follows, assuming that the cytotoxic activity of T cells containing MAGE-A4-zG was 1.00.

[0169] The cytotoxic activity of T cells containing MAGE-A4-zG-m5 was 1.62 when the cell mixing ratio was 3:1 and 1.05 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-Km6 was 1.52 when the cell mixing ratio was 3:1, and 1.61 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-m7 was 1.77 when the cell mixing ratio was 3:1, and 0.99 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-Km8 was 1.47 when the cell mixing ratio was 3:1, and 1.36 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-m9 was 1.61 when the cell mixing ratio was 3:1 and 1.23 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-m10 was 1.77 when the cell mixing ratio was 3:1, and was 0.99 when the cell mixing ratio was 1:1.

[0170] On the other hand, the IFNγ production amount of each mutant was as follows, assuming that the IFNγ production amount of T cells containing MAGE-A4-zG was 1.00.

[0171] The IFNγ production of T cells containing MAGE-A4-zG-m5 was 0.43, The IFNγ production of T cells containing MAGE-A4-zG-Km6 was 0.93, The IFNγ production of T cells containing MAGE-A4-zG-m7 was 0.22, The IFNγ production of T cells containing MAGE-A4-zG-mK8 was 0.91, The IFNγ production of T cells containing MAGE-A4-zG-m9 was 0.29, The IFNγ production level of T cells containing MAGE-A4-zG-m10 was 0.32.

[0172] As shown in Table 8, the cytotoxic activity of each mutant was as follows, assuming that the cytotoxic activity of T cells containing MAGE-A4-zG was 1.00.

[0173] The cytotoxic activity of T cells containing MAGE-A4-zG-Km11 ​​was 2.33 in the case of a cell mixing ratio of 3:1 and 1.68 in the case of a cell mixing ratio of 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-m12 was 1.23 in the case of a cell mixing ratio of 3:1 and was 0.90 in the case of a cell mixing ratio of 1:1.

[0174] On the other hand, the IFNγ production amount of each mutant was as follows, assuming that the IFNγ production amount of T cells containing MAGE-A4-zG was 1.00.

[0175] The IFNγ production of T cells containing MAGE-A4-zG-Km11 ​​was 0.89, The IFNγ production level of T cells containing MAGE-A4-zG-m12 was 0.40.

[0176] The immune cells of this embodiment are CAR-T cells that suppress cytokine production. Furthermore, the cell killing effect is improved in most clones. In MAGE-A4-zG-m12, the cell killing effect is slightly reduced compared to the wild type, but the cytokine production is greatly suppressed. This clone can maintain the cell killing effect higher than or equal to the wild type by increasing the dosage, and can suppress cytokine production better than the wild type.

[0177] Example 3: Preparation of CAR-T cells binding to PRAME / HLA-A24 complex and confirmation of its effect (1) Obtaining a nucleic acid molecule encoding CAR serving as a template As in Example 1, a plasmid DNA for viral vector preparation containing a gene encoding a CAR having a single-chain antibody that binds to the PRAME / HLA-A24 complex is obtained. The complex recognized by the single-chain antibody is a complex of PRAME and HLA-A*24:02. Hereinafter, the CAR encoded by the gene in the plasmid DNA will be referred to as "PRAME-zG". The amino acid sequence of the peptide from PRAME in the above-mentioned PRAME / HLA-A24 complex is LYVDSLFFL (sequence number 2). In order to prepare a nucleic acid molecule encoding a mutant of PRAME-zG by the PCR method, the above-mentioned plasmid DNA is used as a template.

[0178] The structure of the gene encoding PRAME-zG itself is the same as that of the gene encoding MAGE-A4-zG of Example 1. That is, the gene encoding PRAME-zG is linked to a leader sequence, a nucleotide sequence encoding VH, a nucleotide sequence encoding a linker, a nucleotide sequence encoding VL, a nucleotide sequence encoding CL, a nucleotide sequence encoding CD28TM, a nucleotide sequence encoding CD3ζ, and a nucleotide sequence encoding GITRICD in order from the 5' side (see Figure 2A ). PRAME-zG is a CAR that includes a single-chain antibody composed of VH, VL, and a linker connecting them, and CL as an extracellular domain, CD28TM as a transmembrane domain, and CD3ζ and GITRICD as intracellular domains.

[0179] (2) Obtaining nucleic acid molecules encoding PRAME-zG mutants The above-mentioned plasmid DNA is used as a template. A PCR reaction solution containing the template DNA, a primer set (sequence numbers 101 and 102) and PrimeSTAR (registered trademark) Max Premix is ​​prepared to perform a PCR reaction. Thus, a PCR product with restriction sites attached to the 5' end and the 3' end is obtained. DpnI is added to the obtained PCR product to fragment the template plasmid DNA. Using the PCR product treated with DpnI as a template, a nucleic acid molecule encoding a mutant of PRAME-zG is prepared by PCR. Specifically, it is described as follows.

[0180] (2.1) Primer design, PCR and ligation reactions Based on the nucleotide sequence of sequence number 29, a primer set for obtaining a polynucleotide encoding CAR was designed, wherein the CAR comprises a single-chain antibody in which the amino acid residues shown in the following a) or b) in PRAME-zG are replaced by arginine residues. In addition, based on the nucleotide sequence of sequence number 29, a primer set for obtaining a polynucleotide encoding CAR was designed, wherein the amino acid residues shown in the following c) in PRAME-zG are replaced by lysine residues. Using these mutants, the primer sets and the PCR products after the above-mentioned DpnI treatment were used to perform PCR and ligation reactions in the same manner as in Example 1.

[0181] a) amino acid residues 63, 65 and 72 of VL as defined by Kabat; b) amino acid residues at positions 63, 70 and 72 of VL as defined by Kabat; and c) Amino acid residues 65, 70 and 72 of VL defined by Kabat (2.2) Transformation, plasmid DNA extraction and sequencing The solution after the ligation reaction and DH5α were used to obtain E. coli transformants in the same manner as in Example 1. Then, plasmid DNA was extracted from E. coli cultured in a liquid medium and sequenced in the same manner as in Example 1. The sequencing results confirmed that a nucleic acid molecule encoding a mutant of PRAME-zG was obtained.

[0182] Hereinafter, CARs having single-chain antibodies in which the amino acid residues shown in a) or b) above in PRAME-zG are replaced with arginine residues are referred to as "PRAME-zG-m1" and "PRAME-zG-m2", respectively. In addition, CARs having single-chain antibodies in which the amino acid residues shown in c) above are replaced with lysine residues are referred to as "PRAME-zG-Km3". Table 9 shows the correspondence between the sequence numbers of the mutants and the primer sets used for their production.

[0183] [Table 9]

[0184] (3) Production of PRAME-zG and its mutant CAR-T cells As in Example 1, a gene encoding PRAME-zG and its mutants was introduced into PBMC separated and cultured from the blood of a healthy donor using a retrovirus to obtain each CAR-T cell.

[0185] (4) Confirmation of the cell killing effect and cytokine secretion of CAR-T cells The cytotoxic activity and IFNγ production of each CAR-T cell were measured in the same manner as in Example 1. The target cell used was the human melanoma cell line SK-MEL-124. SK-MEL-124 is a PRAME-positive tumor cell. The measurement of PRAME-zG-m1 and PRAME-zG-m2, and the measurement of PRAME-zG-Km3 were performed on different days. The results are shown in Tables 10 and 11.

[0186] [Table 10]

[0187] [Table 11]

[0188] As shown in Table 10, the cytotoxic activity of each mutant was as follows, when the cytotoxic activity of T cells containing PRAME-zG was set to 1.00.

[0189] The cytotoxic activity of T cells containing PRAME-zG-m1 was 2.96 when the cell mixing ratio was 3:1, and 2.37 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing PRAME-zG-m2 was 1.84 in the case of a cell mixing ratio of 3:1 and was 1.54 in the case of a cell mixing ratio of 1:1.

[0190] On the other hand, when the IFNγ production amount of T cells containing PRAME-zG was set to 1.00, the IFNγ production amount of each mutant was as follows.

[0191] The IFNγ production of T cells containing PRAME-zG-m1 was 0.49, The IFNγ production level of T cells containing PRAME-zG-m2 was 0.56.

[0192] As shown in Table 11, the cytotoxic activity of T cells containing PRAME-zG-Km3 was 1.16 when the cell mixing ratio was 3:1, and was 1.71 when the cell mixing ratio was 1:1, when the cytotoxic activity of T cells containing PRAME-zG was set to 1.00. On the other hand, the IFNγ production amount of T cells containing PRAME-zG-Km3 was 0.87 when the IFNγ production amount of T cells containing PRAME-zG was set to 1.00.

[0193] It is suggested that the immune cells of this embodiment are CAR-T cells that suppress the production of cytokines. Furthermore, the cell killing effect of the CAR-T cells is improved.

[0194] Example 4: Preparation of CAR-T cells binding to CD19 and confirmation of their effects (1) Obtaining a nucleic acid molecule encoding CAR serving as a template By gene synthesis, a polynucleotide encoding a single-chain antibody that binds to CD19 (hereinafter also referred to as "anti-CD19-WT") was obtained (SEQ ID NO. 186). In addition, as a mutant of anti-CD19-WT, polynucleotides encoding a single-chain antibody in which the amino acid residue shown in any of the following a) to c) in anti-CD19-WT was substituted with an arginine residue were synthesized (SEQ ID NOs. 187, 188, and 190).

[0195] Hereinafter, single-chain antibodies in which the amino acid residues shown in a) to c) below are changed are referred to as “anti-CD19-m1”, “anti-CD19-m2”, and “anti-CD19-m4”, respectively.

[0196] a) amino acid residues 63, 65, 67 and 70 of VL as defined by Kabat; b) amino acid residues 63, 65, 67 and 72 of VL as defined by Kabat; and c) Amino acid residues 65, 67, 70 and 72 of VL defined by Kabat (2) Obtaining nucleic acid molecules encoding CD19-28z mutants (2.1) Production of the first insert and the second insert The polynucleotides encoding the above-mentioned anti-CD19-WT, anti-CD19-m1, anti-CD19-m2 and anti-CD19-m4 were used as templates, and a PCR reaction solution containing a primer set (sequence numbers 109 and 110) and PrimeSTAR (registered trademark) Max Premix was prepared to perform a PCR reaction. Thus, the first inserts encoding anti-CD19-WT, anti-CD19-m1, anti-CD19-m2 and anti-CD19-m4 were obtained. By gene synthesis, a polynucleotide encoding the hinge domain, transmembrane domain and intracellular domain of CD28 (sequence number 111) was obtained. The polynucleotide was used as a template, and a PCR reaction solution containing a primer set (sequence numbers 112 and 113) and PrimeSTAR (registered trademark) Max Premix was prepared to perform a PCR reaction. Thus, a second insert encoding a region consisting of the hinge domain, transmembrane domain and intracellular domain of CD28 was obtained.

[0197] (2.2) Production of linearized vector and fusion with insert A plasmid DNA (sequence number 114) for viral vector preparation containing a polynucleotide encoding the transmembrane domain and intracellular domain of CD28 was used as a template, and a PCR reaction solution containing a primer set (sequence numbers 115 and 116) and PrimeSTAR (registered trademark) Max Premix was prepared to perform a PCR reaction. DpnI was added to the obtained PCR product to fragment the template plasmid DNA. Thus, a linearized vector was obtained. The linearized vector was fused with the first insert and the second insert described above using In-Fusion (registered trademark) HD ​​Enzyme Premix (Takara Bio Co., Ltd.). Thus, a plasmid DNA containing genes encoding CARs containing anti-CD19-WT, anti-CD19-m1, anti-CD19-m2, and anti-CD19-m4, respectively, was obtained. Hereinafter, CARs having single-chain antibodies of anti-CD19-WT, anti-CD19-m1, anti-CD19-m2, and anti-CD19-m4 are referred to as “CD19-28z,” “CD19-28z-m1,” “CD19-28z-m2,” and “CD19-28z-m4,” respectively.

[0198] As an example, the structure of the gene encoding CD19-28z is described. Figure 2BThe gene encoding CD19-28z is connected to the leader sequence, the nucleotide sequence encoding VL, the nucleotide sequence encoding the linker, the nucleotide sequence encoding VH, the nucleotide sequence encoding the CD28 hinge domain (CD28 hinge), the nucleotide sequence encoding CD28TM, the nucleotide sequence encoding the CD28 intracellular domain (CD28ICD) and the nucleotide sequence encoding CD3ζ in order from the 5' side. CD19-28z is a CAR that contains a single-chain antibody composed of VH, VL and a linker connecting them, and the hinge domain of CD28 as an extracellular domain, CD28TM as a transmembrane domain, and CD28ICD and CD3ζ as intracellular domains.

[0199] (2.3) Primer design, PCR and ligation reactions According to the nucleotide sequence of sequence number 37, a primer set for obtaining a polynucleotide encoding CAR is designed, wherein the CAR comprises a single-chain antibody in which the amino acid residues shown in any one of the following d), e), g) to k) and m) to q) in CD19-28z are replaced by arginine residues. In addition, based on the nucleotide sequence of sequence number 37, a primer set for obtaining a polynucleotide encoding CAR is designed, wherein the CAR comprises a single-chain antibody in which the amino acid residues shown in the following d), f), l) or m) in CD19-28z are replaced by lysine residues. Using these primer sets and template DNA, PCR and ligation reactions are performed in the same manner as in Example 1.

[0200] d) amino acid residues at positions 63, 65, 70 and 72 of VL as defined by Kabat; e) amino acid residues at positions 60, 63 and 65 of VL as defined by Kabat; f) amino acid residues 63, 65 and 70 of VL as defined by Kabat; g) amino acid residues at positions 63, 65 and 72 of VL as defined by Kabat; h) amino acid residues 63, 67 and 70 of VL as defined by Kabat; i) amino acid residues 63, 70 and 72 of VL as defined by Kabat; j) amino acid residues 65, 67 and 70 of VL as defined by Kabat; k) amino acid residues 65, 67 and 72 of VL as defined by Kabat; l) amino acid residues 65, 67, 70 and 72 of VL as defined by Kabat; m) amino acid residues 63, 65, 67, 70 and 72 of VL as defined by Kabat; n) amino acid residues 70, 72 and 74 of VL as defined by Kabat; o) amino acid residues at positions 60, 63 and 76 of VL as defined by Kabat; p) amino acid residues at positions 60, 74 and 76 of VL as defined by Kabat; and q) Amino acid residues 77, 79 and 81 of VL defined by Kabat (2.2) Transformation, plasmid DNA extraction and sequencing The solution after the ligation reaction and DH5α were used to obtain a transformant of E. coli in the same manner as in Example 1. Then, plasmid DNA was extracted from the E. coli cultured in a liquid medium and sequenced in the same manner as in Example 1. The sequencing results confirmed that a nucleic acid molecule encoding a mutant of CD19-28z was obtained.

[0201] Hereinafter, CARs having a single-chain antibody in which the amino acid residue represented by any of the above d), e), g) to k) and m) to q) in CD19-28z is substituted with an arginine residue will be referred to as “CD19-28z-m3”, “CD19-28z-m5”, “CD19-28z-m7”, “CD19-28z-m8”, “CD19-28z-m10”, “CD19-28z-m11”, “CD19-28z-m12”, “CD19-28z-m16”, “CD19-28z-m17”, “CD19-28z-m18”, “CD19-28z-m19” and “CD19-28z-m20”, respectively. In addition, the CAR having a single-chain antibody in which the amino acid residues shown in the above d), f), l) or m) are replaced with lysine residues is respectively referred to as "CD19-28z-Km13", "CD19-28z-Km6", "CD19-28z-Km14" and "CD19-28z-Km15". The correspondence between the sequence numbers of each mutant and the primer set used for its production is shown in Tables 12 and 13. With respect to CD19-CD28z-m18 in Table 13, "1st" represents a primer set for replacing the amino acid residues at positions 60 and 63 of VL, and "2nd" represents a primer set for replacing the amino acid residue at position 76 of VL. With respect to CD19-28z-m19, "1st" represents a primer set for replacing the amino acid residues at positions 74 and 76 of VL, and "2nd" represents a primer set for replacing the amino acid residue at position 60 of VL.

[0202] [Table 12]

[0203] [Table 13]

[0204] (3) Preparation of CD19-28z and CAR-T cells expressing its mutants As in Example 1, genes encoding CD19-28z and its mutants were introduced into PBMCs isolated and cultured from the blood of healthy donors using retroviruses to obtain various CAR-T cells.

[0205] (4) Confirmation of the cell killing effect and cytokine secretion of CAR-T cells The cytotoxic activity and IFNγ production of each CAR-T cell were measured in the same manner as in Example 1. In addition, in some experiments, the ratio of the number of effector cells to target cells was 2:1 (see Table 16). The target cells used were human B precursor cell leukemia cell line Nalm-6. Nalm-6 is a CD19-positive tumor cell. The results of the measurements are shown in Tables 14-17. The results of each table are based on measurements performed on different dates.

[0206] [Table 14]

[0207] [Table 15]

[0208] [Table 16]

[0209] [Table 17]

[0210] As shown in Table 14, the cytotoxic activity of each mutant was as follows when the cytotoxic activity of T cells containing CD19-28z was set to 1.00.

[0211] The cytotoxic activity of T cells containing CD19-28z-m1 was 1.03 when the cell mixing ratio was 3:1, and 1.30 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m2 was 0.90 when the cell mixing ratio was 3:1, and was 1.19 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m3 was 0.91 in the case of a cell mixing ratio of 3:1 and was 1.26 in the case of a cell mixing ratio of 1:1.

[0212] On the other hand, the IFNγ production amount of each mutant when the IFNγ production amount of T cells containing CD19-28z was set to 1.00 was as follows.

[0213] The IFNγ production of T cells containing CD19-28z-m1 was 0.25, The IFNγ production of T cells containing CD19-28z-m2 was 0.24, The IFNγ production of T cells containing CD19-28z-m3 was 0.26.

[0214] As shown in Table 15, the cytotoxic activity of each mutant was as follows when the cytotoxic activity of T cells containing CD19-28z was set to 1.00.

[0215] The cytotoxic activity of T cells containing CD19-28z-m1 was 1.00 when the cell mixing ratio was 3:1, and was 1.56 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m3 was 1.09 when the cell mixing ratio was 3:1, and 1.37 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m4 was 1.17 in the case of a cell mixing ratio of 3:1 and was 1.49 in the case of a cell mixing ratio of 1:1.

[0216] On the other hand, the IFNγ production amount of each mutant when the IFNγ production amount of T cells containing CD19-28z was set to 1.00 was as follows.

[0217] The IFNγ production of T cells containing CD19-28z-m1 was 0.31, The IFNγ production of T cells containing CD19-28z-m3 was 0.27, The IFNγ production of T cells containing CD19-28z-m4 was 0.29.

[0218] As shown in Table 16, the cytotoxic activity of each mutant was as follows when the cytotoxic activity of T cells containing CD19-28z was set to 1.00.

[0219] The cytotoxic activity of T cells containing CD19-28z-m5 was 1.09 when the cell mixing ratio was 2:1, and 0.95 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-Km6 was 1.50 in the case of a cell mixing ratio of 2:1 and 1.13 in the case of a cell mixing ratio of 1:1. The cytotoxic activity of T cells containing CD19-28z-m7 was 1.58 when the cell mixing ratio was 2:1, and 1.11 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m8 was 1.61 in the case of a cell mixing ratio of 2:1 and 1.19 in the case of a cell mixing ratio of 1:1. The cytotoxic activity of T cells containing CD19-28z-m10 was 1.59 when the cell mixing ratio was 2:1, and 0.98 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m11 was 1.40 in the case of a cell mixing ratio of 1:1 and 1.06 in the case of a cell mixing ratio of 1:1. The cytotoxic activity of T cells containing CD19-28z-m12 was 1.47 when the cell mixing ratio was 2:1, and 1.39 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-Km13 was 1.68 in the case of a cell mixing ratio of 2:1 and 1.37 in the case of a cell mixing ratio of 1:1. The cytotoxic activity of T cells containing CD19-28z-Km14 was 1.72 in the case of a cell mixing ratio of 2:1 and 1.34 in the case of a cell mixing ratio of 1:1. The cytotoxic activity of T cells containing CD19-28z-Km15 was 1.29 in the case of a cell mixing ratio of 2:1 and 1.23 in the case of a cell mixing ratio of 1:1. The cytotoxic activity of T cells containing CD19-28z-m16 was 1.93 in the case of a cell mixing ratio of 2:1 and was 1.32 in the case of a cell mixing ratio of 1:1.

[0220] On the other hand, the IFNγ production amount of each mutant when the IFNγ production amount of T cells containing CD19-28z was set to 1.00 was as follows.

[0221] The IFNγ production of T cells containing CD19-28z-m5 was 0.34, The IFNγ production of T cells containing CD19-28z-m6 was 0.20, The IFNγ production of T cells containing CD19-28z-m7 was 0.13, The IFNγ production of T cells containing CD19-28z-m8 was 0.08, The IFNγ production of T cells containing CD19-28z-m10 was 0.21, The IFNγ production of T cells containing CD19-28z-m11 was 0.17, The IFNγ production of T cells containing CD19-28z-m12 was 0.16, The IFNγ production of T cells containing CD19-28z-Km13 was 0.13, The IFNγ production of T cells containing CD19-28z-Km14 was 0.13, The IFNγ production of T cells containing CD19-28z-Km15 was 0.14, The IFNγ production of T cells containing CD19-28z-m16 was 0.15.

[0222] As shown in Table 17, the cytotoxic activity of each mutant was as follows when the cytotoxic activity of T cells containing CD19-28z was set to 1.00.

[0223] The cytotoxic activity of T cells containing CD19-28z-m17 was 1.43 when the cell mixing ratio was 3:1, and 2.43 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m18 was 1.27 when the cell mixing ratio was 3:1, and 1.42 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m19 was 1.38 when the cell mixing ratio was 3:1, and 1.76 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m20 was 1.57 when the cell mixing ratio was 3:1, and was 1.90 when the cell mixing ratio was 1:1.

[0224] On the other hand, the IFNγ production amount of each mutant when the IFNγ production amount of T cells containing CD19-28z was set to 1.00 was as follows.

[0225] The IFNγ production of T cells containing CD19-28z-m17 was 0.34, The IFNγ production of T cells containing CD19-28z-m18 was 0.28, The IFNγ production of T cells containing CD19-28z-m19 was 0.68, The IFNγ production of T cells containing CD19-28z-m20 was 0.39.

[0226] As shown in Examples 1-4, the IFNγ production of the immune cells of the present invention showed a value of 0.08-0.93 when the wild type was set to 1.00, which was lower than that of the wild type. The cytotoxic activity of the CAR-T cells showed a value of 0.90-2.96 when the wild type was 1.00, and the cytotoxic activity was maintained or improved in most clones compared to the wild type.

[0227] Example 5: In vivo cancer treatment model experiment based on CAR-T cell administration (1) Materials and methods (1.1) Experimental animals NOG mice (NOD / Shi-scid, IL-2RγKO Jic) were purchased from CLEA Co., Ltd. 7-8 week old female mice were used in the experiment.

[0228] (1.2) Human tumor transplantation and CAR-T cell administration NW-MEL-38 was used as tumor cells. CAR-T cells containing MAGE-A4-zG-s1 and CAR-T cells containing MAGE-A4-zG-m1-s1 prepared in Example 1 were used as effector cells. 5×10 6 cells / mouse subcutaneously injected with NW-MEL-38. Four days after tumor cell transplantation, 5×10 6 cells / PBS, T cells containing MAGE-A4-zG-s1, or CAR-T cells containing MAGE-A4-zG-m1-s1 were infused only from the tail vein. The groups administered with PBS, administered with CAR-T cells containing MAGE-A4-zG-s1, and administered with CAR-T cells containing MAGE-A4-zG-m1-s1 were all n=4. Tumor diameter was measured every 2 or 3 days.

[0229] (2) Results The average tumor area of ​​each group is shown in the figure Figure 3 As shown. Figure 3 It can be seen that the tumor diameter was reduced in the group administered with CAR-T cells containing MAGE-A4-zG-s1 and the group administered with CAR-T cells containing MAGE-A4-zG-m1-s1 compared with the group administered with PBS. In addition, the tumor diameter was reduced in the group administered with CAR-T cells containing MAGE-A4-zG-m1-s1 compared with the group administered with CAR-T cells containing MAGE-A4-zG-s1. Therefore, it is suggested that the anti-tumor activity of immune cells containing CAR can be improved by changing at least 3 amino acid residues of the light chain FR3 of the single-chain antibody contained in CAR to basic amino acid residues.

Claims

1. A nucleic acid molecule having a nucleotide sequence encoding a chimeric antigen receptor, The nucleic acid molecule comprises a segment encoding an extracellular domain, a segment encoding a transmembrane domain and a segment encoding an intracellular domain, The segment encoding the extracellular domain comprises a nucleotide sequence encoding an antigen binding region comprising a light chain variable region and a heavy chain variable region, In the nucleotide sequence encoding the framework region 3 of the light chain variable region defined by the Kabat method, at least three codons are codons encoding basic amino acid residues.

2. The nucleic acid molecule according to claim 1, wherein The at least 3 codons include at least 3 of the group consisting of a codon encoding the amino acid residue at position 60 of the light chain variable region, a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, a codon encoding the amino acid residue at position 70, a codon encoding the amino acid residue at position 72, a codon encoding the amino acid residue at position 74, a codon encoding the amino acid residue at position 76, a codon encoding the amino acid residue at position 77, a codon encoding the amino acid residue at position 79, and a codon encoding the amino acid residue at position 81.

3. The nucleic acid molecule according to claim 1, wherein Three or more and five or less codons selected from the group consisting of a codon encoding the 60th amino acid residue of the light chain variable region defined by the Kabat method, a codon encoding the 63rd amino acid residue, a codon encoding the 65th amino acid residue, a codon encoding the 67th amino acid residue, a codon encoding the 70th amino acid residue, a codon encoding the 72nd amino acid residue, a codon encoding the 74th amino acid residue, a codon encoding the 76th amino acid residue, a codon encoding the 77th amino acid residue, a codon encoding the 79th amino acid residue and a codon encoding the 81st amino acid residue are codons encoding basic amino acid residues.

4. The nucleic acid molecule according to claim 1, wherein The antigen-binding region comprises a single-chain antibody that binds to a complex of a peptide derived from MAGE-A4 and HLA-A2, a complex of a peptide derived from PRAME and HLA-A24, CD19, BCMA or CEA.

5. The nucleic acid molecule according to claim 1, wherein The transmembrane domain comprises a transmembrane region of any one protein selected from the group consisting of an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS and GITR.

6. The nucleic acid molecule according to claim 1, wherein The intracellular domain comprises a signal transduction domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ and FcRβ.

7. The nucleic acid molecule according to claim 6, wherein The segment encoding the intracellular domain further comprises a nucleotide sequence encoding a costimulatory domain, The co-stimulatory domain is a co-stimulatory domain of at least one protein selected from the group consisting of 4-1BB, CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 and ICOS.

8. The nucleic acid molecule according to claim 1, wherein A segment encoding a hinge domain is also included between the nucleotide sequence encoding the antigen binding region and the segment encoding the transmembrane domain.

9. The nucleic acid molecule according to claim 1, wherein The nucleic acid molecule is DNA or RNA.

10. A vector comprising the nucleic acid molecule according to any one of claims 1 to 9.

11. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein: The extracellular domain includes an antigen binding region comprising a light chain variable region and a heavy chain variable region, In the framework region 3 of the light chain variable region defined by the Kabat method, at least 3 amino acid residues are basic amino acid residues.

12. The chimeric antigen receptor according to claim 11, wherein The at least 3 amino acid residues include at least 3 selected from the group consisting of the amino acid residue at position 60, the amino acid residue at position 63, the amino acid residue at position 65, the amino acid residue at position 67, the amino acid residue at position 70, the amino acid residue at position 72, the amino acid residue at position 74, the amino acid residue at position 76, the amino acid residue at position 77, the amino acid residue at position 79 and the amino acid residue at position 81 of the light chain variable region.

13. The chimeric antigen receptor according to claim 11, wherein Three or more and five or less amino acid residues selected from the group consisting of amino acid residue at position 60, amino acid residue at position 63, amino acid residue at position 65, amino acid residue at position 67, amino acid residue at position 70, amino acid residue at position 72, amino acid residue at position 74, amino acid residue at position 76, amino acid residue at position 77, amino acid residue at position 79 and amino acid residue at position 81 of the light chain variable region defined by the Kabat method are basic amino acid residues.

14. The chimeric antigen receptor according to claim 11, wherein The antigen-binding region comprises a single-chain antibody that binds to a complex of a peptide derived from MAGE-A4 and HLA-A2, a complex of a peptide derived from PRAME and HLA-A24, CD19, BCMA or CEA.

15. The chimeric antigen receptor according to claim 11, wherein The transmembrane domain comprises a transmembrane region of any one protein selected from the group consisting of an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS and GITR.

16. The chimeric antigen receptor according to claim 11, wherein The intracellular domain comprises a signal transduction domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ and FcRβ.

17. The chimeric antigen receptor according to claim 16, wherein The intracellular domain further comprises a co-stimulatory domain, The co-stimulatory domain is a co-stimulatory domain of at least one protein selected from the group consisting of 4-1BB, CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 and ICOS.

18. The chimeric antigen receptor according to claim 11, wherein A hinge domain is also included between the antigen binding region and the transmembrane domain.

19. An immune cell comprising the chimeric antigen receptor according to any one of claims 11 to 18.

20. A pharmaceutical composition for treating malignant tumors, comprising the immune cells according to claim 19.

21. A method for preparing an immune cell comprising a chimeric antigen receptor, comprising: The nucleic acid molecule according to any one of claims 1 to 9 is introduced into immune cells, so that the immune cells express a chimeric antigen receptor.

22. A method for preparing an immune cell comprising a chimeric antigen receptor, comprising: The vector according to claim 10 is introduced into immune cells, so that the immune cells express the chimeric antigen receptor.

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