A nucleic acid molecule encoding CAR and a vector containing the same, CAR, immune cells containing CAR and a pharmaceutical composition containing those cells, and a method for producing immune cells containing CAR.

Abstract

The present invention relates to a nucleic acid molecule having a nucleotide sequence that encodes a chimeric antigen receptor (CAR). The present invention relates to a vector containing said nucleic acid molecule. The present invention relates to a CAR. The present invention relates to an immune cell containing a CAR. The present invention relates to a pharmaceutical composition containing an immune cell containing a CAR. The present invention relates to a method that produces an immune cell containing a CAR.

Description

[Technical Field]

[0001] This invention relates to a nucleic acid molecule having a nucleotide sequence encoding a chimeric antigen receptor (hereinafter also referred to as "CAR"). This invention relates to a vector containing the nucleic acid molecule. This invention relates to CAR. This invention relates to immune cells containing CAR. This invention relates to a pharmaceutical composition for treating malignant tumors, comprising immune cells containing CAR. This invention relates to a method for producing immune cells containing CAR. [Background technology]

[0002] CARs are receptor proteins created by genetically engineering the fusion of an extracellular domain containing an antigen-binding region, a transmembrane domain, and an intracellular domain that transmits activation signals to immune cells. For example, as described in Patent Document 1, the antigen-binding region of a CAR uses a single-chain antibody that recognizes antigens expressed on tumor cells. In recent years, cancer immunotherapy has attracted attention in which immune cells expressing CARs on their surface are transplanted into patients to treat cancer by introducing the gene encoding the CAR into immune cells. When immune cells expressing CARs recognize antigens on tumor cells in the body, they become activated and express cytotoxic molecules and cytokines such as Fas ligand, perforin, and granzyme, thereby exerting an antitumor effect. On the other hand, it is known that cytokine production by immune cells expressing CARs can cause cytokine release syndrome (CRS). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent No. 7,741,465 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The present invention aims to provide a CAR that, when expressed in immune cells, can suppress cytokine production by said immune cells. Furthermore, the present invention aims to provide a nucleic acid molecule encoding such a CAR and a vector containing such a nucleic acid molecule. Moreover, the present invention aims to provide immune cells containing such a CAR, a pharmaceutical composition containing said immune cells, and a method for producing said immune cells. [Means for solving the problem]

[0005] The present inventors have discovered that by changing a predetermined amino acid residue in the framework region 3 (FR3) of the light chain variable region of the antigen-binding region of a CAR to a basic amino acid residue, the production of cytokines by immune cells containing CARs can be suppressed, and have completed the present invention. Accordingly, the inventions described in [1] to

[21] below are provided.

[0006] [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 comprising a nucleotide sequence encoding an antigen-binding region including a light chain variable region and a heavy chain variable region, and at least three codons in the nucleotide sequence encoding FR3 of the light chain variable region as defined by the Kabat method are codons encoding basic amino acid residues.

[0007] [2] The nucleic acid molecule according to [1] above, wherein at least three codons are selected from the group consisting of a codon encoding the 60th amino acid residue of the light chain variable region, 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.

[0008] [3] The nucleic acid molecule according to [1] or [2] above, wherein 3 to 5 codons selected from the group consisting of a codon encoding the 60th amino acid residue of the light chain variable region as 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.

[0009] [4] A nucleic acid molecule according to any one of [1] to [3] above, wherein the antigen-binding region contains a single-chain antibody, and the single-chain antibody is a single-chain antibody that binds to a complex of MAGE-A4-derived peptide and HLA-A2, a complex of PRAME-derived peptide and HLA-A24, CD19, BCMA, or CEA.

[0010] [5] A nucleic acid molecule according to any one of [1] to [4] above, wherein the transmembrane domain comprises the transmembrane region of any one 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, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS, and GITR.

[0011] [6] A nucleic acid molecule according to any one of [1] to [5] above, wherein the intracellular domain comprises a signaling domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ, and FcRβ.

[0012] [7] The nucleic acid molecule described in [6] above, wherein the segment encoding the intracellular domain further comprises a nucleotide sequence encoding the costimulatory domain, and the costimulatory domain is the costimulatory 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.

[0013] [8] A nucleic acid molecule according to any one of [1] to [7] above, further comprising a segment encoding a hinge domain between a nucleotide sequence encoding an antigen-binding region and a segment encoding a transmembrane domain.

[0014] [9] A nucleic acid molecule according to any one of [1] to [8] above, wherein the nucleic acid molecule is DNA or RNA.

[0015]

[10] A vector containing any one of the nucleic acid molecules described in [1] to [9] above.

[0016]

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

[0017]

[12] The CAR according to

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

[0018]

[13] The CAR described in

[11] or

[12] above, wherein 3 to 5 amino acid residues selected from the group consisting of the 60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th, and 81st amino acid residues of the light chain variable region defined by the Kabat method are basic amino acid residues.

[0019]

[14] The CAR according to any one of

[11] to

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

[0020]

[15] The CAR according to any one of

[11] to

[14] above, wherein the transmembrane domain comprises the transmembrane region of any one 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, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS and GITR.

[0021]

[16] The CAR according to any one of

[11] to

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

[0022]

[17] The CAR according to

[16] above, wherein the intracellular domain further comprises a co-stimulatory domain, and the co-stimulatory domain is the 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.

[0023]

[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 domain.

[0024]

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

[11] to

[18] above.

[0025]

[20] A pharmaceutical composition for treating a malignant tumor, comprising the immune cell according to

[19] above.

[0026]

[21] A method for producing an immune cell comprising a chimeric antigen receptor, comprising introducing the nucleic acid molecule according to any one of [1] to [9] above or the vector according to

[10] above into an immune cell to express a chimeric antigen receptor in the immune cell.

Advantages of the Invention

[0027] According to the present invention, immune cells containing CARs in which cytokine production is suppressed can be obtained. [Brief explanation of the drawing]

[0028] [Figure 1A] This figure schematically represents an example of a nucleic acid molecule of this embodiment. An enlarged view of the segment (VL) encoding the light chain variable region in the nucleic acid molecule is also shown. In the figure, A represents the segment encoding the extracellular domain, B represents the segment encoding the transmembrane domain, and C represents the segment encoding the intracellular domain. D represents the segment encoding the antigen-binding region included in the extracellular domain. Among these segments, VH represents the segment encoding the heavy chain variable region, L represents the segment encoding the linker, HD represents the segment encoding the hinge domain, TMD represents the segment encoding the transmembrane domain, co-STD represents the segment encoding the costimulatory domain, and SD represents the segment encoding the signal transduction domain. In the enlarged view of the VL, the dashed lines marked with * indicate codons encoding basic amino acid residues. In Figure 1A, there are three codons encoding basic amino acid residues in the segment encoding FR3, but the present invention is not limited thereto. [Figure 1B] This figure schematically represents an example of a CAR in this embodiment. In the figure, VH represents the heavy chain variable region, VL represents the light chain variable region, HD represents the hinge domain, TMD represents the transmembrane domain, co-STD represents the costimulatory domain, and SD represents the signal transduction domain. The curve connecting VH and VL represents the linker. In VL, the dashed line marked with an asterisk (*) represents a basic amino acid residue. In Figure 1B, there are three basic amino acid residues in FR3, but the present invention is not limited to this. [Figure 2A] This figure schematically represents the nucleic acid molecules encoding CARs in Examples 1-3. [Figure 2B] This figure schematically represents the nucleic acid molecule encoding the CAR in Example 4. [Figure 3]This graph shows the average tumor area of ​​mice after transplantation of tumor cells and administration of PBS or CAR-T cells. [Modes for carrying out the invention]

[0029] 1. Nucleic acid molecules The nucleic acid molecule of this embodiment has a nucleotide sequence that codes for CAR. In this specification, "nucleotide sequence" is synonymous with "base sequence" and "nucleic acid sequence." A nucleotide sequence refers to the one-dimensional arrangement (order) of nucleotides within a nucleic acid molecule. Therefore, a nucleic acid molecule that codes for a polypeptide has a nucleotide sequence that codes for the polypeptide. In this specification, the expression "having a nucleotide sequence" means both consisting of the nucleotide sequence and containing the nucleotide sequence. In this specification, the term "polypeptide" includes protein molecules, parts of a protein molecule, and fragments of protein molecules. In this specification, parts of a nucleic acid molecule may be referred to as a "segment," and parts of a protein molecule may be referred to as a "domain."

[0030] As illustrated in Figure 1A, the nucleic acid molecule of this embodiment includes, in order from the 5' end, 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 including a light chain variable region and a heavy chain variable region. In this specification, "antigen-binding region including a light chain variable region and a heavy chain variable region" means a domain that includes at least one light chain variable region and at least one heavy chain variable region, and can bind to a predetermined antigen via these. An example of an antigen-binding region is a single-chain antibody. A single-chain antibody, also called scFv, has a light chain variable region and a heavy chain variable region linked via a peptide linker, and is a part of the CAR construct of this embodiment. In the nucleic acid molecule of Figure 1A, the segment encoding the antigen-binding region includes a nucleotide sequence encoding a single-chain antibody consisting of VH, L, and VL. Details of each segment of the nucleic acid molecule of this embodiment and the CAR encoded by this nucleic acid molecule will be described later.

[0031] As illustrated in Figure 1A, the nucleic acid molecule of this embodiment is characterized in that at least three codons in the nucleotide sequence encoding FR3 of the light chain variable region (hereinafter also referred to as "light chain FR3") are codons encoding basic amino acid residues. That is, the CAR encoded by the nucleic acid molecule of this embodiment has an antigen-binding region in which at least three amino acid residues of light chain FR3 are basic amino acid residues. This antigen-binding region will also be referred to below as the "modified antigen-binding region." Furthermore, a CAR having a modified antigen-binding region will also be referred to below as the "modified CAR." The nucleic acid molecule of this embodiment can be said to be a nucleic acid molecule encoding a modified CAR. In this specification, "codon" means three consecutive nucleotides in DNA or RNA.

[0032] Nucleic acid molecules encoding a modified antigen-binding region can be obtained by modifying the codons of the original nucleic acid molecule encoding the antigen-binding region, as described below. In this specification, "original antigen-binding region" refers to the antigen-binding region before modification, in which the number of basic amino acid residues in the light chain FR3 is two 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 two or less. Hereinafter, a CAR having the original antigen-binding region will also be referred to as the "unmodified CAR".

[0033] The framework region (FR) is a region in the variable region of an antibody's light chain and heavy chain that is not the complementarity-determining region (CDR). The FR acts as a scaffold linking the three CDRs and contributes to the structural stability of the CDRs. Therefore, the amino acid sequence of the FR is highly conserved among antibodies of the same species. The variable regions of the heavy and light chains each contain three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR4). These are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus of the variable region. Hereafter, the CDR of the heavy chain is sometimes referred to as "HCDR," and the CDR of the light chain as "LCDR."

[0034] In this technical field, a method for numbering amino acid residues in a CDR (hereinafter also referred to as the "numbering method") is known for defining the boundaries and length of the CDR. When amino acid residues of a CDR are numbered using the numbering method, amino acid residues of the FR (free chain) are also numbered. The numbers assigned to amino acid residues by the numbering method indicate the position of that amino acid residue in the amino acid sequence of the light chain or heavy chain. Examples of numbering methods 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 Honegger 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.

[0035] With respect to the antigen-binding region, the boundaries and lengths of the CDR and FR in the variable regions of the light and heavy chains may be defined by any numbering method. In this specification, the boundaries and lengths of the CDR and FR are defined by the Kabat method. For example, if the antigen-binding region contains or consists of a single-chain antibody, according to the Kabat method, the light chain FR1 of the single-chain antibody is defined as the region consisting of amino acid residues 1 to 23 of the light chain variable region. The light chain FR2 of the single-chain antibody is defined as the region consisting of amino acid residues 35 to 49 of the light chain variable region. The light chain FR3 of the single-chain antibody is defined as the region consisting of amino acid residues 57 to 88 of the light chain variable region. The light chain FR4 of the single-chain antibody is defined as the region consisting of amino acid residues 98 to 109 of the light chain variable region. In this specification, when the position of an amino acid residue in the light chain variable region of the antigen-binding region is described, unless otherwise specified, the position of the amino acid residue represents the position defined by the Kabat method.

[0036] The nucleic acid molecule of this embodiment can be obtained by linking 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, in that order from the 5' end. This linking of nucleic acid molecules can be carried out by known genetic recombination techniques and other molecular biological techniques. The nucleic acid molecule encoding the extracellular domain may consist of a nucleic acid molecule encoding a modified antigen-binding region. Preferably, the nucleic acid molecule encoding the extracellular domain is obtained by linking a nucleic acid molecule encoding a modified antigen-binding region and a nucleic acid molecule encoding a hinge domain using the techniques described above.

[0037] In the nucleotide sequence encoding the light chain FR3 of the original antigen-binding region, changing three or more codons encoding amino acid residues that are not basic amino acid residues to codons encoding basic amino acid residues is hereinafter also referred to as "codon modification" or "codon alteration." A nucleic acid molecule encoding the modified antigen-binding region can be obtained by performing codon modifications on the nucleic acid molecule encoding the original antigen-binding region. Amino acid residues that are not basic amino acid residues are neutral amino acid residues and / or acidic amino acid residues, and are preferably neutral amino acid residues. It is preferable that the nucleotide sequence encoding the modified antigen-binding region and the nucleotide sequence encoding the original antigen-binding region are the same except for the locations where the codons have been modified.

[0038] Such codon modification allows for the acquisition of a nucleic acid molecule encoding a modified antigen-binding region, in which 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. Codon modification can be performed by substituting or inserting codons in the nucleic acid molecule encoding the original antigen-binding region.

[0039] Basic amino acid residues are lysine residues, arginine residues, and histidine residues. Among these, arginine residues and lysine residues are preferred. Neutral amino acid residues are 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 are aspartic acid residues and glutamic acid residues.

[0040] In a nucleotide sequence encoding light chain FR3, at least three codons introduced by codon modification may all be codons encoding arginine residues, or they may all be codons encoding lysine residues. Alternatively, in a nucleotide sequence encoding light chain FR3, at least three codons introduced by codon modification may some be codons encoding arginine residues and the rest be codons encoding lysine residues.

[0041] The nucleic acid molecule in this embodiment may be DNA or RNA. In the nucleotide sequence encoding the light chain FR3, the type of codon introduced by codon modification is not particularly limited as long as it encodes a basic amino acid residue. When the nucleic acid molecule in this embodiment is DNA, examples of codons encoding basic amino acid residues include AGA, AGG, CGA, CGC, CGG, and CGT encoding arginine residues, AAA and AAG encoding lysine residues, and CAC and CAT encoding histidine residues. When the nucleic acid molecule in this embodiment is RNA, examples of codons encoding basic amino acid residues include AGA, AGG, CGA, CGC, CGG, and CGU encoding arginine residues, AAA and AAG encoding lysine residues, and CAC and CAU encoding histidine residues.

[0042] In the nucleic acid molecule of this embodiment, the number of codons encoding basic amino acid residues in the nucleotide sequence encoding the light chain FR3 is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. Preferably, the number of codons encoding basic amino acid residues in the nucleotide sequence encoding the light chain FR3 is 3 to 6, and more preferably 3 to 5.

[0043] In the modified light chain FR3 of the antigen-binding region, it is preferable that at least three basic amino acid residues derived from codon modification are located at the amino acid residues obtained by excluding vernier zone residues and non-exposed residues from the amino acid sequence of light chain FR3. "Vernier zone residues" are amino acid residues in the amino acid sequence of FR that contribute to the structural stability of CDR. "Non-exposed residues" are amino acid residues that are folded into the molecule and not exposed on the surface. Modifying non-exposed residues is expected to have little or no effect. For example, the amino acid residues obtained by excluding vernier zone residues and non-exposed residues from the amino acid sequence of light chain FR3 are the amino acid residues at positions 57, 58, 59, 60, 61, 62, 63, 65, 67, 70, 72, 74, 76, 77, 79, 80, and 81 in the light chain variable region.

[0044] In the nucleotide sequence encoding the modified antigen-binding region, it is preferable that at least three codons selected from the group consisting of the codon encoding the 60th amino acid residue of the light chain variable region, the codon encoding the 63rd amino acid residue, the codon encoding the 65th amino acid residue, the codon encoding the 67th amino acid residue, the codon encoding the 70th amino acid residue, the codon encoding the 72nd amino acid residue, the codon encoding the 74th amino acid residue, the codon encoding the 76th amino acid residue, the codon encoding the 77th amino acid residue, the codon encoding the 79th amino acid residue, and the codon encoding the 81st amino acid residue are codons encoding basic amino acid residues. More preferably, three to six codons selected from the above group are codons encoding basic amino acid residues. Particularly more preferably, three to five 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.

[0045] 1) Codons encoding the 60th amino acid residue, the 63rd amino acid residue, and the 65th amino acid residue in the light chain variable region; 2) Codons encoding the 60th amino acid residue, the 63rd amino acid residue, and the 76th amino acid residue in the light chain variable region; 3) Codons encoding the 60th amino acid residue, the 74th amino acid residue, and the 76th amino acid residue in the light chain variable region; 4) Codons encoding the 63rd amino acid residue, the 65th amino acid residue, and the 67th amino acid residue in the light chain variable region; 5) Codons encoding the 63rd amino acid residue, the 65th amino acid residue, and the 70th amino acid residue in the light chain variable region; 6) Codons encoding the 63rd amino acid residue, the 65th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 7) Codons encoding the 63rd amino acid residue, the 67th amino acid residue, and the 70th amino acid residue in the light chain variable region; 8) Codons encoding the 63rd amino acid residue, the 67th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 9) Codons encoding the 63rd amino acid residue, the 70th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 10) Codons encoding the 65th amino acid residue, the 67th amino acid residue, and the 70th amino acid residue in the light chain variable region; 11) Codons encoding the 65th amino acid residue, the 67th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 12) Codons encoding the 65th amino acid residue, the 70th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 13) Codons encoding the 67th amino acid residue, the 70th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 14) Codons encoding the 70th amino acid residue, the 72nd amino acid residue, and the 74th amino acid residue in the light chain variable region; 15) Codons encoding the 77th amino acid residue, the 79th amino acid residue, and the 81st amino acid residue in the light chain variable region; 16) Codons encoding the 63rd amino acid residue, the 65th amino acid residue, the 67th amino acid residue, and the 70th amino acid residue in the light chain variable region; 17) Codons encoding the 63rd amino acid residue, the 65th amino acid residue, the 67th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 18) Codons encoding the 63rd amino acid residue, the 65th amino acid residue, the 70th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 19) Codons encoding the 63rd amino acid residue, the 67th amino acid residue, the 70th amino acid residue, and the 72nd amino acid residue in the light chain variable region; 20) Codons encoding the 65th amino acid residue, the 67th amino acid residue, the 70th amino acid residue, and the 72nd amino acid residue in the light chain variable region; and 21) Codons encoding the 63rd amino acid residue of the light chain variable region, the 65th amino acid residue, the 67th amino acid residue, the 70th amino acid residue, and the 72nd amino acid residue.

[0046] In the nucleotide sequence encoding the modified antigen-binding region, it is preferable that the codons encoding at least three amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 in the light chain variable region, and the codon encoding at least one amino acid residue selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 in the light chain variable region, are codons encoding basic amino acid residues.

[0047] In the nucleotide sequence encoding the light chain FR3 of the modified antigen-binding region, when three to six codons are codons encoding basic amino acid residues, it is preferable that these three to six codons include codons encoding three, four, or five amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 of the light chain variable region, and codons encoding one, two, or three amino acid residues selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 of the light chain variable region.

[0048] In the nucleotide sequence encoding the light chain FR3 of the modified antigen-binding region, when three to five codons are codons encoding basic amino acid residues, it is preferable that these three to five codons include three or four codons selected from the group consisting of positions 63, 65, 67, 70, and 72 of the light chain variable region, and one or two codons selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 of the light chain variable region.

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

[0050] In the nucleotide sequence encoding the light chain FR3 of the modified antigen-binding region, when three to six codons are codons encoding basic amino acid residues, it is preferable that these three to six codons include codons encoding three, four, or five amino acid residues selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 of the light chain variable region, and codons encoding one, two, or three amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 of the light chain variable region.

[0051] In the nucleotide sequence encoding the light chain FR3 of the modified antigen-binding region, when three to five codons are codons encoding basic amino acid residues, it is preferable that these three to five codons include three or four codons selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 of the light chain variable region, and one or two codons selected from the group consisting of positions 63, 65, 67, 70, and 72 of the light chain variable region.

[0052] In the nucleotide sequence encoding the modified antigen-binding region, if codons other than those encoding amino acid residues at positions 60, 63, 65, 67, 70, 72, 74, 76, 77, 79, and 81 of the light chain variable region are modified, it is preferable that the modified codons are those encoding amino acid residues at a site selected from the group consisting of positions 57, 58, 59, 61, and 62.

[0053] Since the CDR is involved in the affinity of the antigen-binding region to the antigen, it is preferable not to change the nucleotide sequence encoding the CDR of the antigen-binding region in the nucleic acid molecule of this embodiment. That is, it is preferable that the amino acid sequence of the modified antigen-binding region CDR and the nucleotide sequence encoding it are the same as the amino acid sequence of the original antigen-binding region CDR and the nucleotide sequence encoding it.

[0054] Hereinafter, immune cells containing CAR before modification will also be referred to as "unmodified immune cells," and immune cells containing CAR after modification will also be referred to as "modified immune cells." When the amino acid sequence of the modified CAR is the same as that of the unmodified CAR, except that at least three amino acid residues in the light chain FR3 are basic amino acid residues, the modified immune cells and the unmodified immune cells can be compared in terms of cytokine production and cytotoxic effects. In modified immune cells, cytokine production is suppressed compared to unmodified immune cells. The types of cytokines are not particularly limited, but examples include IFNγ, tumor necrosis factor (TNF)-α, and interleukin (IL)-6. In this specification, "cytokine production is suppressed" means that when tumor cells and immune cells are mixed in a predetermined ratio, and a predetermined period of time has elapsed since mixing, the amount of cytokine released by modified immune cells is less than the amount released by unmodified immune cells. 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 modification. The amount of cytokines released by immune cells containing CAR can be determined, for example, by co-culturing the immune cells with tumor cells and then measuring the cytokines in the culture supernatant using a known method such as enzyme-linked immunosorbent assay (ELISA). Specifically, the method in Example 1 described below is an example.

[0055] Preferably, the modified immune cells maintain a cytotoxic effect against tumor cells compared to the unmodified immune cells, and more preferably, this cytotoxic effect is improved. In this specification, "maintained cytotoxic effect" means that when tumor cells and immune cells are mixed in a predetermined ratio, and a predetermined period of time has elapsed since mixing, the cytotoxic effects of the unmodified immune cells and the modified immune cells are substantially equivalent. In a preferred embodiment, "maintained cytotoxic effect" means that the cytotoxic effect of the modified immune cells is 95% to 105% of the cytotoxic effect of the unmodified immune cells. Even if the cytotoxic effect of the modified immune cells is lower than that of the unmodified immune cells, a better therapeutic effect may be observed if the amount of cytokines is significantly reduced. When such immune cells are administered to a patient, increasing the dose of immune cells improves the cytotoxic effect against tumor cells, and cytokine levels may remain low. That is, as long as cytokine levels can be maintained at a low level, the dose of immune cells can be appropriately adjusted to maintain or improve the cytotoxic effect. "Cytotoxic effect" can be evaluated in vitro. For example, the cytotoxic effect can be evaluated by indicators such as the number of surviving tumor cells and their survival rate when immune cells containing CAR are co-cultured with tumor cells. Specifically, the method in Example 1 described later is an example. In the case of solid tumors, it is also possible to evaluate the effect by transplanting tumor cells into non-human animals such as mice, administering immune cells containing CAR, and measuring the size of the tumor. Specifically, the method in Example 5 described later is an example. In the case of hematological cancers, it is also possible to evaluate the effect by transplanting tumor cells into non-human animals such as mice, administering immune cells containing CAR, and measuring the number of tumor cells in the blood using a microscope or flow cytometer.

[0056] As described above, nucleic acid molecules encoding a modified antigen-binding region can be obtained from the original nucleic acid molecule encoding the antigen-binding region using known genetic engineering techniques and other molecular biological techniques. First, a primer set for modifying codons is prepared based on the nucleotide sequence of the original nucleic acid molecule encoding the antigen-binding region. For example, if the codon modification is codon substitution, a primer set is prepared designed so that at least three codons in the nucleotide sequence encoding the light chain FR3 are replaced with codons encoding basic amino acid residues. Then, by amplifying the original nucleic acid molecule encoding the antigen-binding region using this primer set as a template via PCR, a nucleic acid molecule encoding an antigen-binding region in which at least three amino acid residues of the light chain FR3 are replaced with basic amino acid residues can be obtained. Alternatively, if the codon modification is codon insertion, a primer set is prepared designed so that at least three codons encoding basic amino acid residues are inserted at the same positions in the nucleotide sequence encoding the light chain FR3. Then, by using this primer set in a PCR method, the nucleic acid molecule encoding the original antigen-binding region can be amplified using the original nucleic acid molecule as a template to obtain a nucleic acid molecule encoding an antigen-binding region in which basic amino acid residues are inserted at at least three positions in the light chain FR3.

[0057] As described above, the nucleic acid molecule encoding the modified antigen-binding region is obtained based on the nucleic acid molecule encoding the original antigen-binding region; therefore, it is preferable that the original antigen-binding region is obtained from a nucleic acid molecule containing the nucleotide sequence encoding it. For example, if an E. coli clone having plasmid DNA encoding the original antigen-binding region is available, the nucleic acid molecule containing the nucleotide sequence encoding the original antigen-binding region can be obtained by extracting the plasmid DNA from the E. coli clone. Furthermore, for the preparation of the primer set described above, it is preferable that the nucleotide sequence encoding the original antigen-binding region is publicly known or verifiable. If the antigen-binding region contains or consists of a single-chain antibody, the nucleotide sequence encoding the single-chain antibody can be found in known databases such as PDB, GeneBank, abYsis, and IMGT. If a nucleic acid molecule containing the nucleotide sequence encoding the original antigen-binding region is possessed, the nucleotide sequence encoding the original antigen-binding region can be found by sequencing the nucleic acid molecule.

[0058] The modified antigen-binding region and the original antigen-binding region are preferably capable of binding to antigens expressed on tumor cells. These antigens may be expressed on normal cells or specifically on tumor cells. The antigens include not only the full-length antigen, but also fragments of the antigen and complexes of these fragments with MHC (major histocompatibility complex) proteins. Examples of antigen fragments include a portion of the antigen presented by antigen-presenting cells or tumor cells themselves, and synthetic peptides consisting of a portion of the amino acid sequence of the antigen. In humans, MHC proteins are called HLA (human leukocyte antigens), and examples include HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP. For example, if the antigen is a protein expressed within tumor cells, the modified antigen-binding region and the original antigen-binding region can bind to complexes of these antigen fragments with MHC proteins.

[0059] The antigens recognized by the antigen-binding region are preferably antigens present on the surface of tumor cells and antigens present inside tumor cells. Examples of such antigens include 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 3 (GPC3), B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), and ganglioside GM2. The antigen recognized by the antigen-binding region may also be a complex of antigen fragments and MHC proteins. This complex is formed when the antigen is fragmented within the tumor cell, and these fragments bind to the MHC of the tumor cell. The formed complex is then presented on the surface of the tumor cell. Examples of such complexes include the complex of MAGE-A4-derived peptide and HLA-A2 (hereinafter also referred to as the "MAGE-A4 / HLA-A2 complex"), and the complex of PRAME-derived peptide and HLA-A24 (hereinafter also referred to as the "PRAME / HLA-A24 complex"). Examples of HLA-A2 that form a complex with MAGE-A4-derived peptide 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. Of these, HLA-A*02:01 is preferred. Examples of HLA-A24 that form a complex with PRAME-derived peptide include HLA-A*24:02 and HLA-A*24:03. Of these, HLA-A*24:02 is preferred. MAGE-A4-derived peptides and PRAME-derived peptides may form complexes with HLAs different from those mentioned above, depending on the sequence of the fragmented peptides. MAGE-A4-derived peptides and PRAME-derived peptides are oligopeptides consisting of a portion of the amino acid sequence of MAGE-A4 and PRAME, respectively (e.g., 8 to 20 amino acids). An example of a MAGE-A4-derived peptide is the oligopeptide consisting of the amino acid sequence of GVYDGREHTV (SEQ ID NO: 1).Examples of PRAME-derived peptides include the oligopeptide consisting of the amino acid sequence LYVDSLFFL (SEQ ID NO: 2).

[0060] The nucleic acid molecule encoding the original antigen-binding region can be obtained by known genetic engineering techniques and other molecular biological techniques. If the antigen-binding region contains or consists of a single-chain antibody, the nucleic acid molecule encoding the single-chain antibody that binds to the tumor cell antigen can be isolated, for example, by phage display using an antibody phage library. Alternatively, a hybridoma that produces an antibody that binds to the tumor cell antigen can be created, and the nucleic acid molecule encoding the single-chain antibody can be produced using RNA extracted from the hybridoma by reverse transcription and PCR. Hybridomas can be prepared by known methods, such as those described in Kohler G. and Milstein C., Nature, vol.256, pp.495-497, 1975.

[0061] 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 the light chain variable region and a nucleotide sequence encoding the heavy chain variable region. The nucleotide sequence encoding the antigen-binding region may also include a nucleotide sequence encoding part or all of the constant region. The constant region may be either the heavy chain or the light chain constant region, but is preferably the 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, in order from the 5' end, a nucleotide sequence encoding the light chain variable region and a nucleotide sequence encoding the heavy chain variable region. Alternatively, the nucleotide sequence encoding the antigen-binding region may include, in order from the 5' end, a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region. If a nucleotide sequence encoding part or all of the light chain constant region is included, it is preferable that this nucleotide sequence follows the nucleotide sequence encoding the light chain variable region. If the nucleotide sequence includes a nucleotide sequence that encodes part or all of the constant region of the heavy chain, it is preferable that the nucleotide sequence follows the nucleotide sequence that encodes the variable region of the heavy chain.

[0062] The segment encoding the antigen-binding region includes or consists of a nucleotide sequence encoding a single-chain antibody that binds to any one of the above-mentioned tumor cell antigens. Preferably, the segment encoding the antigen-binding region includes or consists of a nucleotide sequence encoding a single-chain antibody that binds to a complex of MAGE-A4-derived peptide and HLA-A2, a complex of PRAME-derived peptide and HLA-A24, CD19, or CEA.

[0063] Preferably, the nucleotide sequence encoding a single-chain antibody that binds to a complex of MAGE-A4-derived peptide and HLA-A2 includes the nucleotide sequences shown in SEQ ID NOs. 153 represents the nucleotide sequence encoding HCDR1, 154 represents the nucleotide sequence encoding HCDR2, 155 represents the nucleotide sequence encoding HCDR3, 156 represents the nucleotide sequence encoding LCDR1, 157 represents the nucleotide sequence encoding LCDR2, and 158 represents the nucleotide sequence encoding LCDR3. A specific example of a nucleotide sequence encoding a single-chain antibody including SEQ ID NOs. 153-158 is shown in SEQ ID NO. 159. Based on SEQ ID NO. 159, nucleotide sequences encoding a single-chain antibody with modified FR3 are shown, for example, in SEQ ID NOs. 160-169.

[0064] Preferably, the nucleotide sequence encoding a single-chain antibody that binds to a complex of PRAME-derived peptide and HLA-A24 includes the nucleotide sequences shown in SEQ ID NOs: 170-175. SEQ ID NO: 170 is a nucleotide sequence encoding HCDR1, SEQ ID NO: 171 is a nucleotide sequence encoding HCDR2, SEQ ID NO: 172 is a nucleotide sequence encoding HCDR3, SEQ ID NO: 173 is a nucleotide sequence encoding LCDR1, SEQ ID NO: 174 is a nucleotide sequence encoding LCDR2, and SEQ ID NO: 175 is a nucleotide sequence encoding LCDR3. A specific example of a nucleotide sequence encoding a single-chain antibody including SEQ ID NOs: 170-175 is shown in SEQ ID NO: 176. Based on SEQ ID NO: 176, a nucleotide sequence encoding a single-chain antibody with modified FR3 is shown, for example, in SEQ ID NOs: 177-179.

[0065] Preferably, the nucleotide sequence encoding a single-chain antibody that binds to CD19 includes the nucleotide sequences shown in SEQ ID NOs. 180 is the nucleotide sequence encoding HCDR1, 181 is the nucleotide sequence encoding HCDR2, 182 is the nucleotide sequence encoding HCDR3, 183 is the nucleotide sequence encoding LCDR1, 184 is the nucleotide sequence encoding LCDR2, and 185 is the nucleotide sequence encoding LCDR3. A specific example of a nucleotide sequence encoding a single-chain antibody including SEQ ID NOs. 180-185 is shown in SEQ ID NO. 186. Based on SEQ ID NO. 186, nucleotide sequences encoding a single-chain antibody with modified FR3 are shown, for example, in SEQ ID NOs. 187-205.

[0066] The nucleotide sequence encoding a 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 the portion that connects the light chain variable region and the heavy chain variable region. The amino acid sequence of the peptide linker is not particularly limited, but generally, a sequence of 15 to 20 amino acids in length containing repeats of an amino acid sequence consisting of glycine residues and serine residues (e.g., GGGGS: SEQ ID NO: 206) is used. For example, the nucleotide sequence encoding a single-chain antibody may include, in order from the 5' end, a nucleotide sequence encoding the light chain variable region, a nucleotide sequence encoding the peptide linker, and a nucleotide sequence encoding the heavy chain variable region. Alternatively, the nucleotide sequence encoding a single-chain antibody may include, in order from the 5' end, a nucleotide sequence encoding the heavy chain variable region, a nucleotide sequence encoding the peptide linker, and a nucleotide sequence encoding the light chain variable region.

[0067] In a CAR, the transmembrane domain is the site for fixing the CAR to the cell membrane of an immune cell. The transmembrane domain of a CAR may be derived from a transmembrane protein. For example, in the nucleic acid molecule of this embodiment, the segment having a nucleotide sequence encoding the transmembrane domain (hereinafter also referred to as the "transmembrane segment") may include a nucleotide sequence encoding the full length of a transmembrane protein. Alternatively, the transmembrane segment may include a nucleotide sequence encoding a portion of a transmembrane protein, as long as its function as a transmembrane domain is maintained. Preferably, the portion of the transmembrane protein includes all or part of the transmembrane region of the protein. Transmembrane proteins are not particularly limited, but examples include 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 known as CD137), ICOS (Inducible T-cell co-stimulator), and GITR (Glucocorticoid-induced TNF receptor). Preferably, the transmembrane segment contains a nucleotide sequence encoding the transmembrane region of any one protein selected from this group of transmembrane proteins. Among these, the nucleotide sequence encoding the transmembrane region of CD8α or CD28 is particularly preferred. A specific example of the nucleotide sequence encoding the transmembrane region of CD28 is shown in SEQ ID NO: 207.

[0068] The nucleic acid molecule of this embodiment preferably includes a segment encoding a hinge domain (hereinafter also referred to as the "hinge segment") between the nucleotide sequence encoding the antigen-binding region and the transmembrane segment. In a CAR, the hinge domain can confer length and flexibility to the extracellular domain to allow the antigen-binding region to access the antigen and to avoid steric hindrance. The hinge domain is also called the spacer region. The hinge domain of a CAR may be derived, for example, from a transmembrane protein or IgG. Specifically, in the nucleic acid molecule of this embodiment, the hinge segment may include a nucleotide sequence encoding all of the extracellular region of a transmembrane protein or all of the constant region of IgG. Alternatively, the hinge segment may include a nucleotide sequence encoding part of the extracellular region of a transmembrane protein or part of the constant region of IgG, as long as its function as a hinge domain is maintained. The portion of the extracellular region of a membrane protein that can be used as a hinge domain is hereinafter also referred to as the "hinge region". Preferably, the hinge segment includes a nucleotide sequence encoding one of the following regions selected from the group consisting of the constant region of the IgG light chain, the CD8α hinge region, and the CD28 hinge region. Preferably, the IgG is IgG4. A specific example of the nucleotide sequence encoding the constant region of the IgG light chain is shown in SEQ ID NO: 208. A specific example of the nucleotide sequence encoding the CD28 hinge region is shown in SEQ ID NO: 209.

[0069] In a CAR, the intracellular domain includes a signaling domain. The signaling domain of a CAR is a site for inducing signal transduction that activates immune cells expressing the CAR when the antigen-binding region binds to an antigen. In the nucleic acid molecule of this embodiment, the segment encoding the intracellular domain (hereinafter also referred to as the "intracellular segment") may include a nucleotide sequence encoding the signaling domain. The signaling domain of a CAR may originate from a membrane protein having an intracellular domain, such as a cell membrane receptor or a transmembrane protein. The intracellular domain of such a membrane protein may contain a signaling domain. That is, the intracellular domain of a CAR may include the signaling 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 include a nucleotide sequence encoding a portion of the above-mentioned membrane protein, as long as its function as a signaling domain is maintained. Preferably, the portion of the membrane protein includes all or part of the signaling domain of that protein. The membrane proteins are not particularly limited, but examples include CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ, and FcRβ. Preferably, the intracellular segment contains a nucleotide sequence encoding the signaling domain of at least one protein selected from this group of membrane proteins. Among these, CD3ζ is preferred. A specific example of the nucleotide sequence encoding the signaling domain of CD3ζ is shown in SEQ ID NO: 210.

[0070] The intracellular segment preferably has a nucleotide sequence encoding a costimulatory domain in addition to the nucleotide sequence encoding the signaling domain described above. It is known that when signals from the costimulatory domain are transmitted to immune cells (especially T cells) together with signals from the signaling domain, the proliferation, cytotoxic activity, and viability of the immune cells are improved. The costimulatory domain of CAR may originate from membrane proteins having an intracellular domain, such as cell membrane receptors and transmembrane proteins. The intracellular domain of such membrane proteins may contain a costimulatory domain. That is, the intracellular domain of CAR may include the 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 include a nucleotide sequence encoding a portion of the membrane protein, as long as its function as a costimulatory domain is maintained. It is preferable that the portion of the membrane protein includes all or part of the costimulatory domain of that protein. The membrane proteins are not particularly limited, but examples include 4-1BB (also known as CD137), CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, and ICOS. Preferably, the intracellular segment contains a nucleotide sequence encoding the costimulatory domain of at least one protein selected from this group of membrane proteins. Among these, at least one selected from the group consisting of 4-1BB, CD28, and GITR is preferred. A specific example of the nucleotide sequence encoding the costimulatory domain of CD28 is shown in SEQ ID NO: 211. A specific example of the nucleotide sequence encoding the costimulatory domain of GITR is shown in SEQ ID NO: 212.

[0071] In the intracellular domain segment, the order of the nucleotide sequences encoding the signaling domain and the nucleotide sequences encoding the co-stimulatory domain is not particularly limited. For example, the nucleotide sequence encoding the intracellular domain may include, in order from the 5' end, the nucleotide sequence encoding the signaling domain and the nucleotide sequence encoding the co-stimulatory domain. Alternatively, the nucleotide sequence encoding the intracellular domain may include, in order from the 5' end, the nucleotide sequence encoding the co-stimulatory domain and the nucleotide sequence encoding the signaling domain.

[0072] The nucleic acid molecule of this embodiment may, if necessary, contain various nucleotide sequences in addition to the nucleotide sequence encoding CAR. Examples of such nucleotide sequences include a leader sequence, a restriction enzyme recognition sequence, a nucleotide sequence encoding a peptide tag, and a stop codon. The peptide tag can be appropriately selected from known peptide tags such as histidine tag, glutathione-S-transferase (GST) tag, and FLAG® tag.

[0073] Further embodiments of the present invention relate to vectors containing the nucleic acid molecule described in 1. above. Specifically, the vector of this embodiment may be in which the nucleic acid molecule of this embodiment is incorporated into a known vector. The type of vector is not particularly limited and includes, for example, plasmid vectors and viral vectors. The vector may be linear or circular. The type of plasmid vector is not particularly limited and includes, for example, expression vectors, vectors for constructing viral vectors, transposon vectors, and cloning vectors. An expression vector is a vector that enables the expression of a protein encoded by the nucleotide sequence of the nucleic acid molecule incorporated into the vector in a suitable host cell such as mammalian cells, insect cells, yeast, or Escherichia coli. A transposon vector is a vector that, when introduced into a suitable host together with an expression vector into which a gene encoding a transposase is incorporated, enables the integration of the nucleic acid molecule incorporated into the transposon vector into the genome of the host cell.

[0074] The type of viral vector is not particularly limited and includes, for example, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, and Epstein-Barr virus (EBV) vectors. It is preferable that the viral vector lacks replication ability so that the virus does not self-replicate within the infected cell.

[0075] The vector may include appropriate control sequences as needed. Examples of such control sequences include promoter sequences, operator sequences, enhancer sequences, nucleotide sequences encoding drug resistance markers, and multicloning sites.

[0076] As described above, the nucleic acid molecule in this embodiment may be either DNA or RNA. DNA is a more stable substance than RNA, and various DNA vectors are commercially available. Therefore, the nucleic acid molecule in this embodiment, which is DNA, is advantageous in that it is easy to store and handle. RNA that encodes a protein is known to be able to express the protein without being affected by the transcriptional regulatory process when introduced into cells. Therefore, the nucleic acid molecule in this embodiment, which is RNA, is advantageous in that it enables rapid CAR expression in immune cells.

[0077] 2. CAR Further embodiments of the present invention relate to CARs. The CAR of this embodiment is a protein molecule encoded by the nucleotide sequence of the nucleic acid molecule described in 1. above. The CAR of this embodiment is a protein produced by genetic engineering, comprising, in order from the N-terminus, an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes an antigen-binding region, which includes a light chain variable region and a heavy chain variable region. The CAR of this embodiment is characterized in that at least three 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 includes an antigen-binding region in which at least three amino acid residues of the light chain FR3 are basic amino acid residues. This antigen-binding region is the same as the modified antigen-binding region described above. Hereinafter, the antigen-binding region of the CAR of this embodiment will also be referred to as the "modified antigen-binding region". The CAR of this embodiment can also be described as a variant 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 the immune cells. In immune cells expressing the CAR, cytokine production is suppressed compared to immune cells expressing the original CAR containing the antigen-binding region. Preferably, the cytotoxic effect of these immune cells against tumor cells is maintained, and more preferably, this cytotoxic effect is improved.

[0078] In the CAR of this embodiment, at least three basic amino acid residues of the light chain FR3 are derived from the modification of the codon described above. In the light chain FR3 of the modified antigen-binding region, the at least three basic amino acid residues derived from the modification of the codon may all be arginine residues, or they may all be lysine residues. Alternatively, the at least three basic amino acid residues derived from the modification of the codon may be partly arginine residues and the remainder be lysine residues.

[0079] The amino acid residues in the light chain FR3 before at least three amino acid residues are converted to basic amino acid residues are neutral amino acid residues or acidic amino acid residues, preferably neutral amino acid residues. That is, the at least three basic amino acid residues in the light chain FR3 of the modified antigen-binding region are residues that have been changed from at least three residues selected from neutral amino acid residues and / or acidic amino acid residues in the light chain FR3 of the original antigen-binding region.

[0080] In the light chain FR3 of the modified antigen-binding region, the number of at least three basic amino acid residues derived from the codon modification 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 codon modification in the light chain FR3 of the modified antigen-binding region is preferably 3 to 6, and more preferably 3 to 5. As described above, the at least three basic amino acid residues in the light chain FR3 of the modified antigen-binding region are preferably located at positions selected from amino acid residues 57, 58, 59, 60, 61, 62, 63, 65, 67, 70, 72, 74, 76, 77, 79, 80, and 81 in the light chain variable region.

[0081] In the modified antigen-binding region, it is preferable that at least three amino acid residues selected from the group consisting of amino acid residues 60, 63, 65, 67, 70, 72, 74, 76, 77, 79, and 81 in the light chain variable region are basic amino acid residues derived from the modification of the codon. More preferably, three to six amino acid residues selected from the above group are basic amino acid residues. Particularly more preferably, three to 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 the following 1) to 21) are basic amino acid residues derived from the modification of the codon.

[0082] 1) The 60th, 63rd, and 65th amino acid residues in the light chain variable region; 2) The 60th, 63rd, and 76th amino acid residues in the light chain variable region; 3) The 60th, 74th, and 76th amino acid residues in the light chain variable region; 4) Amino acid residues 63, 65, and 67 in the light chain variable region; 5) Amino acid residues 63, 65, and 70 in the light chain variable region; 6) Amino acid residues 63, 65, and 72 in the light chain variable region; 7) Amino acid residues 63, 67, and 70 in the light chain variable region; 8) The 63rd, 67th, and 72nd amino acid residues in the light chain variable region; 9) Amino acid residues 63, 70, and 72 in the light chain variable region; 10) Amino acid residues 65, 67, and 70 in the light chain variable region; 11) Amino acid residues 65, 67, and 72 in the light chain variable region; 12) Amino acid residues 65, 70, and 72 in the light chain variable region; 13) Amino acid residues 67, 70, and 72 in the light chain variable region; 14) The 70th, 72nd, and 74th amino acid residues in the light chain variable region; 15) Amino acid residues 77, 79, and 81 in the light chain variable region; 16) Amino acid residues 63, 65, 67 and 70 in the light chain variable region; 17) Amino acid residues 63, 65, 67, and 72 in the light chain variable region; 18) Amino acid residues 63, 65, 70, and 72 in the light chain variable region; 19) Amino acid residues 63, 67, 70 and 72 in the light chain variable region; 20) The 65th, 67th, 70th and 72nd amino acid residues in the light chain variable region; and 21) The 63rd, 65th, 67th, 70th and 72nd amino acid residues in the light chain variable region.

[0083] In the modified antigen-binding region, it is preferable that at least three amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 in the light chain variable region, and at least one amino acid residue selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 in the light chain variable region, are basic amino acid residues.

[0084] In the modified antigen-binding region light chain FR3, when three to six amino acid residues are basic amino acid residues, it is preferable that these three to six amino acid residues include three, four, or five amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 in the light chain variable region, and one, two, or three amino acid residues selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 in the light chain variable region.

[0085] In the modified antigen-binding region light chain FR3, when three to five amino acid residues are basic amino acid residues, it is preferable that these three to five amino acid residues include three or four amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 in the light chain variable region, and one or two amino acid residues selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 in the light chain variable region.

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

[0087] In the modified antigen-binding region light chain FR3, when three to six amino acid residues are basic amino acid residues, it is preferable that these three to six amino acid residues include three, four, or five amino acid residues selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 in the light chain variable region, and one, two, or three amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 in the light chain variable region.

[0088] In the modified antigen-binding region light chain FR3, when three to five amino acid residues are basic amino acid residues, it is preferable that these three to five amino acid residues include three or four amino acid residues selected from the group consisting of positions 60, 74, 76, 77, 79, and 81 in the light chain variable region, and one or two amino acid residues selected from the group consisting of positions 63, 65, 67, 70, and 72 in the light chain variable region.

[0089] In the modified antigen-binding region, if amino acid residues other than those at positions 60, 63, 65, 67, 70, 72, 74, 76, 77, 79, and 81 of the light chain variable region are modified, it is preferable that the modified amino acid residues are amino acid residues selected from the group consisting of positions 57, 58, 59, 61, and 62.

[0090] In the CAR of this embodiment, the extracellular domain includes an antigen-binding region that includes a light chain variable region and a heavy chain variable region. An example of the antigen-binding region is a single-chain antibody. The antigen-binding region may include part or all of the constant region of the antibody. The constant region may be the constant region of either the heavy chain or the light chain, but is preferably the constant region of the light chain. 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 the light chain variable region and the heavy chain variable region in order from the N-terminus. Alternatively, the antigen-binding region may include the heavy chain variable region and the light chain variable region in order from the N-terminus. In the antigen-binding region, it is preferable that part or all of the constant region of the light chain is included after the light chain variable region. In the antigen-binding region, it is preferable that part or all of the constant region of the heavy chain is included after the heavy chain variable region.

[0091] In the CAR of this embodiment, the antigen-binding region may include or consist of a single-chain antibody that binds to either an antigen present on the surface of tumor cells or an antigen present inside tumor cells. Preferably, the antigen-binding region includes or consists of a single-chain antibody that binds to a complex of MAGE-A4-derived peptide and HLA-A2, a complex of PRAME-derived peptide and HLA-A24, CD19, or CEA.

[0092] Preferably, the amino acid sequence of a single-chain antibody that binds to a complex of MAGE-A4-derived peptide and HLA-A2 includes the amino acid sequences shown in SEQ ID NOs. 213 is the amino acid sequence of HCDR1, SEQ ID NOs. 214 is the amino acid sequence of HCDR2, SEQ ID NOs. 215 is the amino acid sequence of HCDR3, SEQ ID NOs. 216 is the amino acid sequence of LCDR1, SEQ ID NOs. 217 is the amino acid sequence of LCDR2, and SEQ ID NOs. 218 is the amino acid sequence of LCDR3. A specific example of the amino acid sequence of a single-chain antibody including SEQ ID NOs. 213-218 is shown in SEQ ID NOs. 219. Based on SEQ ID NOs. 219, the amino acid sequences of a single-chain antibody with modified FR3 are shown, for example, in SEQ ID NOs. 220-229.

[0093] Preferably, the amino acid sequence of a single-chain antibody that binds to a complex of PRAME-derived peptide and HLA-A24 includes the amino acid sequences shown in SEQ ID NOs. 230 is the amino acid sequence of HCDR1, 231 is the amino acid sequence of HCDR2, 232 is the amino acid sequence of HCDR3, 233 is the amino acid sequence of LCDR1, 234 is the amino acid sequence of LCDR2, and 235 is the amino acid sequence of LCDR3. A specific example of the amino acid sequence of a single-chain antibody including SEQ ID NOs. 230 to 235 is shown in SEQ ID NO. 236. Based on SEQ ID NO. 236, the amino acid sequences of a single-chain antibody with modified FR3 are shown, for example, in SEQ ID NOs. 237 to 239.

[0094] Preferably, the amino acid sequence of the single-chain antibody that binds to CD19 includes the amino acid sequences shown in SEQ ID NOs. 240 is the amino acid sequence of HCDR1, SEQ ID NOs. 241 is the amino acid sequence of HCDR2, SEQ ID NOs. 242 is the amino acid sequence of HCDR3, SEQ ID NOs. 243 is the amino acid sequence of LCDR1, SEQ ID NOs. 244 is the amino acid sequence of LCDR2, and SEQ ID NOs. 245 is the amino acid sequence of LCDR3. A specific example of the amino acid sequence of a single-chain antibody including SEQ ID NOs. 240 to 245 is shown in SEQ ID NOs. 246. Based on SEQ ID NOs. 246, the amino acid sequences of a single-chain antibody with modified FR3 are shown, for example, in SEQ ID NOs. 247 to 265.

[0095] It is preferable that a single-chain antibody contains a peptide linker between the light chain variable region and the heavy chain variable region. The peptide linker is as described above. For example, a single-chain antibody may contain, in order from the N-terminus, a light chain variable region, a peptide linker, and a heavy chain variable region. Alternatively, a single-chain antibody may contain, in order from the N-terminus, a heavy chain variable region, a peptide linker, and a light chain variable region.

[0096] In the CAR of this embodiment, the transmembrane domain may include the entire length of the transmembrane protein. Alternatively, the transmembrane domain may include a portion of the transmembrane protein, as long as its function is maintained. Preferably, the portion of the transmembrane protein includes all or part of the transmembrane region of the protein. Examples of transmembrane proteins that can be used for the transmembrane domain include the proteins exemplified in 1. above. Preferably, the transmembrane domain is the transmembrane region of any one 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 known as CD137), ICOS, and GITR. Among these, the transmembrane region of CD8α or CD28 is preferred. A specific example of the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 266.

[0097] In this embodiment, the CAR preferably includes a hinge domain between the antigen-binding domain and the transmembrane domain. The hinge domain may include the entire extracellular region of the transmembrane protein or the entire constant region of IgG. Alternatively, the hinge domain may include a portion of the extracellular region of the transmembrane protein or a portion of the constant region of IgG, as long as its function is maintained. Examples of proteins that can be used for the hinge domain include the proteins exemplified in 1. above. Preferably, the hinge domain is one region selected from the group consisting of, for example, the constant region of the IgG light chain, the CD8α hinge region, and the CD28 hinge region. IgG is preferably IgG4. A specific example of the amino acid sequence of the constant region of the IgG light chain is shown in SEQ ID NO: 267. A specific example of the amino acid sequence of the CD28 hinge region is shown in SEQ ID NO: 268.

[0098] In the CAR of this embodiment, the intracellular domain may include the full length of a membrane protein having an intracellular region. Alternatively, the intracellular domain may include a portion of the above-mentioned membrane protein, as long as its function as a signaling domain is maintained. The signaling domain may reside in the intracellular region of the membrane protein. It is preferable that a portion of a membrane protein having an intracellular region includes all or part of the signaling domain of that protein. Examples of membrane proteins that can be used for the intracellular domain include the proteins exemplified in 1. above. Preferably, the intracellular domain includes, for example, the signaling 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 these, the signaling domain of CD3ζ is preferred. A specific example of the amino acid sequence of the signaling domain of CD3ζ is shown in SEQ ID NO: 269.

[0099] In the CAR of this embodiment, the intracellular domain preferably has a co-stimulatory domain in addition to the signal transduction domain described above. The intracellular domain may include the full length of a membrane protein having an intracellular region. Alternatively, the intracellular domain may include a portion of the membrane protein described above, as long as its function as a co-stimulatory domain is maintained. The co-stimulatory domain may be present in the intracellular region of each of the proteins described above. It is preferable that a portion of a membrane protein having an intracellular region includes all or part of the co-stimulatory domain of that protein. Examples of membrane proteins that can be used as a co-stimulatory domain include the proteins exemplified in 1. above. Preferably, the co-stimulatory domain includes the co-stimulatory domain of at least one protein selected from the group consisting of 4-1BB (also called CD137), CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, and ICOS. Among these, the co-stimulatory domains of 4-1BB, CD28, and GITR are preferred. A specific example of the amino acid sequence of the CD28 co-stimulatory domain is shown in SEQ ID NO: 270. A specific example of the amino acid sequence of the GITR co-stimulatory domain is shown in SEQ ID NO: 271.

[0100] In the intracellular domain, the order of the signal transduction domain and the co-stimulation domain is not particularly limited. For example, the intracellular domain may include the signal transduction domain and the co-stimulation domain in order from the N-terminus. Alternatively, the intracellular domain may include the co-stimulation domain and the signal transduction domain in order from the N-terminus.

[0101] The CAR of this embodiment may optionally contain additional oligopeptides or polypeptides. Examples of such oligopeptides and polypeptides include signal peptides and peptide tags. Peptide tags can be appropriately selected from known peptide tags such as histidine tags, GST tags, and FLAG® tags.

[0102] The CAR of this embodiment can be produced by a protein expression system using the nucleic acid molecule of this embodiment. The protein expression system may be an expression system using a host cell or a cell-free protein synthesis system. In production using an expression system using a host cell, for example, the CAR of this embodiment can be expressed by introducing the nucleic acid molecule of this embodiment, which is incorporated into an expression vector suitable for the host cell, into the host cell. Examples of cell-free protein synthesis systems include a wheat germ-derived synthesis system, an E. coli-derived synthesis system, and a reconstituted cell-free protein synthesis system. If the CAR is produced in the host cell, the host cell may be dissolved in a solution containing a suitable solubilizer to release the CAR into the solution. In a cell-free protein synthesis system, the synthesized CAR is contained in the reaction solution. The CAR released into the liquid can be recovered by known methods such as column chromatography. For example, if the produced 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 (nitrilotriacetic acid chelated with nickel ions) or glutathione. If necessary, the recovered CAR may be purified by known methods such as gel filtration or dialysis.

[0103] 3. Immune cells including CAR A further embodiment of the present invention relates to immune cells containing CAR. The immune cells containing CAR in this embodiment are immune cells that express the CAR described in 2. above by introducing the nucleic acid molecule described in 1. above into the immune cells. Details of the CAR and nucleic acid molecule are as described above. Examples of immune cells before the introduction of the nucleic acid molecule described in 1. above include immune cells collected from mammals including humans, immune cells prepared from stem cells, and immune cells established as cell lines. Specifically, examples include immune cells obtained by leukocyte apheresis and immune cells isolated from blood.

[0104] Examples of immune cells isolated from biological samples include peripheral blood mononuclear cells (PBMCs), T cells, and natural killer (NK) cells. Among these, 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. Preferred cell lines for immune cells are lymphocyte-derived cell lines, such as Jurkat cells, MOLT-4 cells, and U-937 cells. Hereinafter, T cells expressing CAR may be referred to as "CAR-T cells."

[0105] The method for introducing the nucleic acid molecules described in item 1 above into immune cells is not particularly limited and can be appropriately selected from known gene transfer methods. Examples of gene transfer methods include lipofection, electroporation, calcium phosphate, gene transfer using cationic polymers, gene transfer using viral vectors, and gene transfer using transposons. For lipofection and gene transfer using cationic polymers, commercially available transfection reagents such as FuGENE® and JetPEI® may be used.

[0106] Immune cells containing CAR according to this embodiment can be cultured in the same way as immune cells before the introduction of the nucleic acid molecule according to this embodiment. The culture medium, serum, additives, etc., can be appropriately determined depending on the type of immune cell used. Examples of culture media include MEM, DMEM, and RPMI-1640. If the immune cells are lymphocytes, commercially available lymphocyte culture media such as GT-T502, GT-T503, and GT-T551 (Takara Bio Inc.) may be used. Examples of serum include fetal bovine serum (FBS) and human type AB serum. Examples of additives include L-glutamine, insulin, and IL-2. Examples of immune cell culture conditions include 37°C under a 5% CO2 atmosphere.

[0107] In the immune cells containing CAR in this embodiment, CAR is expressed as a transmembrane protein. When the extracellular domain of the CAR binds to the antigen of a tumor cell, the immune cell is activated by a signal from the intracellular domain of the CAR. The activated immune cells containing the CAR in this embodiment then exert a cytotoxic effect by releasing cytotoxic proteins (e.g., perforin, granzyme, etc.) and antitumor cytokines (e.g., tumor necrosis factor (TNF)-α, lymphokines, etc.), and by expressing cell surface molecules that induce cell death, such as Fas ligand. Preferably, the immune cells containing the CAR in this embodiment maintain their cytotoxic effect, and more preferably, their cytotoxic effect is improved, compared to immune cells containing the original antigen-binding domain. The cytotoxic effect of immune cells containing CAR can be investigated by known methods such as cytotoxicity assays. Specifically, the method in Example 3 described later is an example. Furthermore, as in Example 4 described later, the cytotoxic effect can also be evaluated by administering immune cells containing CAR to non-human animals transplanted with tumor cells and measuring the size of the tumor. On the other hand, immune cells containing CARs in this embodiment release reduced amounts of cytokines that cause CRS (e.g., IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), etc.) compared to immune cells containing CARs that retain the original antigen-binding domain. The amount of cytokines released by immune cells containing CARs can be investigated by known methods such as enzyme-linked immunosorbent assay (ELISA). Specifically, the method described in Example 5 below is an example.

[0108] 4. Method for producing immune cells containing CAR Further embodiments of the present invention relate to a method for producing immune cells containing CAR (hereinafter also referred to as "the production method of this embodiment"). In the production method of this embodiment, immune cells containing the CAR described in 3 are produced by introducing the nucleic acid molecule described in 1. above into immune cells and causing the CAR described in 2. above to be expressed in the immune cells. Details of the CAR and nucleic acid molecule are as described above. Details of the immune cells before the introduction of the nucleic acid molecule described in 1. above are also as described above. Preferably, these are PBMCs, T cells and NK cells. When transplanting immune cells containing CAR obtained by the production method of this embodiment into a living organism, it is preferable to use immune cells isolated from the living organism itself or from a biological sample taken from another living organism of the same type.

[0109] The method for introducing the nucleic acid molecule described in 1. above into immune cells is as described above. After introducing the nucleic acid molecule into immune cells, it is preferable to culture the immune cells for a predetermined period. The method for culturing immune cells is as described above. The predetermined period may be at least the period until CAR is expressed in the immune cells. The period until CAR is expressed is determined according to the method of introducing the nucleic acid molecule, but is for example 3 hours or more and 72 hours or less, preferably 6 hours or more and 48 hours or less. If the method of introducing the nucleic acid molecule is a method that enables stable expression of CAR in immune cells, the predetermined period may be a period sufficient for the immune cells expressing CAR to proliferate. Such a period is not particularly limited, but may be for example 48 hours or more and 20 days or less, preferably 72 hours or more and 14 days or less.

[0110] By introducing the nucleic acid molecule described in 1. above into immune cells, the CAR described in 2. above is expressed in those immune cells. If necessary, the expression of CAR in immune cells may be confirmed. The expression of CAR in immune cells may be confirmed by known protein detection methods such as ELISA, flow cytometry, immunoprecipitation, polyacrylamide gel electrophoresis, and Western blotting.

[0111] 5. Pharmaceutical Compositions Further embodiments of the present invention relate to a pharmaceutical composition for treating malignant tumors (hereinafter also referred to as "the pharmaceutical composition of this embodiment") comprising immune cells containing CAR. The pharmaceutical composition of this embodiment contains immune cells containing the CAR described in 3. above as an active ingredient. Details of the CAR and the immune cells containing it are as described above. The pharmaceutical composition of this embodiment may further contain pharmaceutically acceptable additives. Such additives include aqueous media for the stable storage of immune cells, D-glucose, dextran, serum albumin, and dimethyl sulfoxide (DMSO). Examples of aqueous media include physiological saline, phosphate-buffered saline (PBS), and complex electrolyte solutions. The pharmaceutical composition of this embodiment is preferably administered to the patient parenterally. That is, the pharmaceutical composition is preferably in a form suitable for parenteral administration, such as an injection or an infusion.

[0112] The malignant tumor targeted for treatment by the pharmaceutical composition of this embodiment is a tumor containing tumor cells having antigens recognized by CAR. The malignant tumor may be a hematological cancer or a solid tumor. Examples include acute leukemia (acute myeloid leukemia, B-cell acute lymphoblastic leukemia, acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, etc.), chronic leukemia (chronic myeloid leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia, etc.), lymphoma (diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, Hodgkin 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, stomach cancer, esophageal cancer, ovarian cancer, osteosarcoma, and neuroblastoma.

[0113] 6.Treatment method Further embodiments of the present invention relate to a method for treating malignant tumors using immune cells containing CAR as described in 3. above (hereinafter also referred to as "the treatment method of this embodiment"). This method includes the step of administering the above-described pharmaceutical composition to a patient with a malignant tumor. The malignant tumors to be treated are as described above.

[0114] In the administration process, immune cells are preferably administered to the patient parenterally. Parenteral administration methods include, for example, intravenous, intra-arterial, intramuscular, intraperitoneal, and subcutaneous administration. Among these, intravenous administration is preferred.

[0115] The dosage of the pharmaceutical composition of this embodiment can be appropriately determined according to the type of cancer, the patient's condition, age, weight, etc. For example, for an adult weighing 50 kg or more, the dosage is 1 × 10¹⁶ CAR-containing immune cells per dose. 4 More than 1 x 10 cells 10 Cells or smaller, preferably 1 × 10 5 More than 1 x 10 cells 9 Below cells, more preferably 1 × 10 6 cells or more: 5 × 10 8 The cells are smaller than or equal to the number of cells. The number of administrations may be a single dose or multiple doses. After administration, immune cells including CAR engraft and proliferate in the patient's body, so usually only one dose is administered. However, if the engraftment or proliferation of immune cells is judged to be insufficient, multiple doses may be administered. The administration interval can be, for example, 1 to 4 times a day, every week, every 10 to 30 days, every month, every 3 to 6 months, every year, etc.

[0116] 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 item 5 above may be on the same day or on different days. Examples of other anticancer agents include, but are not limited to, alkylating agents such as cyclophosphamide, antimetabolites such as pentostatin, molecular targeted drugs such as rituximab, kinase inhibitors such as imatinib, proteasome inhibitors such as bortezomib, calcineurin inhibitors such as cyclosporine, anticancer antibiotics such as idarubicin, plant alkaloids such as irinotecan, platinum-based drugs such as cisplatin, hormone therapy drugs such as tamoxifen, and immunosuppressants such as nivolumab and pembrolizumab.

[0117] The patient's white blood cell count may be reduced by performing lymphocyte apheresis chemotherapy as a pre-administration treatment for the pharmaceutical composition of this embodiment. Examples of lymphocyte apheresis chemotherapy include fludarabine, cyclophosphamide, and bendamustine.

[0118] 7. Methods to reduce cytokine production by immune cells Further embodiments of the present invention relate to a method for reducing cytokine production in immune cells containing a CAR (hereinafter also referred to as "the reduction method of this embodiment"). The reduction method of this embodiment includes the steps of producing the nucleic acid molecule described in 1. above, and introducing the nucleic acid molecule described in 1. above into immune cells to cause the immune cells to express the CAR described in 2. above. The obtained immune cells containing the CAR express a CAR containing a modified antigen-binding region. Immune cells expressing this CAR produce reduced cytokines compared to immune cells expressing the original CAR containing the antigen-binding region. On the other hand, the cytotoxic effect of immune cells expressing the modified CAR containing the antigen-binding region is maintained or improved compared to immune cells expressing the original CAR containing the antigen-binding region. The reduction method of this embodiment may contribute to further reducing cytokine-induced side effects in therapeutic methods using immune cells expressing a CAR. Details of the nucleic acid molecule and the CAR are as described above. A method for introducing a nucleic acid molecule into immune cells and a method for measuring cytokines produced by immune cells containing the CAR are as described above.

[0119] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0120] Example 1: Production of CAR-T cells that bind to the MAGE-A4 / HLA-A2 complex and confirmation of their effects (1)

[0121] (1) Obtaining nucleic acid molecules encoding CARs to be used as templates Two plasmid DNAs for viral vector construction were obtained, each containing a gene encoding a CAR with a single-chain antibody that binds to the MAGE-A4 / HLA-A2 complex, in a manner similar to the example in US2020 / 0276237 (which is incorporated herein by reference). The complex recognized by the single-chain antibody was the complex of MAGE-A4 and HLA-A*02:01. Hereinafter, the CAR encoded by the gene in one plasmid DNA will be referred to as "MAGE-A4-zG," and the CAR encoded by the gene in the other plasmid DNA will be referred to as "MAGE-A4-zG-s1." The amino acid sequence of the MAGE-A4-derived peptide in the above MAGE-A4 / HLA-A2 complex was GVYDGREHTV (SEQ ID NO: 1). The above plasmid DNAs were used as templates to construct nucleic acid molecules encoding variants of MAGE-A4-zG and MAGE-A4-zG-s1 by PCR.

[0122] Referring to Figure 2A, the gene encoding MAGE-A4-zG was linked from the 5' end in the following order: Leader sequence, nucleotide sequence encoding the heavy chain variable region (VH), nucleotide sequence encoding the linker (L), nucleotide sequence encoding the light chain variable region (VL), nucleotide sequence encoding the light chain constant region (CL), nucleotide sequence encoding the transmembrane domain of CD28 (CD28TM), nucleotide sequence encoding CD3ζ, and nucleotide sequence encoding the intracellular domain of GITR (GITRICD). VH, L, VL, and CL constituted a segment containing nucleotide sequences encoding the extracellular domain of CAR. VH, L, and VL constituted a segment containing nucleotide sequences encoding a single-chain antibody that specifically binds to the MAGE-A4 / HLA-A2 complex. CD3ζ and GITRICD constituted a segment containing nucleotide sequences encoding the intracellular domain of CAR. CL was a segment containing nucleotide sequences encoding the hinge domain of CAR.

[0123] The gene encoding MAGE-A4-zG-s1 had the same structure as the gene encoding MAGE-A4-zG, except that an extra nucleotide sequence was added to the 3' end (the 3' side of GITRICD). Searching known databases confirmed that this extra nucleotide sequence did not match any nucleotide sequence encoding any protein. Furthermore, computer analysis predicted that the amino acid sequence corresponding to this extra nucleotide sequence would form a linear polypeptide without any secondary structure.

[0124] MAGE-A4-zG and MAGE-A4-zG-s1 were CARs containing a single-chain antibody consisting of VH, VL and a linker connecting them as extracellular domains, and CL, as well as a transmembrane domain CD28TM, and as intracellular domains CD3ζ and GITRICD.

[0125] (2) Obtaining nucleic acid molecules encoding variants of MAGE-A4-zG and MAGE-A4-zG-s1 [reagent] QIAprep Spin Miniprep Kit (QIAGEN) PrimeSTAR® Max Premix (Takara Bio Inc.) Ligation high ver.2 (Toyobo Co., Ltd.) T4 Polynucleotide Kinase (Toyobo Co., Ltd.) Dpn I (Toyobo Co., Ltd.) Competent high DH5α (Toyobo Co., Ltd.)

[0126] (2.1) Primer design and PCR A primer set was designed based on the nucleotide sequence of Sequence ID No. 3 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which the amino acid residues shown in a) or b) below in MAGE-A4-zG are substituted with arginine residues. Furthermore, a primer set was designed based on the nucleotide sequence of Sequence ID No. 9 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which the amino acid residues shown in a) below in MAGE-A4-zG-s1 are substituted with arginine residues.

[0127] a) The 63rd, 65th, 67th and 70th amino acid residues of VL as defined by Kabat; and b) The 63rd, 65th, 67th, and 72nd amino acid residues of VL as defined by Kabat

[0128] The plasmid DNAs described above, each containing the genes encoding MAGE-A4-zG and MAGE-A4-zG-s1 respectively, were used as template DNA. A PCR reaction mixture was prepared containing the template DNA, primer sets (SEQ ID NOs. 77 and 78), and PrimeSTAR® Max Premix, and a PCR reaction was performed. This yielded PCR products with restriction enzyme recognition sites (hereinafter referred to as restriction sites) added to the 5' and 3' ends. DpnI was added to the obtained PCR products to fragment the template plasmid DNA. Using the DpnI-treated PCR products as templates, a PCR reaction mixture containing a primer set for mutant generation and PrimeSTAR® Max Premix was prepared, and a PCR reaction was performed. The obtained PCR products, restriction enzyme-treated plasmid DNA for viral vector production (empty vector), 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, CARs containing single-chain antibodies 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, CARs containing single-chain antibodies in which the amino acid residues shown in a) above in MAGE-A4-zG-s1 are replaced with arginine residues will be referred to as "MAGE-A4-zG-m1-s1." Table 1 shows the correspondence between each variant and the sequence numbers of the primer sets used to produce them. In the table, "F" indicates the sequence number of the nucleotide sequence of the forward primer, and "R" indicates the sequence number of the nucleotide sequence of the reverse primer. The explanation of the table is the same for the tables in the following examples.

[0130] [Table 1]

[0131] (2.2) Transformation, plasmid DNA extraction and sequencing E. coli transformants were obtained by heat shock using the ligation reaction solution and DH5α. Single colonies on agar plates were cultured in ampicillin-containing LB liquid medium. Plasmid DNA was extracted from the obtained E. coli using the QIAprep Spin Miniprep kit. Sequences were performed on each obtained plasmid DNA. Sequencing results confirmed the acquisition of nucleic acid molecules encoding variants of MAGE-A4-zG and MAGE-A4-zG-s1.

[0132] (3) Production of T cells expressing MAGE-A4-zG, MAGE-A4-zG-s1, and their variants. (3.1) Preparation of human lymphocytes PBMCs were isolated from blood donated by healthy donors using Ficoll-Paque® PLUS (GE Healthcare). The obtained PBMCs were used as human lymphocytes. 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, and all procedures were approved by the Research Ethics Committee of Mie University School of Medicine, with written consent obtained from each subject. The collected samples were encrypted to prevent identification of the individual and stored in a refrigerator and liquid nitrogen tank with theft prevention measures. The personal information of the subjects was anonymized, and strict precautions and measures were taken to ensure that individual privacy and the results of the genetic analysis were not leaked to external parties.

[0133] (3.2) Isolation and culture of PBMCs Anti-CD3 antibody OKT-3 (eBioscience) and RetroNectin® (Takara Bio Inc.) were added to ACD-A solution (Terumo Corporation) to final concentrations of 5 μg / mL and 25 μg / mL, respectively. 400 μL of the resulting solution was placed in each well of a 12-well plate (Nunc) and left to stand overnight at 4°C. The solution in each well was removed, and the plates were washed three times with PBS to prepare plates for PBMC culture. AB serum (300 μL, Veritas) was added to lymphocyte medium GT-T503 (50 mL, Takara Bio Inc.), and then human IL-2 (NIPRO) was added to a final concentration of 300 IU / mL to prepare PBMC culture medium. 2.5 × 10⁶ PBMCs obtained in (3.1) above were placed in the prepared medium. 5 ~3.0×10 5 The cells were suspended to a concentration of cells / mL. 2 mL of the cell suspension was placed in each well of a PBMC culture plate, and the cells were incubated in a CO2 incubator at 37°C for 3 days. On the third day, 1 mL of the culture medium was replaced, and the cells were incubated for another day.

[0134] (3.3) Introduction of CAR genes into immune cells by retroviruses (i) Production of retroviruses using packaging cells plat-A The plasmid DNA for preparing each viral vector containing the genes encoding MAGE-A4-zG, MAGE-A4-zG-s1, and their variants obtained in (2) above was introduced into the packaging cell plat-A using FuGENE (registered trademark) (Promega) and cultured for 2 days. Thereafter, the culture supernatant was collected to obtain a liquid containing a viral vector (hereinafter also referred to as "virus solution").

[0135] (ii) Preparation of retrovirus diluent and washing solution Albumin (registered trademark) 25% (CSL Behring K.K.) was added to a mixed solution of PBS (40 mL) and ACD-A solution (2.4 mL) to a final concentration of 2.5% to prepare a retrovirus diluent. Also, Albumin (registered trademark) 25% 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 retrovirus infection RetroNectin (registered trademark) (Takara Bio Inc.) was diluted with the retrovirus diluent to a final concentration of 20 μg / mL. 250 μL of the diluted RetroNectin (registered trademark) solution was placed in each well of a 24-well plate and left standing overnight at 4°C. The liquid in each well was removed and washed twice with the retrovirus washing solution. 1 mL of the virus solution prepared in (3.1) above was placed in each well and centrifuged at 2000×g for 2 hours at 32°C to coat the retrovirus in each well. The virus solution in each well was removed and washed twice with the retrovirus washing solution to prepare plates for retrovirus infection.

[0137] (iv) Preparation of CAR-T cells by retrovirus infection The PBMC cultured in (3.2) above was collected, 1.3×10 5The cells were suspended in PBMC culture medium to a concentration of cells / mL. 1.5 mL of the cell suspension was placed in each well of a retrovirus infection plate and centrifuged at 1000 × g for 10 minutes at 32°C. The plates were then placed in a CO2 incubator, and the cells were cultured at 37°C. This yielded T cells expressing various CARs, including MAGE-A4-zG, MAGE-A4-zG-s1, and their variants (CAR-T cells). The CAR-T cells were used in various assays 12 days after PBMC isolation.

[0138] (4) Confirmation of the cytotoxic effect of CAR-T cells and cytokine secretion levels. (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.). Human melanoma cell lines SK-MEL-37 or NW-MEL-38 were used as target cells. These target cells were A2-positive MAGE-A4-positive tumor cells.

[0139] The measurement principle of the above assay kit was as follows: When the BM-HT Reagent included with the assay kit is added to target cells, the BM-HT Reagent is hydrolyzed by intracellular esterases, generating HT chelates within the target cells. When the target cells are killed by effector cells, the HT chelates leak into the culture supernatant. When the Eu Solution included with the assay kit is added to the culture supernatant containing the HT chelates, an Eu / HT complex is formed. When this complex is excited with laser light, time-resolved fluorescence is generated. Since the leakage of HT chelates depends on the cytotoxic activity of the effector cells, the cytotoxic effect can be quantitatively measured by measuring time-resolved fluorescence.

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

[0141] (4.2) Measurement 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, A2-positive MAGE-A4-positive tumor cells SK-MEL-37 and NW-MEL-38 were used. Each of the effector cells and target cells was 1 × 10⁶ 5 The cells were suspended in culture medium to a concentration of cells / mL and placed in each well of a 96-well plate, where they were co-cultured at 37°C for 12 hours. The culture supernatant was collected and used as a sample.

[0142] (ii) Preparation of reagents For the measurement of IFNγ in each sample, invitrogen® Human IFN gamma Uncoated ELISA with Plates (Thermo Fisher Scientific) was used. Coating buffer was prepared by diluting 10× Coating Buffer 10-fold with purified water. A diluted solution of the capture antibody was prepared by adding capture antibody (anti-human IFNγ antibody) (48 μL) to Coating buffer (12 mL). 100 μL of this diluted capture antibody solution was added to each well of a 96-well flat-bottom plate, Costar® 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®-20). Assay diluent was prepared by diluting 5× Assay diluent 5-fold with purified water. Assay Diluent was added to each well at 200 μL and blocked at room temperature for 1 hour. The Assay Diluent was removed from the wells, and the wells were washed five times with 0.05% PBS-T to obtain plates coated with capture antibody. Recombinant human IFN-γ was dissolved in Assay Diluent to prepare a final concentration solution of 1000 pg / mL. This solution was diluted seven times by a factor of two to prepare standards. A diluted solution of the detection antibody was prepared by adding detection antibody (biotin-labeled anti-human IFNγ antibody) (48 μL) to coating buffer (12 mL). A diluted solution of the enzyme was prepared by adding enzyme (streptavidin-HRP) (48 μL) to coating buffer (12 mL).

[0143] (iii) Measurement of IFNγ The sample and standard 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 diluted detection antibody was added to each well. This 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 diluted enzyme was added to each well. This 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. This plate was incubated in the dark at room temperature for 15 minutes. 50 μL of 0.18 M 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 created from the measurement results of the standard. The absorbance of each sample was fitted to a standard curve to obtain the concentration of IFNγ in each sample. The obtained values ​​were used as the IFNγ secretion amount 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 the target cell and T cells containing MAGE-A4-zG or its variant were used as effector cells. Table 3 also shows the results of cytotoxic activity and IFNγ production when SK-MEL-37 was used as the target cell and T cells containing MAGE-A4-zG-s1 or its variant were used as effector cells. In the tables, "CAR type" refers to the code assigned to each CAR by the inventors and includes information about CAR mutations. "WT" indicates an unmodified CAR (hereinafter, WT CARs are also referred to as "wild-type"). In "CAR type," codes including "R" indicate a CAR in which the amino acid residue shown in "mutation site" in the table has been replaced with an arginine residue. In the "Sequence Number" column, "NA" indicates the sequence number of the nucleotide sequence encoding the CAR, and "AA" indicates the sequence number of the amino acid sequence of the CAR. The "cytotoxic activity" value is expressed as a ratio with the percentage of target cells killed by CAR-T cells of type WT set to 1.00. "3:1" and "1:1" represent the ratio of the number of effector cells to the number of target cells (hereinafter, this ratio will also be referred to as the "cell mixing ratio"). The "IFNγ production" value is expressed as a ratio with the amount of IFNγ produced by CAR-T cells of type WT set to 1.00. The explanations for the tables are the same for the tables in the following examples.

[0145] As shown in Table 2, the cytotoxic activity of each mutant was as follows, with the cytotoxic activity of T cells containing MAGE-A4-zG set to 1.00. 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 when the cell mixing ratio was 3:1 and 1.60 when the cell mixing ratio was 1:1. Regarding cytotoxic activity, T cells containing MAGE-A4-zG-m1 or MAGE-A4-zG-m2 showed higher activity than T cells containing MAGE-A4-zG. On the other hand, when the IFNγ production of T cells containing MAGE-A4-zG was set to 1.00, the IFNγ production of each mutant was as follows: The IFNγ production of T cells containing MAGE-A4-zG-m1 was 0.67. The IFNγ production of T cells containing MAGE-A4-zG-m2 was 0.47. Regarding IFNγ production, T cells containing MAGE-A4-zG-m1 or MAGE-A4-zG-m2 produced lower levels than T cells containing MAGE-A4-zG.

[0146] As shown in Table 3, when the cytotoxic activity of T cells containing MAGE-A4-zG-s1 is set to 1.00, the cytotoxic activity of T cells containing MAGE-A4-zG-m1-s1 was 2.34 when the cell mixing ratio was 3:1 and 2.25 when the cell mixing ratio was 1:1. On the other hand, when the IFNγ production of T cells containing MAGE-A4-zG-s1 is set to 1.00, the IFNγ production of T cells containing MAGE-A4-zG-m1-s1 was 0.66. In terms of cytotoxic activity, T cells containing MAGE-A4-zG-m1-s1 were higher than T cells containing MAGE-A4-zG-s1, but their IFNγ production was lower than that of T cells containing MAGE-A4-zG-s1.

[0147] [Table 2]

[0148] [Table 3]

[0149] 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 effector cells. As shown in Table 4, when the cytotoxic activity 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 1.35 when the cell mixing ratio was 3:1 and 1.65 when the cell mixing ratio was 1:1. In terms of cytotoxic activity, T cells containing MAGE-A4-zG-m1-s1 showed higher activity than T cells containing MAGE-A4-zG-s1. On the other hand, when the IFNγ production of T cells containing MAGE-A4-zG-s1 was set to 1.00, the IFNγ production of T cells containing MAGE-A4-zG-m1-s1 was 0.40. T cells containing MAGE-A4-zG-m1-s1 showed lower levels than T cells containing MAGE-A4-zG-s1 alone.

[0150] [Table 4]

[0151] The immune cells in this example were suggested to be CAR-T cells with suppressed cytokine production. Furthermore, these CAR-T cells exhibited enhanced cytotoxicity.

[0152] Example 2: Production of CAR-T cells that bind to the MAGE-A4 / HLA-A2 complex and confirmation of their effects (2)

[0153] (1) Obtaining nucleic acid molecules encoding variants of MAGE-A4-zG Using the nucleic acid molecule encoding MAGE-A4-zG prepared in Example 1 as template DNA, a nucleic acid molecule encoding a different variant from that in Example 1 was prepared by PCR. Specifically, it was as follows.

[0154] (1.1) Primer design, PCR and ligation reaction A primer set was designed based on the nucleotide sequence of SEQ ID NO: 3 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which the amino acid residues shown in a), c), e), f), or h) below in MAGE-A4-zG are substituted with arginine residues. A primer set was also designed based on the nucleotide sequence of SEQ ID NO: 3 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which the amino acid residues shown in b), d), or g) below in MAGE-A4-zG are substituted with lysine residues. Using these primer sets and template DNA, PCR and ligation reactions were performed in the same manner as in Example 1.

[0155] a) The 63rd, 65th, and 72nd amino acid residues of VL as defined by Kabat; b) The 63rd, 67th, and 70th amino acid residues of VL as defined by Kabat; c) The 63rd, 70th, and 72nd amino acid residues of VL as defined by Kabat; d) The 65th, 67th, and 70th amino acid residues of VL as defined by Kabat; e) The 65th, 70th, and 72nd amino acid residues of VL as defined by Kabat; f) The 67th, 70th, and 72nd amino acid residues of VL as defined by Kabat; g) The 63rd, 65th, 67th, 70th and 72nd amino acid residues of VL as defined by Kabat; and h) The 60th, 74th, and 76th amino acid residues of VL as defined by Kabat

[0156] (1.2) Transformation, plasmid DNA extraction and sequencing In the same manner as in Example 1, transformed E. coli cells were obtained using the ligation reaction solution and DH5α. Then, in the same manner as in Example 1, plasmid DNA was extracted from E. coli cultured in liquid medium and sequenced. The sequencing results confirmed the acquisition of a nucleic acid molecule encoding the MAGE-A4-zG variant.

[0157] Hereinafter, CARs having single-chain antibodies in which the amino acid residues shown in a), c), e), f), or h) above in MAGE-A4-zG are replaced with arginine residues will be 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 single-chain antibodies in which the amino acid residues shown in b), d), or g) above are replaced with lysine residues will be referred to as "MAGE-A4-zG-Km6", "MAGE-A4-zG-Km8", and "MAGE-A4-zG-Km11", respectively. Tables 5 and 6 show the correspondence between each mutant and the sequence number of the primer set used to create it. In Table 6, "1st time" refers to the primer set for substituting amino acid residues 74 and 76 of VL, and "2nd time" refers to the primer set for substituting amino acid residue 60 of VL.

[0158] [Table 5]

[0159] [Table 6]

[0160] (2) Production of CAR-T cells expressing MAGE-A4-zG and its variants In the same manner as in Example 1, PBMCs isolated and cultured from the blood of a healthy donor were introduced with a retrovirus to produce genes encoding MAGE-A4-zG and its variants, thereby obtaining each type of CAR-T cell.

[0161] (3) Confirmation of the cytotoxic effect of CAR-T cells and cytokine secretion levels. 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 measurements 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 were performed on different days than the measurements of MAGE-A4-zG-Km11 ​​and MAGE-A4-zG-m12. The results are shown in Tables 7 and 8. In the "CAR type" column of the table, the letter "K" indicates a CAR in which the amino acid residue shown in the "mutation site" column of the table has been replaced with a lysine residue. This also applies to the tables in subsequent examples.

[0162] [Table 7]

[0163] [Table 8]

[0164] As shown in Table 7, the cytotoxic activity of each mutant was as follows, with the cytotoxic activity of T cells containing MAGE-A4-zG set to 1.00. 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 0.99 when the cell mixing ratio was 1:1. On the other hand, when the IFNγ production of T cells containing MAGE-A4-zG was set to 1.00, the IFNγ production of each mutant was as follows: 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 amount of IFNγ produced by T cells containing MAGE-A4-zG-m7 was 0.22. The amount of IFNγ produced by T cells containing MAGE-A4-zG-Km8 was 0.91. The amount of IFNγ produced by 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.

[0165] As shown in Table 8, the cytotoxic activity of each mutant was as follows, with the cytotoxic activity of T cells containing MAGE-A4-zG set to 1.00. The cytotoxic activity of T cells containing MAGE-A4-zG-Km11 ​​was 2.33 when the cell mixing ratio was 3:1 and 1.68 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing MAGE-A4-zG-m12 was 1.23 when the cell mixing ratio was 3:1 and 0.90 when the cell mixing ratio was 1:1. On the other hand, when the IFNγ production of T cells containing MAGE-A4-zG was set to 1.00, the IFNγ production of each mutant was as follows: The amount of IFNγ produced by 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.

[0166] The immune cells in this example were suggested to be CAR-T cells with suppressed cytokine production. Furthermore, cytotoxic effects were improved in many clones. In MAGE-A4-zG-m12, the cytotoxic effect was slightly reduced compared to the wild type, but cytokine production was significantly suppressed. This suggests that by increasing the dose, this clone can improve or maintain the same level of cytotoxicity as the wild type, while suppressing cytokine production more effectively than the wild type.

[0167] Example 3: Production of CAR-T cells that bind to the PRAME / HLA-A24 complex and confirmation of their effects.

[0168] (1) Obtaining nucleic acid molecules encoding CARs to be used as templates Plasmid DNA for viral vector construction was obtained in the same manner as in Example 1, containing a gene encoding a CAR with a single-chain antibody that binds to the PRAME / HLA-A24 complex. The complex recognized by the single-chain antibody was the 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 PRAME-derived peptide in the PRAME / HLA-A24 complex was LYVDSLFFL (SEQ ID NO: 2). The plasmid DNA was used as a template to construct nucleic acid molecules encoding variants of PRAME-zG by PCR.

[0169] The structure of the gene encoding PRAME-zG was similar to that of the gene encoding MAGE-A4-zG in Example 1. Specifically, the gene encoding PRAME-zG had the following sequences linked together in this order from the 5' end: a leader sequence, a nucleotide sequence encoding VH, a nucleotide sequence encoding the 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 (see Figure 2A). PRAME-zG was a CAR containing a single-chain antibody consisting of VH, VL, and a linker linking them as an extracellular domain, CL, CD28TM as a transmembrane domain, and CD3ζ and GITRICD as intracellular domains.

[0170] (2) Obtaining nucleic acid molecules encoding variants of PRAME-zG The plasmid DNA described above was used as a template. A PCR reaction mixture containing the template DNA, primer sets (SEQ ID NOs. 101 and 102), and PrimeSTAR® Max Premix was prepared, and a PCR reaction was performed. This yielded PCR products with restriction sites added to the 5' and 3' ends. DpnI was added to the obtained PCR products to fragment the template plasmid DNA. Using the DpnI-treated PCR products as a template, nucleic acid molecules encoding variants of PRAME-zG were constructed by PCR. Specifically, the results were as follows.

[0171] (2.1) Primer design, PCR and ligation reaction A primer set was designed based on the nucleotide sequence of SEQ ID NO: 29 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which the amino acid residue shown in a) or b) below in PRAME-zG is substituted with an arginine residue. A primer set was also designed based on the nucleotide sequence of SEQ ID NO: 29 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which the amino acid residue shown in c) below in PRAME-zG is substituted with a lysine residue. Using these variant generation primer sets and the DpnI-treated PCR product described above, PCR and ligation reactions were performed in the same manner as in Example 1.

[0172] a) The 63rd, 65th, and 72nd amino acid residues of VL as defined by Kabat; b) The 63rd, 70th and 72nd amino acid residues of VL as defined by Kabat; and c) The 65th, 70th, and 72nd amino acid residues of VL as defined by Kabat

[0173] (2.2) Transformation, plasmid DNA extraction and sequencing In the same manner as in Example 1, transformed E. coli cells were obtained using the ligation reaction solution and DH5α. Then, in the same manner as in Example 1, plasmid DNA was extracted from E. coli cultured in liquid medium and sequenced. The sequencing results confirmed the acquisition of a nucleic acid molecule encoding a variant of PRAME-zG.

[0174] 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 will be referred to as "PRAME-zG-m1" and "PRAME-zG-m2," respectively. Furthermore, a CAR having a single-chain antibody in which the amino acid residue shown in c) above is replaced with a lysine residue will be referred to as "PRAME-zG-Km3." Table 9 shows the correspondence between each variant and the sequence number of the primer set used to produce it.

[0175] [Table 9]

[0176] (3) Production of CAR-T cells expressing PRAME-zG and its variants In the same manner as in Example 1, PBMCs isolated and cultured from the blood of a healthy donor were introduced with a retrovirus to produce genes encoding PRAME-zG and its variants, thereby obtaining each type of CAR-T cell.

[0177] (4) Confirmation of the cytotoxic effect of CAR-T cells and cytokine secretion levels. The cytotoxic activity and IFNγ production of each CAR-T cell were measured in the same manner as in Example 1. The target cells used were the human melanoma cell line SK-MEL-124. SK-MEL-124 were PRAME-positive tumor cells. The measurements of PRAME-zG-m1 and PRAME-zG-m2 were performed on different days from the measurement of PRAME-zG-Km3. The results are shown in Tables 10 and 11.

[0178] [Table 10]

[0179] [Table 11]

[0180] As shown in Table 10, the cytotoxic activity of each mutant was as follows, with the cytotoxic activity of T cells containing PRAME-zG set to 1.00. 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 when the cell mixing ratio was 3:1 and 1.54 when the cell mixing ratio was 1:1. On the other hand, when the IFNγ production of T cells containing PRAME-zG was set to 1.00, the IFNγ production of each mutant was as follows: The amount of IFNγ produced by T cells containing PRAME-zG-m1 was 0.49. The IFNγ production level of T cells containing PRAME-zG-m2 was 0.56.

[0181] As shown in Table 11, when the cytotoxic activity of T cells containing PRAME-zG is set to 1.00, the cytotoxic activity of T cells containing PRAME-zG-Km3 was 1.16 when the cell mixing ratio was 3:1 and 1.71 when the cell mixing ratio was 1:1. On the other hand, when the IFNγ production of T cells containing PRAME-zG is set to 1.00, the IFNγ production of T cells containing PRAME-zG-Km3 was 0.87.

[0182] The immune cells in this example were suggested to be CAR-T cells with suppressed cytokine production. Furthermore, these CAR-T cells exhibited enhanced cytotoxicity.

[0183] Example 4: Production of CAR-T cells that bind to CD19 and confirmation of their effects.

[0184] (1) Obtaining nucleic acid molecules encoding CARs to be used as templates Through gene synthesis, a polynucleotide (SEQ ID NO: 186) encoding a single-chain antibody that binds to CD19 (hereinafter also referred to as "anti-CD19-WT") was obtained. Furthermore, as variants of anti-CD19-WT, polynucleotides (SEQ ID NOs: 187, 188, and 190) encoding single-chain antibodies in which one of the amino acid residues shown in a) to c) below in anti-CD19-WT was replaced with an arginine residue were synthesized. Hereinafter, single-chain antibodies with modified amino acid residues as shown in a) to c) below will also be referred to as "anti-CD19-m1," "anti-CD19-m2," and "anti-CD19-m4," respectively.

[0185] a) The 63rd, 65th, 67th, and 70th amino acid residues of VL as defined by Kabat; b) The 63rd, 65th, 67th and 72nd amino acid residues of VL as defined by Kabat; and c) The 65th, 67th, 70th, and 72nd amino acid residues of VL as defined by Kabat

[0186] (2) Obtaining nucleic acid molecules encoding variants of CD19-28z (2.1) Preparation of the first and second inserts Using the polynucleotides encoding anti-CD19-WT, anti-CD19-m1, anti-CD19-m2, and anti-CD19-m4 respectively as templates, a PCR reaction mixture containing primer sets (SEQ ID NOs. 109 and 110) and PrimeSTAR® Max Premix was prepared, and a PCR reaction was performed. This yielded the first inserts encoding anti-CD19-WT, anti-CD19-m1, anti-CD19-m2, and anti-CD19-m4 respectively. Gene synthesis yielded a polynucleotide (SEQ ID NOs. 111) encoding the hinge domain, transmembrane domain, and intracellular domain of CD28. Using this polynucleotide as a template, a PCR reaction mixture containing primer sets (SEQ ID NOs. 112 and 113) and PrimeSTAR® Max Premix was prepared, and a PCR reaction was performed. This yielded the second insert encoding the region consisting of the hinge domain, transmembrane domain, and intracellular domain of CD28.

[0187] (2.2) Preparation of linearization vectors and fusion with inserts Using plasmid DNA for viral vector construction (SEQ ID NO: 114) containing polynucleotides encoding the transmembrane and intracellular domains of CD28 as a template, a PCR reaction mixture containing primer sets (SEQ ID NOs: 115 and 116) and PrimeSTAR® Max Premix was prepared, and a PCR reaction was performed. DpnI was added to the obtained PCR product to fragment the template plasmid DNA. This yielded a linearized vector. This linearized vector was fused with the first and second inserts described above using In-Fusion® HD Enzyme Premix (Takara Bio Inc.). This yielded plasmid DNA containing genes encoding CARs, including anti-CD19-WT, anti-CD19-m1, anti-CD19-m2, and anti-CD19-m4, respectively. Hereinafter, CARs possessing single-chain antibodies against CD19-WT, CD19-m1, CD19-m2, and CD19-m4 will be referred to as "CD19-28z," "CD19-28z-m1," "CD19-28z-m2," and "CD19-28z-m4," respectively.

[0188] As an example, the structure of the gene encoding CD19-28z will be described. Referring to Figure 2B, the gene encoding CD19-28z had the following sequences linked together in this order from the 5' end: a leader sequence, a nucleotide sequence encoding VL, a nucleotide sequence encoding the linker, a nucleotide sequence encoding VH, a nucleotide sequence encoding the hinge domain of CD28 (CD28 hinge), a nucleotide sequence encoding CD28TM, a nucleotide sequence encoding the intracellular domain of CD28 (CD28ICD), and a nucleotide sequence encoding CD3ζ. CD19-28z was a CAR containing a single-chain antibody consisting of VH, VL and the linker linking them as an extracellular domain, the hinge domain of CD28, CD28TM as a transmembrane domain, and CD28ICD and CD3ζ as intracellular domains.

[0189] (2.3) Primer design, PCR and ligation reaction A primer set was designed based on the nucleotide sequence of SEQ ID NO: 37 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which one of the amino acid residues indicated in d), e), g)~k) and m)~q) below in CD19-28z is substituted with an arginine residue. A primer set was also designed based on the nucleotide sequence of SEQ ID NO: 37 to obtain a polynucleotide encoding a CAR containing a single-chain antibody in which one of the amino acid residues indicated in d), f), l) or m) below in CD19-28z is substituted with a lysine residue. Using these primer sets and template DNA, PCR and ligation reactions were performed in the same manner as in Example 1.

[0190] d) The 63rd, 65th, 70th, and 72nd amino acid residues of VL as defined by Kabat; e) The 60th, 63rd, and 65th amino acid residues of VL as defined by Kabat; f) The 63rd, 65th, and 70th amino acid residues of VL as defined by Kabat; g) The 63rd, 65th, and 72nd amino acid residues of VL as defined by Kabat; h) The 63rd, 67th, and 70th amino acid residues of VL as defined by Kabat; i) The 63rd, 70th, and 72nd amino acid residues of VL as defined by Kabat; j) The 65th, 67th, and 70th amino acid residues of VL as defined by Kabat; k) The 65th, 67th, and 72nd amino acid residues of VL as defined by Kabat; l) The 65th, 67th, 70th, and 72nd amino acid residues of VL as defined by Kabat; m) The 63rd, 65th, 67th, 70th, and 72nd amino acid residues of the VL as defined by Kabat; n) The 70th, 72nd, and 74th amino acid residues of VL as defined by Kabat; o) The 60th, 63rd, and 76th amino acid residues of VL as defined by Kabat; p) The 60th, 74th, and 76th amino acid residues of VL as defined by Kabat; and q) The 77th, 79th, and 81st amino acid residues of VL as defined by Kabat

[0191] (2.2) Transformation, plasmid DNA extraction and sequencing In the same manner as in Example 1, transformed E. coli cells were obtained using the ligation reaction solution and DH5α. Then, in the same manner as in Example 1, plasmid DNA was extracted from E. coli cultured in liquid medium and sequenced. The sequencing results confirmed the acquisition of a nucleic acid molecule encoding the CD19-28z mutant.

[0192] Hereinafter, CARs having single-chain antibodies in which any of the amino acid residues shown in d), e), g)~k) and m)~q) above for CD19-28z are replaced with arginine residues 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". Furthermore, CARs having single-chain antibodies in which the amino acid residues shown in d), f), l), or m) above are substituted with lysine residues are called "CD19-28z-Km13," "CD19-28z-Km6," "CD19-28z-Km14," and "CD19-28z-Km15," respectively. Tables 12 and 13 show the correspondence between each mutant and the sequence numbers of the primer sets used to produce them. For CD19-28z-m18 in Table 13, "1st time" refers to the primer set for substituting the 60th and 63rd amino acid residues of VL, and "2nd time" refers to the primer set for substituting the 76th amino acid residue of VL. For CD19-28z-m19, "1st time" refers to the primer set for substituting the 74th and 76th amino acid residues of VL, and "2nd time" refers to the primer set for substituting the 60th amino acid residue of VL.

[0193] [Table 12]

[0194] [Table 13]

[0195] (3) Production of CAR-T cells expressing CD19-28z and its variants In the same manner as in Example 1, PBMCs isolated and cultured from the blood of a healthy donor were introduced with a retrovirus to produce genes encoding CD19-28z and its variants, thereby obtaining each type of CAR-T cell.

[0196] (4) Confirmation of the cytotoxic effect of CAR-T cells and cytokine secretion levels. The cytotoxic activity and IFNγ production of each CAR-T cell were measured in the same manner as in Example 1. In some experiments, the ratio of effector cells to target cells was 2:1 (see Table 16). The target cells used were the human B-cell precursor leukemia cell line Nalm-6. Nalm-6 were CD19-positive tumor cells. The measurement results are shown in Tables 14-17. The results in each table were based on measurements taken on different days.

[0197] [Table 14]

[0198] [Table 15]

[0199] [Table 16]

[0200] [Table 17]

[0201] As shown in Table 14, the cytotoxic activity of each mutant was as follows, with the cytotoxic activity of T cells containing CD19-28z set to 1.00. 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 1.19 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m3 was 0.91 when the cell mixing ratio was 3:1 and 1.26 when the cell mixing ratio was 1:1. On the other hand, when the IFNγ production of T cells containing CD19-28z was set to 1.00, the IFNγ production of each mutant was as follows: The amount of IFNγ produced by T cells containing CD19-28z-m1 was 0.25. The amount of IFNγ produced by T cells containing CD19-28z-m2 was 0.24. The IFNγ production of T cells containing CD19-28z-m3 was 0.26.

[0202] As shown in Table 15, the cytotoxic activity of each mutant was as follows, with the cytotoxic activity of T cells containing CD19-28z set to 1.00. The cytotoxic activity of T cells containing CD19-28z-m1 was 1.00 when the cell mixing ratio was 3:1 and 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 when the cell mixing ratio was 3:1 and 1.49 when the cell mixing ratio was 1:1. On the other hand, when the IFNγ production of T cells containing CD19-28z was set to 1.00, the IFNγ production of each mutant was as follows: The amount of IFNγ produced by T cells containing CD19-28z-m1 was 0.31. The amount of IFNγ produced by T cells containing CD19-28z-m3 was 0.27. The IFNγ production level of T cells containing CD19-28z-m4 was 0.29.

[0203] As shown in Table 16, the cytotoxic activity of each mutant, with the cytotoxic activity of T cells containing CD19-28z set to 1.00, was as follows: 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 when the cell mixing ratio was 2:1 and 1.13 when the cell mixing ratio was 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 when the cell mixing ratio was 2:1 and 1.19 when the cell mixing ratio was 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 when the cell mixing ratio was 2:1 and 1.06 when the cell mixing ratio was 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 when the cell mixing ratio was 2:1 and 1.37 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-Km14 was 1.72 when the cell mixing ratio was 2:1 and 1.34 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-Km15 was 1.29 when the cell mixing ratio was 2:1 and 1.23 when the cell mixing ratio was 1:1. The cytotoxic activity of T cells containing CD19-28z-m16 was 1.93 when the cell mixing ratio was 2:1 and 1.32 when the cell mixing ratio was 1:1. When the IFNγ production of T cells containing CD19-28z was set to 1.00, the IFNγ production of each variant was as follows. 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.

[0204] As shown in Table 17, when the cytotoxic activity of T cells containing CD19-28z was set to 1.00, the cytotoxic activity of each variant was as follows. The cytotoxic activity of T cells containing CD19-28z-m17 was 1.43 at a cell mixing ratio of 3:1 and 2.43 at a cell mixing ratio of 1:1, The cytotoxic activity of T cells containing CD19-28z-m18 was 1.27 at a cell mixing ratio of 3:1 and 1.42 at a cell mixing ratio of 1:1, The cytotoxic activity of T cells containing CD19-28z-m19 was 1.38 at a cell mixing ratio of 3:1 and 1.76 at a cell mixing ratio of 1:1, The cytotoxic activity of T cells containing CD19-28z-m20 was 1.57 at a cell mixing ratio of 3:1 and 1.90 at a cell mixing ratio of 1:1. On the other hand, when the IFNγ production of T cells containing CD19-28z was set to 1.00, the IFNγ production of each mutant was as follows: The amount of IFNγ produced by T cells containing CD19-28z-m17 was 0.34. The amount of IFNγ produced by T cells containing CD19-28z-m18 was 0.28. The amount of IFNγ produced by T cells containing CD19-28z-m19 was 0.68. The IFNγ production of T cells containing CD19-28z-m20 was 0.39.

[0205] As shown in Examples 1-4, the IFNγ production of the immune cells of the present invention ranged from 0.08 to 0.93 compared to the wild type (set at 1.00), and was lower in all cases compared to the wild type. The cytotoxic activity of the CAR-T cells ranged from 0.90 to 2.96 compared to the wild type (set at 1.00), and cytotoxic activity was maintained or improved in many clones compared to the wild type.

[0206] Example 5: In vivo cancer treatment model experiment using CAR-T cell administration (1) Materials and methods (1.1) Laboratory animals NOG mice (NOD / Shi-scid, IL-2RγKO Jic) were purchased from CREA Japan Co., Ltd. Female mice aged 7-8 weeks were used for the experiment.

[0207] (1.2) Transplantation of human tumors and administration of CAR-T cells NW-MEL-38 was used as the tumor cell. As effector cells, CAR-T cells containing MAGE-A4-zG-s1 and CAR-T cells containing MAGE-A4-zG-m1-s1, which were prepared in Example 1, were used. NW-MEL-38 was placed on the side of 12 NOG mice in a 5 × 10⁶ arrangement. 6 Cells / animal were subcutaneously injected. Four days after tumor cell transplantation, 5 × 10⁶ cells of PBS, T cells containing MAGE-A4-zG-s1, or CAR-T cells containing MAGE-A4-zG-m1-s1 were administered. 6Cells were infused via tail vein at a dose per animal. The group administered PBS, the group administered CAR-T cells containing MAGE-A4-zG-s1, and the group administered CAR-T cells containing MAGE-A4-zG-m1-s1 all had n=4. Tumor diameter was measured every 2 or 3 days.

[0208] (2) Results Figure 3 shows a graph recording the average tumor area for each group. As can be seen from Figure 3, compared to the group administered PBS, the tumor diameter was reduced in the group administered CAR-T cells containing MAGE-A4-zG-s1 and the group administered CAR-T cells containing MAGE-A4-zG-m1-s1. Furthermore, the tumor diameter was reduced more in the group administered CAR-T cells containing MAGE-A4-zG-m1-s1 than in the group administered CAR-T cells containing MAGE-A4-zG-s1. Therefore, it was suggested that the antitumor activity of immune cells containing CAR can be improved by modifying at least three amino acid residues of the light chain FR3 of the single-chain antibody contained in CAR to basic amino acid residues.

Claims

1. A T cell comprising a chimeric antigen receptor including an extracellular domain, a transmembrane domain, and an intracellular domain, 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 three amino acid residues are arginine residues or lysine residues. The at least three amino acid residues include at least three selected from the group consisting of the 60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th, and 81st amino acid residues in the light chain variable region. The antigen-binding region is - A heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 213, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 214, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 215, and a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 216, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 217, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 218, - A heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 230, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 231, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 232, and a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 233, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 234, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 235, or - A heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 240, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 241, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 242, and a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 243, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 244, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 245, Compared to T cells containing a chimeric antigen receptor having the same amino acid sequence as the chimeric antigen receptor except that at least three amino acid residues are neutral or acidic amino acid residues, the T cells exhibit suppressed IFNγ production. T cells containing chimeric antigen receptors.

2. The T cell according to claim 1, wherein three to five amino acid residues selected from the group consisting of the 60th, 63rd, 65th, 67th, 70th, 72nd, 74th, 76th, 77th, 79th, and 81st amino acid residues in the light chain variable region defined by the Kabat method are arginine residues or lysine residues.

3. The T cell according to claim 1, wherein the antigen-binding region comprises a single-chain antibody, and the single-chain antibody is a complex of MAGE-A4-derived peptide and HLA-A2, a complex of PRAME-derived peptide and HLA-A24, or a single-chain antibody that binds to CD19.

4. The T cell according to claim 1, wherein the transmembrane domain comprises the transmembrane region of any one 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, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS, and GITR.

5. The T cell according to claim 1, wherein the intracellular domain comprises a signaling domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ, and FcRβ.

6. The intracellular domain further comprises a co-stimulatory domain, The T cell according to claim 5, wherein 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.

7. The T cell according to claim 1, further comprising a hinge domain between the antigen-binding region and the transmembrane domain.

8. The T cell according to claim 1, wherein the antigen-binding region, in which at least three amino acid residues are neutral amino acid residues or acidic amino acid residues, comprises an amino acid sequence shown in SEQ ID NO: 219, 236, or 246.

9. A pharmaceutical composition comprising T cells according to any one of claims 1 to 8.

10. A pharmaceutical composition for treating malignant tumors, wherein the extracellular domain of a chimeric antigen receptor contained in the T cell comprises a single-chain antibody in the antigen-binding region, and the single-chain antibody is a complex of MAGE-A4-derived peptide and HLA-A2, a complex of PRAME-derived peptide and HLA-A24, or a single-chain antibody that binds to CD19, according to claim 9.

11. A method for producing T cells comprising a chimeric antigen receptor according to any one of claims 1 to 8, The manufacturing method includes culturing T cells containing a nucleic acid molecule having a nucleotide sequence encoding the 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 includes a nucleotide sequence encoding an antigen-binding region that includes 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 as defined by the Kabat method, at least three codons are codons encoding arginine residues or lysine residues. The at least three codons include at least three selected from the group consisting of a codon encoding the 60th amino acid residue of the light chain variable region, 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. The nucleotide sequence encoding the antigen-binding region is, - A nucleotide sequence encoding a heavy chain variable region including a nucleotide sequence encoding HCDR1 shown in SEQ ID NO: 153, a nucleotide sequence encoding HCDR2 shown in SEQ ID NO: 154, and a nucleotide sequence encoding HCDR3 shown in SEQ ID NO: 155, and a nucleotide sequence encoding a light chain variable region including a nucleotide sequence encoding LCDR1 shown in SEQ ID NO: 156, a nucleotide sequence encoding LCDR2 shown in SEQ ID NO: 157, and a nucleotide sequence encoding LCDR3 shown in SEQ ID NO: 158, - A nucleotide sequence encoding a heavy chain variable region, including a nucleotide sequence encoding HCDR1 shown in SEQ ID NO: 170, a nucleotide sequence encoding HCDR2 shown in SEQ ID NO: 171, and a nucleotide sequence encoding HCDR3 shown in SEQ ID NO: 172, and a nucleotide sequence encoding a light chain variable region, including a nucleotide sequence encoding LCDR1 shown in SEQ ID NO: 173, a nucleotide sequence encoding LCDR2 shown in SEQ ID NO: 174, and a nucleotide sequence encoding LCDR3 shown in SEQ ID NO: 175, or - A nucleotide sequence encoding a heavy chain variable region, including a nucleotide sequence encoding HCDR1 shown in SEQ ID NO: 180, a nucleotide sequence encoding HCDR2 shown in SEQ ID NO: 181, and a nucleotide sequence encoding HCDR3 shown in SEQ ID NO: 182, and a nucleotide sequence encoding a light chain variable region, including a nucleotide sequence encoding LCDR1 shown in SEQ ID NO: 183, a nucleotide sequence encoding LCDR2 shown in SEQ ID NO: 184, and a nucleotide sequence encoding LCDR3 shown in SEQ ID NO:

185. The manufacturing method wherein T cells containing the chimeric antigen receptor have suppressed IFNγ production compared to T cells containing the chimeric antigen receptor having the same amino acid sequence as the chimeric antigen receptor except that at least three amino acid residues are neutral or acidic amino acid residues.

12. The method for producing nucleic acid molecules, wherein three to five codons selected from the group consisting of a codon encoding the 60th amino acid residue, the 63rd amino acid residue, the 65th amino acid residue, the 67th amino acid residue, the 70th amino acid residue, the 72nd amino acid residue, the 74th amino acid residue, the 76th amino acid residue, the 77th amino acid residue, the 79th amino acid residue, and the 81st amino acid residue are codons encoding arginine residues or lysine residues.

13. In the nucleic acid molecule, the nucleotide sequence encoding the antigen-binding region includes a nucleotide sequence encoding a single-chain antibody. The method for producing the product according to claim 11, wherein the single-chain antibody is a complex of MAGE-A4 derived peptide and HLA-A2, a complex of PRAME derived peptide and HLA-A24, or a single-chain antibody that binds to CD19.

14. The method for producing a protein according to claim 11, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the transmembrane domain of any one 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, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS, and GITR.

15. The method for producing a nucleic acid molecule according to claim 11, wherein the segment encoding the intracellular domain comprises a nucleotide sequence encoding a signaling domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ, and FcRβ.

16. In the nucleic acid molecule, the segment encoding the intracellular domain further comprises a nucleotide sequence encoding the costimulatory domain, The method for producing a protein according to claim 15, wherein 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.

17. The manufacturing method according to claim 11, wherein the nucleic acid molecule further comprises a segment encoding a hinge domain between the nucleotide sequence encoding the antigen-binding region and the segment encoding the transmembrane domain.

18. The manufacturing method according to claim 11, wherein the nucleic acid molecule is DNA or RNA.

19. The manufacturing method according to claim 11, wherein the nucleotide sequence encoding an antigen-binding region in which at least three amino acid residues are neutral amino acid residues or acidic amino acid residues includes the nucleotide sequence shown in SEQ ID NO: 159, 176, or 186.

20. A method for producing T cells containing a chimeric antigen receptor, comprising culturing T cells containing a vector comprising the nucleic acid molecule described in claim 11.

Images