Chimeric antigen receptor

Abstract

Provided is a novel chimeric antigen receptor. This chimeric antigen receptor includes an scFv region having such a structure that the C-terminus of a heavy-chain variable region and the N-terminus of a light-chain variable region are linked to each other through a linker, wherein: the heavy-chain variable region includes a heavy-chain CDR1 having the amino acid sequence represented by SEQ ID NO: 1, a heavy-chain CDR2 having the amino acid sequence represented by SEQ ID NO: 2, and / or a heavy-chain CDR3 having the amino acid sequence represented by SEQ ID NO: 3, and / or the light-chain variable region includes a light-chain CDR1 having the amino acid sequence represented by SEQ ID NO: 5, a light-chain CDR2 having the amino acid sequence represented by SEQ ID NO: 6, and / or a light-chain CDR3 having the amino acid sequence represented by SEQ ID NO: 7; and the number of amino acid residues constituting the linker is 15-50.

Description

Chimeric Antigen Receptor

[0001] A new chimeric antigen receptor and its use are disclosed.

[0002] The present inventors identified the R8H283 antibody, which specifically binds to myeloma cells in the bone marrow of human multiple myeloma patients (Patent Document 1). The R8H283 antibody was isolated as an antibody that recognizes the human CD98 heavy chain (CD98hc). It was subsequently confirmed that the antibody recognizes an epitope that contains the N-glycosylation site of human CD98hc and is exposed by the inhibition of N-glycosylation.

[0003] Although several antibodies against CD98 are known, none have yet been marketed, and there is currently limited information regarding their use in CAR-T cells.

[0004] International Publication No. 2017 / 026497

[0005] J Immunol. 2011 Feb 1; 186(3): 1840-1848J Immunol. 2009 Nov 1; 183(9): 5563-74.N Engl J Med. 2014 Oct 16; 371(16): 1507-17.Nat Med. 2017 23, 1436-1443.

[0006] The present invention aims to provide a novel chimeric antigen receptor. One of the objectives is to provide a more effective means for treating malignant tumors and the like.

[0007] CAR-T cells containing scFv derived from the R8H283 antibody were generated, and as shown in the Examples below, it was found that CAR-T cells containing scFv in which the light chain variable region (VL) and heavy chain variable region (VH) were arranged in this order from the N-terminus exhibited limited proliferation and also had limited in vivo antitumor effects. On the other hand, it was confirmed that CAR-T cells containing scFv in which the VH and VL were arranged in this order from the N-terminus exhibited superior proliferation and in vivo antitumor effects. Based on these findings, further research and investigation led to the provision of the following representative inventions.

[0008] Item 1. A chimeric antigen receptor comprising an scFv region having a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are linked via a linker, wherein the heavy chain variable region comprises a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and / or a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and / or the light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 5, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 6, and / or a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 7; and the number of amino acid residues comprising the linker is 15 to 50. Item 2. A chimeric antigen receptor according to Aspect 1, wherein the amino acid sequence of the heavy chain FR region of the heavy chain variable region comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of the heavy chain FR region of the amino acid sequence shown in SEQ ID NO: 4, and / or the amino acid sequence of the light chain FR region of the light chain variable region comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of the light chain FR region of the amino acid sequence shown in SEQ ID NO: 8. A chimeric antigen receptor according to Aspect 3, wherein the amino acid sequence of the heavy chain variable region comprises an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 4, and / or the amino acid sequence of the light chain variable region comprises an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 8; and wherein the number of amino acid residues constituting the linker is 15 to 50. A chimeric antigen receptor according to any one of Aspects 1 to 3, wherein the chimeric antigen receptor has a structure in which the following is arranged in order from the N-terminus: heavy chain variable region-linker-light chain variable region-hinge sequence-transmembrane domain-intracellular signaling domain. Aspect 5. Item 6. A polynucleotide encoding the chimeric antigen receptor of any one of Items 1 to 4. Item 6. A cell comprising the polynucleotide of Item 5. Item 7. The cell of Item 6, which is a chimeric antigen receptor T cell. Item 8. A pharmaceutical composition comprising the cell of Item 6 or 7. Item 9. The pharmaceutical composition of Item 8, which is used for the treatment and / or prevention of cancer.Item 10. A method for producing chimeric antigen receptor T cells, comprising culturing T cells introduced with a polynucleotide encoding the chimeric antigen receptor of any one of Items 1 to 4 in the presence of dasatinib. Item 11. Chimeric antigen receptor T cells produced by the production method of Item 10. Item 12. A pharmaceutical composition comprising the cells of Item 11. Item 13. The pharmaceutical composition of Item 12, which is used for the treatment and / or prevention of cancer.

[0009] A novel chimeric antigen receptor and a cell containing a polynucleotide encoding the receptor are provided. Use of the chimeric antigen receptor makes it possible to effectively treat or prevent diseases such as malignant tumors.

[0010] A conceptual diagram of the chimeric antigen receptor construct produced is shown. The results of evaluating the proliferation of CAR-T cells are shown. The results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells are shown. The results of evaluating the IFN-γ production ability of CAR-T cells are shown. The results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells are shown. The results of evaluating the proliferation of CAR-T cells are shown. The results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells are shown. The results of evaluating the IFN-γ production ability of CAR-T cells are shown. The results of evaluating the change in CAR-T cell number after repeated antigen stimulation are shown. The results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells after repeated antigen stimulation are shown. The results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells are shown. The results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells are shown. The results of evaluating the IFN-γ production ability of CAR-T cells are shown. The results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells are shown. The results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells are shown.

[0033] Figure 1 shows the results of evaluating the aggregation score of Fv structures.

[0034] Figure 1 shows the results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells.

[0035] Figure 2 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0036] Figure 3 shows the results of evaluating the antitumor effect (luminescence value) of CAR-T cells.

[0037] Figure 4 shows the results of evaluating the antitumor effect (luminescence value) of CAR-T cells.

[0038] Figure 5 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0039] Figure 6 shows the results of evaluating the antitumor effect (luminescence value) of CAR-T cells.

[0039] Figure 7 shows the schedule for evaluating the antitumor effect (luminescence value) of CAR-T cells.

[0039] Figure 8 shows the results of evaluating the antitumor effect (luminescence value) of CAR-T cells.

[0039] Figure 9 shows the results of evaluating the antitumor effect (luminescence value) of CAR-T cells.

[0039] Figure 10 shows the results of evaluating the antitumor effect (luminescence value) of CAR-T cells.

[0039] Figure 11 shows the results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells. Figure 1 shows the results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells. Figure 2 shows the results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells. Figure 3 shows the results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells. Figure 4 shows the results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells. Figure 5 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0033] Figure 1 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0034] Figure 1 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0035] Figure 2 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0036] Figure 3 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0037] Figure 4 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0038] Figure 5 shows the results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells.

[0039] Figure 6 shows the results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells.

[0039] Figure 7 shows the results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells.

[0039] Figure 8 shows the results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells.

[0039] Figure 9 shows the results of evaluating the cytotoxic activity (luminescence value) of CAR-T cells.

[0039] Figure 10 shows the results of evaluating the IFN-γ production ability of CAR-T cells.

[0039] Figure 11 shows the results of evaluating the anti-tumor effect (luminescence value) of CAR-T cells.

[0011] 1. Chimeric Antigen Receptors (CARs) Chimeric antigen receptors are generally known as artificial T cell receptor (TCR)-like proteins. They are constructed by replacing the antigen recognition site (corresponding to the extracellular domain) expressed on the cell membrane of T cells with a desired antigen recognition site, and by enabling the T cells to more effectively exert their inherent functions, such as cytotoxic activity. Chimeric antigen receptors generally have a single-chain fragment (scFv) at the N-terminus, in which the light and heavy chain variable regions of an antibody are linked in tandem, and a T cell receptor (TCR) zeta chain at the C-terminus. T cells expressing chimeric antigen receptors recognize antigens via the scFv region and then transmit the recognition signal to the T cells via the zeta chain.

[0012] In one embodiment, the chimeric antigen receptor preferably has a structure in which an scFv region, a hinge / spacer sequence, a transmembrane domain, and an intracellular signaling domain are arranged in this order from the N-terminus.

[0013] In one embodiment, the chimeric antigen receptor preferably comprises an scFv region having a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are linked via a linker.

[0014] The number of amino acid residues constituting the linker is preferably 15 to 50. The lower limit of this range may be 16, 17, or 18, and the upper limit may be 45, 40, 35, 30, 25, 24, 23, or 22.

[0015] The type of linker is not particularly limited, and examples include a linker composed of glycine and serine, such as a G4S linker (GGGGS; SEQ ID NO: 22), a 218 linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 21), etc., and the linker may have a structure in which these linkers are repeated. Furthermore, the sequence of the linker may be the same as or different from the hinge / spacer sequence provided between the scFv region and the transmembrane domain.

[0016] CD98 (also known as "4F2") is a heterodimeric protein of approximately 120 kDa that is expressed on the cell membrane and is known to function as an amino acid transporter. CD98hc, also known as "4F2hc" or "SLC3A2," is an approximately 80 kDa type II transmembrane protein that constitutes CD98. Human CD98hc exists in several isoforms (e.g., b, c, e, f), but the extracellular domain amino acid sequence is common to all isoforms. The amino acid sequence of a representative human CD98hc, isoform f, is shown in the Sequence Listing as SEQ ID NO: 17. Human CD98hc has an N-glycan structure at positions 264, 280, 323, and 405 of the amino acid sequence shown in SEQ ID NO: 17. While the amino acid sequence of isoform f of human CD98hc is referred to herein as a representative example, those skilled in the art will understand that corresponding sequences / amino acid residues exist in other isoforms and function equivalently.

[0017] Myeloma cells, myeloma progenitor cells, and other tumor cells are thought to have altered N-glycosylation on CD98hc (e.g., impaired N-glycosylation). This is thought to be due to the constant endoplasmic reticulum stress in myeloma cells, myeloma progenitor cells, and other tumor cells, which inhibits N-glycosylation. As a result, amino acid sequences on CD98hc that are inaccessible to antibodies and other substances on other normal cells are thought to be exposed on the cell surface of myeloma cells, myeloma progenitor cells, and other tumor cells in an accessible state to antibodies and other substances. Such regions of CD98hc specific to myeloma cells, myeloma progenitor cells, and other tumor cells are sometimes referred to herein as "regions containing the N-glycosylation site of human CD98hc and exposed by inhibited N-glycosylation" or "regions affected by the presence of N-glycans." The presence of epitopes within these regions enables antibodies and other substances to specifically recognize myeloma cells, myeloma progenitor cells, and other tumor cells. The R8H283 antibody is an antibody that recognizes an epitope that contains the N-glycosylation site of human CD98hc and is exposed by inhibition of N-glycosylation. For details, see Hasegawa et al., Sci. Transl. Med. 14, eaax7706 (2022).

[0018] "N-glycan attachment site of human CD98hc" refers to an amino acid residue to which an N-glycan is attached among the amino acid residues constituting human CD98hc. In one embodiment, the N-glycan attachment site is the asparagine residue at position 264, 280, 323, or 405 in the amino acid sequence shown in SEQ ID NO: 17. In a preferred embodiment, the N-glycan attachment site is the asparagine residue at position 405 in the amino acid sequence shown in SEQ ID NO: 17.

[0019] In one embodiment, the epitope of the scFv is preferably located within a region on human CD98hc corresponding to positions 203 to 427 of SEQ ID NO: 17. In a preferred embodiment, the epitope of the scFv is preferably located within a region on human CD98hc corresponding to positions 365 to 427 of SEQ ID NO: 17. In another preferred embodiment, the epitope of the scFv is preferably located within a region on human CD98hc corresponding to positions 365 to 409 of SEQ ID NO: 17. Here, the term "corresponding" is used because, in human CD98hc other than isoform f, the location of the above regions, counting the N-terminal amino acid residue as the first amino acid, is not necessarily positions 203 to 427, 365 to 427, or 365 to 409. The amino acid sequence from positions 365 to 427 of SEQ ID NO: 17 is as follows: FSYGDEIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQR (SEQ ID NO: 18). The amino acid sequence from positions 365 to 409 of SEQ ID NO: 17 is as follows: FSYGDEIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVK (SEQ ID NO: 19). Thus, in a preferred embodiment, the epitope of the scFv is preferably located within the region consisting of the amino acid sequence of human CD98hc shown in SEQ ID NO: 18, and more preferably within the region consisting of the amino acid sequence of SEQ ID NO: 19. The amino acid sequence from positions 203 to 427 of SEQ ID NO: 17 is shown in the Sequence Listing as SEQ ID NO: 20.

[0020] In one embodiment, the epitope of the scFv is preferably present in the region of human CD98hc at positions 365 to 376 and the region of positions 395 to 409. In one embodiment, the epitope of the scFv preferably comprises the amino acid residues at positions 374, 375, 395, 396, 397, 400, and / or 401 of human CD98hc. In this specification, the positions of amino acid residues in human CD98hc refer to the positions in the amino acid sequence of SEQ ID NO: 17, unless otherwise specified.

[0021] Whether an scFv has an epitope in the region of the amino acid sequence shown in SEQ ID NO: 19 can be confirmed by the following procedure. Specifically, a polynucleotide encoding a human / mouse chimeric CD98hc is constructed in which the portion other than the amino acid sequence shown in SEQ ID NO: 19 is replaced with the amino acid sequence of mouse-derived CD98hc, and the polynucleotide is forcedly expressed in appropriate cells. Separately, cells of the same species are prepared by forcibly expressing mouse CD98hc. The binding of the scFv to these cells is then measured. If the scFv binds to cells expressing the chimeric CD98hc but not to mouse CD98hc, it can be determined that the scFv has an epitope in the region. Note that the amino acid sequence of SEQ ID NO: 19 is used as an example here, but similar measurements can be made for other regions or sites.

[0022] In one embodiment, the scFv preferably has a complementarity-determining region identical to at least one selected from the group consisting of the three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and the three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3) of the R8H283 antibody. The amino acid sequences of the heavy and light chain complementarity-determining regions (CDRs 1 to 3) of the R8H283 antibody are shown in Table 1 below (Kabat).

[0023]

[0024] More specifically, it is preferred that the scFv has a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and / or a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 5, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 6, and / or a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 7.

[0025] In one embodiment, CDR-H1 may have one or two amino acid substitutions, preferably one, in the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, CDR-H2 may have one to three amino acid substitutions, preferably one or two, and more preferably one, in the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, CDR-H3 may have one or two amino acid substitutions, preferably one, in the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, CDR-L1 may have one to three amino acid substitutions, preferably one or two, and more preferably one, in the amino acid sequence set forth in SEQ ID NO: 5. In one embodiment, CDR-L2 may have one or two amino acid substitutions, preferably one, in the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, CDR-L3 may have one or two amino acid substitutions, preferably one, in the amino acid sequence set forth in SEQ ID NO: 7. The type of amino acid substitution is not particularly limited, but in one embodiment, the amino acid substitution is preferably a conservative amino acid substitution.

[0026] In one embodiment, the scFv has one or more CDRs selected from the group consisting of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the R8H283 antibody, preferably two or more, preferably three or more, preferably four or more, preferably five or more, and preferably all six. In one embodiment, the scFv preferably has at least CDR-H3 and / or CDR-L3 of the R8H283 antibody.

[0027] In one embodiment, the scFv preferably comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, in order from the N-terminus of the heavy chain variable region. In one embodiment, the scFv preferably comprises light chain CDR1, light chain CDR2, and light chain CDR3, in order from the N-terminus of the light chain variable region. In one embodiment, the scFv preferably comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, in order from the N-terminus of the heavy chain variable region, and light chain CDR1, light chain CDR2, and light chain CDR3, in order from the N-terminus of the light chain variable region.

[0028] In one embodiment, the heavy chain variable region of the scFv may have the same amino acid sequence (SEQ ID NO: 4) as the heavy chain variable region of the R8H283 antibody. In another embodiment, the light chain variable region of the scFv may have the same amino acid sequence (SEQ ID NO: 8) as the light chain variable region of the R8H283 antibody. In one embodiment, the scFv may have the same amino acid sequence (SEQ ID NO: 4) as the heavy chain variable region of the R8H283 antibody and the same amino acid sequence (SEQ ID NO: 8) as the light chain variable region of the R8H283 antibody. Here, "same" refers not only to a complete amino acid sequence match, but also to a sequence that differs by one or several amino acid residues (e.g., 15 or fewer, preferably 10 or fewer, preferably 5 or fewer, preferably 3 or fewer, and preferably 2) but still retains the ability to bind to substantially the same epitope as the R8H283 antibody. In one embodiment, the heavy chain variable region of the scFv may have the amino acid sequence of SEQ ID NO: 4, with the isoleucine at position 95 substituted with arginine (I95R). In one embodiment, the light chain variable region of the scFv may have the amino acid sequence of SEQ ID NO: 8, except that proline at position 12 is substituted with arginine (P12R). In one embodiment, the light chain variable region of the scFv may have the amino acid sequence of SEQ ID NO: 8, except that isoleucine at position 90 is substituted with arginine (I90R). In one embodiment, the light chain variable region of the scFv may have the amino acid sequence of SEQ ID NO: 8, except that proline at position 12 is substituted with glutamine (P12Q). These amino acid substitutions can be combined in any manner. For example, an scFv may have an I95R substitution in the heavy chain variable region and P12R and I90R substitutions in the light chain variable region, or an I95R substitution in the heavy chain variable region and P12Q and I90R substitutions in the light chain variable region.

[0029] In one embodiment, the amino acid sequence of the heavy chain variable region of the scFv has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the light chain variable region of the scFv has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence set forth in SEQ ID NO: 8. Amino acid identity is measured by the method described below.

[0030] When an scFv has one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 8, any mutations may be present in the amino acid sequence, as long as the epitope is located within a region affected by the presence of N-glycans on human CD98hc. In one embodiment, it is preferred that no mutations are made in the complementarity-determining regions of the scFv, as mutations in complementarity-determining regions are generally thought to affect the epitope. Therefore, in one embodiment, mutations in the variable region are preferably present in the heavy chain FR region and / or the light chain FR region.

[0031] In one embodiment, the amino acid sequence of the heavy chain FR region of the heavy chain variable region of the scFv has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the heavy chain FR region of the amino acid sequence set forth in SEQ ID NO: 4. For example, the amino acid sequence of the heavy chain FR1 region of the heavy chain variable region of the scFv has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the heavy chain FR1 region of the amino acid sequence set forth in SEQ ID NO: 4. For example, the amino acid sequence of the heavy chain FR2 region of the heavy chain variable region of the scFv has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the heavy chain FR2 region of the amino acid sequence set forth in SEQ ID NO: 4. For example, the amino acid sequence of the heavy chain FR3 region of the heavy chain variable region of the scFv has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the heavy chain FR3 region of the amino acid sequence set forth in SEQ ID NO: 4. For example, the amino acid sequence of the heavy chain FR4 region of the heavy chain variable region of the scFv has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the heavy chain FR4 region of the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, the amino acid sequence of the light chain FR region of the light chain variable region of the scFv has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the light chain FR region of the amino acid sequence set forth in SEQ ID NO: 8. For example, the amino acid sequence of the light chain FR1 region of the scFv light chain variable region has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the light chain FR1 region of the amino acid sequence shown in SEQ ID NO: 8. For example, the amino acid sequence of the light chain FR2 region of the scFv light chain variable region has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the light chain FR2 region of the amino acid sequence shown in SEQ ID NO: 8.For example, the amino acid sequence of the light chain FR3 region of the scFv light chain variable region has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the light chain FR3 region of the amino acid sequence shown in SEQ ID NO: 8. For example, the amino acid sequence of the light chain FR4 region of the scFv light chain variable region has 80% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the amino acid sequence of the light chain FR4 region of the amino acid sequence shown in SEQ ID NO: 8.

[0032] There is no particular limitation on the number of mutations (in terms of amino acid residues) that can be introduced into one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 8. In one embodiment, the identity between the amino acid sequence before and after mutation introduction is 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, or preferably 99% or more.

[0033] Amino acid identity can be calculated using commercially available or internet-available analytical tools (e.g., software such as FASTA, BLAST, PSI-BLAST, and SSEARCH). For example, the main initial conditions commonly used for BLAST searches are as follows: In Advanced BLAST 2.1, the program is blastp, the Expect value is 10, all filters are turned off, the Matrix is ​​BLOSUM62, the Gap existence cost, Per residue gap cost, and Lambda ratio are set to 11, 1, and 0.85 (default values), respectively, and other parameters are also set to default values. The percent identity is rounded off.

[0034] The above-mentioned mutations in the amino acid sequence include, for example, substitutions, deletions, insertions, etc. Specific mutations are not particularly limited as long as they can be achieved by employing conventional methods. The amino acid substitution is preferably a conservative substitution. A conservative substitution is a substitution of an amino acid residue with an amino acid residue having a side chain similar to that of the original amino acid residue.

[0035] Specific conservative amino acid substitutions include substitutions of amino acid residues with basic side chains, such as lysine, arginine, and histidine; substitutions of amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; substitutions of amino acid residues with uncharged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions of amino acid residues with nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions of amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and substitutions of amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0036] In one embodiment, the scFv is preferably a humanized scFv. Humanized scFvs can be obtained, for example, by replacing the amino acid sequence of a region other than the complementarity-determining regions of a mouse antibody with the amino acid sequence of a human antibody. The regions other than the complementarity-determining regions present in the variable region are called FR regions. The heavy chain variable region and the light chain variable region each contain the FR1 region between the N-terminus of the variable region and CDR1, the FR2 region between CDR1 and CDR2, the FR3 region between CDR2 and CDR3, and the FR4 region between CDR3 and the C-terminus. Humanized scFvs can be obtained by humanizing one or more or all of these regions. The amino acid sequence of a humanized scFv is usually about 90% or more, but is not limited to this.

[0037] In one embodiment, the scFv is preferably a human scFv. A human scFv is an scFv whose entire structure is derived from a human. A human scFv is sometimes called a "fully humanized scFv."

[0038] The length of the hinge / spacer sequence and the types of amino acid residues that make it up are not limited as long as they do not inhibit the function of the chimeric antigen receptor. For example, the hinge / spacer sequence can be designed to have about 10 to 250, about 10 to 200, about 10 to 100, about 10 to 50, or about 10 to 25 amino acid residues. For example, the hinge / spacer sequence is not particularly limited, and a portion of a factor used in a transmembrane domain (e.g., CD28, CD8α, IgG1, IgG4, IgD, CD34, 4-1BB, etc.) as described below, or a constant region containing the hinge region of an antibody (e.g., IgG4 hinge-CH2-CH3) can be used.

[0039] The type of transmembrane domain is not limited as long as it does not inhibit the function of the chimeric antigen receptor. For example, CD28, CD3ε, CD8α, CD3, CD4, 4-1BB, etc. can be used. These transmembrane domains may be appropriately mutated as long as they do not inhibit the function of the chimeric antigen receptor. In one embodiment, the transmembrane domain is preferably CD28 or CD8α.

[0040] The intracellular signaling domain may be, for example, an intracellular signaling domain derived from CD3, also known as the TCR ζ chain. Appropriate mutations may be introduced as long as they do not inhibit the function of CD3 or the chimeric antigen receptor. When introducing a mutation into CD3, it is preferable to introduce the mutation so that an ITAM (immunoreceptor tyrosine-based activation motif) is included. In one embodiment, the intracellular signaling domain is preferably CD28-CD3ζ, 4-1BB-CD3ζ, or CD28-4-1BB-CD3ζ.

[0041] Any combination of hinge / spacer sequences, transmembrane domains, and intracellular signal domains may be used. In one embodiment, it is preferable to use any of the following combinations:

[0042] In one embodiment, the chimeric antigen receptor may contain regions other than those described above, such as a costimulatory factor domain, a leader sequence (signal peptide) that promotes transport of the chimeric antigen receptor onto the cell membrane, a spacer / linker sequence (e.g., between the transmembrane domain and the intracellular signal domain), etc.

[0043] The costimulatory factor is not particularly limited as long as it is a costimulatory factor possessed by T cells, etc. For example, one or more factors selected from the group consisting of OX40, 4-1BB, CD28, etc. may be appropriately selected and used. These costimulatory factors may be appropriately mutated as long as they do not inhibit the function of the chimeric antigen receptor.

[0044] Techniques for producing chimeric antigen receptors are known, including, for example, the methods described in Non-Patent Documents 1 to 4. Chimeric antigen receptors can be produced according to or in accordance with known methods.

[0045] 2. Polynucleotides In one embodiment, a polynucleotide encoding the chimeric antigen receptor is provided. A "polynucleotide" is a polymeric compound in which nucleotides (e.g., ribonucleotides or deoxyribonucleotides) are polymerized in a linear chain. A polynucleotide may be single-stranded or double-stranded.

[0046] The base sequence of the polynucleotide is not particularly limited and may be any as long as it encodes the chimeric antigen receptor.

[0047] The base sequences of polynucleotides encoding the amino acid sequences of the heavy and light chain complementarity determining regions and variable regions of the R8H283 antibody are shown in SEQ ID NOs: 9 to 16. The correspondence between these sequences is shown in Table 2 below.

[0048]

[0049] The polynucleotide encoding the scFv has one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 9 to 16 (when there are two or more sequences, the combination is arbitrary). In a preferred embodiment, the polynucleotide encoding the scFv has one or more nucleotide sequences (preferably two or more, more preferably all) selected from the group consisting of SEQ ID NOs: 9 to 11, and / or one or more nucleotide sequences (preferably two or more, more preferably all) selected from the group consisting of SEQ ID NOs: 13 to 15. In a preferred embodiment, the polynucleotide encoding the scFv has the nucleotide sequences of SEQ ID NOs: 9 to 11 and the nucleotide sequences of SEQ ID NOs: 13 to 15. In a preferred embodiment, the polynucleotide encoding the scFv has the nucleotide sequence of SEQ ID NO: 12 and / or SEQ ID NO: 16.

[0050] The nucleotide sequence of a polynucleotide encoding an scFv does not need to be completely identical to SEQ ID NOs: 9 to 16, as long as it encodes the scFv. For example, the nucleotide sequence of a polynucleotide encoding an scFv can have 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, or preferably 99% or more identity to SEQ ID NOs: 9 to 16. Here, "identity" can be calculated using analytical tools that are commercially available or available via telecommunications lines (Internet). For example, the nucleotide sequence homology (%) can be calculated by searching using Advanced BLAST 2.1, using the blastn program with various parameters set to default values. The codon usage of the polynucleotide may be optimized depending on the type of vector or cell in which it is expressed.

[0051] The state of the polynucleotide is not particularly limited, and may be, for example, isolated or incorporated into a vector. The type and use of the vector are not particularly limited. For example, the vector may be a plasmid vector or a viral vector (e.g., adenovirus, retrovirus, lentivirus, etc.). The vector may also be, for example, a cloning vector or an expression vector. Examples of expression vectors include vectors for prokaryotic cells such as Escherichia coli or actinomycetes, and vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells.

[0052] 3. Cells In one embodiment, a cell is provided that contains a polynucleotide encoding a chimeric antigen receptor. Any type of cell may be used, but cells with cytotoxic activity are preferred. Examples of cells with cytotoxic activity include T cells, NK cells, and NK-T cells, with killer T cells (also known as cytotoxic T cells (CTLs)) being preferred.

[0053] The cell may have the polynucleotide in the form of a vector, or may have the polynucleotide integrated into the genomic DNA of the cell. The cell may or may not express the polynucleotide encoding the chimeric antigen receptor. When the polynucleotide encoding the chimeric antigen receptor is expressed in the cell, it is preferred that the scFv region constituting the chimeric antigen receptor is exposed to the outside of the cell, and the transmembrane domain and intracellular signal domain are present in the cell membrane or inside the cell.

[0054] When the scFv region recognizes its epitope, it activates a signal that induces cytotoxic activity within the cell via the transmembrane domain, and in conjunction with this, the cell attacks or exerts cytotoxic activity against other cells or tissues that express the same epitope.

[0055] Cells containing a polynucleotide encoding a chimeric antigen receptor (particularly cells having cytotoxic activity) are useful as an active ingredient in pharmaceutical compositions.

[0056] Cells (e.g., CAR-T cells) containing a polynucleotide encoding a chimeric antigen receptor can be produced with reference to the methods described in Non-Patent Documents 1 to 4. For example, a retroviral vector, a lentiviral vector, or the like may be used to introduce a polynucleotide encoding a chimeric antigen receptor into cells.

[0057] Cells containing polynucleotides encoding such chimeric antigen receptors specifically recognize myeloma cells, myeloma progenitor cells, and other tumor cells by recognizing an epitope containing the N-glycosylation site of human CD98hc and exposed by inhibiting N-glycosylation, making them useful for the treatment and / or prevention of tumors and other conditions. The type of tumor is not particularly limited, and includes solid cancers and hematologic cancers. Examples of solid cancers include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumors, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma. Examples of hematologic cancers include leukemia, diseases associated with neoplastic proliferation of plasma cells (e.g., multiple myeloma), and malignant lymphoma. In one embodiment, the preferred leukemia is lymphocytic leukemia.

[0058] 4. Pharmaceutical Composition and Treatment and / or Prevention Method In one embodiment, there are provided a pharmaceutical composition comprising cells comprising a polynucleotide encoding the chimeric antigen receptor, and a method for treating and / or preventing a disease using cells comprising a polynucleotide encoding the chimeric antigen receptor.

[0059] The content of the cells in the pharmaceutical composition can be appropriately determined taking into consideration the type of disease to be treated, the desired therapeutic effect, the administration method, the treatment period, the age and body weight of the patient, etc. The content of the cells in the pharmaceutical composition can be, for example, 10 1 cells / mL ~ 10 7 It can be on the order of cells / mL.

[0060] The administration form of the pharmaceutical composition is not particularly limited as long as the desired effect is obtained, and it can be administered to mammals, including humans, by either oral or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration). Because the active ingredient is cells, the preferred administration form is parenteral administration, and more preferably intravenous injection. Dosage forms for oral and parenteral administration and their production methods are well known to those skilled in the art, and can be produced according to standard methods by mixing cells containing a polynucleotide encoding the chimeric antigen receptor with a pharmaceutically acceptable carrier or the like.

[0061] Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, etc.), topical preparations (e.g., ointments, poultices, lotions, etc.), suppositories, inhalants, ophthalmic preparations, eye ointments, nasal drops, ear drops, liposomes, etc. For example, injectable preparations are prepared by dissolving cells in distilled water for injection, and solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, etc. can be added as needed. The pharmaceutical composition can also be a lyophilized preparation for immediate preparation.

[0062] The pharmaceutical composition may further contain other drugs effective for treating or preventing diseases. In addition, the pharmaceutical composition may also contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids, as needed.

[0063] Carriers used in formulating pharmaceutical compositions include excipients, binders, disintegrants, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, and the like that are commonly used in the art.

[0064] 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, for example, tumors. The type of tumor is not particularly limited and includes solid cancers and blood cancers. Examples of solid cancers include lung cancer, colon 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. Examples of blood cancers include leukemia, diseases accompanied by neoplastic proliferation of plasma cells (e.g., multiple myeloma), and malignant lymphoma. In one embodiment, a preferred leukemia is lymphocytic leukemia. In one embodiment, a preferred disease is a disease accompanied by neoplastic proliferation of plasma cells. A "disease accompanied by neoplastic proliferation of plasma cells" is a disease characterized by neoplastic proliferation of abnormal plasma cells and an increase in abnormal proteins secreted by them. Examples of such diseases include multiple myeloma, plasma cell leukemia, plasmacytoma, H-chain disease, systemic AL-type amyloidosis, etc. In one embodiment, the preferred target disease is multiple myeloma.

[0065] The subject (subject) to which the pharmaceutical composition is administered is, for example, an animal that is afflicted with or may be afflicted with the above-mentioned disease. The term "possibly afflicted" can be determined, for example, by the diagnostic method described below. The animal is, for example, a mammal, preferably a human.

[0066] The dosage of the pharmaceutical composition can be determined by a clinician based on various factors, such as the route of administration, the type of disease, the degree of symptoms, the age, sex, and weight of the patient, the severity of the disease, pharmacological findings such as pharmacokinetics and toxicological characteristics, whether a drug delivery system is used, and whether the pharmaceutical composition is administered as part of a combination of other drugs. ４ cells / kg (body weight) ~10 ９ The dosage can be approximately 1000 cells / kg (body weight). The administration schedule of the pharmaceutical composition can also be determined taking into account factors similar to those for the dosage. For example, the above daily dosage can be administered once a day to once a month.

[0067] As described above, cells containing a polynucleotide encoding the chimeric antigen receptor can be used to produce the pharmaceutical composition.

[0068] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0069] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0070] The subject matter encompassed by the present disclosure will be described in more detail below, but the subject matter is not limited to the following examples. In the following, unless otherwise specified, experiments were conducted under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "volume percentage concentration (v / v%)."

[0071] Example 1: Effect of VL and VH order on CD98 CAR-T cell activity The inventors previously identified the R8H283 antibody, which specifically binds to myeloma cells in the bone marrow of human multiple myeloma patients (Patent Document 1). R8H283 is an antibody that recognizes human CD98hc, and the amino acid sequences of the complementarity-determining regions (CDR1-3) of its heavy and light chains are as follows (note that the complementarity-determining regions were determined by Kabat):

[0072]

[0073] The use of scFv derived from R8H283 enables CD98 CAR-T cells to capture target cells, and proper capture is necessary for the drug to exert its therapeutic effect on target cells. We evaluated the effect of the VH (heavy chain variable region) and VL (light chain variable region) order used in creating the scFv on the efficacy of CD98 CAR-T cells. Commercially available CAR-T cell therapies include CD19 CAR-T cells and BCMA CAR-T cells, and the scFvs used in these CAR-T cells are all configured in the VL-VH order (e.g., KYMRIAH, YESCARTA, Breyanzi, Abecma (all product names)).

[0074] The CAR-T cells generated using retroviral vectors to evaluate the order of VH and VL are shown in Table 4 below and Figure 1.

[0075]

[0076] The method of Hosen et al. (Non-Patent Document 4) was used for the preparation of retroviral vectors and the generation of CAR-T cells using retroviral vectors. Specifically, 293T (ATCC: CRL-3216) cells were cultured in a 10-cm dish at 37°C and 5% CO2 using D-MEM containing 10% Fetal Bovine Serum (FBS) (NICHIREI, Lot 19C00A or Lot S173012) and 1% penicillin-streptomycin. After detaching the cells, the cell number was counted using trypan blue staining, and 5 × 10 cells were collected. 6Cells were seeded at 1000 x g / dish. For retroviral vector production, one day after cell seeding, Gag-pol vector, VSV-G vector, CAR vector, Opti-MEM, and Lipofectamin 2000 (Thermo Fisher Scientific, 11668019) were mixed and added dropwise to the cells for transfection. Viruses were harvested 48 hours after transfection and cryopreserved at -80°C. Cryopreserved commercially available human PBMCs (PRECISION for medicine, 39000-10M, Lot#3010116166) were rapidly thawed in a 37°C incubator and suspended in GT-T551 (TAKARA, WK551S) containing 5% human albumin serum and 1% penicillin-streptomycin mixture. The supernatant was removed by centrifugation (400 x g, 5 minutes, 25°C). Commercially available human PBMCs were stimulated using RetroNectin (TAKARA, T100B) in a 48-well plate coated with anti-CD3 antibody (Clone: ​​OKT3) and anti-CD28 antibody. One day after PBMC stimulation, T cells were expanded in a medium containing IL-2 (100 U / mL). Two days after PBMC stimulation, CAR sequences were introduced using a retroviral vector using RetroNectin, following the protocol provided with the product. Subsequently, cells were cultured every 2–3 days until the cell density reached 5×10 5 The cells were replated in fresh medium at 100 cells / mL and expanded. On day 9 after PBMC stimulation, the cells were collected and cryopreserved at -80°C.

[0077] CAR-T 1B, which uses scFvs constructed in the VL-VH order, showed limited CAR-T cell proliferation compared to CAR-T 1A, which uses scFvs constructed in the VH-VL order (Figure 2). The inventors attempted to improve performance by constructing CAR constructs in the VL-VH order with two different leader sequences, but the proliferation was comparable to that of CAR-T 1B. One of the leader sequences has the amino acid sequence of SEQ ID NO: 23. Note that the amino acid sequence of SEQ ID NO: 23 does not contain an initiating methionine residue.

[0078] Example 2: In vitro activity evaluation Next, the activity of the CAR-T cells prepared in Example 1 was evaluated as follows.

[0079] Cytotoxicity was measured using the following procedure. 5 NALM6-Luc cells (NALM6, clone G5 (ATCC, CRL-3273) transfected to stably express luciferase) and CAR-T cells were incubated in a 96-well plate for 4 hours. VivoGlo-Luciferin (Promega) was then added to the cell culture medium to a final concentration of 140 μg / mL, and the suspension was transferred to a 1 / 2 area OptiPlate-96 (Revvity). Luminescence was measured using an INFINITE M1000 PRO (TECAN). Cytotoxicity was then calculated using the following formula: specific lysis (%) = {1 - Luminescence (CAR-T:NALM6-Luc = 1) / Luminescence (CAR-T:NALM6-Luc = 0)} × 100.

[0080] The interferon (IFN)-γ production ability was evaluated as follows: NALM6-Luc cells and CAR-T cells were cultured in a 96-well plate at 1 × 10 5 After 4 hours, the mixture was centrifuged at 1500 rpm for 3 minutes at 4°C, and 100 μL of the supernatant was collected. The collected supernatant was used to measure cytokine levels according to the protocol of the Human IFN-gamma Quantikine ELISA Kit (R&D SYSTEMS).

[0081] As a result, CAR-T 1A and 1B had similar cytotoxic activity (Figure 3), but CAR-T 1B had superior IFN-γ production ability compared to CAR-T 1A (Figure 4).

[0082] Example 3: In vivo evaluation Although the proliferation ability was poor, the CAR-T 1B showed some activity in vitro. Therefore, in vivo evaluation of the CAR-T 1B was performed using a NALM6-Luc xenograft model as follows: NOD / Shi-scid, IL-2RγKO Jic (NOG) mice (Invivo Science Co., Ltd.) produced by CLEA Japan, Inc. were delivered at 6 weeks of age. At 7 weeks of age, 20 mg / kg of Busulfex (Otsuka Pharmaceutical) was administered intraperitoneally the day before transplantation. The following day, 1 x 10 NALM6-Luc cells were injected into the tail vein as tumor cells. 5 Tumor-bearing mice were generated by transplanting 0.1 mL of CAR-T cells per body. Three days after transplantation, luminescence values ​​were measured using an IVIS Lumina X5 (Revvity). Using these values ​​as an index, mice were assigned to groups using stratified random selection with SAS software R9.4 (SAS Institute Japan). CAR-T cells were administered once, on the same day as group assignment, via the tail vein at 0.1 mL / body. Drug efficacy was evaluated using tumor luminescence values ​​measured by the IVIS Lumina X5. Tumor luminescence values ​​were measured 20 minutes after intraperitoneal administration of 15 mg / mL VivoGlo-luciferin at a dose of 10 mL / kg based on pre-measured body weight. Total flux [p / s] of tumor luminescence values ​​refers to photons / sec.

[0083] The dose of CAR-T cells was 1 × 10 6 The cells were prepared as CAR-T cells and administered once via the tail vein at 0.1 mL / body on the same day as group allocation. As a result, the activity of CAR-T 1B, which used scFvs engineered in the VL-VH order, was limited. On the other hand, CAR-T 1A, which was engineered in the VH-VL order, demonstrated clear antitumor activity (Figure 5). This confirms that the VL-VH scFv alone, as with conventional CAR-T cells, is not sufficient for CD98 CAR-T cells to exert their therapeutic effects.

[0084] Example 4: Effect of Linker Length on CD98 CAR-T Cell Activity 1 Next, we evaluated the effect of the linker length connecting the VL and VH of scFv on the efficacy of CD98 CAR-T cells. Conventional linkers include the 218 linker (18 amino acids) and the G4S linker (15 amino acids). However, based on previous results, it was determined that the efficacy of CD98 CAR-T cells could not be predicted using conventional methods. Therefore, we evaluated the efficacy of CD98 CAR-T cells using the 218 linker or G4S linkers (10, 15, 20, and 25 amino acids) with different linker lengths. CAR-T cells generated using lentiviral vectors to evaluate the effect of linker length on CD98 CAR-T cells are shown in Table 5.

[0085]

[0086] Lentiviral vectors were produced according to the instructions provided with Takara Bio's Lentiviral High Titer Packaging Mix (Takara, 6194) or LVpro Packaging Mix with pLVpro (Takara, 6195). Specifically, 293T (ATCC) cells were cultured in a 10-cm dish at 37°C and 5% CO2 using D-MEM containing 10% FBS and 1% penicillin-streptomycin. After detaching the cells, the cell number was counted using trypan blue staining and then 5 × 10 cells were cultured. 6Cells were seeded at 1000 cells / dish. For lentiviral vector production, one day after cell seeding, Lentiviral High Titer Packaging Mix or LVpro Packaging Mix with pLVpro, CAR vector, serum-free D-MEM, and TransIT-293 Transfection Reagent (Takara, V2700) were mixed and added dropwise to the cells for transfection. When using LVpro Packaging Mix with pLVpro, the medium was replaced with D-MEM supplemented with 10% FBS and 1% penicillin-streptomycin 24 hours after transfection. Virus was collected 48 hours after transfection, concentrated, and frozen at -80°C. The protocol for generating CAR-T cells using lentiviral vectors was then performed according to Miltenyi Biotech's Engineering of CAR-T cells for research use. Specifically, cryopreserved, commercially available human PBMCs were rapidly thawed in a 37°C incubator, suspended in TexMACS (Miltenyi Biotec) containing 5% Human Albumin Serum and 1% penicillin-streptomycin mixture, and the supernatant was removed by centrifugation (400 × g, 5 minutes, 25°C). Commercially available human PBMCs were stimulated with TransAct (Miltenyi Biotec) and further expanded in medium containing 12.5 ng / mL IL-7 (Miltenyi Biotec) and 12.5 ng / mL IL-15 (Miltenyi Biotec) to induce T cell development. Two days after PBMC stimulation, a sequence carrying the CD98 CAR construct was introduced via a lentiviral vector. Subsequently, the cells were cultured every 2–3 days until the cell density reached 5×10 5 During the process of reseeding in fresh medium at 100 cells / mL, dasatinib (TCI Chemicals) was added to a final concentration of 1 μM from day 6 after PBMC stimulation. Cells were harvested on day 9 after PBMC stimulation and stored frozen at -80°C.

[0087] The continuous antigen stimulation test was carried out as follows: 0.8 × 10 6cells and 1.6 × 10 CAR-positive T cells 6 NALM6-Luc cells were suspended in a mixed medium (a 1:1 mixture of TexMACS containing 5% Human Albumin Serum and 1% penicillin-streptomycin mixed solution and RPMI1640 containing 10% FBS (NICHIREI, Lot 19C00A) and 1% penicillin-streptomycin) and co-cultured in a T25 flask. After 72-96 hours, the cells were harvested and adjusted to the above-mentioned number of CAR-positive cells. Then, 1.6 × 10 cells were cultured separately. 6 The cells were then co-cultured with NALM6-Luc cells. A portion of the cells was used for cytotoxicity testing. The point at which no further proliferation of CAR-T cells was detected was set as the end point, and these tests were continued.

[0088] The results of this study showed no significant differences in cell proliferation during CAR-T cell generation due to linker length (Figure 6). Compared to CAR-T 2A with a 218-amino acid linker, CAR-T 2B and 2C, with linker lengths of 10 and 15 amino acids, had reduced cytotoxic activity and IFN-γ production (Figures 7 and 8). Similar trends were observed in the changes in CAR-T cell number (Figure 9) and cytotoxic activity (Figure 10) after repeated antigen stimulation. In particular, CAR-T 2B with a 10-amino acid linker length showed no proliferation ability and no cytotoxic activity on Day 6.

[0089] Next, the in vivo efficacy of these CAR-T cell preparations was evaluated (Figure 11A). The evaluation method was the same as that described in Example 3, and 1 × 10 6CAR-T cells were administered once via the tail vein at 0.1 mL / body on the same day as group allocation. Figure 11B shows the time course of CAR-T cell efficacy as a function of linker length. CAR-T 2B activity was weak, with limited activity at 10 amino acids. CAR-T cells with 15 to 25 amino acids subsequently exhibited strong growth inhibition, with clear tumor regression observed by Day 14. While the 15 amino acid linker exhibited strong regression activity, tumor growth continued to grow slightly by Day 7, indicating that increasing the linker length enhanced antitumor activity (Figure 11B).

[0090] These results suggest that using a linker of 15 amino acids or more is important for CD98 CAR-T cells to exert their therapeutic effects.

[0091] Example 5: Effect of linker length on CD98 CAR-T cell activity 2 Furthermore, the effect of the linker length connecting the VL and VH of scFv on the efficacy of CD98 CAR-T cells was evaluated. CAR-T cells prepared by the method described in Example 4 to evaluate the effect of linker length on CD98 CAR-T cells are shown in Table 6.

[0092]

[0093] As a result of this study, CAR-T 3B-3G, which had linker lengths of 20, 25, 30, 35, 40, and 50 amino acids, had reduced cytotoxic activity and IFN-γ production compared to CAR-T 2A with a 218 linker (Figures 12 and 13).

[0094] The in vivo efficacy of CAR-T cells with different linker lengths was evaluated. The evaluation method was the same as that described in Example 3, and 3 × 10 5 CAR-T cells were administered once via the tail vein at 0.1 mL / body on the same day as group allocation.

[0095] The efficacy of CAR-T cells with different linker lengths is shown in Figures 14 and 15. CAR-T 3G showed gradual growth inhibition up to Day 14, after which tumor regression was evident, and linker lengths of up to 50 amino acids had antitumor activity and a regression effect (Figure 15).

[0096] Example 6: Effect of CAR aggregation on CD98 CAR-T cell activity Considering the possibility that CAR aggregation may affect the efficacy of CAR-T cells, we used BioLuminate (Schrodinger) to predict Fv (fragment variable) structures with low aggregation potential, and evaluated the efficacy of CAR-T cells produced based on the resulting structures.

[0097] To evaluate the aggregation potential of CAR, the VH and VL sequences of CD98 CAR were entered into Bioluminate and the AggScore (aggregation score) of the predicted structure was calculated. In addition, residues that affect the AggScore were identified and the AggScore was calculated for structures with alternative substitutions.

[0098] As a result of this study, compared to the structure before the amino acid substitution (hereinafter referred to as CAR-T 2A), a decrease in AggScore was observed in the structure in which isoleucine at position 95 of the VH sequence was substituted with arginine, proline at position 12 of the VL sequence was substituted with arginine, and isoleucine at position 90 of the VL sequence was substituted with arginine (hereinafter referred to as CAR-T 4B), and in the structure in which isoleucine at position 95 of the VH sequence was substituted with arginine, proline at position 12 of the VL sequence was substituted with glutamine, and isoleucine at position 90 of the VL sequence was substituted with arginine (hereinafter referred to as CAR-T 4C) (Figure 16).

[0099] Table 7 shows CAR-T cells prepared by the method described in Example 4 to evaluate the effect of the degree of aggregation in the CAR structure on CD98 CAR-T cells.

[0100]

[0101] The results of this study showed that CAR-T 4B and 4C had similar cytotoxic activity compared to CAR-T 2A before amino acid substitution (Figure 17). CAR-T 4B and 4C had improved IFN-γ production (Figure 18).

[0102] The in vivo efficacy of CAR-T cells with the amino acid substitution in scFv was evaluated. The evaluation method was the same as that described in Example 3, and 3 × 10 5 CAR-T cells were administered once via the tail vein at 0.1 mL / body on the same day as group allocation.

[0103] The efficacy of CAR-T cells with scFv amino acid substitutions is shown in Figures 19 and 20. 5 In terms of the number of CAR-T cells administered, CAR-T 4C showed growth suppression up to Day 14 compared to CAR-T 2A, and thereafter showed significant tumor regression, demonstrating a regression effect from the time of group division (Figure 20).

[0104] Example 7: Comparison of the efficacy of CD98 CAR-T cells and CD19 CAR-T cells The efficacy of CD98 CAR-T cells was compared with that of CD19 CAR-T cells. KYMRIAH, YESCARTA, Breyanzi, and other CAR-T cell therapies on the market are all CAR-T cells that target CD19. To evaluate the efficacy of CD98 CAR-T cells, their efficacy was compared with that of CD19 CAR-T cells.

[0105] To compare the efficacy of CD98 CAR-T cells (CAR-T 2A) and CD19 CAR-T cells (CAR-T 5B), CAR-T cells were generated using lentiviral vectors, as shown in Table 8. CD19 CAR-T cells were generated using a lentiviral vector based on the KYMRIAH CAR sequence.

[0106]

[0107] CAR-T cells were evaluated using the same method as described in Example 2.

[0108] In vitro comparison of the efficacy of CAR-T 2A and CD19 CAR-T cells showed comparable cytotoxic activity (Figure 21). There was no significant difference in IFN-γ production (Figure 22).

[0109] The in vivo efficacy of CAR-T 2A and CD19 CAR-T cells was compared. The evaluation method was the same as that described in Example 3, and 1 × 10 6 CAR-T cells were administered once via the tail vein at 0.1 mL / body on the same day as group allocation.

[0110] Comparing the in vivo efficacy of CAR-T 2A and CD19 CAR-T cells, CAR-T 2A demonstrated strong growth inhibition and tumor regression immediately after administration. Control CD19 CAR-T cells inhibited tumor growth but did not result in significant regression, and the difference between CAR-T 2A and CD19 CAR-T cells was significant (Figure 23).

[0111] Example 8: In vivo evaluation against other cancer types. The antitumor effect against other cancer types was evaluated using luciferase gene-transduced cell lines and three cancer types. For hematological cancers, U937-luc cells (ATCC, CRL-1593.2), an acute myelogenous leukemia (AML) cell line, and RPMI8226-luc cells (ATCC, CCL-155), a multiple myeloma (MM) cell line, were used. For solid cancers, NCI-H460-luc cells (ATCC, HTB-177), a non-small cell lung cancer (NSCLC) cell line, were used. The in vivo efficacy of CAR-T 2A was evaluated as follows: NOD / Shi-scid, IL-2RγKO Jic (NOG) mice produced by CLEA Japan, Inc. were delivered at 6 weeks of age. At 7 weeks of age, U937-luc cells were administered at 1 × 10 5 cells / 0.1 mL / body, RPMI8226-luc cells were 5 × 10 5 cells / 0.1 mL / body, NCI-H460-luc cells were 2 × 10 5Tumor-bearing mice were created by transplanting 0.1 mL of CAR-T cells per body via the tail vein. However, for hematological cancer transplants, 20 mg / kg of Busulfex was administered intraperitoneally the day before transplantation. Following the schedule shown in Figure 24 below, luminescence values ​​were measured using an IVIS Lumina X5 after transplantation, and the luminescence values ​​were used as an index for group allocation using stratified random sampling with SAS software R9.4. The CAR-T cells administered were 2 x 10 for the AML and MM models. 6 cells CAR-T cells, NSCLC model 3 × 10 6 CAR-T cells were administered once via the tail vein at 0.1 mL / body volume on the same day as group allocation. Drug efficacy was evaluated using tumor luminescence values ​​measured with an IVIS Lumina X5. Tumor luminescence values ​​were measured after intraperitoneal administration of 15 mg / mL VivoGlo-luciferin at a dose of 10 mL / kg based on the body weight measured in advance. Total Flux [p / s] of tumor luminescence values ​​means photons / sec.

[0112] Regarding the antitumor effects against other cancer types, in blood cancer, the compound showed a complete growth suppression effect against AML cells (Figure 25), and a regression effect against MM cells (Figure 26).Furthermore, its effectiveness was confirmed against NSCLC cells, a solid cancer (Figure 27), and significant growth suppression effects were observed in both cases.

[0113] Example 9: Evaluation of the effect of domain combinations on CD98 CAR-T cell activity In addition to the scFv sequence and linker length that had been found to affect CD98 CAR-T cell activity in previous studies, CAR structures were created that combined signal domains, and the effect on CD98 CAR-T cell activity was evaluated.

[0114] Tables 9 to 13 show the CAR structures of CAR-T cells prepared by the method described in Example 4 to evaluate the effect of the combination of each CAR domain on CD98 CAR-T cells.

[0115] The results of this study showed that all constructs in the test shown in Table 9 exhibited cytotoxic activity compared to the control, with improved activity observed in constructs with CD28-derived hinge / spacer, CD28-derived transmembrane, and signal domains other than the CD28-CD3ζ combination (Figures 28 and 29). This trend was also observed when amino acid substitutions were made in the scFv in the combinations shown in Tables 10, 11, 12, and 13 (Figures 30, 31, 32, and 33). Among the constructs for which cytotoxic activity was compared simultaneously, no significant differences in cytotoxic activity were observed between constructs with equivalent structures below the hinge.

[0116] The evaluation of IFN-γ production showed that all constructs produced higher IFN-γ levels than the control. In particular, constructs with structures other than those containing a CD28-derived hinge / spacer, CD28-derived transmembrane, and CD28-CD3ζ signal domain tended to produce higher IFN-γ levels than those containing these combinations (Figures 34, 35, 36, 37, 38, and 39).

[0117] The in vivo efficacy of CAR-T cells in various combinations shown in Tables 9, 10, 11, 12, and 13 was evaluated. The evaluation method was the same as that described in Example 3, and 3 × 10 5 CAR-T cells were administered once via the tail vein at 0.1 mL / body on the same day as group allocation.

[0118] The in vivo efficacy of CAR-T cells in the various combinations shown in Tables 9, 10, 11, 12, and 13 is shown in Figures 40, 41, 42, 43, and 44. All combinations demonstrated growth inhibition compared to the vehicle, with CAR-T 6B, 6C, 6E, and CAR-T 6H in particular demonstrating a significant tumor growth inhibition effect, with all demonstrating a strong regression effect on Day 14 (Figure 40).

[0119] Example 10: Effect of dasatinib on CD98 CAR-T cell activity The effect of dasatinib, which was used when producing CAR-T cells, on the efficacy of CD98 CAR-T cells was evaluated.

[0120] To evaluate the effect of dasatinib on CD98 CAR-T cells, CAR-T cells with and without dasatinib were prepared using the method described in Example 4. The prepared CAR-Ts are shown in Table 14.

[0121]

[0122] The results of this study showed that the cytotoxic activity of CAR-T 11A without dasatinib was improved compared to that of CAR-T 2A with dasatinib (Figure 45). IFN-γ production was comparable between CAR-T 2A and CAR-T 11A (Figure 46).

[0123] The efficacy of dasatinib in vivo was compared between CAR-T 2A and CAR-T 11A. The evaluation method was the same as that described in Example 3, and 1 × 10 6 CAR-T cells were administered once via the tail vein at 0.1 mL / body on the same day as group allocation.

[0124] In the in vivo efficacy of CAR-T 2A and CAR-T 11A, CAR-T 2A with dasatinib showed clear growth inhibition up to Day 14 compared to CAR-T 11A without dasatinib, and then showed a significant antitumor effect by Day 28, down to a tumor burden below that at the start of CAR-T cell administration (Figure 47).

Claims

1. A chimeric antigen receptor comprising an scFv region having a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are linked via a linker, wherein the heavy chain variable region comprises a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and / or a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and / or the light chain variable region comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 5, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 6, and / or a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 7; and the number of amino acid residues constituting the linker is 15 to 50.

2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the heavy chain FR region of the heavy chain variable region comprises an amino acid sequence having 80% or more identity with the amino acid sequence of the heavy chain FR region of the amino acid sequence shown in SEQ ID NO:4, and / or the amino acid sequence of the light chain FR region of the light chain variable region comprises an amino acid sequence having 80% or more identity with the amino acid sequence of the light chain FR region of the amino acid sequence shown in SEQ ID NO:

8.

3. A chimeric antigen receptor, wherein the amino acid sequence of the heavy chain variable region comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:4, and / or the amino acid sequence of the light chain variable region comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:8; and the number of amino acid residues constituting the linker is 15 to 50.

4. The chimeric antigen receptor according to any one of claims 1 to 3, wherein the chimeric antigen receptor has a structure in which the following are arranged in order from the N-terminus: heavy chain variable region-linker-light chain variable region-hinge sequence-transmembrane domain-intracellular signal domain.

5. A polynucleotide encoding the chimeric antigen receptor of any one of claims 1 to 4.

6. A cell comprising the polynucleotide of claim 5.

7. The cell of claim 6, which is a chimeric antigen receptor T cell.

8. A pharmaceutical composition comprising the cells according to claim 6 or 7.

9. The pharmaceutical composition according to claim 8, for use in the treatment and / or prevention of cancer.

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