Nucleic acid molecule encoding CAR, carrier containing same, CAR, immune cell containing CAR, manufacturing method of immune cell, pharmaceutical composition containing cell, and method for improving cell injury activity

Abstract

The purpose of the present invention is to provide: an improved chimeric antigen receptor (CAR) that makes it possible to increase the cell damage activity of an immune cell containing the CAR; and a nucleic acid molecule encoding the same. The aforementioned problem is solved by a nucleic acid molecule encoding a CAR in which at least three amino acid residues in a framework region 3 of a light chain variable region of an antigen-binding region included in the CAR are used as acidic amino acid residues.

Description

[Technical field] The present invention relates to a nucleic acid molecule having a nucleotide sequence encoding a chimeric antigen receptor (hereinafter, also referred to as "CAR"). The present invention relates to a vector containing the nucleic acid molecule. The present invention relates to CAR. The present invention relates to immune cells containing CAR. The present invention relates to a pharmaceutical composition for treating malignant tumors containing immune cells containing CAR. The present invention relates to a method for enhancing the cell damage activity of immune cells containing CAR. The present invention relates to a method for manufacturing immune cells containing CAR. [Background technology] CAR refers to a receptor protein produced 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 of immune cells. For example, as described in Patent Document 1, in the antigen binding region of CAR, a single-chain antibody that binds to an antigen expressed by tumor cells is used. In recent years, attention has been paid to cancer immunotherapy, which involves introducing genes encoding CAR into immune cells and transplanting immune cells that express CAR on the cell surface into patients to treat cancer. Immune cells expressing CAR recognize antigens of tumor cells in vivo, and then activate and express cell-damaging molecules or cytokines such as Fas ligand, perforin, and granzymes, exerting anti-tumor effects. [Prior art literature] [Patent Literature] [Patent Document 1] U.S. Patent No. 7,741,465 [Summary of the Invention] [Problems to be solved by the invention] The present invention aims to provide an improved CAR that makes it possible to enhance the cell-damaging activity of immune cells expressing CAR. In addition, the present invention aims to provide a nucleic acid molecule encoding such a CAR and a vector containing the nucleic acid molecule. Furthermore, the present invention aims to provide an immune cell containing such a CAR, a pharmaceutical composition containing the immune cell, a method for enhancing the cell-damaging activity of the immune cell, and a method for producing the immune cell.

Methods for solving the problem

[20] are provided. Hereinafter, the framework region 3 is also referred to as "FR3".

[0001] A nucleic acid molecule having a nucleotide sequence encoding a CAR, wherein the nucleic acid molecule comprises a segment encoding an extracellular domain, a segment encoding a transmembrane domain, and a segment encoding an intracellular domain, the segment encoding the extracellular domain comprises a nucleotide sequence encoding an antigen binding region comprising a light chain variable region and a heavy chain variable region, at least three codons in the nucleotide sequence encoding FR3 of the light chain variable region defined by the Chothia method are codons encoding acidic amino acid residues, and at least three codons containing the light chain variable region are selected from the codon encoding the amino acid residue at position 60, the codon encoding the amino acid residue at position 63, the codon encoding the amino acid residue at position 65, the codon encoding the amino acid residue at position 67, the codon encoding the amino acid residue at position 70, the codon encoding the amino acid residue at position 72, the codon encoding the amino acid residue at position 74, and at least three codons encoding the amino acid residue at position 76.

[0002] The nucleic acid molecule described in [1] above, wherein three or more and five or less codons selected from the group consisting of a codon encoding an amino acid residue at position 60, a codon encoding an amino acid residue at position 63, a codon encoding an amino acid residue at position 65, a codon encoding an amino acid residue at position 67, a codon encoding an amino acid residue at position 70, a codon encoding an amino acid residue at position 72, a codon encoding an amino acid residue at position 74, and a codon encoding an amino acid residue at position 76 in the light chain variable region are codons encoding acidic amino acid residues.

[0003] The nucleic acid molecule described in [1] or [2] above, wherein the antigen binding region is a single-chain antibody that binds to a complex of a MAGE-A4-derived peptide and HLA-A2, CD19, BCMA or CEA.

[0004] The nucleic acid molecule described in any one of [1] to [3] above, wherein the transmembrane domain comprises a 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.

[0005] The nucleic acid molecule described in any one of [1] to [4] above, wherein the intracellular domain contains a signal transduction domain of at least one protein selected from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ and FcRβ.

[0006] The nucleic acid molecule described in [5] above, wherein the segment encoding the intracellular domain also contains a nucleotide sequence encoding a co-stimulatory domain, and the co-stimulatory domain is a co-stimulatory domain of at least one protein selected from 4-1BB, CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 and ICOS.

[0007] The nucleic acid molecule described in any one of [1] to [6] above further contains a segment encoding a hinge domain between the nucleotide sequence encoding the antigen-binding region and the segment encoding the transmembrane domain.

[0008] The nucleic acid molecule described in any one of [1] to [7] above, wherein the nucleic acid molecule is DNA or RNA.

[0009] A vector comprising the nucleic acid molecule described in any one of [1] to [8] above.

[0010] A CAR comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain contains an antigen binding region comprising a light chain variable region and a heavy chain variable region, at least three amino acid residues in FR3 of the light chain variable region defined by the Chothia method are acidic amino acid residues, and at least three amino acid residues contain at least three of the amino acid residues at position 60, the amino acid residue at position 63, the amino acid residue at position 65, the amino acid residue at position 67, the amino acid residue at position 70, the amino acid residue at position 72, the amino acid residue at position 74 and the amino acid residue at position 76 of the light chain variable region.

[0011] The CAR described in

[10] above, wherein 3 or more and 5 or less of the amino acid residues selected from the group consisting of amino acid residue at position 60, amino acid residue at position 63, amino acid residue at position 65, amino acid residue at position 67, amino acid residue at position 70, amino acid residue at position 72, amino acid residue at position 74 and amino acid residue at position 76 in the light chain variable region are acidic amino acid residues.

[0012] The CAR described in

[10] or

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

[0013] The CAR described in any one of

[10] to

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

[0014] The CAR described in any one of

[10] to

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

[0015] The CAR described in

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

[0016] The CAR described in any one of

[10] to

[15] above further comprises a hinge domain between the antigen binding region and the transmembrane domain.

[0017] An immune cell comprising the chimeric antigen receptor described in any one of

[10] to

[16] above.

[0018] A pharmaceutical composition for treating malignant tumors, comprising the immune cells described in

[17] above.

[0019] A method for enhancing the cell-damaging activity of immune cells containing CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain and an intracellular domain, the extracellular domain comprising an antigen-binding region comprising a light chain variable region and a heavy chain variable region, the method comprising enhancing the cell-damaging activity of immune cells containing the CAR by setting at least three amino acid residues of FR3 of the light chain variable region defined by the Chothia method to acidic amino acid residues, compared to the CAR before setting the at least three amino acid residues to acidic amino acid residues.

[0020] A method for producing an immune cell containing CAR, comprising introducing the nucleic acid molecule described in any one of [1] to [8] above or the vector described in [9] above into the immune cell, so that the immune cell expresses CAR. [Effects of the invention] The present invention can obtain immune cells containing CAR with enhanced cell-damaging activity. [Brief description of the attached drawings]

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Claims

1. A nucleic acid molecule having a nucleotide sequence encoding a chimeric antigen receptor, wherein The nucleic acid molecule contains 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 contains a nucleotide sequence encoding an antigen binding region containing a light chain variable region and a heavy chain variable region, At least three codons in the nucleotide sequence encoding the framework region 3 of the light chain variable region defined by the Chothia method are codons encoding acidic amino acid residues, The at least 3 codons containing the light chain variable region are selected from at least 3 of the following: a codon encoding the amino acid residue at position 60, a codon encoding the amino acid residue at position 63, a codon encoding the amino acid residue at position 65, a codon encoding the amino acid residue at position 67, a codon encoding the amino acid residue at position 70, a codon encoding the amino acid residue at position 72, a codon encoding the amino acid residue at position 74, and a codon encoding the amino acid residue at position 76.

2. The nucleic acid molecule of claim 1, wherein more than 3 but less than 5 codons selected from the following in the light chain variable region are codons encoding acidic amino acid residues: a codon encoding an amino acid residue at position 60, a codon encoding an amino acid residue at position 63, a codon encoding an amino acid residue at position 65, a codon encoding an amino acid residue at position 67, a codon encoding an amino acid residue at position 70, a codon encoding an amino acid residue at position 72, a codon encoding an amino acid residue at position 74, and a codon encoding an amino acid residue at position 76.

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

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

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

6. The nucleic acid molecule of claim 5, wherein The segment encoding the intracellular domain also contains a nucleotide sequence encoding a co-stimulatory domain. The co-stimulatory domain is a co-stimulatory domain of at least one protein selected from the group consisting of 4-1BB, CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, and ICOS.

7. The nucleic acid molecule of claim 1, further comprising a segment encoding a hinge domain between the nucleotide sequence encoding the antigen binding region and the segment encoding the transmembrane domain.

8. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is DNA or RNA.

9. A vector comprising the nucleic acid molecule according to any one of claims 1 to 8.

10. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein The extracellular domain contains an antigen binding region comprising a light chain variable region and a heavy chain variable region, At least three amino acid residues in the framework region 3 of the light chain variable region defined by the Chothia method are acidic amino acid residues, The at least 3 amino acid residues contain at least 3 of the following selected from the light chain variable region: the amino acid residue at position 60, the amino acid residue at position 63, the amino acid residue at position 65, the amino acid residue at position 67, the amino acid residue at position 70, the amino acid residue at position 72, the amino acid residue at position 74 and the amino acid residue at position 76.

11. The chimeric antigen receptor of claim 10, wherein at least 3 but no more than 5 amino acid residues selected from the group consisting of the amino acid residue at position 60, the amino acid residue at position 63, the amino acid residue at position 65, the amino acid residue at position 67, the amino acid residue at position 70, the amino acid residue at position 72, the amino acid residue at position 74, and the amino acid residue at position 76 in the light chain variable region are acidic amino acid residues.

12. The chimeric antigen receptor of claim 10, wherein The antigen binding region comprises a single chain antibody, The single-chain antibody is a single-chain antibody that binds to a complex of a MAGE-A4-derived peptide and HLA-A2, CD19, BCMA, or CEA.

13. The chimeric antigen receptor of claim 10, wherein the transmembrane domain comprises a transmembrane region of any one of the following proteins: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD154, 4-1BB, ICOS, and GITR.

14. The chimeric antigen receptor of claim 10, wherein the intracellular domain comprises a signal transduction domain of at least one protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD66d, CD79a, CD79b, FcRγ, and FcRβ.

15. The chimeric antigen receptor of claim 14, wherein The intracellular domain also contains a co-stimulatory domain, The co-stimulatory domain is a co-stimulatory domain of at least one protein selected from the group consisting of 4-1BB, CD28, GITR, CD2, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, and ICOS.

16. The chimeric antigen receptor of claim 10, further comprising a hinge domain between the antigen binding region and the transmembrane domain.

17. An immune cell comprising the chimeric antigen receptor according to any one of claims 10 to 16.

18. A pharmaceutical composition for treating malignant tumors, comprising the immune cell according to claim 17.

19. A method for enhancing the cell damaging activity of immune cells containing chimeric antigen receptors, wherein The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises an antigen binding region comprising a light chain variable region and a heavy chain variable region. The method comprises setting at least three amino acid residues of the framework region 3 of the light chain variable region defined by the Chothia method as acidic amino acid residues, thereby improving the cell damaging activity of the immune cell containing the chimeric antigen receptor compared to the chimeric antigen receptor before setting the at least three amino acid residues as acidic amino acid residues.

20. A method for producing an immune cell containing a chimeric antigen receptor, comprising introducing the nucleic acid molecule according to any one of claims 1 to 8 into an immune cell, so that the immune cell expresses the chimeric antigen receptor.

21. A method for producing an immune cell containing a chimeric antigen receptor, comprising introducing the vector according to claim 9 into an immune cell, so that the immune cell expresses the chimeric antigen receptor.

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