TWI756223B - Chimeric antigen receptors, and their utilization - Google Patents

Abstract

Provide novel CAR and CAR-T cells that are effective in the treatment of diseases such as cancer.

Chimeric antigen receptors selected from the group consisting of chimeric antigen receptors A to F and CAR-T cells expressing them.

Description

Chimeric antigen receptors, and their utilization

Techniques are disclosed for chimeric antigen receptors, chimeric antigen receptor T cells and antibodies.

With the development of immunology, the development of various immunotherapies for cancer has progressed. Among them, adoptive immunotherapy in which T cells capable of recognizing cancer cells and normal cells and killing cancer cells are transferred into patients is expected as a treatment strategy with few side effects that can respond to cancer metastasis/recurrence. However, it is difficult to obtain T cell lines of sufficient quality and quantity to exert therapeutic effects from cancer patients, and this is one of the reasons that hinders the clinical application of this therapy. As a method to overcome this problem, in recent years, a large number of T cells that can attack cancer cells by introduction of a chimeric antigen receptor (CAR) gene are formulated, and the CAR-expressing T (CAR-T) cells are used as cells Adoptive immunotherapy of medicine has attracted attention.

At present, the target diseases of CAR-T cell therapy with advanced research are mostly hematopoietic cancer, especially for B-cell lymphoma, which targets CD19 CAR-T cell therapy. Significant effects that cannot be obtained in therapy. However, there are few clinical reports showing the effectiveness of CAR-T cell therapy on solid tumors. This accreditation This is because the transplanted CAR-T cells are easy to contact with cancer cells in hematopoietic tumors. In contrast, in order to directly contact cancer cells in solid tumors, they must infiltrate outside the blood vessels and then pass through the interstitial tissue. The attack rate of blood cancer is less than 5% of all cancer patients, and solid tumors account for the majority of other cancer types. Thus, the development of CAR-T cell therapy for solid tumors is in demand.

[Prior Art Literature] [Non-patent literature]

[Non-Patent Document 1] Kanagawa, N. et al., Cancer Gene Ther., 20(1): 57-64 (2013)

[Non-Patent Document 2] N Engl J Med. 2014 Oct 16; 371(16): 1507-17.

Under the current situation as described above, providing novel CAR and CAR-T cells that are effective in the treatment of cancer and other diseases is one of the issues.

As a result of repeated efforts to solve the above-mentioned problems, CAR and CAR-T cells with specific CDR sequences have been discovered. Improvements and investigations have been repeated on the basis of this knowledge, and inventions represented by the following are provided.

Item 1.

A chimeric antigen receptor, which is selected from the group formed by the chimeric antigen receptors A～F of any one of the following: chimeric antigen receptor A, comprising: comprising the amino acid with SEQ ID NO: 1 The light chain CDR1 of the amino acid sequence of the sequence, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 2, and the light chain comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 3 The heavy chain variable region of CDR3, and/or the heavy chain CDR1 comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 5, and the heavy chain CDR1 comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 6 Chain CDR2, and the light chain variable region of the heavy chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 7; Chimeric Antigen Receptor B, comprising: comprising the amino acid of SEQ ID NO: 21 The light chain CDR1 of the amino acid sequence of the sequence, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 22, and the light chain comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 23 CDR3 The variable region of the heavy chain, and/or containing: the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 25, the heavy chain comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 26 CDR2, and the light chain variable region of the heavy chain CDR3 comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 27; The light chain CDR1 of the amino acid sequence of SEQ ID NO: 42, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 42, and the light chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 43 The variable region of the heavy chain, and/or containing: the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 45, the heavy chain comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 46 CDR2, and the light chain variable region of the heavy chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 47; Chimeric Antigen Receptor D, comprising Contains: the light chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 61, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 62, and the amine comprising the amino acid sequence of SEQ ID NO: 63 The heavy chain variable region of the light chain CDR3 of the amino acid sequence of the amino acid sequence, and/or containing: the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 65, including the heavy chain CDR1 of the amino acid sequence of SEQ ID NO: 66 The heavy chain CDR2 of the amino acid sequence of the amino acid sequence, and the light chain variable region comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 67; chimeric antigen receptor E, which comprises Contains: the light chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 81, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 82, and the amine comprising the amino acid sequence of SEQ ID NO: 83 The heavy chain variable region of the light chain CDR3 of the amino acid sequence of the amino acid sequence, and/or the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 85, The heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 86, and the light chain variable region comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 87; or chimeric Antigen receptor F, which comprises: the light chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 101, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 102, and The heavy chain variable region comprising the light chain CDR3 having the amino acid sequence of SEQ ID NO: 103, and/or comprising: the heavy chain CDR1 comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 105 , a heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 106, and a light chain variable region comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 107.

Item 2.

A chimeric antigen receptor A according to item 1.

Item 3.

A chimeric antigen receptor T cell or a chimeric antigen receptor NK cell, which Has a chimeric antigen receptor according to item 1 or 2.

Item 4.

A polynucleotide encoding the chimeric antigen receptor of item 1 or 2.

Item 5.

A pharmaceutical composition comprising the chimeric antigen receptor T cell or the chimeric antigen receptor NK cell according to item 3.

Item 6.

The pharmaceutical composition of item 5 is for the treatment or prevention of cancer.

Means are provided for the treatment of cancer, preferably solid cancer.

Fig. 1 shows the amino acid sequence of scFV possessed by Chimeric Antigen Receptor A. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. The area without the offline line is the linker. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

Fig. 2 shows the amino acid sequence of scFV possessed by Chimeric Antigen Receptor B. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

[ Fig. 3] Fig. 3 shows the amino acid sequence of scFV possessed by Chimeric Antigen Receptor C. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

[ Fig. 4] Fig. 4 shows the amino acid sequence of scFV possessed by Chimeric Antigen Receptor D. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

Fig. 5 shows the amino acid sequence of scFV possessed by chimeric antigen receptor E. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

Fig. 6 shows the amino acid sequence of scFV possessed by Chimeric Antigen Receptor F. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

[ Fig. 7] Fig. 7 shows the base sequence encoding the amino acid sequence of scFV possessed by chimeric antigen receptor A. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The area indicated by the large type of bold font is shown below, CDR1~3 of light chain and heavy chain.

[ Fig. 8] Fig. 8 shows the base sequence encoding the amino acid sequence of scFV possessed by chimeric antigen receptor B. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

[ Fig. 9] Fig. 9 shows the base sequence encoding the amino acid sequence of scFV possessed by chimeric antigen receptor C. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

Fig. 10 shows the base sequence encoding the amino acid sequence of scFV possessed by chimeric antigen receptor D. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

Fig. 11 shows the base sequence encoding the amino acid sequence of scFV possessed by chimeric antigen receptor E. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. Areas without downlines are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

[ Fig. 12] Fig. 12 shows the base sequence encoding the amino acid sequence of scFV possessed by Chimeric Antigen Receptor F. [Fig. The region indicated by the line under the solid line is the light chain variable region. The region indicated by the line under the dashed line is the heavy chain variable region. No offline zone Domains are linkers. The regions indicated by the large font in bold, as shown below, are CDRs 1-3 of the light and heavy chains.

[ Fig. 13 ] The construction procedure of pMXs-IG/CAR[mV-(h28)-h28-h3Z] is shown.

[Fig. 14] shows the construction steps of pMXs-IG/CAR[hV-(h28)-h28-h3Z].

[ Fig. 15 ] The construction procedure of pMXs-IG/CAR[mV-(h8α)-h137-h3Z] is shown.

[ Fig. 16 ] The construction procedure of pMXs-IG/CAR[hV-(h8α)-h137-h3Z] is shown.

[ Fig. 17 ] The structure of the pMXs-IG vector encoding each CAR is shown.

Fig. 18 shows the results of measuring the antitumor effect of in-vivo by CAR-T cells.

1. Chimeric Antigen Receptor

Chimeric Antigen Receptor (CAR) refers to a single-chain antibody (scFv) obtained by in-line binding of the light chain (VL) and heavy chain (VH) of the variable region of a monoclonal antibody at the N-terminal side, and a T-side at the C-terminal side. A chimeric protein of the cellular receptor (TCR) zeta chain. T cells that express CAR are called CAR-T cells.

The amino acid sequences of the scFV regions possessed by the chimeric antigen receptors A to F and the nucleotide sequences encoding the amino acid sequences are shown in Table 1 below. Numbers in the table refer to serial numbers. AA means amino acid sequence. V means variable region. scFV means the entirety of the scFV region.

Chimeric antigen receptor A, preferably having a light chain CDR1 selected from the group consisting of the amino acid sequence having the amino acid sequence of SEQ ID NO: 1, and the light chain comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 2 CDR2, light chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 3, heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 5, comprising the amino group of SEQ ID NO: 6 The heavy chain CDR2 of the amino acid sequence of the acid sequence and at least one CDR of the group comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 7, more preferably two or more, more It is preferable to have 3 or more types, it is more preferable to have 4 types or more, it is more preferable to have 5 types or more, and it is more preferable to have all CDRs. In a suitable embodiment, the chimeric antigen receptor A preferably has: a light chain variable region having the amino acid sequence of SEQ ID NO: 4 and/or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 . In a preferred embodiment, the chimeric antigen receptor A preferably has an scFV structure having the amino acid sequence shown in SEQ ID NO: 10.

In one embodiment, the light chain variable region of Chimeric Antigen Receptor A The amino acid sequence of the domain is 90% or more, preferably 95% or more, preferably 98% or more, preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 4. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor A is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 8 , preferably more than 99% identity. In one embodiment, Chimeric Antigen Receptor A is 90% or more, preferably 95% or more, preferably 98% or more, preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 10. In one embodiment, the amino acid sequence of the linker of the chimeric antigen receptor A is arbitrary as long as the function as the chimeric antigen receptor is maintained.

Chimeric antigen receptor B, preferably having a light chain CDR1 selected from the group consisting of the amino acid sequence having the amino acid sequence of SEQ ID NO: 21, and the light chain comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 22 CDR2, light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26 The heavy chain CDR2 of the amino acid sequence of the acid sequence and at least one CDR of the group comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 27, more preferably two or more, more It is preferable to have 3 or more types, it is more preferable to have 4 types or more, it is more preferable to have 5 types or more, and it is more preferable to have all CDRs. In a suitable embodiment, the chimeric antigen receptor B preferably has: a light chain variable region having the amino acid sequence of SEQ ID NO: 24 and/or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 . In a suitable embodiment, the chimeric antigen receptor B preferably has: SEQ ID NO: 30 The scFV construct of the amino acid sequence.

In one embodiment, the amino acid sequence of the light chain variable region of Chimeric Antigen Receptor B is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 24 , preferably more than 99% identity. In one embodiment, the amino acid sequence of the heavy chain variable region of Chimeric Antigen Receptor B is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 28 , preferably more than 99% identity. In one embodiment, Chimeric Antigen Receptor B is 90% or more, preferably 95% or more, preferably 98% or more, preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 30. In one embodiment, the amino acid sequence of the linker of the chimeric antigen receptor B is arbitrary as long as the function as the chimeric antigen receptor is maintained.

Chimeric antigen receptor C, preferably having a light chain CDR1 selected from the group consisting of the amino acid sequence having the amino acid sequence of SEQ ID NO: 41, and the light chain comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 42 CDR2, light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 43, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 45, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 46 The heavy chain CDR2 of the amino acid sequence of the acid sequence and at least one CDR of the group comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 47, more preferably two or more, more It is preferable to have 3 or more types, it is more preferable to have 4 types or more, it is more preferable to have 5 types or more, and it is more preferable to have all CDRs. In a suitable embodiment, the chimeric antigen receptor C preferably has: a light chain variable region having the amino acid sequence of SEQ ID NO: 44 and/or having Heavy chain variable region of the amino acid sequence of SEQ ID NO: 48. In a suitable embodiment, the chimeric antigen receptor C preferably has an scFV structure having the amino acid sequence represented by SEQ ID NO: 50.

In one embodiment, the amino acid sequence of the light chain variable region of Chimeric Antigen Receptor C is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 44 , preferably more than 99% identity. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor C is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 48 , preferably more than 99% identity. In one embodiment, the chimeric antigen receptor C is 90% or more, preferably 95% or more, preferably 98% or more, preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 50. In one embodiment, the amino acid sequence of the linker of the chimeric antigen receptor C is arbitrary as long as the function as the chimeric antigen receptor is maintained.

Chimeric antigen receptor D, preferably having a light chain CDR1 selected from the group consisting of the amino acid sequence having the amino acid sequence of SEQ ID NO: 61, and the light chain comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 62 CDR2, light chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 63, heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 65, comprising the amino group of SEQ ID NO: 66 The heavy chain CDR2 of the amino acid sequence of the acid sequence and at least one CDR of the group comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 67, more preferably two or more, more It is preferable to have 3 or more types, more preferably to have 4 or more types, more preferably to have 5 types or more, and more preferably to have all the CDRs. In a suitable embodiment, the chimeric antigen receptor D preferably has: a light chain variable region having the amino acid sequence of SEQ ID NO: 64 and/or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 68 . In a preferred embodiment, the chimeric antigen receptor D preferably has an scFV structure having the amino acid sequence represented by SEQ ID NO: 70.

In one embodiment, the amino acid sequence of the light chain variable region of Chimeric Antigen Receptor D is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 64 , preferably more than 99% identity. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor D is equal to or more than 90%, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 68 , preferably more than 99% identity. In one embodiment, the chimeric antigen receptor D has an identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more with the amino acid sequence of SEQ ID NO: 70. In one embodiment, the amino acid sequence of the linker of the chimeric antigen receptor D is arbitrary as long as the function as the chimeric antigen receptor is maintained.

Chimeric antigen receptor E, preferably having a light chain CDR1 selected from the group consisting of the amino acid sequence having the amino acid sequence of SEQ ID NO: 81, and the light chain comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 82 CDR2, light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 83, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 85, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 86 At least one of the group consisting of the heavy chain CDR2 of the amino acid sequence of the acid sequence and the heavy chain CDR3 of the amino acid sequence comprising the amino acid sequence of SEQ ID NO: 87 More preferably, there are two or more types of CDRs, more preferably three or more types, more preferably four or more types, more preferably five or more types, and more preferably all CDRs. In a suitable embodiment, the chimeric antigen receptor E preferably has: a light chain variable region having the amino acid sequence of SEQ ID NO: 84 and/or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 88 . In a suitable embodiment, the chimeric antigen receptor E preferably has an scFV structure having the amino acid sequence represented by SEQ ID NO: 90.

In one embodiment, the amino acid sequence of the light chain variable region of Chimeric Antigen Receptor E is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 84 , preferably more than 99% identity. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor E is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 88 , preferably more than 99% identity. In one embodiment, the chimeric antigen receptor E is 90% or more, preferably 95% or more, preferably 98% or more, preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 90. In one embodiment, the amino acid sequence of the linker of the chimeric antigen receptor E is arbitrary as long as the function as the chimeric antigen receptor is maintained.

Chimeric antigen receptor F, preferably having a light chain CDR1 selected from the group consisting of the amino acid sequence having the amino acid sequence of SEQ ID NO: 101, and the light chain comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 102 CDR2, light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 103, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 105, heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 106 acid sequence The heavy chain CDR2 of the amino acid sequence of SEQ ID NO: 107 and at least one CDR of the group consisting of the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 107, more preferably two or more, more preferably There are three or more types, more preferably four or more types, more preferably five or more types, and more preferably all CDRs. In a suitable embodiment, the chimeric antigen receptor F preferably has: a light chain variable region having the amino acid sequence of SEQ ID NO: 104 and/or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 108 . In a preferred embodiment, the chimeric antigen receptor F preferably has an scFV structure having the amino acid sequence represented by SEQ ID NO: 110.

In one embodiment, the amino acid sequence of the light chain variable region of Chimeric Antigen Receptor F is 90% or more, preferably 95% or more, preferably 98% or more of the amino acid sequence of SEQ ID NO: 104 , preferably more than 99% identity. In one embodiment, the amino acid sequence of the heavy chain variable region of the chimeric antigen receptor F is equal to or more than 90%, preferably more than 95%, preferably more than 98% of the amino acid sequence of SEQ ID NO: 108 , preferably more than 99% identity. In one embodiment, the Chimeric Antigen Receptor F is 90% or more, preferably 95% or more, preferably 98% or more, preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 110. In one embodiment, the amino acid sequence of the linker of the chimeric antigen receptor F is arbitrary as long as the function as the chimeric antigen receptor is maintained.

Amino acid identity can be calculated using commercially available or Internet-available analytical tools (eg, software such as FASTA, BLAST, PSI-BLAST, SSEARCH, etc.). For example, the main initial conditions generally used in BLAST searches are as follows. That is, at In Advanced BLAST 2.1, the program uses blastp, the Expect value is set to 10, all Filters are set to OFF, the Matrix uses BLOSUM62, and the Gap existence cost, Per residue gap cost and Lambda ratio are set to 11, 1, and 0.85 respectively (default value) , and other various parameters are also set to default values for searching, whereby the identity value (%) of the amino acid sequence can be calculated.

The scFVs (amino acid sequences of SEQ ID NOs: 10, 30 and 50) possessed by chimeric antigen receptors A to C are derived from monoclonal antibodies that specifically recognize vascular endothelial growth factor receptor (VEGFR2). VEGFR2 is highly expressed in tumor angiogenesis. The scFVs (amino acid sequences of SEQ ID NOs: 70, 90 and 110) possessed by the chimeric antigen receptors D~F are derived from monoclonal antibodies that specifically recognize the vascular endothelial cell-specific receptor (Robo4). Robo4 is also known as a tumor vessel specific marker. Therefore, the chimeric antigen receptors A to F can specifically recognize cancer tissue (tumor tissue).

Chimeric antigen receptors A to F, preferably cells having scFv regions, spacer sequences, membrane-penetrating domains, intracellular domains of costimulatory factors, and TCRs sequentially arranged from the N-terminus of the chimeric antigen receptors Construction of the inner domain. The length of the spacer sequence provided between the scFv region and the transmembrane domain and the type of amino acid residues constituting it are not limited as long as they do not hinder the function of the chimeric antigen receptor. For example, the spacer sequence can be designed to be about 10 to 25 amino acid residues.

The type of membrane penetration domain is not limited as long as it does not hinder the function of the chimeric antigen receptor. For example, CD28, CD3ζ, CD4, CD8α, etc. expressed by T cells can be used. As long as the penetrating domains of these membranes do not hinder the function of the chimeric antigen receptor, variations may be introduced as appropriate.

The intracellular domain of the costimulatory factor is not particularly limited as long as it is derived from the intracellular domain of a costimulatory factor possessed by T cells or the like. For example, one or more species selected from the group consisting of OX40, 4-1BB, CD27, CD278, CD28 and the like can be appropriately selected and used. The intracellular domains of these co-stimulatory factors may be appropriately mutated as long as they do not hinder the function of the chimeric antigen receptor.

The intracellular domain of TCR may be, for example, the intracellular domain derived from CD3, also known as TCRζ chain, or the like. As long as CD3 does not interfere with the function of the chimeric antigen receptor, mutations may be introduced appropriately. When introducing a mutation into CD3, it is preferable to carry out in a manner including an ITAM (immunoreceptor tyrosine-based activation motif).

Techniques for utilizing chimeric antigen receptors of specific scFVs and producing CAR-T cells expressing them are well known. For example, the chimeric antigen receptors A to F can be produced by the methods disclosed in Non-Patent Documents 1 and 2.

2. Polynucleotides encoding chimeric antigen receptors

The polynucleotide encoding the chimeric antigen receptor A is not particularly limited as long as it encodes the above-mentioned chimeric antigen receptor A. In one embodiment, the polynucleotide encoding the chimeric antigen receptor A preferably has a region selected from the group consisting of a region encoding the light chain CDR1 having the nucleotide sequence of SEQ ID NO: 11, and a light chain encoding the nucleotide sequence having the SEQ ID NO: 12. The region of CDR2, the region encoding the light chain CDR3 having the nucleotide sequence of SEQ ID NO: 13, the region encoding the heavy chain CDR1 having the nucleotide sequence of SEQ ID NO: 15, the region encoding the heavy chain CDR2 having the nucleotide sequence of SEQ ID NO: 16 region, and coding with SEQ ID NO: 17 At least one region of the group consisting of the regions of the heavy chain CDR3 of the nucleotide sequence, more preferably two or more types, more preferably three or more types, more preferably four or more types, more preferably five or more types, It is more preferable to have the whole area. In a suitable embodiment, the polynucleotide encoding Chimeric Antigen Receptor A preferably has a region encoding the light chain variable region having the nucleotide sequence of SEQ ID NO: 14 and/or encoding the base having SEQ ID NO: 18 The region of the heavy chain variable region of the sequence. In a suitable embodiment, the polynucleotide encoding Chimeric Antigen Receptor A preferably has the nucleotide sequence of SEQ ID NO: 20.

In one embodiment, the polynucleotide encoding Chimeric Antigen Receptor A has 80% or more, preferably 85% or more, preferably 90% or more of the base sequence of SEQ ID NO: 11-20, preferably A nucleotide sequence having an identity of 95% or more, preferably 98% or more, preferably 99% or more.

The polynucleotide encoding the chimeric antigen receptor B is not particularly limited as long as it encodes the above-mentioned chimeric antigen receptor B. In one embodiment, the polynucleotide encoding the chimeric antigen receptor B preferably has a region selected from the group consisting of a region encoding the light chain CDR1 having the nucleotide sequence of SEQ ID NO: 31, and a light chain encoding the nucleotide sequence having the nucleotide sequence of SEQ ID NO: 32 The region of CDR2, the region encoding the light chain CDR3 having the nucleotide sequence of SEQ ID NO:33, the region encoding the heavy chain CDR1 having the nucleotide sequence of SEQ ID NO:35, the region encoding the heavy chain CDR2 having the nucleotide sequence of SEQ ID NO:36 Region and at least one region of the group consisting of regions encoding the heavy chain CDR3 having the nucleotide sequence of SEQ ID NO: 37, more preferably two or more types, more preferably three or more types, more preferably four or more types , It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, the polynucleoside encoding chimeric antigen receptor B The acid preferably has a region encoding the light chain variable region having the nucleotide sequence of SEQ ID NO: 34 and/or a region encoding the heavy chain variable region having the nucleotide sequence of SEQ ID NO: 38. In a suitable embodiment, the polynucleotide encoding chimeric antigen receptor B preferably has the nucleotide sequence of SEQ ID NO: 40.

In one embodiment, the polynucleotide encoding Chimeric Antigen Receptor B has 80% or more, preferably 85% or more, preferably 90% or more of the base sequence of SEQ ID NO: 31-40, preferably A nucleotide sequence having an identity of 95% or more, preferably 98% or more, preferably 99% or more.

The polynucleotide encoding the chimeric antigen receptor C is not particularly limited as long as it encodes the above-mentioned chimeric antigen receptor C. In one embodiment, the polynucleotide encoding chimeric antigen receptor C preferably has a region selected from the group consisting of a region encoding the light chain CDR1 having the nucleotide sequence of SEQ ID NO: 51, and a light chain encoding the light chain having the nucleotide sequence of SEQ ID NO: 52 The region of CDR2, the region encoding the light chain CDR3 having the nucleotide sequence of SEQ ID NO:53, the region encoding the heavy chain CDR1 having the nucleotide sequence of SEQ ID NO:55, the region encoding the heavy chain CDR2 having the nucleotide sequence of SEQ ID NO:56 Region and at least one region of the group consisting of regions encoding the heavy chain CDR3 having the nucleotide sequence of SEQ ID NO: 57, more preferably two or more, more preferably three or more, more preferably four or more , It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor C preferably has a region encoding the light chain variable region having the nucleotide sequence of SEQ ID NO: 54 and/or encoding the base having SEQ ID NO: 58 The region of the heavy chain variable region of the sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor C preferably has the base sequence of SEQ ID NO: 60.

In one embodiment, the polynucleotide encoding the chimeric antigen receptor C has 80% or more, preferably 85% or more, preferably 90% or more of the base sequence of SEQ ID NO: 51-60, preferably A nucleotide sequence having an identity of 95% or more, preferably 98% or more, preferably 99% or more.

The polynucleotide encoding the chimeric antigen receptor D is not particularly limited as long as it encodes the above-mentioned chimeric antigen receptor D. In one embodiment, the polynucleotide encoding the chimeric antigen receptor D preferably has a region selected from the group consisting of a region encoding the light chain CDR1 having the nucleotide sequence of SEQ ID NO: 71, and a light chain encoding the light chain having the nucleotide sequence of SEQ ID NO: 72 The region of CDR2, the region encoding the light chain CDR3 having the nucleotide sequence of SEQ ID NO:73, the region encoding the heavy chain CDR1 having the nucleotide sequence of SEQ ID NO:75, the region encoding the heavy chain CDR2 having the nucleotide sequence of SEQ ID NO:76 Region and at least one region of the group consisting of regions encoding the heavy chain CDR3 having the nucleotide sequence of SEQ ID NO: 77, more preferably two or more, more preferably three or more, more preferably four or more , It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor D preferably has a region encoding the light chain variable region having the nucleotide sequence of SEQ ID NO: 74 and/or encoding the base having SEQ ID NO: 78 The region of the heavy chain variable region of the sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor D preferably has the nucleotide sequence of SEQ ID NO: 80.

In one embodiment, the polynucleotide encoding the chimeric antigen receptor D has 80% or more, preferably 85% or more, preferably 90% or more of the nucleotide sequence of SEQ ID NO: 71-80, preferably A nucleotide sequence having an identity of 95% or more, preferably 98% or more, preferably 99% or more.

The polynucleotide encoding the chimeric antigen receptor E is not particularly limited as long as it encodes the above-mentioned chimeric antigen receptor E. In one embodiment, the polynucleotide encoding the chimeric antigen receptor E preferably has a region selected from the group consisting of a region encoding the light chain CDR1 having the nucleotide sequence of SEQ ID NO: 91, and a light chain encoding the nucleotide sequence having the nucleotide sequence of SEQ ID NO: 92 The region of CDR2, the region encoding the light chain CDR3 having the nucleotide sequence of SEQ ID NO:93, the region encoding the heavy chain CDR1 having the nucleotide sequence of SEQ ID NO:95, the region encoding the heavy chain CDR2 having the nucleotide sequence of SEQ ID NO:96 Region and at least one region of the group consisting of regions encoding the heavy chain CDR3 having the nucleotide sequence of SEQ ID NO: 97, more preferably two or more, more preferably three or more, more preferably four or more , It is more preferable to have 5 or more types, and it is more preferable to have all the regions. In a suitable embodiment, the polynucleotide encoding chimeric antigen receptor E preferably has a region encoding the light chain variable region having the base sequence of SEQ ID NO: 94 and/or encoding the base having SEQ ID NO: 98 The region of the heavy chain variable region of the sequence. In a suitable embodiment, the polynucleotide encoding the chimeric antigen receptor E preferably has the base sequence of SEQ ID NO: 100.

In one embodiment, the polynucleotide encoding the chimeric antigen receptor E has 80% or more, preferably 85% or more, preferably 90% or more of the base sequence of SEQ ID NO: 91-100, preferably A nucleotide sequence having an identity of 95% or more, preferably 98% or more, preferably 99% or more.

The polynucleotide encoding the chimeric antigen receptor F is not particularly limited as long as it encodes the above-mentioned chimeric antigen receptor F. In one embodiment, the polynucleotide encoding Chimeric Antigen Receptor F preferably has a region selected from a region encoding the light chain CDR1 having the nucleotide sequence of SEQ ID NO: 111, encoding a region having The region encoding the light chain CDR2 of the nucleotide sequence of SEQ ID NO: 112, the region encoding the light chain CDR3 having the nucleotide sequence of SEQ ID NO: 113, the region encoding the heavy chain CDR1 having the nucleotide sequence of SEQ ID NO: 115, the region encoding the heavy chain CDR1 having the nucleotide sequence of SEQ ID NO: 115 At least one region of the group consisting of the heavy chain CDR2 region of the nucleotide sequence of 116 and the region encoding the heavy chain CDR3 of the nucleotide sequence of SEQ ID NO: 117, more preferably two or more, more preferably three more than one type, more preferably four or more types, more preferably five or more types, more preferably all regions. In a suitable embodiment, the polynucleotide encoding Chimeric Antigen Receptor F preferably has a region encoding the light chain variable region having the base sequence of SEQ ID NO: 114 and/or encoding the base having SEQ ID NO: 118 The region of the heavy chain variable region of the sequence. In a suitable embodiment, the polynucleotide encoding chimeric antigen receptor F preferably has the base sequence of SEQ ID NO: 120.

In one embodiment, the polynucleotide encoding the chimeric antigen receptor F has 80% or more, preferably 85% or more, preferably 90% or more of the nucleotide sequence of SEQ ID NO: 111-120, preferably A nucleotide sequence having an identity of 95% or more, preferably 98% or more, preferably 99% or more.

The identity of the base sequence can be calculated using a commercially available analysis tool or an analytical tool available through a telecommunication circuit (Internet). For example, specifically, in Advanced BLAST 2.1, the program uses blastn, and searches are performed with various parameters set to default values, whereby the value (%) of nucleotide sequence identity can be calculated. A polynucleotide can be optimized for codon usage frequency depending on the type of vector or cell in which it is expressed.

The state of the polynucleotide is not particularly limited, for example, it can be isolated or inserted into a vector. The type and use of the carrier are not specific Special restrictions. For example, the vector can be a plastid vector or a viral vector (eg, adenovirus or retrovirus). In addition, the vector may be, for example, a vector for colonization or a vector for expression. Examples of expression vectors include those for prokaryotic cells such as Escherichia coli and actinomycetes, and those for eukaryotic cells such as yeast cells, insect cells, and mammalian cells. The polynucleotide may be modified arbitrarily, for example, a base sequence encoding a message peptide may be appropriately added to the 5' terminal side.

The polynucleotide can also be in a state in which it has been taken up into a host cell. The form in which the host cell contains the polynucleotide is not particularly limited. For example, the host cell can have the polynucleotide in the form of a vector, and can also have the polynucleotide in the form of genomic DNA integrated into the host cell. The type of the host cell is arbitrary and not particularly limited. For example, host cells can be eukaryotic cells such as yeast cells, insect cells and mammalian cells, and prokaryotic cells such as Escherichia coli and actinomycetes. In one embodiment, the host cells are preferably eukaryotic cells (eg, mammalian, human), such as T cells or NK cells. A host cell containing a polynucleotide can be obtained, for example, by introducing the polynucleotide (for example, in the form of a vector) into any host cell.

The host cell may express the polynucleotide encoding the chimeric antigen receptor, or may not express it. When the polynucleotide encoding the chimeric antigen receptor is expressed in the host cell, it is preferable that the scFv region constituting the chimeric antigen receptor is exposed on the outside of the cell, and the transmembrane domain, the costimulatory factor, and the intracellular structure of the TCR Domain lines exist in the cell membrane or within the cell.

3. CAR-T cells

T cells (CAR-T cells) expressing any one of the above-mentioned chimeric antigen receptors A to F are provided. The T cells expressing the chimeric antigen receptor recognize the antigen with the scFv region, and then transmit the recognition information to the inside of the T cells and the like through the zeta chain. When the scFv region recognizes its epitope, through the transmembrane domain and co-stimulatory factors, the message that induces cytotoxic activity is actuated in the cell, and in conjunction with it, the cell line acts on other cells or tissues that express the same epitope. Aggressive or cytotoxic activity.

When the cells that perform such functions are CTLs, they are called chimeric antigen receptor T cells (CAR-T cells). Similar to chimeric antigen receptor T cells, cells that have the potential to exhibit cytotoxic activity, such as NK cells, can exhibit cytotoxic activity by binding the scFv region to its epitope. Therefore, host cells containing polynucleotides encoding chimeric antigen receptors (especially host cells with cytotoxic activity) are useful as active ingredients in pharmaceutical compositions. Host cells (eg, CAR-T cells) containing a polynucleotide encoding a chimeric antigen receptor can be produced by referring to known methods described in Non-Patent Documents 1 and 2 and the like.

Such CAR-T cells and the like specifically recognize cancer tissues (tumor tissues), and are therefore useful for the treatment or prevention of tumors and the like. The type of tumor is not particularly limited, including solid cancer and blood cancer. Examples of solid cancer include lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma.

4. Pharmaceutical composition

A pharmaceutical composition containing the above-mentioned CAR-T cells, etc., and a method for treating or preventing diseases using the same are provided. The content of the above-mentioned CAR-T cells in the pharmaceutical composition can be determined in consideration of the type of the target disease (for example, solid cancer), the intended therapeutic effect, the method of administration, the treatment period, the age of the patient, and the weight of the patient. appropriate settings. For example, when the total pharmaceutical composition is 100 parts by weight, the content of the antibody in the pharmaceutical composition may be about 0.001 parts by weight to 10 parts by weight. The content of cells in the pharmaceutical composition may be, for example, about 1 cell/mL to 10 ⁴ cells/mL.

The administration form of the pharmaceutical composition is not particularly limited as long as the desired effect can be obtained, and oral administration and parenteral administration (for example, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, Any route of administration (dermal administration, topical administration) is administered to mammals, including humans. Since the active ingredient is cells, the preferred form of administration is parenteral administration, more preferably intravenous injection. Dosage forms for oral administration and parenteral administration and their manufacturing methods are well known to those with ordinary knowledge in the technical field, and the present invention can be produced by mixing with pharmaceutically acceptable pharmaceutical carriers, etc., in accordance with general methods. Invented antibodies or cells.

Dosage forms for parenteral administration include injection preparations (for example, drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (for example, ointments, ointments, lotions), suppositories Inhalants, eye drops, eye ointments, nasal drops, ear drops, liposomes, etc. For example, preparations for injection can be prepared by dissolving or suspending antibodies or cells in distilled water for injection, and adding dissolution aids, buffers, pH adjusters, isotonic agents, pain relievers, preservatives, and stabilizers as needed agent, etc. The pharmaceutical composition can also be used as a freeze-dried preparation for preparation at the time of use.

The pharmaceutical composition may further contain other agents effective for the treatment or prevention of diseases. In addition, the pharmaceutical composition may contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids as required.

The pharmaceutical carriers used in the formulation of pharmaceutical compositions can be excipients, binders, disintegrants, lubricants, colorants, flavoring and odorants commonly used in this technical field, or stabilizers, Emulsifier, absorption enhancer, surfactant, pH adjuster, preservative, antioxidant, extender, wetting agent, surfactant, dispersant, buffer, preservative, dissolution aid, pain reliever Wait.

The type of disease to be treated or prevented using the pharmaceutical composition is not particularly limited as long as the treatment or prevention can be achieved. Specific target diseases include tumors, for example. The type of tumor is not particularly limited, including solid cancer and blood cancer. Examples of solid cancer include lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma.

The administration object (subject) of the pharmaceutical composition is, for example, an animal suffering from the above-mentioned disease or an animal likely to suffer from it. "Possible to suffer from" can be determined, for example, by a diagnostic method described later. Animal refers to eg mammals, preferably humans.

The dosage of the pharmaceutical composition can be based on, for example, the route of administration, the type of disease, the degree of symptoms, the patient's age, sex, body weight, severity of the disease, pharmacokinetics, and toxicological characteristics, etc. Pharmacological insights, Whether the drug delivery system is utilized or not, and whether it is administered as part of a combination with other drugs, is up to the clinician to decide. The dosage of the pharmaceutical composition, for example, when the active ingredient is an antibody, can be about 1 μg/kg (body weight) to 10 g/kg (body weight) per day. In addition, when the active ingredient is cells (VI), it may be about 10 ⁴ cells/kg (body weight) to 10 ⁹ cells/kg (body weight). The administration schedule of the pharmaceutical composition can also be determined by considering the same factors as the administration amount. For example, it is possible to administer the above-mentioned daily dose once a day to a month.

[Example]

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

1. Construction of pMXs-IG/CAR[hV-(h28)-h28-h3Z]

Human CD3zeta chain cDNA and Human CD28 cDNA, hCD3zeta STD and hCD28 HD-hCD28 TMD-hCD28 STD genes were purchased from Open Biosystems, using the Primer group and KOD Plus (TOYOBO, Inc) shown in Table 2, and the hCD3zeta STD Each gene fragment encoding these was produced so that the 5' end had the same nucleotide sequence as the 3' end of hCD28 HD-hCD28 TMD-hCD28 STD. Next, assembly PCR was performed using these gene fragments and KOD Plus to prepare a gene fragment obtained by linking hCD28 HD-hCD28 TMD-hCD28 STD and CD3zeta STD genes with restriction enzyme sites of Sac II in the upstream and Not I in the downstream. (Insert 1) (Figure 13). Will have anti-mVEGFR2 scFv and pMXs-IG/anti-mVEGFR2 scFv with platelet-derived growth factor receptor (PDGFR) as the membrane penetrating domain to restrict the cleavage of enzymes Sac II and Not I, by using DNA Ligation kit Ver.2.1 (TAKARA The ligation reaction of BIO, Inc) was inserted into Insert 1 to construct pMXs-IG/CAR[mV-(h28)-h28-h3Z].

For pET15b-mouse anti-hVEGFR2-scFv (aV2-95h) with the gene sequence of anti-hVEGFR2 scFv (SEQ ID NO: 20), PCR was performed using the Primer group shown in Table 2 and KOD Plus to obtain Sfi I upstream and downstream Gene fragment with restriction enzyme site for Sac II (Insert 2) (Fig. 14). The pMXs-IG/CAR[mV-(h28)-h28-h3Z] was cleaved by restriction enzymes Sfi I and Sac II, and inserted into Insert 2 by the ligation reaction using DNA Ligation kit Ver.2.1 to construct pMXs- IG/CAR [hV-(h28)-h28-h3Z].

2. Construction of pMXs-IG/CAR[hV-(h8α)-h137-h3Z]

Using plasmid pCR4-TOPO/hCD8αHD-TMD-hCD137-hCD3zeta having a sequence obtained by linking the genes of hCD8αHD and TMD with the gene of CD137 STD, and the Primer group and KOD Plus shown in Table 2, PCR was performed to produce upstream Sac II and downstream possess restriction enzyme sites for Not I, and encode hCD8αHD and gene fragments (Insert 3) of TMD and CD137 STD (FIG. 15). The pMXs-IG/CAR[mV-(h28)-h28-h3Z] was cleaved with restriction enzymes Sac II and Not I, and inserted into Insert 3 by the ligation reaction using DNA Ligation kit Ver.2.1 to construct pMXs- IG/CAR [mV-(h8α)-h137-h3Z].

The pMXs-IG/CAR[hV-(h28)-h28-h3Z] was cut with restriction enzymes EcoR I and Sac II to obtain a gene fragment (Insert 4) with the gene sequence of anti-hVEGFR2 scFv (SEQ ID NO: 20) ( Figure 16). pMXs-IG/CAR[mV-(h8α)-h137-h3Z] was cleaved with restriction enzymes EcoR I and Sac II, and ligated using DNA Ligation kit Ver.2.1 The reaction was inserted into Insert 4 to construct pMXs-IG/CAR[hV-(h8α)-h137-h3Z].

CAR[mV-(m28)-m28-m3Z], CAR[mV-(h28)-h28-h3Z], CAR[hV-(h28)-h28-h3Z], CAR[mV- (h8α)-h137-h3Z], CAR[hV-(h8α)-h137-h3Z] gene plastids, using the primers shown in Table 2 and DNA polymerase KOD plus (Toyobo, Inc) PCR was performed to prepare the plasmids containing The DNA template of T7 promoter sequence and CAR gene was purified using mMESSAGE mMACHINE(R) T7 ULTRA Transcription Kit (Ambion, Inc) according to a general method, suspended in Nuclease Free Water (Ambion, Inc), and stored at -20°C. CAR-mRNA is obtained in this way.

3. Introduction of CAR-mRNA

The CD8+ T cells were suspended in Opti ^- MEM (Life Technology, Inc) for 3 times, centrifuged, and the supernatant was removed to completely remove the protein contained in the serum. 10 ⁷ cells/mL. In addition, 25 μL of Nuclease Free Water containing 80pg-4 μg/μL of CAR-mRNA was mixed with 175 μL of cell suspension, and just before electroporation, a 4 mm colorimetric tube (BEX, Inc) was filled so that air bubbles would not enter. 200 μL. The colorimetric tube was inserted into the colorimetric tube electrode chamber (CU500; Nepagene, Inc.), and the resistance was measured using an electroporator (CUY21Pro-Vitro; Nepagene, Inc.). After confirming that there was no abnormality, EP was performed. After 3 hours and 6 hours After 12 hours, 24 hours, 36 hours, 48 hours, and 72 hours, the CAR expression intensity was measured by FCM, and the cell viability was measured by trypan blue exclusion method.

4. In vivo anti-tumor effect

3×10 ⁵ melanoma B16BL6 cells were intradermally transplanted into the abdomen of C57BL/6 mice. Seven days after tumor inoculation, when the length of the tumor reached about 5.0-6.0 mm, the expression of CAR [mV-( m28)-m28-m3Z] mouse CAR-T cells were administered at 5×10 ⁶ cells/500 μL/mouse as a single administration, or additionally administered twice every 2 days, and then the tumor length was measured over time. The diameter and the short diameter were used to calculate the tumor volume according to the formula shown below. In addition, a non-treatment group and a group to which CAR-mRNA-unintroduced CD8+ T cells were administered were prepared as a control group. The mouse CAR-T cell line was suspended in RPMI1640 for administration. Tumor volume (mm ³ )=(longer diameter of tumor; mm)×(shorter diameter of tumor; mm) ² ×0.5236

The test results are shown in FIG. 18 . In cancer-bearing mice administered with CAR-T cells once, the tumor proliferation was significantly inhibited compared with the control group, and a stronger anti-tumor effect was obtained when additional administration was performed twice. . Therefore, the CAR-T cells we produced suggest the following possibility: although the performance of CAR is transient, it is effective only after a single administration, and if it is administered multiple times, it can become a Cell medicine with full effect.

<110> MEDINET CO.,LTD.

<120> Chimeric Antigen Receptor, and Its Utilization

<130> P17-079WO

<150> JP 2016-095125

<151> 2016-05-11

<160> 128

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> Mus musculus

<400> 1

<210> 2

<211> 7

<212> PRT

<213> Mus musculus

<400> 2

<210> 3

<211> 9

<212> PRT

<213> Mus musculus

<400> 3

<210> 4

<211> 107

<212> PRT

<213> Mus musculus

<400> 4

<210> 5

<211> 10

<212> PRT

<213> Mus musculus

<400> 5

<210> 6

<211> 17

<212> PRT

<213> Mus musculus

<400> 6

<210> 7

<211> 8

<212> PRT

<213> Mus musculus

<400> 7

<210> 8

<211> 134

<212> PRT

<213> Mus musculus

<400> 8

<210> 9

<211> 15

<212> PRT

<213> Mus musculus

<400> 9

<210> 10

<211> 256

<212> PRT

<213> Mus musculus

<400> 10

<210> 11

<211> 30

<212> DNA

<213> Mus musculus

<400> 11

<210> 12

<211> 21

<212> DNA

<213> Mus musculus

<400> 12

<210> 13

<211> 27

<212> DNA

<213> Mus musculus

<400> 13

<210> 14

<211> 321

<212> DNA

<213> Mus musculus

<400> 14

<210> 15

<211> 30

<212> DNA

<213> Mus musculus

<400> 15

<210> 16

<211> 51

<212> DNA

<213> Mus musculus

<400> 16

<210> 17

<211> 24

<212> DNA

<213> Mus musculus

<400> 17

<210> 18

<211> 402

<212> DNA

<213> Mus musculus

<400> 18

<210> 19

<211> 45

<212> DNA

<213> Mus musculus

<400> 19

<210> 20

<211> 768

<212> DNA

<213> Mus musculus

<400> 20

<210> 21

<211> 11

<212> PRT

<213> Mus musculus

<400> 21

<210> 22

<211> 7

<212> PRT

<213> Mus musculus

<400> 22

<210> 23

<211> 9

<212> PRT

<213> Mus musculus

<400> 23

<210> 24

<211> 108

<212> PRT

<213> Mus musculus

<400> 24

<210> 25

<211> 10

<212> PRT

<213> Mus musculus

<400> 25

<210> 26

<211> 17

<212> PRT

<213> Mus musculus

<400> 26

<210> 27

<211> 9

<212> PRT

<213> Mus musculus

<400> 27

<210> 28

<211> 118

<212> PRT

<213> Mus musculus

<400> 28

<210> 29

<211> 15

<212> PRT

<213> Mus musculus

<400> 29

<210> 30

<211> 241

<212> PRT

<213> Mus musculus

<400> 30

<210> 31

<211> 33

<212> DNA

<213> Mus musculus

<400> 31

<210> 32

<211> 21

<212> DNA

<213> Mus musculus

<400> 32

<210> 33

<211> 27

<212> DNA

<213> Mus musculus

<400> 33

<210> 34

<211> 324

<212> DNA

<213> Mus musculus

<400> 34

<210> 35

<211> 30

<212> DNA

<213> Mus musculus

<400> 35

<210> 36

<211> 51

<212> DNA

<213> Mus musculus

<400> 36

<210> 37

<211> 27

<212> DNA

<213> Mus musculus

<400> 37

<210> 38

<211> 354

<212> DNA

<213> Mus musculus

<400> 38

<210> 39

<211> 45

<212> DNA

<213> Artificial

<220>

<223> Linker

<400> 39

<210> 40

<211> 723

<212> DNA

<213> Artificial

<220>

<223> ScFV

<400> 40

<210> 41

<211> 11

<212> PRT

<213> Mus musculus

<400> 41

<210> 42

<211> 7

<212> PRT

<213> Mus musculus

<400> 42

<210> 43

<211> 9

<212> PRT

<213> Mus musculus

<400> 43

<210> 44

<211> 108

<212> PRT

<213> Mus musculus

<400> 44

<210> 45

<211> 10

<212> PRT

<213> Mus musculus

<400> 45

<210> 46

<211> 17

<212> PRT

<213> Mus musculus

<400> 46

<210> 47

<211> 12

<212> PRT

<213> Mus musculus

<400> 47

<210> 48

<211> 121

<212> PRT

<213> Mus musculus

<400> 48

<210> 49

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Linker

<400> 49

<210> 50

<211> 244

<212> PRT

<213> Artificial

<220>

<223> Linker

<400> 50

<210> 51

<211> 33

<212> DNA

<213> Mus musculus

<400> 51

<210> 52

<211> 21

<212> DNA

<213> Mus musculus

<400> 52

<210> 53

<211> 27

<212> DNA

<213> Mus musculus

<400> 53

<210> 54

<211> 324

<212> DNA

<213> Mus musculus

<400> 54

<210> 55

<211> 30

<212> DNA

<213> Mus musculus

<400> 55

<210> 56

<211> 51

<212> DNA

<213> Mus musculus

<400> 56

<210> 57

<211> 36

<212> DNA

<213> Mus musculus

<400> 57

<210> 58

<211> 363

<212> DNA

<213> Mus musculus

<400> 58

<210> 59

<211> 45

<212> DNA

<213> Artificial

<220>

<223> Linker

<400> 59

<210> 60

<211> 732

<212> DNA

<213> Artificial

<220>

<223> scFV

<400> 60

<210> 61

<211> 10

<212> PRT

<213> Mus musculus

<400> 61

<210> 62

<211> 7

<212> PRT

<213> Mus musculus

<400> 62

<210> 63

<211> 9

<212> PRT

<213> Mus musculus

<400> 63

<210> 64

<211> 107

<212> PRT

<213> Mus musculus

<400> 64

<210> 65

<211> 10

<212> PRT

<213> Mus musculus

<400> 65

<210> 66

<211> 17

<212> PRT

<213> Mus musculus

<400> 66

<210> 67

<211> 9

<212> PRT

<213> Mus musculus

<400> 67

<210> 68

<211> 118

<212> PRT

<213> Mus musculus

<400> 68

<210> 69

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Linker

<400> 69

<210> 70

<211> 240

<212> PRT

<213> Artificial

<220>

<223> scFV

<400> 70

<210> 71

<211> 30

<212> DNA

<213> Mus musculus

<400> 71

<210> 72

<211> 21

<212> DNA

<213> Mus musculus

<400> 72

<210> 73

<211> 27

<212> DNA

<213> Mus musculus

<400> 73

<210> 74

<211> 321

<212> DNA

<213> Mus musculus

<400> 74

<210> 75

<211> 30

<212> DNA

<213> Mus musculus

<400> 75

<210> 76

<211> 51

<212> DNA

<213> Mus musculus

<400> 76

<210> 77

<211> 27

<212> DNA

<213> Mus musculus

<400> 77

<210> 78

<211> 354

<212> DNA

<213> Mus musculus

<400> 78

<210> 79

<211> 45

<212> DNA

<213> Artificial

<220>

<223> Linker

<400> 79

<210> 80

<211> 720

<212> DNA

<213> Artificial

<220>

<223> scFV

<400> 80

<210> 81

<211> 10

<212> PRT

<213> Mus musculus

<400> 81

<210> 82

<211> 7

<212> PRT

<213> Mus musculus

<400> 82

<210> 83

<211> 9

<212> PRT

<213> Mus musculus

<400> 83

<210> 84

<211> 107

<212> PRT

<213> Mus musculus

<400> 84

<210> 85

<211> 10

<212> PRT

<213> Mus musculus

<400> 85

<210> 86

<211> 17

<212> PRT

<213> Mus musculus

<400> 86

<210> 87

<211> 13

<212> PRT

<213> Mus musculus

<400> 87

<210> 88

<211> 122

<212> PRT

<213> Mus musculus

<400> 88

<210> 89

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Linker

<400> 89

<210> 90

<211> 244

<212> PRT

<213> Artificial

<220>

<223> scFV

<400> 90

<210> 91

<211> 30

<212> DNA

<213> Mus musculus

<400> 91

<210> 92

<211> 21

<212> DNA

<213> Mus musculus

<400> 92

<210> 93

<211> 27

<212> DNA

<213> Mus musculus

<400> 93

<210> 94

<211> 321

<212> DNA

<213> Mus musculus

<400> 94

<210> 95

<211> 30

<212> DNA

<213> Mus musculus

<400> 95

<210> 96

<211> 51

<212> DNA

<213> Mus musculus

<400> 96

<210> 97

<211> 39

<212> DNA

<213> Mus musculus

<400> 97

<210> 98

<211> 366

<212> DNA

<213> Mus musculus

<400> 98

<210> 99

<211> 45

<212> DNA

<213> Artificial

<220>

<223> Linker

<400> 99

<210> 100

<211> 732

<212> DNA

<213> Artificial

<220>

<223> scFV

<400> 100

<210> 101

<211> 11

<212> PRT

<213> Mus musculus

<400> 101

<210> 102

<211> 7

<212> PRT

<213> Mus musculus

<400> 102

<210> 103

<211> 9

<212> PRT

<213> Mus musculus

<400> 103

<210> 104

<211> 108

<212> PRT

<213> Mus musculus

<400> 104

<210> 105

<211> 10

<212> PRT

<213> Mus musculus

<400> 105

<210> 106

<211> 17

<212> PRT

<213> Mus musculus

<400> 106

<210> 107

<211> 8

<212> PRT

<213> Mus musculus

<400> 107

<210> 108

<211> 117

<212> PRT

<213> Mus musculus

<400> 108

<210> 109

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Linker

<400> 109

<210> 110

<211> 240

<212> PRT

<213> Artificial

<220>

<223> scFV

<400> 110

<210> 111

<211> 33

<212> DNA

<213> Mus musculus

<400> 111

<210> 112

<211> 21

<212> DNA

<213> Mus musculus

<400> 112

<210> 113

<211> 27

<212> DNA

<213> Mus musculus

<400> 113

<210> 114

<211> 324

<212> DNA

<213> Mus musculus

<400> 114

<210> 115

<211> 30

<212> DNA

<213> Mus musculus

<400> 115

<210> 116

<211> 51

<212> DNA

<213> Mus musculus

<400> 116

<210> 117

<211> 24

<212> DNA

<213> Mus musculus

<400> 117

<210> 118

<211> 351

<212> DNA

<213> Mus musculus

<400> 118

<210> 119

<211> 45

<212> DNA

<213> Artificial

<220>

<223> Linker

<400> 119

<210> 120

<211> 720

<212> DNA

<213> Artificial

<220>

<223> scFV

<400> 120

<210> 121

<211> 49

<212> DNA

<213> artificial

<220>

<223> primer

<400> 121

<210> 122

<211> 38

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 122

<210> 123

<211> 50

<212> DNA

<213> artificial

<220>

<223> primer

<400> 123

<210> 124

<211> 19

<212> DNA

<213> artificial

<220>

<223> primer

<400> 124

<210> 125

<211> 43

<212> DNA

<213> artificial

<220>

<223> primer

<400> 125

<210> 126

<211> 45

<212> DNA

<213> artificial

<220>

<223> primer

<400> 126

<210> 127

<211> 30

<212> DNA

<213> artificial

<220>

<223> primer

<400> 127

<210> 128

<211> 31

<212> DNA

<213> artificial

<220>

<223> primer

<400> 128

Claims (6)

A kind of chimeric antigen receptor selected from the group of chimeric antigen receptor A～F of following any one: Chimeric antigen receptor A, it comprises and contains: comprises the amino acid sequence with SEQ ID NO: 1 The light chain CDR1 of the amino acid sequence, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 2, and the light chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 3 Heavy chain variable region, and/or containing: heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 5, heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 6 , and the light chain variable region of the heavy chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 7; Chimeric Antigen Receptor B, comprising: comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 21 The light chain CDR1 of the amino acid sequence, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 22, and The heavy chain variable region comprising the light chain CDR3 of the amino acid sequence of SEQ ID NO: 23, and/or the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 25 , the heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprising the heavy chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 27; Chimeric Antigen receptor C, which comprises: the light chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 41, the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 42, and The heavy chain variable region comprising the light chain CDR3 of the amino acid sequence of SEQ ID NO: 43, and/or the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 45 , the heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 46, and the heavy chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 47 The light chain variable region; Chimeric Antigen Receptor D, which comprises: a light chain CDR1 comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 61, an amine comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 62 The light chain CDR2 of the amino acid sequence and the heavy chain variable region of the light chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 63, and/or the heavy chain variable region comprising: the amino acid sequence of the amino acid sequence of SEQ ID NO: 65 The heavy chain CDR1 of the amino acid sequence of SEQ ID NO: 66, the heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 66, and the heavy chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 67 The light chain variable region; Chimeric Antigen Receptor E, which comprises: the light chain CDR1 comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 81, the amine comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 82 The light chain CDR2 of the amino acid sequence and the heavy chain variable region of the light chain CDR3 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 83, and/or Contains: the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 85, the heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 86, and the amine comprising the amino acid sequence of SEQ ID NO: 87 The light chain variable region of the heavy chain CDR3 of the amino acid sequence of the amino acid sequence; or the chimeric antigen receptor F, which comprises: the light chain CDR1 comprising the amino acid sequence having the amino acid sequence of SEQ ID NO: 101 , the light chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 102, and the heavy chain variable region comprising the light chain CDR3 of the amino acid sequence of the amino acid sequence of SEQ ID NO: 103, and/ Or contain: the heavy chain CDR1 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 105, the heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 106, and the heavy chain CDR2 comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 107 The light chain variable region of the heavy chain CDR3 of the amino acid sequence of the amino acid sequence. A chimeric antigen receptor A as described in claim 1. A chimeric antigen receptor T cell or chimeric antigen receptor NK cell having the chimeric antigen receptor as claimed in claim 1 or 2. A polynucleotide encoding the chimeric antigen receptor as claimed in item 1 or 2, and the polynucleotide has a region encoding a light chain variable region having the base sequence of SEQ ID NO: 14 and encoding a light chain variable region having the nucleotide sequence of SEQ ID NO: 18 A polynucleotide encoding the chimeric antigen receptor A in the region of the heavy chain variable region of the nucleotide sequence of SEQ ID NO: 34; The polynucleotide encoding the chimeric antigen receptor B in the region of the heavy chain variable region of the base sequence, the region encoding the light chain variable region having the base sequence of SEQ ID NO: 54, and the base encoding the base having SEQ ID NO: 58 A polynucleotide encoding the chimeric antigen receptor C in the region of the heavy chain variable region of the base sequence, a region encoding the light chain variable region having the nucleotide sequence of SEQ ID NO: 74, and a region encoding the base having SEQ ID NO: 78 The polynucleotide encoding the chimeric antigen receptor D in the region of the heavy chain variable region of the sequence, the region encoding the light chain variable region having the nucleotide sequence of SEQ ID NO: 94, and the region encoding the nucleotide sequence having the SEQ ID NO: 98 The polynucleotide encoding the chimeric antigen receptor E in the region of the heavy chain variable region of the The polynucleotide encoding the chimeric antigen receptor F of the variable region of the heavy chain. A pharmaceutical composition comprising the chimeric antigen receptor T cell or the chimeric antigen receptor NK cell according to claim 3. The pharmaceutical composition of claim 5 is for the treatment or prevention of cancer.

Images