WO2022211046A1 - Ccr8を抗原として認識するキメラ抗原受容体 - Google Patents
Ccr8を抗原として認識するキメラ抗原受容体 Download PDFInfo
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Definitions
- the present invention relates to a chimeric antigen receptor that recognizes CCR8 as an antigen, cells expressing the chimeric antigen receptor, and pharmaceutical compositions containing the cells.
- Non-Patent Document 1 Potent negative regulatory mechanisms, including immunosuppression mediated by regulatory T cells (Treg cells) within the tumor microenvironment, are a major obstacle to tumor therapy.
- tumor-infiltrating CD4-positive Treg cells can potently inhibit anti-tumor immune responses and can be a major obstacle to effective cancer therapy.
- Tumor immunosuppression mediated by CD4-positive FoxP3-positive Treg cells has been well documented in animal tumor models, and systemic Treg cell depletion, including intratumoral, provides anti-tumor effects, although only as much as 50% It has been reported that removal of tumor-infiltrating Treg cells has no effect (Non-Patent Document 2).
- Non-Patent Documents 3-8) In humans, an increase in the ratio of CD4+CD25+ Treg cells in the total CD4+ T cell population (cell population containing Treg cells) has been detected in tumors of various cancer patients, including lung, breast and ovarian tumors. , it has been reported that there is a negative correlation between the abundance ratio and the patient survival rate (Non-Patent Documents 3-8).
- CCR8 also known as CY6, CKR-L1 or TER1
- CY6, CKR-L1 or TER1 is a G protein-coupled 7-transmembrane CC chemokine receptor protein expressed in the thymus, spleen, etc., and its gene is located at 3p21 in human chromosomes.
- Human CCR8 consists of 355 amino acids (Non-Patent Document 9).
- CCL1 is known as an endogenous ligand for CCR8 (Non-Patent Document 10).
- Human CCR8 cDNA is available from Genbank ACC No. Consisting of the base sequence shown in NM_005201.3, mouse CCR8 cDNA is Genbank ACC No. It consists of the base sequence shown in NM_007720.2.
- CCR8 is also specifically expressed in tumor-infiltrating Treg cells, and when breast cancer cells were transplanted into CCR8-deficient mice and wild-type mice, the growth and metastasis of breast cancer in CCR8-deficient mice were significantly higher than in wild-type mice. It has been shown that it was suppressed (Patent Document 1 and Non-Patent Document 11).
- Non-Patent Documents 12 to 16 also describe that CCR8 is involved in cancer pathology. Furthermore, it has been disclosed that administration of an anti-CCR8 antibody to a cancer model animal showed an antitumor effect (Patent Documents 2 to 12).
- T-cell receptors expressed in lymphocytes that function as effector cells such as T cells and NK cells, including extracellular domains, transmembrane domains, and intracellular domains that bind to tumor antigens
- CAR chimeric antigen receptor
- An object of the present invention is to provide a chimeric antigen receptor that recognizes CCR8 as an antigen.
- a further object is to provide chimeric antigen receptor-expressing cells that recognize CCR8 as an antigen and are useful for cancer therapy.
- the present inventors discovered a chimeric antigen receptor that recognizes CCR8 as an antigen. Furthermore, it was found that the chimeric antigen receptor-expressing cells of the present invention that recognize CCR8 as an antigen have cytotoxic activity against CCR8-expressing cells. That is, the cells expressing the chimeric antigen receptor that recognizes CCR8 as an antigen of the present invention are useful for cancer therapy.
- the present invention relates to the following.
- a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain that bind to CCR8, the intracellular domain comprising an intracellular signal domain of a signaling molecule expressed in lymphocytes that function as effector cells.
- the chimeric antigen receptor according to (1) which comprises an anti-CCR8-scFv region in the CCR8-binding extracellular region.
- the anti-CCR8-scFv region is 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 25 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 26, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 27; A heavy chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 28 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 29; 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 30; a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 31 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 33; A heavy chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 28 and CDR3 consisting of the amino acid sequence of S
- the anti-CCR8-scFv region is 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 25 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 26, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 27; A heavy chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 28 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 29; 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 30; a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 31 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 33; 4) a heavy chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 28 and CDR3 consisting of the amino acid
- the anti-CCR8-scFv region is a CDR1 consisting of the amino acid sequence of SEQ ID NO: 24; a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 25 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 26, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 27;
- the anti-CCR8-scFv region is a CDR1 consisting of the amino acid sequence of SEQ ID NO:30; a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 31 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 33;
- the anti-CCR8-scFv region is 1) a light chain variable region having the amino acid sequence of SEQ ID NO: 12, and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; 2) a light chain variable region having the amino acid sequence of SEQ ID NO: 14, and 3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; or 3) a light chain variable region having the amino acid sequence of SEQ ID NO: 16, and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17;
- the anti-CCR8-scFv region is a light chain variable region having the amino acid sequence of SEQ ID NO: 14; and The chimeric antigen receptor of (8), comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:15.
- the effector cells Effector cells expressing the chimeric antigen receptor according to any one of (1) to (31). (33) The cell according to (32), wherein the effector cells are NK cells. (34) The cell according to (32), wherein the effector cell is a T cell. (35) The cell of (32), wherein the effector cell is a macrophage. (36) The cell according to any one of (32) to (35), wherein the effector cell is derived from peripheral blood. (37) The cell according to any one of (32) to (35), wherein the effector cell is differentiated from a stem cell. (38) The cell according to (36), wherein the stem cell is an iPS cell. (39) A pharmaceutical composition containing the cells of any one of (32) to (38).
- An expression vector comprising the polynucleotide of (43).
- the chimeric antigen receptor-expressing cells of the present invention that recognize CCR8 as an antigen have cytotoxic activity against CCR8-expressing cells, they are very useful as pharmaceuticals, particularly as pharmaceuticals for the treatment or prevention of CCR8-related diseases. is.
- anti-CCR8-CAR chimeric antigen receptor
- the cytotoxic activity of KHYG-1 cells introduced with the mRNA of each anti-CCR8-CAR ( ⁇ CCR8-2-CAR-1 to 6) having the same anti-CCR8-scFv region but different transmembrane and intracellular regions was evaluated by human CCR8.
- Expressing Rat-1 cells were used as target cells for evaluation.
- Toxic activity was assessed using human CCR8-expressing Rat-1 cells as target cells.
- A The cytotoxic activity of KHYG-1 cells introduced with the mRNA of each anti-CCR8-CAR ( ⁇ CCR8-2-CAR-11 to 13) having the same anti-CCR8-scFv region but different transmembrane and intracellular regions , using human CCR8-expressing Rat-1 cells as target cells.
- B Culture supernatant of KHYG-1 cells transfected with mRNA of each anti-CCR8-CAR ( ⁇ CCR8-2-CAR-11 to 13) having the same anti-CCR8-scFv region but different transmembrane and intracellular regions The amount of interferon- ⁇ secreted into the body was evaluated.
- the cytotoxic activity of KHYG-1 cells introduced with the mRNA of each anti-CCR8-CAR ( ⁇ CCR8-1-CAR to ⁇ CCR8-6-CAR) having different anti-CCR8-scFv regions and the same transmembrane region and intracellular region was examined. , using human CCR8-expressing Rat-1 cells as target cells.
- the cytotoxic activity of KHYG-1 cells transfected with anti-CCR8-CAR ( ⁇ CCR8-1-CAR and ⁇ CCR8-2-CAR) mRNA was examined using human CCR8-expressing RAMOS cells (C5 cells and C6 cells) as target cells. evaluated.
- B The cytotoxic activity of the negative control and ⁇ CCR8-2-CAR mRNA-introduced KHYG-1 cells was evaluated using human CCR8-expressing Treg cells as target cells.
- the cytotoxic activity of KHYG-1 cells transfected with anti-CCR8-CAR ( ⁇ CCR8-7-CAR) mRNA was evaluated using human CCR8-expressing Rat-1 cells as target cells.
- the cytotoxic activity of human peripheral blood-derived NK cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2) mRNA was evaluated using human CCR8-expressing Rat-1 cells as target cells.
- the cytotoxic activity of human peripheral blood-derived T cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2 and ⁇ CCR8-2-CAR-12) mRNA was investigated using ATL cell line-derived ATN-1 cells as target cells. was evaluated using The cytotoxic activity of human peripheral blood-derived T cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2 and ⁇ CCR8-2-CAR-12) mRNA was investigated using ALL cell line-derived TALL-1 cells as target cells.
- the cytotoxic activity of KHYG-1 cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-12) mRNA was evaluated using ATL cell line-derived ATN-1 cells as target cells. Evaluation of inhibitory activity of anti-CCR8 antibody against cytotoxic activity of KHYG-1 cells into which anti-CCR8-CAR ( ⁇ CCR8-2-CAR-12) mRNA was introduced, using ATL cell line-derived ATN-1 cells as target cells did.
- the antitumor effect of KHYG-1 cells introduced with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-12) mRNA was evaluated in mice transplanted with human CCR8-expressing Rat-1 cells.
- cytotoxic activity of human iPS cell-derived NK cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2) mRNA was evaluated using human CCR8-expressing Rat-1 cells as target cells.
- B The cytotoxic activity of human iPS cell-derived macrophages transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2) mRNA was evaluated using human CCR8-expressing Rat-1 cells as target cells.
- the cytotoxic activity of KHYG-1 cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2) mRNA was evaluated using human ovarian cancer-derived tumor-infiltrating Treg cells as target cells.
- the cytotoxic activity of human peripheral blood-derived NK cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2) mRNA was evaluated using human ovarian cancer-derived tumor-infiltrating Treg cells as target cells.
- the cytotoxic activity of human peripheral blood-derived NK cells transfected with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-2) mRNA was evaluated using human colon cancer-derived tumor-infiltrating Treg cells as target cells.
- the antitumor effect of KHYG-1 cells into which anti-CCR8-CAR ( ⁇ CCR8-2-CAR-12) mRNA was introduced was evaluated in ATL cell line-derived ATN-1 cell-transplanted mice.
- the anti-tumor effect of KHYG-1 cells introduced with anti-CCR8-CAR ( ⁇ CCR8-2-CAR-12) mRNA in ATL cell line-derived ATN-1 cell-transplanted mice was shown by luminescence imaging data.
- the anti-tumor effect of anti-CCR8-CAR ( ⁇ CCR8-2-CAR-12) mRNA-introduced KHYG-1 cells in colon cancer-derived CT26 cell-transplanted mice was evaluated.
- the cytotoxic activity of anti-CCR8-CAR ( ⁇ CCR8-2-CAR-12) mRNA-introduced KHYG-1 cells against tumor-infiltrating Treg cells in colon cancer-derived CT26 cell-transplanted mice was evaluated.
- the chimeric antigen receptor of the present invention comprises an extracellular region, a transmembrane region, and an intracellular region that bind to CCR8, and the intracellular region contains an intracellular signal region of a signaling molecule expressed in lymphocytes that function as effector cells.
- the entire amino acid sequence is determined based on the amino acid sequence information of the "extracellular domain that binds to CCR8", the "transmembrane domain” and the “intracellular domain”, and an expression vector is constructed.
- the expression vector can be used to transform effector cells for expression.
- extracellular domain that binds to CCR8 refers to an extracellular domain that is a part of an anti-CCR8 antibody and includes a fragment of the anti-CCR8 antibody that specifically binds to CCR8 like the anti-CCR8 antibody.
- the "extracellular region that binds to CCR8” may further include a signal sequence region at the N-terminus. Examples of the signal sequence region include the CD8A signal sequence region, the CD28 signal sequence region, and the like, but the CD8A signal sequence is preferred.
- the CD8A signal sequence region preferably has the amino acid sequence of SEQ ID NO:1.
- Anti-CCR8 antibodies can be produced by antibody preparation techniques known in the art. Examples thereof include the method described in Immunochemistry in Practice (Blackwell Scientific Publications). In addition, genetic engineering techniques known in the art are available. For example, methods described in Molecular Cloning, A Laboratory Manual, Forth Edition, Cold Spring Harbor Laboratory Press (2012), Current Protocols Essential Laboratory Techniques, Current Protocols 2, etc. (2).
- the amino acid sequence of human CCR8 is shown in UniProtKB/Swiss-Prot: P51685 (SEQ ID NO: 49).
- the extramembrane domain of human CCR8 corresponds to the N-terminal region consisting of amino acids 1 to 35, the loop1 region consisting of amino acids 94 to 107, the loop2 region consisting of amino acids 172 to 202, and the loop3 region consisting of amino acids 264 to 280. do.
- Hybridomas that produce anti-CCR8 antibodies can be prepared using human CCR8 proteins, genes encoding full-length human CCR8, human CCR8-expressing cells, etc. as immunogens.
- a gene encoding full-length human CCR8 is used as an immunogen, for example, a hybridoma producing an anti-CCR8 antibody can be obtained by fusing mouse myeloma cells with spleen cells of a mouse DNA-immunized using the gene as an antigen. can be done.
- the anti-CCR8 antibody includes monoclonal antibodies having the CDRs or heavy chain variable regions/light chain variable regions described herein.
- the antibody or antibody fragment may be from any class (eg IgG, IgE, IgM, IgD or IgA, preferably IgG) or subclass of immunoglobulin molecule, such as mouse, rat, shark, rabbit, It may be obtained from any species including pigs, hamsters, camels, llamas, goats or humans.
- the antibody or antibody fragment is preferably a mouse-derived monoclonal antibody or a humanized monoclonal antibody.
- Anti-CCR8 antibodies are characterized by inhibiting binding of CCR8 to any of the CCR8 ligands.
- CCR8 ligand is not particularly limited as long as it is a substance that binds to CCR8, such as CCL1, CCL8, and CCL18, but CCL1 and CCL18 are preferred, and CCL1 is particularly preferred.
- CCL1 is particularly preferred.
- human CCR8-expressing 293 cells were used to measure Ca 2+ influx with the addition of human CCL1, and the signal in the absence of human CCL1 was evaluated as an inhibition rate of 100%.
- Human CCL1 is UniProtKB/Swiss-Prot No. It has the amino acid sequence shown in P22362 and others.
- Human CCL8 is available from GenBank No. It has an amino acid sequence shown in AAI26243.1 and the like.
- Human CCL18 is available from GenBank No. It has the amino acid sequence shown in EAW80102.1 and the like.
- Anti-CCR8 antibodies also include chimeric, humanized, and fully human antibodies. Since humanized monoclonal antibodies have reduced antigenicity in the human body, they are useful when administered to humans for therapeutic purposes.
- a humanized monoclonal antibody is obtained by grafting the complementarity determining region (CDR) of a non-human mammalian antibody, such as a mouse antibody, to the framework region (FR) of a human antibody. Therefore, the FRs of humanized monoclonal antibodies are of human origin.
- a suitable FR is described in Kabat E. A. The selection can be made by referring to these documents. In this case, the FR is selected so that the CDR can form a good antigen-binding site.
- amino acids in the FRs of the variable region of the antibody may be substituted so that the CDRs of the reshaped humanized monoclonal antibody form appropriate antigen-binding sites (Sato, K. et al., Cancer Res. 1993). 1999, vol. 53, p. 851).
- the ratio of FR amino acids to be substituted is 0-15%, preferably 0-5%, of the total FR region.
- the anti-CCR8 antibody is preferably a CCR8 neutralizing antibody.
- a CCR8 neutralizing antibody means an antibody having neutralizing activity against CCR8. Whether or not it has neutralizing activity against CCR8 can be determined, for example, by measuring whether or not it suppresses the physiological action of any one of the CCR8 ligands (eg, CCL1) on CCR8.
- the anti-CCR8 antibody is preferably an antibody that strongly recognizes human CCR8.
- To select an antibody or an antibody fragment thereof that recognizes human CCR8 more strongly by selecting an antibody or an antibody fragment thereof based on the intensity of neutralizing activity when selecting an antibody that strongly recognizes human CCR8. can be done.
- the anti-CCR8 antibody fragment contained in the "extracellular region that binds to CCR8" is preferably a single-chain antibody that specifically binds to CCR8 (hereinafter referred to as anti-CCR8-scFv).
- Anti-CCR8-scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate linker sequence (hereinafter referred to as P). , antibody fragments with CCR8 binding activity. Preferred are VH-P-VL polypeptides.
- the VH and VL contained in the scFv used in the present invention may be those of the monoclonal antibody of the present invention.
- the scFv used in the present invention is expressed by constructing an scFv expression vector using cDNAs encoding the VH and VL of the anti-CCR8 antibody of the present invention, and introducing it into Escherichia coli, yeast or animal cells. can be manufactured.
- the linker sequence As the linker sequence, the linker sequence of SEQ ID NO: 2 is preferable.
- the "extracellular region that binds to CCR8" includes an anti-CCR8-scFv region and a signal sequence region
- the signal sequence region preferably exists on the N-terminal side of the anti-CCR8-scFv region.
- a peptide containing CDRs such as anti-CCR8-scFv, comprises at least one or more regions of CDRs of VH or VL. Multiple CDRs can be joined directly or through suitable linker sequences.
- the peptide containing CDR used in the present invention is obtained by constructing CDR-encoding DNA using cDNA encoding VH and VL of the monoclonal antibody of the present invention, inserting the DNA into an expression vector for animal cells, It can be produced by introducing the vector into Escherichia coli, yeast or animal cells and expressing the vector.
- Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
- extracellular region that binds to CCR8 is characterized by binding to human CCR8. In particular, those that specifically bind to human CCR8 are preferred.
- Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 ⁇ 10 ⁇ 6 M or less (eg, a lower Kd indicates tighter binding).
- the Kd value is preferably 1 ⁇ 10 ⁇ 7 M or less, more preferably 1 ⁇ 10 ⁇ 8 M or less, and even more preferably 1 ⁇ 10 ⁇ 9 M or less.
- amino acid sequence of the "extracellular region that binds to CCR8" can be determined using the amino acid sequence information of the CDRs or heavy chain variable region/light chain variable region of the anti-CCR8 antibody described herein.
- the anti-CCR8-scFv region included in the "extracellular region that binds to CCR8" preferably contains any of the following sequences. 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 25 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 26, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 27; A heavy chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 28 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 29; 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 30; a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 31 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 33; A heavy chain variable region comprising CDR2 consisting
- a CDR1 consisting of the amino acid sequence of SEQ ID NO:56; a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO:57 and CDR3 consisting of the amino acid sequence of SEQ ID NO:58 and CDR1 consisting of the amino acid sequence of SEQ ID NO:59; A heavy chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 60 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 61 More preferably, it contains any of the following sequences.
- CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 25 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 26, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 27;
- CDR1 consisting of the amino acid sequence of SEQ ID NO: 30; a light chain variable region comprising CDR2 consisting of the amino acid sequence of SEQ ID NO: 31 and CDR3 consisting of the amino acid sequence of SEQ ID NO: 32 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 33;
- the anti-CCR8-scFv region included in the "extracellular region that binds to CCR8" preferably contains any of the following sequences. 1) a light chain variable region having the amino acid sequence of SEQ ID NO: 12, and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; 2) a light chain variable region having the amino acid sequence of SEQ ID NO: 14, and 3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; or 3) a light chain variable region having the amino acid sequence of SEQ ID NO: 16, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 More preferably, it contains any of the following sequences.
- the "extracellular region that binds to CCR8" may further include a spacer sequence at the C-terminus, and the anti-CCR8-scFv region and the "transmembrane region” may be linked via the spacer sequence.
- the spacer sequence region preferably includes a Flag epitope-containing sequence region and/or a CD8A hinge region. Further, the Flag epitope-containing sequence region preferably has the amino acid sequence of SEQ ID NO:3 and the CD8A hinge region preferably has the amino acid sequence of SEQ ID NO:4.
- Transmembrane region means a region intervening between the extracellular region and the intracellular region.
- the region is not particularly limited as long as it is a region interposed between the extracellular region and the intracellular region of the membrane protein, but the transmembrane region of a signaling molecule expressed in lymphocytes that function as effector cells is preferred. Examples of such signaling molecules include CD8A, CD28, NKG2D, and the like.
- the "transmembrane domain” preferably includes the CD8A transmembrane domain or the CD28 transmembrane domain.
- the CD8A transmembrane region preferably has the amino acid sequence of SEQ ID NO:5 and the CD28 transmembrane region preferably has the amino acid sequence of SEQ ID NO:6.
- the intracellular domain preferably also includes the CD28 intracellular signal domain.
- the CD28 intracellular signal region has the amino acid sequence of SEQ ID NO:7.
- Intracellular domain includes an intracellular signal domain of a signaling molecule expressed in lymphocytes that function as effector cells, and when the "extracellular domain that binds to CCR8" binds to CCR8, activation of effector cells It means a region capable of transducing necessary signals into cells.
- “Lymphocytes that function as effector cells” are exemplified by NK cells and T cells, preferably NK cells or T cells. Examples of the signaling molecule include 4-1BB, 2B4, CD3 ⁇ , DAP10, DAP12, and the like.
- the "intracellular domain” preferably includes at least one, more preferably at least two, of the intracellular signal domains of 4-1BB, 2B4, CD3 ⁇ and DAP10. More preferably, it contains an intracellular signal region or an intracellular signal region of 2B4 and CD3 ⁇ , more preferably an intracellular signal region of 4-1BB and CD3 ⁇ , and a CD3 ⁇ intracellular signal region at the end of the 4-1BB intracellular signal region. It particularly preferably contains a region where the signal regions are linked, and most preferably contains a region where the 4-1BB intracellular signal region, the DAP10 intracellular signal region and the CD3 ⁇ intracellular signal region are linked in this order.
- the 4-1BB intracellular signal region preferably has the amino acid sequence of SEQ ID NO:8.
- the 2B4 intracellular signal domain preferably has the amino acid sequence of SEQ ID NO:9.
- the CD3 ⁇ intracellular signal region preferably has the amino acid sequence of SEQ ID NO:10.
- the DAP10 intracellular signal domain preferably has the amino acid sequence of SEQ ID NO:11.
- each of the "extracellular region that binds to CCR8", "linker sequence”, “spacer sequence region”, “transmembrane region” and “intracellular region” has an amino acid sequence identity of 90% or more, , Preferably, if there is 95% or more identity, the same effect can be exhibited.
- the chimeric antigen receptor of the present invention has cytotoxic activity against CCR8-expressing cells when expressed in effector cells.
- Effector cells expressing the chimeric antigen receptor of the present invention are constructed by inserting the cDNA encoding the chimeric antigen receptor of the present invention into an appropriate expression vector to construct a chimeric antigen receptor expression vector, and constructing the chimeric antigen receptor expression vector.
- it can be produced by introducing the mRNA of the chimeric antigen receptor of the present invention synthesized using the expression vector into effector cells and expressing them.
- the "cytotoxic activity" includes cell phagocytic activity by macrophages and the like.
- Effector cells that express the chimeric antigen receptor of the present invention include NK cells, T cells, macrophages, etc., but NK cells or T cells are preferred, and NK cells are more preferred.
- the effector cells can be obtained from peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, etc. collected from humans.
- the effector cells may also be differentiated from stem cells such as iPS cells. Examples of stem cells include somatic stem cells such as hematopoietic stem cells, embryonic stem cells such as ES cells, and pluripotent stem cells such as iPS cells, with iPS cells being preferred.
- the effector cells may be used for autologous transplantation or for production of cell preparations by allograft.
- the medium for effector cells expressing the chimeric antigen receptor of the present invention includes DMEM medium, RPMI1640 medium, MEM medium, Ham's F-12 medium, and the like.
- Various organic or inorganic substances such as serum such as FCS (fetal bovine serum) and HS (horse serum), cytokines such as IL-2, amino acids, and hormones are added to the medium to optimize the culture conditions.
- CCR8-expressing cells in which effector cells expressing the chimeric antigen receptor of the present invention exhibit cytotoxic activity include Treg cells and tumor cells, with Treg cells being preferred, and tumor-infiltrating Treg cells being more preferred.
- the tumor is preferably a solid tumor.
- breast cancer endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, gastric (gastric adenocarcinoma) cancer, non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma , colon cancer, renal cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, liver cancer, skin cancer, brain tumor, and the like.
- gastric gastric adenocarcinoma
- non-small cell lung cancer pancreatic cancer
- head and neck squamous cell carcinoma esophageal cancer
- bladder cancer melanoma
- colon cancer renal cancer
- non-Hodgkin's lymphoma urothelial cancer
- sarcoma bile duct cancer
- gallbladder cancer gallblad
- CCR8-expressing cells are tumor-infiltrating Treg cells
- CCR8 may not be expressed in the tumor cells.
- the tumor is preferably melanoma or hematological cancer.
- Hematologic cancers expressing CCR8 include leukemias such as acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), adult T-cell leukemia-lymphoma (ATL), peripheral T-cell lymphoma (PTCL), and cutaneous T-cell lymphoma. (CTCL), diffuse large B-cell lymphoma (DLBCL), and Hodgkin's lymphoma.
- AML acute myeloid leukemia
- ALL acute lymphoblastic leukemia
- ATL adult T-cell leukemia-lymphoma
- PTCL peripheral T-cell lymphoma
- CTCL diffuse large B-cell lymphoma
- Hodgkin's lymphoma Hodgkin's lymphoma.
- effector cells expressing the chimeric antigen receptor of the present invention exhibit cytotoxic activity
- the effector cells increase the amount of cytokine secretion and may exhibit cytotoxic activity.
- the cytokine include IL-2 and interferon, with interferon- ⁇ being preferred.
- Effector cells expressing the chimeric antigen receptor of the present invention are useful as pharmaceutical compositions. Therefore, pharmaceutical compositions containing effector cells expressing the chimeric antigen receptor of the present invention can be administered systemically or locally.
- an administration route for example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like can be selected.
- the pharmaceutical composition of the present invention may be a composition for autologous transplantation containing cells in which the chimeric antigen receptor of the present invention is expressed in autologous effector cells, or a composition for autologous transplantation containing the chimeric antigen receptor of the present invention in allogeneic effector cells. It may be a cell preparation obtained by allograft containing cells expressing the.
- the pharmaceutical composition of the present invention is very useful as a medicament for treating and/or preventing CCR8-related diseases.
- it is very useful as a medicament for treating and/or preventing cancer in which CCR8-expressing Treg cells infiltrate into the tumor or cancer in which CCR8 is expressed.
- breast cancer endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, stomach (gastric adenocarcinoma) cancer, non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colon cancer , kidney cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, liver cancer, skin cancer, brain tumor, etc.
- stomach gastric adenocarcinoma
- non-small cell lung cancer pancreatic cancer
- head and neck squamous cell carcinoma esophageal cancer
- bladder cancer melanoma
- colon cancer e.ophageal cancer
- kidney cancer non-Hodgkin's lymphoma
- urothelial cancer s
- breast cancer endometrial cancer
- ovarian cancer lung cancer, bladder cancer, colon cancer, renal cancer, sarcoma, liver cancer, skin cancer and brain tumor. It is also very useful as a drug for treating and/or preventing leukemia and lymphoma.
- Cancers according to the present invention include all solid cancers and blood cancers. Specifically, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, gastric (gastric adenocarcinoma) cancer, non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma , colorectal cancer, renal cancer, non-Hodgkin's lymphoma, urothelial cancer, sarcoma, blood cell cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, liver cancer, skin cancer, brain tumor, etc.
- Breast cancer, endometrial cancer, ovarian cancer, lung cancer, bladder cancer, colon cancer, renal cancer, sarcoma, liver cancer, skin cancer and brain tumor are preferred. Also preferred are leukemia and lymphoma.
- the cancer according to the present invention means not only epithelial malignant tumors such as ovarian cancer and gastric cancer, but also non-epithelial malignant tumors including hematopoietic cancers such as chronic lymphocytic leukemia and Hodgkin's lymphoma.
- epithelial malignant tumors such as ovarian cancer and gastric cancer
- non-epithelial malignant tumors including hematopoietic cancers such as chronic lymphocytic leukemia and Hodgkin's lymphoma.
- the pharmaceutical composition of the present invention contains a therapeutically effective amount of effector cells expressing the chimeric antigen receptor of the present invention. contains.
- the pharmaceutical composition of the present invention is not limited to these doses.
- the administration period can be appropriately selected depending on the patient's age and symptoms.
- the pharmaceutical composition of the present invention may contain both pharmaceutically acceptable carriers and additives depending on the route of administration. Examples of such carriers and additives include dimethyl sulfoxide (DMSO), serum albumin, various antibiotics, vitamins, cytokines, growth factors, cell activating components such as steroids, and surfactants acceptable as pharmaceutical additives. agents, and the like. Additives to be used are appropriately selected from the above or in combination according to the dosage form, but are not limited to these.
- compositions or methods of the present invention can be used alone or in combination with other compositions or methods for treatment or prevention of cancer treatment or prevention.
- compositions or methods of the present invention can be utilized in conjunction with compositions or methods for chemotherapeutic or immunological therapeutic methods.
- physiochemical treatment methods such as radiotherapy, proton beam therapy, and hyperthermia can be used in combination.
- the invention includes polynucleotides encoding the amino acid sequences of the chimeric antigen receptors of the invention.
- the invention further includes an expression vector comprising said polynucleotide.
- the polynucleotide is not particularly limited as long as it encodes the chimeric antigen receptor of the present invention, and is a polymer composed of multiple nucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It may contain non-natural bases.
- the polynucleotide of the present invention can be used to produce the chimeric antigen receptor of the present invention and effector cells expressing the chimeric antigen receptor by genetic engineering techniques.
- Test Example Preparation of anti-human CCR8 antibody
- Anti-human CCR8 antibody was obtained based on the methods described in Examples 1-2, 4-7 and 9 of WO2020/138489.
- the amino acid sequence of each variable region of the obtained antibody is shown in FIG. 1, and the sequence numbers of the light chain variable region, heavy chain variable region and CDRs are shown in Table 1.
- ⁇ CCR8-1 and ⁇ CCR8-2 are humanized monoclonal antibodies
- ⁇ CCR8-3-6 are mouse-derived monoclonal antibodies
- ⁇ CCR8-7 is a rat-derived monoclonal antibody.
- Example 1 mRNA Synthesis of Chimeric Antigen Receptor (Anti-CCR8-CAR) Recognizing CCR8 as Antigen Based on the amino acid sequences of the light and heavy chain variable regions of the anti-human CCR8 antibody obtained in the test example. , constructed an anti-CCR8-CAR-loaded vector. The structure of anti-CCR8-CAR is shown in FIG.
- the CD8A signal sequence region SEQ ID NO: 1
- anti-CCR8-scFv region anti-human CCR8 antibody heavy chain variable region, G4S peptide linker (SEQ ID NO: 2) and anti- Human CCR8 antibody light chain variable region
- spacer sequence region Flag epitope-containing sequence region (SEQ ID NO: 3)
- CD8A hinge region SEQ ID NO: 4
- the CD8A transmembrane domain (SEQ ID NO: 5) or the CD28 transmembrane domain (SEQ ID NO: 6) was used.
- the intracellular domain was linked to the C-terminal side of the transmembrane domain.
- the intracellular regions include 4-1BB intracellular signal region (SEQ ID NO: 8), 2B4 intracellular signal region (SEQ ID NO: 9), CD3 ⁇ intracellular signal region (SEQ ID NO: 10) and DAP10 intracellular signal region (SEQ ID NO: 11 ) were used by ligating multiple intracellular signal regions.
- the CD28 intracellular signal region (SEQ ID NO: 7) was simultaneously inserted into the N-terminal side of the intracellular region.
- the DNA encoding the anti-CCR8-CAR was made by custom synthesis at Fasmac.
- the DNA was cloned into the SmaI site of the pUC19 vector with the EcoRI site oriented upstream of the gene.
- the anti-CCR8-CAR gene region was amplified by PCR using pUC19-T7 primer (SEQ ID NO: 50) and pUC19-R1 primer (SEQ ID NO: 51).
- PCR conditions 10 ng of template DNA was used, and KOD polymerase was used to perform 35 cycles of 94° C. ⁇ 65° C. ⁇ 72° C. to amplify the target region.
- the amount of amplified anti-CCR8-CAR gene DNA was quantified, iScribe T7 ARCA mRNA kit (manufactured by NEW ENGLAND BioLabs) was used to synthesize mRNA according to the method described in the kit, and poly A was added to obtain anti-CCR8-CAR mRNA.
- T7-AMP-F2 primer SEQ ID NO: 52
- AMP-R1 primer SEQ ID NO: 53
- Example 2 Culture of Human NK Cell Line KHYG-1 Cells and Introduction of mRNA Human IL-2 (PeproTech) was added to human NK cell line KHYG-1 cells at a final concentration of 100 units/ml.
- RPMI1640/10% FCS was used to culture at 37° C., 5% CO 2 in the presence of antibiotics. It was used for mRNA introduction when the cell concentration reached 2-3 ⁇ 10 5 /ml.
- KHYG-1 cells were washed twice with OPTI-MEM and suspended at 2.5 ⁇ 10 6 cells/100 ⁇ l.
- Example 3 Preparation of human CCR8-expressing cells (1) Preparation of Rat-1 cells and RAMOS cells expressing human CCR8 (SEQ ID NO: 49) was cloned by a standard method, and the expression human CCR8 expression vector was transformed into Rat-1 cells (pQCXIP vector) and RAMOS cells (pQEF vector) by standard method using Lipofectamine 3000. Two days later, Puromycin was added at 1 ⁇ g/ml to select for transformed cells. The human CCR8 expression rate of cells was confirmed using a flow cytometer using a PE-labeled anti-human CCR8 antibody (clone name: L263G8, BioLegend). In the case of RAMOS cells, cell cloning was performed to further single cells after drug selection.
- Example 4 Cytotoxic activity of human CCR8-expressing cells by anti-CCR8-CAR-expressing KHYG-1 cells
- An anti-CCR8-CAR ( ⁇ CCR8-1-CAR, respectively, ⁇ CCR8-1-CAR, ⁇ CCR8-2-CAR) mRNA was introduced.
- a negative control mRNA prepared by the method of Example 1 was introduced. 20 hours after the transformation of the KHYG-1 cells, the calcein-labeled human CCR8-expressing Rat-1 cells prepared in Example 3 as target cells or the parental Rat-1 cells, the transformed KHYG-1 cells, and the target The cells were mixed in 160 ⁇ l of cytotoxic activity evaluation buffer so that the cell ratio (E/T cell ratio) was 3:1.
- Cytotoxic activity was assessed using 96-well round bottom dishes. After mixing the transformed KHYG-1 cells and the target cells, they were centrifuged at 1000 rpm for 2 minutes using a plate centrifuge to sediment the cells. After mixing, the cells were cultured at 37° C., 5% CO 2 for 2 hours. After that, 60 ⁇ l of cell supernatant was slowly removed from the plate. The collected supernatant was quantified for the amount of fluorescence contained in the supernatant using a fluorometer within 30 minutes. The spontaneous release (BG) of calcein from each target cell was determined by similarly measuring the supernatant of each target cell-only well.
- the total amount of calcein released was determined by adding 1% Triton X-100 to each target cell-only well so that Triton X-100 was 0.1%, and measuring the same amount of supernatant. Decided.
- the method of calculating cytotoxic activity using the amount of calcein released from killed target cells as an index is as follows.
- the killing rate (%) was calculated by setting the amount of fluorescence in the supernatant as 100% when all cells were killed (in this case, the amount of fluorescence released into the supernatant due to complete cell lysis by triton). .
- the results are shown in FIG. Assuming that the killing rate is 1 when negative control mRNA-introduced KHYG-1 cells are mixed with parental Rat-1 cells and human CCR8-expressing Rat-1 cells, ⁇ CCR8-1-CAR-expressing KHYG-1 cells are killed at a multiple of 1.
- Example 5 Examination of transmembrane region and intracellular region (1)
- Each anti-CCR8-CAR mRNA in Table 2 prepared by the method of Example 1 was introduced into KHYG-1 cells.
- a negative control mRNA prepared by the method of Example 1 was introduced. Eighteen hours after KHYG-1 cells were transformed, the expression rate of each anti-CCR8-CAR on the cell surface was quantified using an anti-FLAG antibody (PE-anti-DYKDDDDK Tag Clone: 1.5, BioLegend). did.
- LIVE/DEAD Thermo Fisher
- anti-FLAG antibody for 1 hour at room temperature
- FSC/SSC the main cell population region
- FLAG expression level was analyzed for this cell population.
- a threshold was set at a value at which the FLAG positive rate in control (AMP) transformed cells was about 1 to 2%, and the ratio of cells above that was calculated as the expression rate.
- the expression rate of the cell with the lowest expression rate was adjusted. Specifically, control transformed cells and anti-CCR8-CAR transformed cells were mixed at a certain ratio and adjusted so that the expression rate of anti-CCR8-CAR was about 30%. The expression rate of anti-CCR8-CAR in all cells is around 30%. Cytotoxic activity evaluation was performed using this adjusted KHYG-1 cells. Transformed KHYG-1 cells and calcein-labeled human CCR8-expressing Rat-1 cells were mixed at E/T cell ratios of 10:1, 3:1 and 1:1, and calcein release was evaluated. Calcein release evaluation was performed in the same manner as in Example 4. When the actual cell count is 10:1, 3 ⁇ 10 4 effector cells and 3 ⁇ 10 3 target cells are contained in 150 ⁇ l of cytotoxic activity evaluation buffer.
- the results are shown in FIG.
- the anti-CCR8-CAR with the highest cytotoxic activity was ⁇ CCR8-2-CAR-2.
- a significant difference test was performed between ⁇ CCR8-2-CAR-2 and other anti-CCR8-CARs. Tukey's multiple comparison test was performed, and there was a significant difference of P ⁇ 0.05 for all items between ⁇ CCR8-2-CAR-2 and other anti-CCR8-CARs.
- the sequence of the transmembrane region of CD8A was superior in all cytotoxic activities in KHYG-1 cells. found.
- the chimeric receptor with the intracellular signal domain of 4-1BB had the highest activity regardless of whether the transmembrane domain was CD8A or CD28. It was found that the combination of intracellular signaling regions of 4-1BB and CD3 ⁇ was superior in 1 cells. From the above, it was found that the combination of the transmembrane region of CD8A and the intracellular signaling region of 4-1BB-CD3zeta had the strongest cytotoxic activity in NK cells expressing anti-CCR8-CAR.
- Example 6 Examination of transmembrane region and intracellular region (2) The cytotoxic activity of each anti-CCR8-CAR-expressing KHYG-1 cell in Table 3 was evaluated in the same manner as in Example 5. The conditions were such that the E/T cell ratio was 27:1 and 3:1. The results are shown in FIG. At an E/T cell ratio of 27:1, ⁇ CCR8-2-CAR-12-expressing KHYG-1 cells had a significantly 1.2-fold greater killing activity than ⁇ CCR8-2-CAR-2-expressing KHYG-1 cells. was found to be elevated (P ⁇ 0.05 by Tukey's multiple comparison test). From the above results, it was found that addition of DAP10 to the intracellular domain of ⁇ CCR8-2-CAR-2 provides stronger cytotoxic activity than ⁇ CCR8-2-CAR-2-expressing KHYG-1 cells.
- Example 7 Examination of transmembrane region and intracellular region (3) The cytotoxic activity of each anti-CCR8-CAR-expressing KHYG-1 cell in Table 4 was evaluated in the same manner as in Example 5. The expression rate of anti-CCR8-CAR was adjusted to about 40%, and the conditions were such that the E/T cell ratios were 10:1, 3:1 and 1:1. The results are shown in FIG. 6A. Tukey's multiple comparison test was performed when the E/T cell ratio was 10:1 and 3:1, and between ⁇ CCR8-2-CAR-12 and other anti-CCR8-CAR, either E/T cell The ratios were also significantly different at P ⁇ 0.05. The most killing anti-CCR8-CAR among the three was found to be ⁇ CCR8-2-CAR-12.
- Example 8 Examination of anti-CCR8-scFv region (1) Anti-CCR8-scFv regions were examined in the same manner as in Example 5.
- Six types of anti-CCR8-CAR shown in Table 5 were designed that differed only in the antigen-binding region of the extracellular domain from the variable region amino acid sequence of the anti-human CCR8 antibody. All sequences other than the antibody region were the same, and the CD8A transmembrane region was used as the transmembrane region, and the 2B4-CD3 ⁇ intracellular signal region was used as the intracellular region. The conditions were adjusted so that the expression rate of anti-CCR8-CAR was 36%, and the E/T cell ratios were 10:1, 3:1 and 1:1. The results are shown in FIG.
- ⁇ CCR8-2-CAR-expressing KHYG-1 cells had the strongest cytotoxic activity, followed by ⁇ CCR8-1-CAR-expressing KHYG-1 cells.
- ⁇ CCR8-2-CAR-expressing KHYG-1 cells were significantly different from all other anti-CCR8-CAR-expressing KHYG-1 cells.
- ⁇ CCR8-1-CAR-expressing KHYG-1 cells are anti-CCR8-CAR-expressing KHYG-1 cells other than ⁇ CCR8-2-CAR-expressing KHYG-1 cells under conditions of E/T cell ratios of 10:1 and 3:1. significantly higher than all.
- Example 8-2 Examination of anti-CCR8-scFv region (2) In KHYG-1 cells, the extracellular region that binds to CCR8 including the variable region of ⁇ CCR8-7 of the anti-CCR8 antibody prepared by the method of Example 1, the CD8A transmembrane region, the 2B4 intracellular signal region, and the CD3 ⁇ intracellular An anti-CCR8-CAR (referred to as ⁇ CCR8-7-CAR) mRNA linked with an intracellular region linked with a signal region was introduced. As a control, a negative control mRNA prepared by the method of Example 1 was introduced. ⁇ CCR8-7-CAR-expressing KHYG-1 cells were evaluated for cytotoxic activity in the same manner as in (1) above (FIG. 10). The conditions were such that the E/T cell ratio was 9:1, 3:1, 1:1 and 0.3:1.
- Example 9 Evaluation of cytotoxic activity of anti-CCR8-CAR-expressing KHYG-1 cells against human CCR8-expressing RAMOS cells expressing CCR8 to the same extent as CCR8-expressing tumor-infiltrating Treg cells
- Human CCR8-expressing RAMOS prepared in Example 3 Human CCR8 expression level was confirmed for cells (C5 cells and C6 cells) and parental RAMOS cells by the method of Example 3 using a flow cytometer. Furthermore, the approximate average number of human CCR8-expressing molecules on the cell surface per cell was determined using QIFIKIT (DAKO) and an anti-human CCR8 antibody (unlabeled L263G8, BioLegend) according to the protocol.
- DAKO QIFIKIT
- an anti-human CCR8 antibody unlabeled L263G8, BioLegend
- C5 cells were 2630 molecules/cell for C5 cells and 5950 molecules/cell for C6 cells.
- the average number of CCR8-expressing molecules on the cell surface of CCR8-expressing tumor-infiltrating Treg cells of human lung cancer was evaluated by the same method, and it was about 4000 molecules/cell. It was confirmed that CCR8 was expressed at the same level as infiltrating Treg cells.
- C5 and C6 cells which are human CCR8-expressing RAMOS cells, as target cells, ⁇ CCR8-1-CAR-expressing KHYG-1 cells and ⁇ CCR8-2-CAR-expressing KHYG-1 cells were obtained in the same manner as in Example 5. Cytotoxic activity was assessed (Fig. 8).
- ⁇ CCR8-1-CAR-expressing KHYG-1 cells and ⁇ CCR8-2-CAR-expressing KHYG-1 cells have the same or 50% CCR8 expression level as that expressed on the cell surface of CCR8-expressing tumor-infiltrating Treg cells. It was found to retain sufficient cytotoxic activity against cells with moderate expression levels.
- Example 10 Evaluation of cytotoxic activity of anti-CCR8-CAR-expressing KHYG-1 cells against human Treg cells (1) Preparation of human Treg cells PBMCs were isolated from peripheral blood using Ficoll-Paque PLUS (GEW Healthcare). separated. CD4+ T cells were purified therefrom using the CD4+ T Cell Isolation Kit human (Miltenyi Biotec).
- the CD45RA + CD127-CD4 + CD25 + cells were sorted by the sorting function of FACS_Aria (FACS Aria, BD Company). This sorting separated human Treg cells. Dynabeads Human Treg Expander (Veritas) was added to the isolated Treg cells according to the protocol, and cultured using RPMI1640/10% FCS medium at 37° C., 5% CO 2 for 20 days according to the protocol. The expression rate of human CCR8 protein on the cell surface of cultured Treg cells was detected by a flow cytometer according to the method of Example 3, and the positive rate was found to be about 20%.
- FACS_Aria FACS Aria, BD Company
- the number of each cell in 96 wells was 1 ⁇ 10 4 cells/well for all target Treg cells.
- the number of transformed KHYG-1 cells was 1 ⁇ 10 5 to 1 ⁇ 10 4 according to the E/T cell ratio.
- RPMI1640/10% FCS was used as the culture medium for activity evaluation. 16 hours after the start of evaluation, 1/20 volume of Human TruStain FcX (BioLegend) was added to each well to block Fc receptors, and allowed to stand at room temperature for 15 minutes.
- the cells were washed once with HBSS/2% FCS/10 mM HEPES buffer, suspended in 100 ⁇ l of the same buffer, and then treated with LIVE/DEAD (Thermo Fisher), Bv421-labeled anti-human CD4 (BD Bioscience), PE-labeled anti- Staining was performed at 4° C. for 1 hour using human CCR8 (BioLegend) or PE-labeled isotype control antibody (BioLegend).
- Cells used for isotype antibody staining were cells obtained by mixing Treg cells and transformed KHYG-1 cells at an E/T cell ratio of 3:1.
- the figure in which the cytotoxic activity in FIG. 9A was evaluated is an example of cells mixed at 10:1.
- the cells were washed twice with the same buffer and analyzed by calculating the ratio of surviving human CCR8-expressing cells using a flow cytometer.
- a background threshold was set with an isotype control antibody, and cells with fluorescence intensity higher than the threshold were determined as expressing cells by anti-CCR8 staining.
- Identification of viable human CCR8-expressing cells was performed by gating CD4-expressing cells into a negative fraction with LIVE/DEAD staining, in which a CCR8-positive fraction was isolated from viable human CCR8-expressing cells. cells. Also, the ratio of human CCR8-expressing cells in wells containing only target cells was calculated as 100%.
- FIG. 9A An analysis example of the analyzed flow cytometer is shown in FIG. 9A.
- FIG. 9B In the cells transformed with the negative control mRNA, the killing rate was about 15% at any E/T cell ratio. Cells had a killing rate of 80% or more at all E/T cell ratios, and a significant increase in killing rate was observed compared to the control. (P ⁇ 0.001 for all E/T cell ratios by Student t-test). From the above, it was confirmed that NK cells expressing ⁇ CCR8-2-CAR have cytotoxic activity against Treg cells expressing human CCR8.
- Example 11 Evaluation of cytotoxic activity using peripheral blood-derived NK cells expressing anti-CCR8-CAR (1) Identification of cell lines expressing human CCR8 ATL patient-derived cell line ATN-1 cells and ALL patient-derived cell lines TALL-1 cells and CCRF-HSB2 cells (all manufactured by RIKEN) were stained in the same manner as in Example 9 using a PE-labeled anti-human CCR8 antibody (PE-labeled L263G8, BioLegend) at a final concentration of 4 ⁇ g/ml according to the protocol. . A positive signal was detected in both cells by flow cytometer analysis.
- PE-labeled anti-human CCR8 antibody PE-labeled L263G8, BioLegend
- the CD16 positive rate after culture was stained with PE-anti-human CD16 antibody (3G8, BioLegend) and FITC-anti-human CD56 antibody (HCD56, BioLegend). Both positive cell ratios were 95% or more.
- NEPA21 was used to transform the cells with 5 ⁇ g of ⁇ CCR8-2-CAR-2 mRNA.
- the CAR positive rate was 10.6% 16 hours after transfection. Using these cells and human CCR8-expressing Rat-1 cells as target cells, cytotoxic activity was evaluated by the calcein method.
- peripheral blood-derived NK cells expressing Cytotoxic activity against expressing Rat-1 cells was significantly increased (Fig. 11). From the above, it was found that expression of ⁇ CCR8-2-CAR-2 mRNA in peripheral blood-derived NK cells exhibited cytotoxic activity against human CCR8-expressing Rat-1 cells. Next, cytotoxic activity was evaluated in a similar manner against ALL cell line TALL-1 cells. As a result, it was found that ALL cell line TALL-1 cells can be killed by expressing ⁇ CCR8-2-CAR-2 mRNA in peripheral blood-derived NK cells, similarly to human CCR8-expressing Rat-1 cells.
- Example 12 Cytotoxic Activity Evaluation Using Peripheral Blood-Derived T Cells Expressing Anti-CCR8-CAR (1) Separation of T Cells from Human Peripheral Blood T cells were isolated using an isolation kit (MojoSort, BioLegend). The cells were cultured for 2 weeks in the presence of RPMI1640/10% FCS/Nonessential amino acid+Dynabeads human T-Activator CD3/CD28 (GIBCO)+human IL-2 (200 U/ml) (PeproTech). Each T cell subset of the T cell population after two weeks of culture was evaluated by flow cytometry using various antibodies.
- Detection antibodies include PE-CD16 (B73.1, BioLegend), APC-CD8 (SK1, BioLegend), FITC-CD4 (OKT4, BioLegend), Bv421-CD3 (SK7, BioLegend), LIVE/DEAD (APC-Cy7 ), an isotype antibody (MOPC-21, BioLegend) was used.
- the ratios of CD3-positive, CD4-positive, CD8-positive, and CD16-positive cells were 99.6%, 51.6%, 41.6%, and 0.5%, respectively, and most of them were CD3-positive T cells. , almost no CD16-positive NK cells were observed.
- peripheral blood-derived T cells expressing ⁇ CCR8-2-CAR-2 or ⁇ CCR8-2-CAR-12 showed significantly similar results compared to the negative control Amp mRNA-introduced peripheral blood-derived T cells. It was found to have some degree of cytotoxic activity (Figure 12).
- the E / T cell ratio was set to 5: 1, and ⁇ CCR8-2-CAR-2 or ⁇ CCR8-2-CAR- for human ALL cell lines TALL-1 cells and CCRF-HSB2 cells.
- the cytotoxic activity of peripheral blood-derived T cells expressing 12 was evaluated by the calcein method.
- peripheral blood-derived T cells expressing ⁇ CCR8-2-CAR-2 or ⁇ CCR8-2-CAR-12 were compared to negative control Amp-introduced peripheral blood-derived T cells.
- a significant increase in cytotoxic activity was observed (Fig. 13).
- CCRF-HSB2 cells also tended to show increased cytotoxic activity.
- Example 13 Evaluation of inhibitory activity of anti-CCR8 antibody against cytotoxic activity of anti-CCR8-CAR-expressing KHYG-1 cells (1) Preparation of luciferase gene-expressing ATN-1 cells (Luc/ATN-1 cells) pGL3 vector (Clontech) ) into the SalI/BglII sites of the pMEI-5-Neo vector to create a vector expressing the firefly luciferase gene (pMEI5-FLuc). This expression vector was transduced into ATN-1 cells with NEPA21 by electroporation. Drug selection was performed with G418 from 3 days after the introduction.
- the luciferase activity of the drug-resistant cells was evaluated using Glo kit (Promega) to confirm that the luciferase gene was expressed.
- (2) Introduction of anti-CCR8-CAR gene into KHYG-1 cells by retrovirus The gene fragment of ⁇ CCR8-2-CAR-12-IRES-Puromycin (SEQ ID NO: 62) was converted to NotI of the retroviral vector pMEI-5 vector.
- a vector was constructed that can be inserted into the site and express ⁇ CCR8-2-CAR-12-IRES-Puromysin mRNA.
- Retroviruses were obtained by co-transfection of 293GP2 cells with ⁇ CCR8-2-CAR-12 retroviral vector and pE-Ampho amphotropic env expression vector (TAKARA), a retroviral envelope protein expression vector. .
- the resulting retrovirus-containing 293GP2 cell supernatant was added to KHYG-1 cells together with polybrene (final concentration 10 ⁇ g/ml) to obtain infected cells.
- puromycin 0.5 ⁇ g/ml
- the ⁇ CCR8-2-CAR-12 expression level in these cells was stained with a PPE-labeled anti-Flag antibody and analyzed by a flow cytometer.
- Tumor volumes and body weights were measured on days 8, 13, 16, 17 and 20 after transplantation of target cells.
- the tumor volume (mm 3 ) was measured by major axis (mm) ⁇ minor axis (mm) ⁇ minor axis (mm)/2.
- the tumor volume increase rate was significantly suppressed in the administration group of ⁇ CCR8-2-CAR-12-expressing KHYG-1 cells compared to the administration group of KHYG-1 cells, which are parental control cells ( Figure 16). No difference in body weight change was observed between the two administration groups.
- Example 15 Evaluation of cytotoxic activity using iPS cell-derived NK cells expressing anti-CCR8-CAR
- iPS cell-derived NK cells were prepared by optimizing the culture method with reference to a known method (CancerScience, 2020, Vol. 111, pp. 1478-1490). NK cell-specific surface antigens were confirmed on the cultured differentiated cells by FACS analysis.
- the expression of hematopoietic progenitor markers CD34 and CD45 was confirmed, while in the cell group 5 weeks after differentiation induction, the expression of CD34 decreased, while mature hematopoietic cells also showed expression. increased expression of CD45
- the expression rate of the NK marker CD56 was 50% or more in the cell group after 5 weeks of differentiation induction, and the expression of NKG2D, KIR2D and the like, which are important for cytotoxic activity, was similarly confirmed.
- ppPACKH1 Lentivector Packaging Kit (System Biosciences) it was packaged according to the method described in the kit to obtain an ⁇ CCR8-2-CAR-2-loaded lentiviral vector.
- a negative control only the lentiviral vector pCDH-EF1-MCS was used in the ppPACKH1 Lentivector Packaging Kit (System Biosciences), and a viral vector was synthesized according to the method described in the kit and used as a negative control viral vector.
- the cells were stained with Zombie NIR Fiable Viability Kit (BioLegend), anti-FLAG antibody and anti-CD56 antibody (Brilliant Violet421-anti-human CD56 clone: HCD56, BioLegend) for 1 hour at room temperature, and a buffer for evaluating cytotoxic activity ( After washing twice with DMEM/4% FCS), the cell surface expression rate of anti-CCR8-CAR was evaluated by a flow cytometer. As a result, about 20% of ⁇ CCR8-2-CAR-2 was expressed on the surface of human iPS cell-derived NK cells.
- Cytotoxic activity of human CCR8-expressing cells by anti-CCR8-CAR-introduced iPS cell-derived NK cells 24 hours after gene transfer to iPS cell-derived NK cells, the calcein-labeled human produced in (5) above as target cells
- Target cells Mix CCR8-expressing Rat-1 cells, transgenic iPS cell-derived NK cells and target cells (E/T cell ratio) to a maximum of 10:1 in cytotoxic activity evaluation buffer, Serial dilutions were performed to achieve E/T cell ratios of the same ratio. Cytotoxic activity was assessed using U-bottom 96 wells.
- the cells were sedimented by centrifugation at 2000 rpm for 5 minutes using a plate centrifuge. After mixing the cells were incubated at 37° C., 5% CO 2 for 30 minutes. After that, it was centrifuged at 2000 rpm for 5 minutes using a plate centrifuge, and 70 ⁇ l of the cell supernatant was slowly collected from the plate. The collected supernatant was quantified for the amount of fluorescence contained in the supernatant using a fluorometer within 30 minutes. The spontaneous release (BG) of calcein from each target cell was determined by similarly measuring the supernatant of each target cell-only well.
- BG spontaneous release
- the total amount of calcein released was determined by adding 1% Triton X-100 to each target cell-only well so that Triton X-100 was 0.1%, and measuring the same amount of supernatant. Decided.
- the method of calculating cytotoxic activity using the amount of calcein released from killed target cells as an index is as follows.
- Example 16 Evaluation of cytotoxic activity using iPS cell-derived macrophages expressing anti-CCR8-CAR
- CD34 a hematopoietic progenitor cell marker
- CD45 which is also found in mature hematopoietic cells
- CD11b and CD14 which are surface antigens specific to macrophages
- the recovered cells were washed with a medium and then suspended in a solution of P3 Primary Cell 4D-Nucleofector X Kit (Lonza) at 5 ⁇ 10 5 cells/100 ⁇ l. 0.25 ⁇ l (0.5 ⁇ g) of each of ⁇ CCR8-2-CAR-2 mRNA and negative control mRNA was added to a Single Nucleocuvette (100 ⁇ l) containing 100 ⁇ l of cell solution together with 0.1 ⁇ l (0.1 ⁇ g) of pmaxGFP Vector (Lonza). Then, the normal human macrophage gene transfer protocol of the 4D-Nucleofector device was selected and gene transfer was performed.
- the mixture was added to a 96-well round-bottom plate, spun at 100 g for 2 minutes using a plate centrifuge to settle the cells, and cultured at 37° C., 5% CO 2 for 2 hours.
- the cells were centrifuged at 3000 rpm for 30 seconds, the supernatant was removed, and the cells were suspended in 100 ⁇ l of SFEB medium/1% BSA containing 0.05 ⁇ g/ml DAPI. Cytophagocytic activity against CCR8-expressing cells was detected with a flow cytometer.
- the calculation method of cell phagocytic activity is ⁇ (GFP/Far Red co-positive cells) / (GFP-positive cells) ⁇ x 100 (%) Calculated by As a result, compared with the cells transfected with the negative control, the cells transfected with ⁇ CCR8-2-CAR-2 exhibited human CCR8-expressing cell-dependent and anti-CCR8-CAR-expressing cell-specific cytotoxic activity. was taken.
- cytotoxic activity evaluation was also performed by another method in which (2) and subsequent steps were replaced with the following method.
- 0.2 ⁇ l (0.5 ug) of ⁇ CCR8-2-CAR-2 vector was added together with 0.1 ⁇ l (0.1 ⁇ g) of pmaxGFP Vector (Lonza) to Single Nucleocuvette (100 ⁇ l) containing 100 ⁇ l of cell solution, and the 4D-Nucleofector was added.
- Gene transfer was performed by selecting the normal human macrophage gene transfer protocol of the device. Cells after mRNA transfection were cultured for 16-18 hours at 37° C., 5% CO 2 . The transformed cells were used for evaluation of cytotoxic activity against human CCR8-expressing cells serving as target cells within 20 hours after introduction of the plasmid.
- Functional cells that are the ratio of human CCR8-expressing Rat-1 cells and transformed iPS cell-derived macrophages stained with the CellTrace Far Red staining solution prepared in (3′) above 20 hours after transformation of iPS cell-derived macrophages. They were mixed in 200 ⁇ l of iPS cell-derived macrophage culture medium so that the ratio of cells/target cells (E/T cell ratio) was 3:1 (1.5 ⁇ 10 5 cells:5.0 ⁇ 10 4 cells). The mixture was added to a 96-well round-bottom plate, spun at 100 g for 2 minutes using a plate centrifuge to settle the cells, and cultured at 37° C., 5% CO 2 for 2 hours.
- the cells were centrifuged at 3000 rpm for 30 seconds, the supernatant was removed, and the cells were suspended in 100 ⁇ l of SFEB medium/1% BSA containing 0.05 ⁇ g/ml DAPI. Cytophagocytic activity against CCR8-expressing cells was detected with a flow cytometer. The cell phagocytic activity was calculated as follows for GFP-positive cells transfected with ⁇ CCR8-2-CAR-2 and GFP-negative cells not transfected with ⁇ CCR8-2-CAR-2.
- ⁇ Cytophagocytic activity of GFP-positive cells ⁇ (GFP/Far Red co-positive cells) / (GFP-positive cells) ⁇ x 100 (%)
- ⁇ Cytophagocytic activity of GFP-negative cells ⁇ (GFP-negative/Far Red-positive cells)/(GFP-negative cells) ⁇ 100 (%)
- the results are shown in FIG. 17B.
- a significant increase in cytotoxic activity was observed in ⁇ CCR8-2-CAR-2-transfected GFP-positive cells compared to the negative control GFP-negative cells (Student t-test, P ⁇ 0.005). ).
- Example 17 Evaluation of cytotoxic activity of anti-CCR8-CAR-expressing NK cells against human ovarian cancer-derived tumor-infiltrating Treg cells
- Human ovarian cancer patient-derived tumor tissue was subjected to Tumor Dissociation. Kit, human (Miltenyi Biotec) were used to disperse the cells, and a cell population containing Treg cells, CD8-positive T cells, and CD4-positive T cells other than Treg cells was prepared. Cells immediately after preparation or after cryopreservation were used for experiments.
- the CCR8 positive rate in the tumor-infiltrating Treg cells (CD45/CD3/CD4/Foxp3-positive and Zombie NIR/CD8/CD56-negative fraction) was measured using a flow cytometer.
- (3) Evaluation of human tumor-infiltrating Treg cytotoxic activity by anti-CCR8-CAR-expressing KHYG-1 cells Human ovarian cancer-derived tumor-infiltrating Treg cells obtained in (1) above are target cells, ⁇ CCR8-2-CAR-2 KHYG-1 cells transformed with mRNA or negative control mRNA were used as effector cells.
- the ⁇ CCR8-2-CAR-2 expression rate on the KHYG-1 cell surface 20 hours after transfection was calculated in the same manner as in Example 5, and was 22%.
- Cytotoxic activity evaluation was performed from 20 hours to 44 hours after transfection.
- Target cells and effector cells were seeded in 96-well round-bottom plates at 1 ⁇ 10 5 cells/well, and RPMI1640/10% FCS/1% PS was used as the culture medium.
- 24 hours after the start of evaluation the cells were collected, suspended in HBSS/2% FCS/10 mM HEPES buffer supplemented with Human TruStain FcX (BioLegend) and Zombie NIR TM Fixable Viability Kit (Biolegend), and allowed to stand at 4° C. for 30 minutes. did.
- the ratio of tumor-infiltrating Treg cells (CD45/CD3/CD4/Foxp3-positive and Zombie NIR/CD8/CD56-negative fraction) among surviving CD45-positive cells was quantified using a flow cytometer. .
- a significant reduction in tumor-infiltrating Treg cells was observed in ⁇ CCR8-2-CAR-2-expressing KHYG-1 cells compared to negative control mRNA-introduced cells (FIG. 18, Student t-test, P ⁇ 0. 005).
- the width of the decrease almost coincided with the CCR8-positive rate of tumor-infiltrating Treg cells.
- ⁇ CCR8-2-CAR-2-expressing KHYG-1 cells have potent cytotoxic activity against tumor-infiltrating Treg cells expressing CCR8.
- (4) Evaluation of human tumor-infiltrating Treg cytotoxic activity by peripheral blood-derived NK cells expressing anti-CCR8-CAR The human ovarian cancer-derived tumor-infiltrating Treg cells obtained in (1) above are used as target cells in Example 11. Peripheral blood-derived NK cells transformed with the obtained ⁇ CCR8-2-CAR-2 mRNA or negative control mRNA were used as effector cells.
- Example 18 Evaluation of cytotoxic activity of anti-CCR8-CAR-expressing NK cells against human colorectal cancer-derived tumor-infiltrating Treg cells
- Infiltrating Treg cells were used as target cells, and peripheral blood-derived NK cells transformed with ⁇ CCR8-2-CAR-2 mRNA obtained in Example 11 or negative control mRNA were used as effector cells.
- the abundance ratio of tumor-infiltrating Treg cells and CD4 other than CD8-positive T cells and tumor-infiltrating Treg cells were measured using a flow cytometer on day 5. IFN ⁇ production of positive T cells was quantified.
- Example 19 Evaluation of anti-tumor activity of anti-CCR8-CAR-expressing KHYG-1 cells in mice transplanted with human CCR8-expressing tumor cells (ATL cell line-derived ATN-1 cells)
- Human IL-15 knock-in NOG mice 29 weeks old, 1 ⁇ 10 7 (100 ⁇ l) of ATN-1 cells transfected with the luciferase gene were implanted intraperitoneally in female mice.
- ATN-1 cells are a cell line that naturally expresses human CCR8.
- the ⁇ CCR8-2-CAR-12-expressing KHYG-1 cell administration group received the ⁇ CCR8-2-CAR-12-expressing KHYG-1 cells obtained in Example 13, and the control group received KHYG-1 cells.
- Example 20 Evaluation of anti-tumor activity of anti-CCR8-CAR-expressing KHYG-1 cells in human CCR8 knock-in mice transplanted with colon cancer-derived CT26 cells (hCCR8-KI (KI/KI) mice hereinafter) hCCR8-KI (KI /KI)
- the CT26 cell line was implanted intradermally on the back of mice (13 weeks old, female). 7, 9, and 10 days after tumor transplantation, ⁇ CCR8-2-CAR-12-expressing KHYG-1 cells obtained in Example 13 were administered to the ⁇ CCR8-2-CAR-12-expressing KHYG-1 cell administration group, and the control group was administered the ⁇ CCR8-2-CAR-12-expressing KHYG-1 cells.
- Example 21 Evaluation of cytotoxic activity of anti-CCR8-CAR-expressing KHYG-1 cells against tumor-infiltrating Treg cells in hCCR8-KI (KI/KI) mice implanted with colon cancer-derived CT26 cells hCCR8-KI (KI/KI )
- the CT26 cell line was implanted intradermally on the back of mice (10 weeks old, female).
- Tumors were harvested from mice one day after cell administration. The tumor mass was finely cut with scissors, and a tumor-infiltrating cell suspension was prepared using Tumor Dissociation Kit, mouse (Miltenyi) according to the protocol attached to the kit. The cell suspension was stained with Fixable Viability Dye eFluor 780 reagent (eBioscience) at 4°C for 30 minutes. After washing once with 2% FCS/10 mM HEPES/HBSS, the cells were stained with non-fluorescent-labeled anti-human CCR8 antibody (or isotype control antibody) at 4° C. for 30 minutes.
- Fixable Viability Dye eFluor 780 reagent eBioscience
- the cells were stained with AF647-labeled anti-rat IgG antibody at 4° C. for 30 minutes.
- the cells were stained with Bv510-labeled anti-mouse CD45, Bv605-labeled anti-mouse TCR ⁇ , and Bv785-labeled anti-mouse CD4 antibodies at 4° C. for 30 minutes.
- cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the attached protocol, and intracellular FoxP3 was stained using PE-labeled anti-mouse FoxP3 antibody.
- T cells (CD45+TCR ⁇ +) were gated using a flow cytometer, and the CCR8 positive rate among tumor-infiltrating Treg cells (CD4+Foxp3+) was analyzed.
- the Student t-test method was used for the significance test (significance level: P ⁇ 0.05).
- the staining antibodies used are shown below.
- As the anti-human CCR8 antibody an antibody obtained according to the method described in Example 2 of WO2020/138489 was used.
- rat IgG2a isotype control antibody (BioXcell) AF647 AffiniPure F(ab') Fragment Donkey Anti-Rat IgG(H+L) (Jackson Immuno Research) PE anti-mouse/rat FoxP3 (clone FJK-16s, eBiosciences) Bv510 anti-mouse CD45 (clone 30-F11, Biolegend) Bv605 anti-mouse TCR ⁇ (clone PC61, BioLegend) Bv421 anti-mouse CD25 (clone PC61, BioLegend) Bv785 anti-mouse CD4 (clone RM4-5, BioLegend)
- ⁇ CCR8-2-CAR-12-expressing KHYG-1 cell administration group a significant decrease in CCR8-positive cells among the tumor-infiltrating Treg cells was observed as compared with the control group (Fig. 24). From the above, it was revealed that ⁇ CCR8-2-CAR-12-expressing
- the chimeric antigen receptor of the present invention which recognizes CCR8 as an antigen, has cytotoxic activity against CCR8-expressing cells by expressing it in effector cells. That is, the chimeric antigen receptor-expressing cells that recognize CCR8 as an antigen of the present invention are very useful as pharmaceuticals for the treatment or prevention of CCR8-related diseases.
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Abstract
Description
例えば、腫瘍に浸潤するCD4陽性Treg細胞は、抗腫瘍免疫応答を強力に阻害している可能性があり、効果的な癌治療の大きな障害となりうる。
CD4陽性FoxP3陽性Treg細胞が媒介する腫瘍免疫抑制は、動物腫瘍モデルで十分に立証されており、腫瘍内も含めた全身性のTreg細胞除去により抗腫瘍効果が得られるが、一方で50%程度の腫瘍内浸潤Treg細胞除去では効果が認められないことが報告されている(非特許文献2)。
しかし、CCR8のような、腫瘍内浸潤Treg細胞に発現する抗原に結合する細胞外領域、膜貫通領域及び細胞内領域を含み、細胞内領域に、T細胞やNK細胞などのエフェクター細胞として機能するリンパ球に発現するシグナル伝達分子の細胞内シグナル領域を含むキメラ抗原受容体が癌治療に有用であることは、これまでに示されていない。
(1)CCR8に結合する細胞外領域、膜貫通領域及び細胞内領域を含み、細胞内領域にエフェクター細胞として機能するリンパ球に発現するシグナル伝達分子の細胞内シグナル領域を含むキメラ抗原受容体。
(2)CCR8に結合する細胞外領域に抗CCR8-scFv領域を含む、(1)記載のキメラ抗原受容体。
(3)抗CCR8-scFv領域が、
1)配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域;
2)配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域;
3)配列番号35のアミノ酸配列からなるCDR1、
配列番号36のアミノ酸配列からなるCDR2及び
配列番号37のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号38のアミノ酸配列からなるCDR1、
配列番号39のアミノ酸配列からなるCDR2及び
配列番号40のアミノ酸配列からなるCDR3を含む重鎖可変領域;
4)配列番号30のアミノ酸配列からなるCDR1、
配列番号41のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号42のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号43のアミノ酸配列からなるCDR3を含む重鎖可変領域;
5)配列番号44のアミノ酸配列からなるCDR1、
配列番号45のアミノ酸配列からなるCDR2及び
配列番号46のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号47のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号48のアミノ酸配列からなるCDR3を含む重鎖可変領域;又は
6)配列番号56のアミノ酸配列からなるCDR1、
配列番号57のアミノ酸配列からなるCDR2及び
配列番号58のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号59のアミノ酸配列からなるCDR1、
配列番号60のアミノ酸配列からなるCDR2及び
配列番号61のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、(2)記載のキメラ抗原受容体。
(4)抗CCR8-scFv領域が、
1)配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域;
2)配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域;又は
4)配列番号30のアミノ酸配列からなるCDR1、
配列番号41のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号42のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号43のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、(3)記載のキメラ抗原受容体。
(5)抗CCR8-scFv領域が、
配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、(4)記載のキメラ抗原受容体。
(6)抗CCR8-scFv領域が、
配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、(4)記載のキメラ抗原受容体。
(7)抗CCR8-scFv領域が、
1)配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域;
2)配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域;又は
3)配列番号16のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号17のアミノ酸配列を有する重鎖可変領域;
を含む、(2)又は(3)記載のキメラ抗原受容体。
(8)抗CCR8-scFv領域が、
1)配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域;又は
2)配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域;
を含む、(7)記載のキメラ抗原受容体。
(9)抗CCR8-scFv領域が、
配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域
を含む、(8)記載のキメラ抗原受容体。
(10)抗CCR8-scFv領域が、
配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域
を含む、(8)記載のキメラ抗原受容体。
(11)抗CCR8-scFv領域において、軽鎖可変領域と重鎖可変領域が配列番号2のリンカー配列を介して連結している、(2)~(10)のいずれかに記載のキメラ抗原受容体。
(12)CCR8に結合する細胞外領域にCD8Aシグナル配列領域を含む、(2)~(11)のいずれかに記載のキメラ抗原受容体。
(13)CD8Aシグナル配列領域が、抗CCR8-scFv領域のN末端側に存在する、(12)記載のキメラ抗原受容体。
(14)CD8Aシグナル配列領域が、配列番号1のアミノ酸配列を有する、(12)又は(13)記載のキメラ抗原受容体。
(15)抗CCR8-scFv領域と膜貫通領域がスペーサー配列を介して連結している、(2)~(14)のいずれかに記載のキメラ抗原受容体。
(16)スペーサー配列に、Flagエピトープ含有配列領域を含む、(15)記載のキメラ抗原受容体。
(17)Flagエピトープ含有配列領域が、配列番号3のアミノ酸配列を有する、(16)記載のキメラ抗原受容体。
(18)スペーサー配列に、CD8Aヒンジ領域を含む、(15)~(17)のいずれかに記載のキメラ抗原受容体。
(19)CD8Aヒンジ領域が、配列番号4のアミノ酸配列を有する、(18)記載のキメラ抗原受容体。
(20)膜貫通領域にCD8A膜貫通領域を含む、(1)~(19)のいずれかに記載のキメラ抗原受容体。
(21)CD8A膜貫通領域が、配列番号5のアミノ酸配列を有する、(20)記載のキメラ抗原受容体。
(22)膜貫通領域にCD28膜貫通領域を含み、細胞内領域にCD28細胞内シグナル領域を含む、(1)~(19)のいずれかに記載のキメラ抗原受容体。
(23)CD28膜貫通領域が、配列番号6のアミノ酸配列を有し、CD28細胞内シグナル領域が、配列番号7のアミノ酸配列を有する、(22)記載のキメラ抗原受容体。
(24)細胞内領域に4-1BB、2B4、CD3ζ及びDAP10の細胞内シグナル領域のうち、少なくとも一つを含む、(1)~(23)のいずれかに記載のキメラ抗原受容体。
(25)細胞内領域に4-1BB、2B4、CD3ζ及びDAP10の細胞内シグナル領域のうち、少なくとも二つを含む、(24)記載のキメラ抗原受容体。
(26)細胞内領域に4-1BB及びCD3ζの細胞内シグナル領域を含む、(25)記載のキメラ抗原受容体。
(27)細胞内領域に2B4及びCD3ζの細胞内シグナル領域を含む、(25)記載のキメラ抗原受容体。
(28)細胞内領域に4-1BB細胞内シグナル領域を含み、当該4-1BB細胞内シグナル領域が、配列番号8のアミノ酸配列を有する、(24)~(27)のいずれかに記載のキメラ抗原受容体。
(29)細胞内領域に2B4細胞内シグナル領域を含み、当該2B4細胞内シグナル領域が、配列番号9のアミノ酸配列を有する、(24)~(27)のいずれかに記載のキメラ抗原受容体。
(30)細胞内領域にCD3ζ細胞内シグナル領域を含み、当該CD3ζ細胞内シグナル領域が、配列番号10のアミノ酸配列を有する、(24)~(27)のいずれかに記載のキメラ抗原受容体。
(31)細胞内領域にDAP10細胞内シグナル領域を含み、当該DAP10細胞内シグナル領域が、配列番号11のアミノ酸配列を有する、(24)~(27)記載のキメラ抗原受容体。
(32)(1)~(31)のいずれかに記載のキメラ抗原受容体を発現したエフェクター細胞。
(33)エフェクター細胞がNK細胞である、(32)記載の細胞。
(34)エフェクター細胞がT細胞である、(32)記載の細胞。
(35)エフェクター細胞がマクロファージである、(32)記載の細胞。
(36)エフェクター細胞が末梢血由来である、(32)~(35)のいずれかに記載の細胞。
(37)エフェクター細胞が幹細胞から分化させたものである、(32)~(35)のいずれか記載の細胞。
(38)幹細胞がiPS細胞である、(36)記載の細胞。
(39)(32)~(38)のいずれかに記載の細胞を含有する医薬組成物。
(40)癌を治療するために用いる、(39)記載の医薬組成物。
(41)CCR8を発現する腫瘍内浸潤Treg細胞の除去作用を有する、(40)記載の医薬組成物。
(42)CCR8を発現する腫瘍細胞の除去作用を有する、(40)記載の医薬組成物。
(43)(1)~(31)のいずれかに記載のキメラ抗原受容体のアミノ酸配列をコードするポリヌクレオチド。
(44)(43)記載のポリヌクレオチドを含む発現ベクター。
「CCR8に結合する細胞外領域」には、N末端にさらにシグナル配列領域を含んでいてもよい。シグナル配列領域としては、CD8Aシグナル配列領域、CD28シグナル配列領域、などが例示されるが、CD8Aシグナル配列が好ましい。CD8Aシグナル配列領域としては、配列番号の1のアミノ酸配列を有することが好ましい。
また、当該分野で公知の遺伝子操作的手法が利用可能である。例えば、Molecular Cloning, A Laboratory Manual, Forth Edition, Cold Spring Harbor Laboratory Press (2012)、Current Protocols Essential Laboratory Techniques, Current Protocols (2012)に記載された方法等が挙げられる。
CCR8とCCR8リガンドの結合阻害能については、例えば、ヒトCCL1の場合、ヒトCCR8発現293細胞を用い、ヒトCCL1添加によるCa2+流入を測定し、ヒトCCL1非添加時のシグナルを阻害率100%、ヒトCCL1添加及び抗体非添加時のシグナルを阻害率0%として、IC50値を計算することにより決定することができる。他のCCR8リガンドの結合阻害能についても、上記ヒトCCL1の場合と同様に、決定することができる。
なお、「CCR8に結合する細胞外領域」が抗CCR8-scFv領域及びシグナル配列領域を含む場合、シグナル配列領域は抗CCR8-scFv領域のN末端側に存在するのが好ましい。
1)配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域;
2)配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域;
3)配列番号35のアミノ酸配列からなるCDR1、
配列番号36のアミノ酸配列からなるCDR2及び
配列番号37のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号38のアミノ酸配列からなるCDR1、
配列番号39のアミノ酸配列からなるCDR2及び
配列番号40のアミノ酸配列からなるCDR3を含む重鎖可変領域;
4)配列番号30のアミノ酸配列からなるCDR1、
配列番号41のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号42のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号43のアミノ酸配列からなるCDR3を含む重鎖可変領域;又は
5)配列番号44のアミノ酸配列からなるCDR1、
配列番号45のアミノ酸配列からなるCDR2及び
配列番号46のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号47のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号48のアミノ酸配列からなるCDR3を含む重鎖可変領域。
6)配列番号56のアミノ酸配列からなるCDR1、
配列番号57のアミノ酸配列からなるCDR2及び
配列番号58のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号59のアミノ酸配列からなるCDR1、
配列番号60のアミノ酸配列からなるCDR2及び
配列番号61のアミノ酸配列からなるCDR3を含む重鎖可変領域
より好ましくは、以下のいずれかの配列を含む。
1)配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域;
2)配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域;又は
4)配列番号30のアミノ酸配列からなるCDR1、
配列番号41のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号42のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号43のアミノ酸配列からなるCDR3を含む重鎖可変領域。
1)配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域;
2)配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域;又は
3)配列番号16のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号17のアミノ酸配列を有する重鎖可変領域
より好ましくは、以下のいずれかの配列を含む。
1)配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域;又は
2)配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域
ここで、「膜貫通領域」にCD28膜貫通領域を含む場合、同時に細胞内領域にCD28細胞内シグナル領域も含むのが好ましい。CD28細胞内シグナル領域は配列番号7のアミノ酸配列を有するのが好ましい。
「エフェクター細胞として機能するリンパ球」としては、NK細胞、T細胞などが例示されるが、好ましくはNK細胞又はT細胞である。該シグナル伝達分子としては、4-1BB、2B4、CD3ζ、DAP10、DAP12、などが例示される。
「細胞内領域」に4-1BB細胞内シグナル領域を含む場合、当該4-1BB細胞内シグナル領域は配列番号8のアミノ酸配列を有するのが好ましい。
「細胞内領域」に2B4細胞内シグナル領域を含む場合、当該2B4細胞内シグナル領域は、配列番号9のアミノ酸配列を有するのが好ましい。
「細胞内領域」にCD3ζ細胞内シグナル領域を含む場合、当該CD3ζ細胞内シグナル領域は配列番号10のアミノ酸配列を有するのが好ましい。
「細胞内領域」にDAP10細胞内シグナル領域を含む場合、当該DAP10細胞内シグナル領域は配列番号11のアミノ酸配列を有するのが好ましい。
なお、「細胞傷害活性」には、マクロファージ等による細胞貪食活性を含むものとする。
CCR8発現細胞が腫瘍内浸潤Treg細胞の場合は、該腫瘍は固形癌が好ましい。具体的には、乳癌、子宮体癌、子宮頸癌、卵巣癌、前立腺癌、肺癌、胃(胃腺)癌、非小細胞肺癌、膵臓癌、頭頚部扁平上皮癌、食道癌、膀胱癌、メラノーマ、大腸癌、腎癌、非ホジキンリンパ腫、尿路上皮癌、肉腫、胆管癌、胆のう癌、甲状腺癌、精巣癌、胸腺癌、肝臓癌、皮膚癌、脳腫瘍、等が挙げられる。好ましくは乳癌、子宮体癌、卵巣癌、肺癌、膀胱癌、大腸癌、腎癌、肉腫、肝臓癌、皮膚癌及び脳腫瘍が挙げられる。CCR8発現細胞が腫瘍内浸潤Treg細胞の場合は、腫瘍細胞にCCR8が発現していなくてもよい。
CCR8発現細胞が腫瘍細胞の場合は、該腫瘍はメラノーマ又は血液癌が好ましい。CCR8を発現する血液癌としては、急性骨髄性白血病(AML)、急性リンパ性白血病(ALL)などの白血病や成人T細胞白血病リンパ腫(ATL)、末梢性T細胞リンパ腫(PTCL)、皮膚T細胞リンパ腫(CTCL)、びまん性大細胞型B細胞リンパ腫(DLBCL)、ホジキンリンパ腫などのリンパ腫が例示される。
該ポリヌクレオチドは、本発明のキメラ抗原受容体をコードする限り、特に限定されず、複数のデオキシリボ核酸(DNA)又はリボ核酸(RNA)等のヌクレオチドからなる重合体である。天然以外の塩基を含んでいてよい。本発明のポリヌクレオチドは、本発明のキメラ抗原受容体及び該キメラ抗原受容体を発現するエフェクター細胞を遺伝子工学的な手法により製造するために使用することができる。
WO2020/138489号公報の実施例1~2、4~7及び9記載の方法に基づき、抗ヒトCCR8抗体を得た。得られた抗体の各可変領域のアミノ酸配列を図1に、軽鎖可変領域、重鎖可変領域及びCDRの配列番号を表1に示す。
ここで、αCCR8-1及びαCCR8-2はヒト化モノクローナル抗体、αCCR8-3~6はマウス由来モノクローナル抗体、αCCR8-7はラット由来モノクローナル抗体である。
試験例において得られた抗ヒトCCR8抗体における軽鎖可変領域及び重鎖可変領域のアミノ酸配列を元にして、抗CCR8-CAR搭載ベクターを構築した。抗CCR8-CARの構造を図2に示す。
CCR8に結合する細胞外領域としては、N末端側より、CD8Aシグナル配列領域(配列番号1)、抗CCR8-scFv領域(抗ヒトCCR8抗体重鎖可変領域、G4Sペプチドリンカー(配列番号2)及び抗ヒトCCR8抗体軽鎖可変領域をこの順に連結)、スペーサー配列領域(Flagエピトープ含有配列領域(配列番号3)とCD8Aヒンジ領域(配列番号4)をこの順に連結)の順に連結した。
膜貫通領域は、上記CCR8に結合する細胞外領域のC末端側に連結した。膜貫通領域としては、CD8A膜貫通領域(配列番号5)又はCD28膜貫通領域(配列番号6)を用いた。
細胞内領域は、上記膜貫通領域のC末端側に連結した。細胞内領域としては、4-1BB細胞内シグナル領域(配列番号8)、2B4細胞内シグナル領域(配列番号9)、CD3ζ細胞内シグナル領域(配列番号10)及びDAP10細胞内シグナル領域(配列番号11)のうち複数の細胞内シグナル領域を連結して用いた。
なお、膜貫通領域にCD28膜貫通領域を含む場合は、同時に細胞内領域のN末端側にCD28細胞内シグナル領域(配列番号7)を挿入した。
上記抗CCR8-CARをコードするDNAを、ファスマック社におけるカスタム合成により作成した。該DNAをpUC19ベクターのSmaI部位に、EcoRI部位が遺伝子の上流となる向きでクローニングした。該クローニングベクターを鋳型として、PCR法によりpUC19-T7プライマー(配列番号50)とpUC19-R1プライマー(配列番号51)を用いて、抗CCR8-CAR遺伝子領域を増幅した。PCR条件としては、鋳型DNAを10ng用い、KODポリメラーゼを用いて94℃→65℃→72℃のサイクルを35サイクル行うことにより目的領域を増幅した。増幅した抗CCR8-CAR遺伝子のDNA量を定量し、iScribe T7 ARCA mRNA kit(NEW ENGLAND BioLabs社製)を用い、キット記載の方法に従ってmRNAを合成し、ポリAを付加して抗CCR8-CAR mRNAとした。
なお、陰性コントロールとしては、以下に記載のプライマー(T7-AMP-F2プライマー(配列番号52)、AMP-R1プライマー(配列番号53)を用いて、pUC19プラスミドを鋳型としてアンピシリン遺伝子の反対鎖をPCRにより増幅した。増幅したDNAよりmRNAを上記と同様に合成し、ポリA付加して陰性コントロールmRNAとした。
ヒトNK細胞株であるKHYG-1細胞を、ヒトIL-2(PeproTech社)を最終濃度で100ユニット/mlになるように添加したRPMI1640/10%FCSを用いて、抗生物質存在下、37℃、5%CO2で培養した。細胞濃度が2~3×105/mlになった時点でmRNA導入に使用した。
KHYG-1細胞を2回OPTI-MEMで洗浄し、2.5×106個/100μlになるように懸濁した。各mRNAは3μl(6μg)を100μlの細胞溶液の入ったNEPA21専用キュベット(2mmギャップ)に添加し、以下の条件でNEPA21エレクトロポレーション(ネッパジーン社)を行った。導入条件はプロトコル記載の方法に従った。mRNA導入後の細胞を、16~18時間、37℃、5%CO2で培養した。
該形質転換細胞は、mRNA導入後20時間以内に、標的細胞となるヒトCCR8発現細胞に対する細胞傷害活性評価に使用した。
(1)ヒトCCR8を発現するRat-1細胞及びRAMOS細胞の作製
pQCXIP(又はpQEF、pQCXIPベクターのCMVプロモーターをヒトEFプロモーターに変換したベクター)ベクターにヒトCCR8(配列番号49)を定法でクローニングし、該発現ヒトCCR8発現ベクターを、リポフェクタミン3000を使用して定法通りRat-1細胞(pQCXIPベクター)及びRAMOS細胞(pQEFベクター)に形質転換し、2日後よりピューロマイシンを1μg/ml添加し、形質転換細胞の選択を実施した。細胞のヒトCCR8発現率は、PE標識された抗ヒトCCR8抗体(クローン名:L263G8、BioLegend社)を用いてフローサイトメーターを用いて確認した。
RAMOS細胞の場合は、薬剤選択後にさらに単一細胞になるように細胞クローニングを行なった。ヒトCCR8の発現確認は、上記と同様にPE標識された抗ヒトCCR8抗体を用い、フローサイトメーターを用いて確認した。また細胞あたりのヒトCCR8の発現分子数はQIFIKIT(DAKO社)を用いて、キット記載の方法に従い決定した。
標識するヒトCCR8発現細胞を5×105個/mlとなるようにDMEM/10%FCS培地に懸濁し、DMSOに溶解したカルセイン(Calcein-AM、349-07201、DOJINDO社)を終濃度4μg/mlになるように添加した。DMSOの最終濃度は8%以下となるように調整した。細胞培養液を懸濁後、37℃、5%CO2で30分間培養した。その後、細胞を細胞傷害活性評価バッファー(DMEM/4%FCS)で2回洗浄し、細胞傷害活性評価に使用した。
KHYG-1細胞に、実施例1の方法で作成した、抗CCR8抗体のうちαCCR8-1、αCCR8-2の可変領域を含むCCR8に結合する細胞外領域、CD8A膜貫通領域及び2B4細胞内シグナル領域とCD3ζ細胞内シグナル領域を連結した細胞内領域を連結した抗CCR8-CAR(それぞれ、αCCR8-1-CAR、αCCR8-2-CARとする)のmRNAを導入した。コントロールとしては、実施例1の方法で作成した陰性コントロールmRNAを導入した。
KHYG-1細胞を形質転換した20時間後に、標的細胞として実施例3で作成したカルセイン標識したヒトCCR8発現Rat-1細胞又は親細胞であるRat-1細胞と、形質転換KHYG-1細胞と標的細胞の比率(E/T細胞比率)が3:1になるように、細胞傷害活性評価バッファー160μl中に混合した。
細胞傷害活性は96ウエル丸底皿を使用して評価した。形質転換KHYG-1細胞と標的細胞を混合した後で、プレート遠心機を用いて1000rpmで2分間遠心し、細胞を沈下させた。細胞を混合後、37℃、5%CO2で2時間培養した。その後、プレートより細胞上清60μlをゆっくり分取した。分取した上清は、30分以内に蛍光測定器を用いて上清中に含まれる蛍光量を定量した。
各標的細胞からのカルセインの自然放出量(BG)は、各標的細胞のみのウエルの上清を同様に測定することで決定した。また全カルセイン放出量(TOTAL)はトリトンX-100が0.1%になるように、各標的細胞のみのウエルに1%トリトンX-100を添加し、同等量の上清を測定することで決定した。
殺傷された標的細胞から放出されたカルセイン量を指標にした細胞傷害活性の計算方法は、
{(各ウエルの蛍光値―BG蛍光値)/((TOTAL蛍光値―BG蛍光値)}×100(%)
で算出した。自然放出を差し引いて、全細胞が殺傷された場合(この場合トリトンによる細胞完全溶解により上清に放出された蛍光量)の上清中蛍光量を100%として、殺傷率(%)を算出した。
その結果を図3に示す。親細胞のRat-1細胞及びヒトCCR8発現Rat-1細胞に陰性コントロールmRNA導入KHYG-1細胞を混合した場合の殺傷率を1として、それに対する殺傷倍率でαCCR8-1-CAR発現KHYG-1細胞及びαCCR8-2-CAR発現KHYG-1細胞の殺傷率を示した。陰性コントロールmRNAを形質転換した場合と比較して、αCCR8-1-CAR又はαCCR8-2-CARを形質転換した細胞では、ヒトCCR8発現細胞依存的かつ抗CCR8-CAR発現細胞特異的な細胞傷害活性が認められた。一方、ヒトCCR8を発現していない親細胞(Rat-1)に対しては、抗CCR8-CARによる特異的細胞傷害活性は認められなかった。
KHYG-1細胞に、実施例1の方法で作成した、表2の各抗CCR8-CARのmRNAを導入した。コントロールとしては、実施例1の方法で作成した陰性コントロールmRNAを導入した。
KHYG-1細胞を形質転換した18時間後に、各抗CCR8-CARの細胞表面上での発現率を、抗FLAG抗体(PE-anti-DYKDDDDK Tag Clone:1.5、BioLegend社)を用いて定量した。細胞をLIVE/DEAD(サーモフィッシャー社)及び抗FLAG抗体により、1時間室温で染色し、細胞傷害活性評価用バッファーで1回洗浄後、フローサイトメーターにより抗CCR8-CARの細胞表面発現率を評価した。解析方法はLIVE/DEADにより生細胞にゲートを行い、その後FSC/SSCで主要な細胞集団領域にゲートを行ない、この細胞集団についてFLAGの発現量を解析した。コントロール(AMP)形質転換細胞でFLAG陽性率が1~2%程度になる値に閾値を設定し、それ以上の細胞比率を発現率として算出した。次に各抗CCR8-CARの発現率を揃えるため、最も発現率の低い細胞の発現率に合わせた。具体的にはコントロールの形質転換細胞と抗CCR8-CAR形質転換細胞をある比率で混合し、抗CCR8-CARの発現率が約30%になるように調整した。どの細胞の抗CCR8-CARの発現率は30%前後となっている。この調整されたKHYG-1細胞を用いて細胞傷害活性評価を行った。
形質転換KHYG-1細胞とカルセイン標識したヒトCCR8発現Rat-1細胞を、E/T細胞比率が10:1、3:1及び1:1となる条件で混合し、カルセイン放出評価を行なった。カルセイン放出評価は、実施例4と同様の方法で実施した。実際の細胞数は10:1の場合は150μlの細胞傷害活性評価バッファー中にエフェクター細胞が3×104個、標的細胞が3×103個である。
この実験において膜貫通領域(CD8AまたはCD28)のみを変更し、他の領域が全く同じ組み合わせについて、KHYG-1細胞での細胞傷害活性はいずれもCD8Aの膜貫通領域の配列が優れていることが判明した。
同一の膜貫通領域の抗CCR8-CAR間の比較では、膜貫通領域がCD8AとCD28のどちらの場合でも4-1BBの細胞内シグナル領域を持つキメラ受容体が最も活性が高いことから、KHYG-1細胞においては4-1BBとCD3ζの細胞内シグナル領域の組み合わせが優れていることが判明した。
以上より抗CCR8-CARを発現するNK細胞においては、CD8Aの膜貫通領域と4-1BB-CD3ζの細胞内シグナル領域の組み合わせが最も細胞傷害活性が強いことが判明した。
実施例5と同様の方法で、表3の各抗CCR8-CAR発現KHYG-1細胞の細胞傷害活性を評価した。なお、E/T細胞比率が27:1及び3:1となる条件で行った。
その結果を図5に示す。E/T細胞比率が27:1の場合、αCCR8-2-CAR-12発現KHYG-1細胞はαCCR8-2-CAR-2発現KHYG-1細胞と比較して有意に1.2倍程度殺傷活性が上昇していることが判明した(Tukey多重比較検定でP<0.05)。
以上の結果から、αCCR8-2-CAR-2の細胞内領域にDAP10を付加することで、αCCR8-2-CAR-2発現KHYG-1細胞よりも強い細胞傷害活性が得られることが判明した。
実施例5と同様の方法で、表4の各抗CCR8-CAR発現KHYG-1細胞の細胞傷害活性を評価した。なお、抗CCR8-CARの発現率が約40%になるように調整し、E/T細胞比率が10:1、3:1及び1:1となる条件で行った。
その結果を図6Aに示す。E/T細胞比率が10:1及び3:1の場合に、Tukey多重比較検定を実施し、αCCR8-2-CAR-12それ以外の抗CCR8-CARとの間に、どちらのE/T細胞比率の場合でもP<0.05で有意差があった。3者間でもっとも殺傷活性の優れた抗CCR8-CARは、αCCR8-2-CAR-12であることが判明した。
さらに、同じ細胞傷害活性評価の実験を26時間実施し、26時間後に培養上清に分泌されたインターフェロン-γの量をIFN-γ ELISAキット(BPS社)で測定したところ、表4の各抗CCR8-CAR発現KHYG-1細胞の培養上清において、インターフェロン-γの有意な分泌上昇が認められた(図6B、Student t-test、P<0.001)。
実施例5と同様の方法で、抗CCR8-scFv領域の検討を行った。抗ヒトCCR8抗体の可変領域アミノ酸配列より細胞外領域の抗原結合領域のみ異なる、表5に示す6種類の抗CCR8-CARを設計した。抗体領域以外は全て同じ配列であり、膜貫通領域はCD8A膜貫通領域、細胞内領域は全て2B4-CD3ζ細胞内シグナル領域を用いた。なお、抗CCR8-CARの発現率が36%になるように調整し、E/T細胞比率が10:1、3:1及び1:1となる条件で行った。
その結果を図7に示す。αCCR8-2-CAR発現KHYG-1細胞が最も細胞傷害活性が強く、次にαCCR8-1-CAR発現KHYG-1細胞の活性が強かった。Tukeyの多重検定により、αCCR8-2-CAR発現KHYG-1細胞は、それ以外の抗CCR8-CAR発現KHYG-1細胞全てに対して有意差があった。αCCR8-1-CAR発現KHYG-1細胞は、E/T細胞比率が10:1と3:1の条件において、αCCR8-2-CAR発現KHYG-1細胞以外の抗CCR8-CAR発現KHYG-1細胞全てに対して有意に高かった。
KHYG-1細胞に、実施例1の方法で作成した、抗CCR8抗体のうちαCCR8-7の可変領域を含むCCR8に結合する細胞外領域、CD8A膜貫通領域及び2B4細胞内シグナル領域とCD3ζ細胞内シグナル領域を連結した細胞内領域を連結した抗CCR8-CAR(αCCR8-7-CARとする)のmRNAを導入した。コントロールとしては、実施例1の方法で作成した陰性コントロールmRNAを導入した。
αCCR8-7-CAR発現KHYG-1細胞について、上記(1)と同様の方法で細胞傷害活性を評価した(図10)。なお、E/T細胞比率が9:1、3:1、1:1及び0.3:1となる条件で行った。
実施例3で作成したヒトCCR8発現RAMOS細胞(C5細胞及びC6細胞)及び親細胞RAMOS細胞について、ヒトCCR8発現量を実施例3の方法でフローサイトメーターを用いて確認した。さらに細胞あたりのおおよその細胞表面上の平均のヒトCCR8発現分子数を、QIFIKIT(DAKO社)及び抗ヒトCCR8抗体(未標識L263G8, BioLegend社)を用いてプロトコルに従い決定した。
その結果、C5細胞が2630分子/細胞、C6細胞が5950分子/細胞であった。また、ヒト肺癌のCCR8発現腫瘍内浸潤Treg細胞の細胞表面上の平均CCR8発現分子数を同様の方法で評価し、約4000分子/細胞であったことから、C5細胞及びC6細胞がCCR8発現腫瘍内浸潤Treg細胞と同程度のCCR8を発現していることが確認できた。
このヒトCCR8発現RAMOS細胞であるC5細胞及びC6細胞を標的細胞として使用し、実施例5と同様の方法でαCCR8-1-CAR発現KHYG-1細胞及びαCCR8-2-CAR発現KHYG-1細胞の細胞傷害活性を評価した(図8)。なお、E/T細胞比率が10:1及び3:1となる条件で行った。
その結果、C5細胞及びC6細胞の両方の細胞で、E/T細胞比率10:1及び3:1の両方の比率において、αCCR8-2-CAR発現KHYG-1細胞はαCCR8-1-CAR発現KHYG-1細胞及び陰性コントロールと比較して、有意に細胞傷害活性が上昇した(Tukey多重比較検定でいずれの対でもP<0.05)。αCCR8-1-CAR発現KHYG-1細胞の場合は、C5およびC6細胞の両方で、E/T細胞比率10:1の場合にのみ、陰性コントロールと比較して有意に細胞傷害活性が上昇した(Tukey多重比較検定でどちらの細胞でもP<0.05)。
以上より、αCCR8-1-CAR発現KHYG-1細胞及びαCCR8-2-CAR発現KHYG-1細胞は、CCR8発現腫瘍内浸潤Treg細胞が細胞表面に発現しているCCR8発現量と同程度あるいは50%程度の発現量の細胞に対しても、十分な細胞傷害活性を保持することが判明した。
(1)ヒトTreg細胞の調製
末梢血よりフィコールパック(Ficoll-Paque PLUS, GEW Healthcare社)を用いてPBMCを分離した。そこからCD4+ T Cell Isolation Kit human (Miltenyi Biotec)を用いてCD4+T細胞を精製した。その後FITC標識抗ヒトCD4抗体、PerCP_Cy5.5標識抗ヒトCD45RA抗体、PECy7標識抗ヒトCD25抗体、Bv510標識抗ヒトCD127抗体を用いて、FACS_Ariaのソート機能により、CD45RA+CD127-CD4+CD25+細胞をソーティング(FACS Aria、BD社)した。このソーティングによりヒトTreg細胞を分離した。
分離したTreg細胞をDynabeads Human Treg Expander (Veritas社)をプロトコルに従い添加し、プロトコルに従った方法で、RPMI1640/10%FCS培地を用いて37℃、5%CO2で20日間培養した。培養したTreg細胞の細胞表面上のヒトCCR8タンパク質の発現率を、実施例3の方法でフローサイトメーターにより検出した結果、陽性率は約20%であることが判明した。
(1)で得られたTreg細胞をターゲット細胞とし、エフェクター細胞としてαCCR8-2-CAR mRNAまたは陰性コントロールmRNAを形質転換したKHYG-1細胞を用いた。実施例5と同様の方法で抗FLAG抗体を用いて、形質転換後24時間でのKHYG-1細胞表面上のαCCR8-2-CAR発現率を算定したところ、70%であった。
細胞傷害活性評価は、形質転換後7時間で、96ウエル丸底プレートを用いて評価を開始し、16時間実施した。細胞傷害活性評価は、E/T細胞比率を10:1、3:1、1:1の比率で細胞を混合することで開始した。96ウエル中の各細胞数は、ターゲットのTreg細胞は全て1×104個/wellとした。形質転換したKHYG-1細胞はE/T細胞比率に従って1×105~1×104個とした。活性評価時の培養液はRPMI1640/10%FCSをもちいた。
評価開始16時間後に、Fc受容体のブロックを行うため、各ウエルにHuman TruStain FcX(BioLegend社)を1/20量添加し、15分間室温に静置した。その後HBSS/2%FCS/10mM HEPESバッファーで1回洗浄を行い、100μlの同バッファーに懸濁した後、LIVE/DEAD(サーモフィッシャー社)、Bv421標識抗ヒトCD4(BD Bioscience社)、PE標識抗ヒトCCR8(BioLegend社)またはPE標識アイソタイプコントロール抗体(BioLegend社)を用いて4℃で1時間染色した。アイソタイプ抗体の染色に用いた細胞は、Treg細胞と形質転換KHYG-1細胞をE/T細胞比率3:1で混合した細胞を用いた。また、図9Aの細胞傷害活性を評価した図は、全て10:1で混合した細胞の例である。16時間培養後に同じバッファーで2回洗浄し、フローサイトメーターにより生存しているヒトCCR8発現細胞率を算出することで解析した。ヒトCCR8発現細胞の同定はアイソタイプコントロール抗体でバックグラウンドの閾値を設定し、抗CCR8染色で、その閾値よりも蛍光強度の強い細胞を発現細胞とした。生存しているヒトCCR8発現細胞の同定は、CD4発現細胞をゲートして、それにLIVE/DEAD染色で陰性画分にゲートし、さらにその中でCCR8陽性画分を、生存しているヒトCCR8発現細胞とした。またターゲット細胞のみを入れたウエルでのヒトCCR8発現細胞率を100%として算出した。解析したフローサイトメーターの解析例を図9Aで示す。
その結果、図9Bのように、陰性コントロールmRNAを形質転換した細胞では、どのE/T細胞比率においても殺傷率は約15%であったが、αCCR8-2-CARを形質転換したKHYG-1細胞は、全てのE/T細胞比率において、殺傷率は80%以上であり、Controlと比較して、有意な殺傷率の上昇が認められた。(Student t-testで全てのE/T細胞比率でP<0.001)。
以上より、αCCR8-2-CARを発現させたNK細胞は、ヒトCCR8を発現するTreg細胞に対して細胞傷害活性を有することが確認できた。
(1)ヒトCCR8を発現する細胞株の同定
ATL患者由来細胞株ATN-1細胞ならびにALL患者由来細胞株TALL-1細胞及びCCRF-HSB2細胞(全て理研)を、実施例9と同様の方法でPE標識抗ヒトCCR8抗体(PE標識L263G8、BioLegend社)を用いて最終濃度4μg/mlでプロトコルに従い染色した。フローサイトメーター解析により両細胞とも陽性シグナルを検出した。この陽性シグナルは、染色開始5分前に表1に示される抗CCR8抗体であるαCCR8-1抗体を250倍の濃度(1mg/ml)で添加した場合、陽性シグナルは完全に消失された。
以上より、これら細胞の陽性シグナルはヒトCCR8の発現であることが確認され、これら細胞でヒトCCR8が発現していることが判明した。
(2)末梢血由来NK細胞を用いた細胞傷害活性評価
ヒト末梢血由来NK細胞(バイオセラピー研究所)を、RPMI1640/10%FCS/Nonessential amino acid+ヒトIL-2(200U/ml)(Peprotech)で2週間培養し増殖させた。培養後のCD16陽性率をPE-抗ヒトCD16抗体(3G8、BioLegend)及びFITC-抗ヒトCD56抗体(HCD56、BioLegend)により染色した結果、アイソタイプ抗体染色(MOPC21、BioLegend)との比較で、両マーカーともに陽性の細胞比率は95%以上であった。実施例2及び4に従い、この細胞にNEPA21を用いてαCCR8-2-CAR-2 mRNAを5μg形質転換した。形質転換16時間後のCAR陽性率は10.6%であった。この細胞を用い、ヒトCCR8発現Rat-1細胞をターゲット細胞として、カルセイン法により細胞傷害活性評価を行なった。
その結果、親細胞の未処理の末梢血由来NK細胞や、コントロールのAmp mRNA導入末梢血由来NK細胞と比較して、αCCR8-2-CAR-2を発現する末梢血由来NK細胞は、ヒトCCR8発現Rat-1細胞に対する細胞傷害活性が有意に上昇した(図11)。
以上より、末梢血由来NK細胞にαCCR8-2-CAR-2 mRNAを発現させることで、ヒトCCR8発現Rat-1細胞に対して細胞傷害活性を示すことが判明した。
次に、ALL細胞株TALL-1細胞に対して同様の方法で細胞傷害活性を評価した。その結果、ヒトCCR8発現Rat-1細胞と同様に、末梢血由来NK細胞にαCCR8-2-CAR-2 mRNAを発現させることで、ALL細胞株TALL-1細胞を殺傷できることが判明した。
(1)ヒト末梢血からのT細胞の分離
健常人より末梢血を分離し、ヒトCD8陽性T細胞単離キット(MojoSort、BioLegend)を用いて、T細胞を分離した。この細胞をRPMI1640/10%FCS/Nonessential amino acid+Dynabeads human T-Activator CD3/CD28(GIBCO)+ヒトIL-2(200U/ml)(PeproTech)存在下で2週間培養した。2週間培養後のT細胞集団の各T細胞サブセットについて、各種抗体を用いてフローサイトメーターにより評価した。検出抗体としては、PE-CD16(B73.1、BioLegend)、APC-CD8(SK1、BioLegend)、FITC-CD4(OKT4、BioLegend)、Bv421-CD3(SK7、BioLegend)、LIVE/DEAD(APC-Cy7)、アイソタイプ抗体(MOPC-21、BioLegend)を用いた。
その結果、CD3陽性、CD4陽性、CD8陽性、CD16陽性細胞の各比率はそれぞれ99.6%、51.6%、41.6%、0.5%であり、ほとんどがCD3陽性T細胞であり、CD16陽性のNK細胞はほとんど認められなかった。
(2)末梢血由来T細胞を用いた細胞傷害活性評価
実施例2及び4に従い、この細胞にNEPA21を用いてαCCR8-2-CAR-2又はαCCR8-2-CAR-12のmRNAを5μg形質転換した。実施例2及び4に従い、形質転換16時間後に、カルセイン標識したATL細胞株であるATN-1細胞をターゲット細胞として、細胞傷害活性評価を実施した。なお、形質転換16時間後の末梢血由来T細胞におけるαCCR8-2-CAR-2及びαCCR8-2-CAR-12のCAR陽性率は、それぞれ5.9%、3.3%であった。
その結果、陰性コントロールであるAmp mRNA導入末梢血由来T細胞と比較して、αCCR8-2-CAR-2又はαCCR8-2-CAR-12を発現する末梢血由来T細胞では、どちらも有意に同程度の細胞傷害活性があることが判明した(図12)。
次に同様の方法で、E/T細胞比率を5:1に設定し、ヒトALL細胞株であるTALL-1細胞及びCCRF-HSB2細胞に対するαCCR8-2-CAR-2又はαCCR8-2-CAR-12を発現する末梢血由来T細胞の細胞傷害活性をカルセイン法で評価した。
その結果、TALL-1細胞に対して、αCCR8-2-CAR-2又はαCCR8-2-CAR-12を発現する末梢血由来T細胞は、陰性コントロールのAmp導入末梢血由来T細胞と比較して、有意な細胞傷害活性の増大が認められた(図13)。CCRF-HSB2細胞についても、同様に細胞傷害活性の増大傾向が認められた。
(1)ルシフェラーゼ遺伝子発現ATN-1細胞(Luc/ATN-1細胞)の作製
pGL3ベクター(Clontech社)のSalI/BglII断片をpMEI-5-NeoベクターのSalI/BglII部位に挿入することで、ホタルルシフェラーゼ遺伝子を発現するベクターを作成した(pMEI5-FLuc)。この発現ベクターを、ATN-1細胞にNEPA21を用いてエレクトロポレーションにより形質導入した。導入3日後よりG418で薬剤選択を実施した。薬剤耐性細胞のルシフェラーゼ活性をGlo kit(Promega)を用いて評価し、ルシフェラーゼ遺伝子が発現していることを確認した。
(2)レトロウイルスによるKHYG-1細胞への抗CCR8-CAR遺伝子の導入
αCCR8-2-CAR-12-IRES-Puromycinの遺伝子断片(配列番号62)をレトロウイルスベクターであるpMEI-5ベクターのNotI部位に挿入し、αCCR8-2-CAR-12-IRES-Puromysin mRNAを発現できるベクターを作成した。レトロウイルスは、293GP2細胞を用い、αCCR8-2-CAR-12レトロウイルスベクターとレトロウイルスのエンベロープタンパク質発現ベクターであるpE-Amphoアンフォトロピックenv発現ベクター(TAKARA)を同時にトランスフェクションすることで得た。得られたレトロウイルスを含む293GP2細胞上清をポリブレン(終濃度10μg/ml)とともにKHYG-1細胞に添加し、感染細胞を得た。感染3日後よりピューロマイシン(0.5μg/ml)でそのまま薬剤選択を行い、ピューロマイシン耐性細胞を得た。この細胞におけるαCCR8-2-CAR-12発現量をPPE標識抗Flag抗体で染色し、フローサイトメーターにより解析した。その結果、CAR陽性率は86%であった。
以上より、αCCR8-2-CAR-12がKHYG-1細胞表面に発現していることが判明した。
αCCR8-2-CAR-12発現KHYG-1細胞を用いて、Luc/ATN-1細胞に対する細胞傷害活性を、実施例4と同様にカルセイン法で評価した。
その結果、αCCR8-2-CAR-12発現KHYG-1細胞は、陰性コントロールの親細胞であるKHYG-1細胞と比較して、ATN-1細胞に対して有意差のある強力な細胞傷害活性が認められた(図14)。
(3)抗CCR8抗体の阻害活性評価
αCCR8-2-CAR-12発現KHYG-1細胞によるATN-1細胞に対する傷害活性が、中和活性のある抗CCR8抗体により阻害されるか否か検討した。抗CCR8抗体としては、表1に示される抗CCR8抗体であるαCCR8-1抗体及びBioLegend社抗CCR8抗体(L263G8)を用いた。なお、陰性コントロール抗体としてシナジスを使用した。抗体濃度は全て最終納度が33nMとなるようにした。予めこれら抗体をATN-1細胞に添加しておき、その10分後にエフェクター細胞であるKHYG-1細胞又はαCCR8-2-CAR-12発現KHYG-1細胞を加えることで、細胞傷害活性評価を行った。カルセイン評価法は実施例4と同様に行った。結果の表示は抗体未添加コントロールの殺傷率を100%として表示した。
その結果、KHYG-1細胞によるATN-1細胞の細胞傷害活性は、いかなる抗体によってもほとんど抑制されなかった。一方、αCCR8-2-CAR-12発現KHYG-1細胞によるATN-1細胞に対する細胞傷害活性は、抗体未添加あるいは陰性コントロールのシナジス抗体添加群と比較して、αCCR8-1抗体及びL263G8抗体を添加した場合に、どちらも50%以上有意に抑制された。
以上の結果よりαCCR8-2-CAR-12発現KHYG-1細胞によるATN-1細胞傷害活性は、ATN-1細胞表面に発現するヒトCCR8分子を介していることが判明した(図15)。
免疫不全マウスであるSKID/Beigeマウス(メス、10週齢)の背部皮下に5×106個のヒトCCR8発現Rat-1細胞を移植した。移植後8日目に、腫瘍体積を測定し、平均値が同一になるように2群に分けた後、マウスあたり3×106個の親細胞のKHYG-1細胞又は実施例13で得られたαCCR8-2-CAR-12発現KHYG-1細胞を、移植8、13及び16日目に腫瘍内に30μl投与(N=3)した。ターゲット細胞を移植8、13、16、17及び20日目に腫瘍体積及び体重を測定した。なお、腫瘍体積(mm3)は、長径(mm)×短径(mm)×短径(mm)/2で計量した。
その結果、コントロールの親細胞であるKHYG-1細胞の投与群と比較して、αCCR8-2-CAR-12発現KHYG-1細胞の投与群では、腫瘍体積の増加率が有意に抑制された(図16)。なお、両投与群は体重変化に差が認められなかった。
(1)ヒトiPS細胞由来NK細胞の作製
ヒトiPS細胞(TkDN4-M、東京大学ステムセルバンク)を用いて、公知の方法(CancerScience、2020年、第111巻、1478-1490頁)を参考に、培養方法を最適化してiPS細胞由来のNK細胞を作製した。培養した分化細胞に対し、NK細胞特異的な表面抗原をFACS解析にて確認した。分化誘導2週間後の細胞群では、造血前駆マーカーであるCD34及びCD45の発現が確認された一方、分化誘導5週目の細胞群ではCD34の発現が低下し、一方で成熟した造血細胞にもみられるCD45の発現量が増加した。また、分化誘導5週目の細胞群は、NKマーカーであるCD56の発現率が50%以上であり、細胞傷害活性に重要なNKG2D、KIR2D等の発現も同様に確認された。
(2)抗CCR8-CAR搭載レンチウイルスベクター作製
合成したαCCR8-2-CAR-2をコードするDNAをレンチウイルスベクターpCDH-EF1-MCS(System Biosciences社)のマルチクローニングサイトへクローニングし、αCCR8-2-CAR-2レンチウイルスベクターとした。
10%FCS、Penicillin―Streptomycin(ThermoFisherScientific)を加えたDMEM培地(Sigma)を使用して37°C、5%CO2で培養したLenti―X293T細胞(タカラバイオ)を、Poly―L―Lysine(Sigma)処理したディッシュに播種した。ppPACKH1 Lentivector Packaging Kit(System Biosciences社)を用い、キット記載の方法に従ってパッケージングし、αCCR8-2-CAR-2搭載レンチウイルスベクターとした。
なお、陰性コントロールとしては、レンチウイルスベクターpCDH-EF1-MCSのみをppPACKH1 Lentivector Packaging Kit(System Biosciences社)を用い、キット記載の方法に従ってウイルスベクターを合成し、陰性コントロールウイルスベクターとした。
(3)ヒトiPS細胞由来NK細胞へのレンチウイルスベクター導入
レンチウイルス導入補助剤であるSynperonic F108(SIGMA-Aldrich社)を最終濃度1mg/mlになるように添加し、さらにαCCR8-2-CAR-2搭載レンチウイルスベクターと陰性コントロールウイルスベクターをヒトiPS細胞由来NK細胞に対して20,000vps/cellになるように添加し、およそ24時間、37℃、5%CO2で培養した。
該遺伝子導入細胞は、導入後およそ24時間以内に、標的細胞となるヒトCCR8を発現するRat-1細胞に対する細胞傷害活性評価に使用した。
(4)ヒトiPS細胞由来NK細胞の抗CCR8-CAR発現解析
ヒトiPS細胞由来NK細胞を遺伝子導入したおよそ24時間後に、抗CCR8-CARの細胞表面上での発現率を、抗FLAG抗体(PE-anti-DYKDDDDK Tag Clone:L5、BioLegend社)を用いて定量した。細胞をZombie NIR Fiable Viability Kit(BioLegend社)、抗FLAG抗体及び抗CD56抗体(Brilliant Violet421-anti-human CD56 clone:HCD56、BioLegend社)により、1時間室温で染色し、細胞傷害活性評価用バッファー(DMEM/4%FCS)で2回洗浄後、フローサイトメーターにより抗CCR8-CARの細胞表面発現率を評価した。その結果、ヒトiPS細胞由来NK細胞の表面上にαCCR8-2-CAR-2は約20%発現していた。
(5)カルセインによるヒトCCR8発現細胞の標識
標識するヒトCCR8発現細胞を1.8×106個/3ml以下となるようにDMEM/10%FCS培地に懸濁し、DMSOに溶解したカルセイン(Calcein-AM、349-07201、DOJINDO社)を終濃度10μg/mlになるように添加した。細胞培養液を懸濁後、37℃、5%CO2にて30分間培養した。その後、細胞を細胞傷害活性評価バッファー(DMEM/4%FCS)で2回洗浄し、細胞傷害活性評価に使用した。
(6)抗CCR8-CAR導入iPS細胞由来NK細胞によるヒトCCR8発現細胞の細胞傷害活性
iPS細胞由来NK細胞への遺伝子導入の24時間後に、標的細胞として上記(5)で作製したカルセイン標識したヒトCCR8発現Rat-1細胞と、遺伝子導入iPS細胞由来NK細胞と標的細胞の比率(E/T細胞比率)が最大10:1になるように、細胞傷害活性評価バッファーに混合し、以下3倍公比のE/T細胞比率になるように段階希釈した。
細胞傷害活性はU底96ウエルを使用して評価した。遺伝子導入iPS細胞由来NK細胞と標的細胞を混合した後で、プレート遠心機を用いて2000rpmで5分間遠心し、細胞を沈下させた。細胞を混合後、37℃、5%CO2で30分間培養した。その後、プレート遠心機を用いて2000rpmで5分間遠心し、プレートより細胞上清70μlをゆっくり分取した。分取した上清は、30分以内に蛍光測定器を用いて上清中に含まれる蛍光量を定量した。
各標的細胞からのカルセインの自然放出量(BG)は、各標的細胞のみのウエルの上清を同様に測定することで決定した。また全カルセイン放出量(TOTAL)はトリトンX-100が0.1%になるように、各標的細胞のみのウエルに1%トリトンX-100を添加し、同等量の上清を測定することで決定した。
殺傷された標的細胞から放出されたカルセイン量を指標にした細胞傷害活性の計算方法は、
{(各ウエルの蛍光値―BG蛍光値)/((TOTAL蛍光値―BG蛍光値)}×100(%)
で算出した。自然放出を差し引いて、全細胞が殺傷された場合(この場合トリトンによる細胞完全溶解により上清に放出された蛍光量)の上清中蛍光量を100%として、殺傷率(%)を算出した。
その結果を図17Aに示す。陰性コントロールを遺伝子導入した場合と比較して、αCCR8-2-CAR-2を遺伝子導入したiPS細胞由来NK細胞では、ヒトCCR8発現細胞依存的かつ抗CCR8-CAR発現細胞特異的な細胞傷害活性が認められた。
(1)ヒトiPS細胞由来マクロファージへの培養
ヒトiPS細胞(TkDN4-M、東京大学ステムセルバンク)を公知の方法(Mol. Ther. Nucleic Acids、2018年、第12巻、793-804頁)を参考に最適化し、iPS細胞由来マクロファージを作製した。培養した分化細胞に対し、マクロファージ特異的な表面抗原を、FACS解析にて確認した。分化誘導36日後の細胞群では造血前駆細胞マーカーであるCD34の発現が減少しており、一方では成熟した造血細胞にもみられるCD45の発現量が増加していた。また、マクロファージ特有の表面抗原であるCD11b、CD14の発現が確認された。
(2)ヒトiPS細胞由来マクロファージへの抗CCR8-CARのmRNA導入
iPS細胞由来マクロファージをPBS(-)で洗浄後、0.05% Trypsin-EDTAを20分37℃、5%CO2で処理により回収した。回収した細胞は培地で洗浄後、5×105個/100μlになるようにP3 Primary Cell 4D-Nucleofector X Kit(Lonza)の溶液に懸濁した。αCCR8-2-CAR-2 mRNAと陰性コントロールmRNAそれぞれ0.25μl(0.5μg)をpmaxGFP Vector(Lonza)0.1μl(0.1μg)とともに100μlの細胞溶液の入ったSingle Nucleocuvette(100μl)に添加し、4D-Nucleofector装置の正常ヒトマクロファージ遺伝子導入プロトコルを選択し遺伝子導入を行った。mRNA導入後の細胞を、16~18時間、37℃、5%CO2で培養した。該形質転換細胞は、mRNA導入後20時間以内に、標的細胞となるヒトCCR8発現細胞に対する細胞傷害活性評価に使用した。
(3)ヒトCCR8発現細胞の標識
標識するヒトCCR8発現細胞をCTS TrypLE Select Enzyme(Gibco)により回収し、血清添加のないDMEM培地で1度洗浄し、1×106個/ml以下となるように血清添加のないDMEM培地に懸濁した。1000倍希釈となるようにCellTrace Far Red staining solutionを加え、緩やかに攪拌し、37℃、恒温槽にて20分間静置した。等量のDMEM/10%FCS培地を加え遠心し、iPS細胞由来マクロファージ培養培地で1回洗浄後、1.5×106個/mlとなるようにiPS細胞由来マクロファージ培養培地に懸濁し、細胞傷害活性評価に使用した。
(4)抗CCR8-CAR導入iPS細胞由来マクロファージによるヒトCCR8発現細胞の細胞傷害活性評価
iPS細胞由来マクロファージのヒトCCR8発現Rat-1細胞に対する細胞傷害活性を検証するため、細胞貪食活性を評価した。
iPS細胞由来マクロファージを形質転換した20時間後に標的細胞として上記(3)で作製したCellTrace Far Red staining solutionで染色したヒトCCR8発現Rat-1細胞と形質転換iPS細胞由来マクロファージの比率である機能細胞/標的細胞比率(E/T細胞比率)が3:1(1.5×105個:5.0×104個)になるように、iPS細胞由来マクロファージ培養培地200μl中に混合した。混合液は96ウエル丸底プレートに加え、プレート遠心機を用いて100g, 2分間延伸し、細胞を沈下させ、37℃、5%CO2で2時間培養した。その後、細胞を3000rpm、30秒遠心後に上清を除き、0.05μg/ml DAPIが入った100μlのSFEB培地/1%BSAに懸濁した。CCR8発現細胞に対する細胞貪食活性は、フローサイトメーターで検出した。
細胞貪食活性の計算方法は、
{(GFP・Far Red共陽性細胞)/(GFP陽性細胞)}×100(%)
で算出した。
その結果、陰性コントロールを遺伝子導入した細胞と比較して、αCCR8-2-CAR-2を遺伝子導入した細胞では、ヒトCCR8発現細胞依存的かつ抗CCR8-CAR発現細胞特異的な細胞傷害活性が認められた。
(2’)ヒトiPS細胞由来マクロファージへの抗CCR8-CARのプラスミド導入
iPS細胞由来マクロファージをPBS(-)で洗浄後、0.05% Trypsin-EDTAを20分37℃、5%CO2で処理により回収した。回収した細胞は培地で洗浄後、5×105個/100μlになるようにP3 Primary Cell 4D-Nucleofector X Kit(Lonza)の溶液に懸濁した。 αCCR8-2-CAR-2ベクター0.2μl(0.5ug)をpmaxGFP Vector(Lonza)0.1μl(0.1μg)とともに100μlの細胞溶液の入ったSingle Nucleocuvette(100μl)に添加し、4D-Nucleofector装置の正常ヒトマクロファージ遺伝子導入プロトコルを選択し遺伝子導入を行った。mRNA導入後の細胞を、16~18時間、37℃、5%CO2で培養した。該形質転換細胞は、プラスミド導入後20時間以内に、標的細胞となるヒトCCR8発現細胞に対する細胞傷害活性評価に使用した。
(3’)ヒトCCR8発現細胞の標識
標識するヒトCCR8発現細胞をCTS TrypLE Select Enzyme(Gibco)により回収し、血清添加のないDMEM培地で1度洗浄し、1×106個/ml以下となるように血清添加のないDMEM培地に懸濁した。1000倍希釈となるようにCellTrace Far Red staining solutionを加え、緩やかに攪拌し、37℃、恒温槽にて20分間静置した。等量のDMEM/10%FCS培地を加え遠心し、iPS細胞由来マクロファージ培養培地で1回洗浄後、1.5×106個/mlとなるようにiPS細胞由来マクロファージ培養培地に懸濁し、細胞傷害活性評価に使用した。
(4’)抗CCR8-CAR導入iPS細胞由来マクロファージによるヒトCCR8発現細胞の細胞傷害活性評価
iPS細胞由来マクロファージのヒトCCR8発現Rat-1細胞に対する細胞傷害活性を検証するため、細胞貪食活性を評価した。
iPS細胞由来マクロファージを形質転換した20時間後に標的細胞として上記(3’)で作製したCellTrace Far Red staining solutionで染色したヒトCCR8発現Rat-1細胞と形質転換iPS細胞由来マクロファージの比率である機能細胞/標的細胞比率(E/T細胞比率)が3:1(1.5×105個:5.0×104個)になるように、iPS細胞由来マクロファージ培養培地200μl中に混合した。混合液は96ウエル丸底プレートに加え、プレート遠心機を用いて100g, 2分間延伸し、細胞を沈下させ、37℃、5%CO2で2時間培養した。その後、細胞を3000rpm、30秒遠心後に上清を除き、0.05μg/ml DAPIが入った100μlのSFEB培地/1%BSAに懸濁した。CCR8発現細胞に対する細胞貪食活性は、フローサイトメーターで検出した。
細胞貪食活性の計算方法は、αCCR8-2-CAR-2について遺伝子導入が起こったGFP陽性細胞と遺伝子導入が起こらなかったGFP陰性細胞で以下の様に算出した。
・GFP陽性細胞の細胞貪食活性:
{(GFP・Far Red共陽性細胞)/(GFP陽性細胞)}×100(%)
・GFP陰性細胞の細胞貪食活性:
{(GFP陰性・Far Red陽性細胞)/(GFP陰性細胞)}×100(%)
その結果を図17Bに示す。陰性コントロールであるGFP陰性細胞と比較して、αCCR8-2-CAR-2を遺伝子導入したGFP陽性細胞では、細胞傷害活性の有意な上昇が認められた(Student t-test、P<0.005)。
(1)ヒト卵巣癌由来腫瘍内浸潤Treg細胞の調製
ヒト卵巣癌患者由来腫瘍組織をTumor Dissociation Kit、human(Miltenyi Biotec社)を用いて分散し、Treg細胞やCD8陽性T細胞、Treg細胞以外のCD4陽性T細胞を含む細胞集団を調製した。実験には、調製直後もしくは凍結保存後の細胞を使用した。
(2)ヒト卵巣癌由来腫瘍内浸潤Treg細胞のCCR8陽性率測定
上記(1)で得られた細胞集団について、フローサイトメトリーを用いて腫瘍内浸潤Treg細胞中のCCR8陽性率を測定した。Human TruStain FcX (BioLegend社)およびZombie NIR Fixable Viability Kit(BioLegend社)を添加したHBSS/2%FCS/10mM HEPESバッファーに細胞を懸濁し、4℃に30分間静置した。洗浄後、各種抗体を添加して4℃に30分間静置した(Bv785標識抗ヒトCD45抗体、AlexaFlour700標識抗ヒトCD3抗体、Bv711標識抗ヒトCD4抗体、PE標識抗ヒトCCR8抗体、Bv605標識抗ヒトCD56抗体、Bv510標識抗ヒトCD8抗体(全てBioLegend社))。洗浄後、eBioscience Foxp3/Transcription Factor Staining Buffer Set(サーモフィッシャー社)を用いて固定および膜透過処理後、APC標識抗ヒトFoxp3(サーモフィッシャー社)を添加して4℃に30分間静置した。洗浄後、フローサイトメーターを用いて、腫瘍内浸潤Treg細胞(CD45・CD3・CD4・Foxp3陽性かつ、Zombie NIR・CD8・CD56陰性画分)中のCCR8陽性率を測定した。
(3)抗CCR8-CAR発現KHYG-1細胞によるヒト腫瘍内浸潤Treg細胞傷害活性評価
上記(1)で得られたヒト卵巣癌由来腫瘍内浸潤Treg細胞をターゲット細胞、αCCR8-2-CAR-2 mRNAまたは陰性コントロールmRNAを形質転換したKHYG-1細胞をエフェクター細胞として用いた。実施例5と同様の方法で、形質転換後20時間でのKHYG-1細胞表面上のαCCR8-2-CAR-2発現率を算定したところ、22%であった。
細胞傷害活性評価は、形質転換後20時間から44時間にかけて実施した。96ウエル丸底プレートにターゲット細胞およびエフェクター細胞を1×105個/wellずつ播種し、培養液はRPMI1640/10%FCS/1%PSを用いた。
評価開始24時間後に細胞を回収、Human TruStain FcX (BioLegend社)およびZombie NIRTM Fixable Viability Kit(Biolegend社)を添加したHBSS/2%FCS/10mM HEPESバッファーに懸濁し、4℃に30分間静置した。洗浄後、各種抗体を添加して4℃に30分間静置した(Bv785標識抗ヒトCD45抗体、AlexaFlour700標識抗ヒトCD3抗体、Bv605標識抗ヒトCD56抗体、Bv711標識抗ヒトCD4、Bv510標識抗ヒトCD8抗体(全てBiolegend社))。洗浄後、eBioscienceTM Foxp3 / Transcription Factor Staining Buffer Set(サーモフィッシャー社)を用いて固定および膜透過処理後、APC標識抗ヒトFoxp3(サーモフィッシャー社)を添加して4℃に30分間静置した。
洗浄後、フローサイトメーターを用いて、生存しているCD45陽性細胞中の腫瘍内浸潤Treg細胞(CD45・CD3・CD4・Foxp3陽性かつ、Zombie NIR・CD8・CD56陰性画分)の割合を定量した。
その結果、陰性コントロールmRNA導入細胞に比べ、αCCR8-2-CAR-2発現KHYG-1細胞では腫瘍内浸潤Treg細胞の有意な減少が認められた(図18、Student t-test、P<0.005)。また、その減少幅は腫瘍内浸潤Treg細胞のCCR8陽性率とほぼ一致した。
以上より、αCCR8-2-CAR-2発現KHYG-1細胞は、CCR8を発現する腫瘍内浸潤Treg細胞に対して強力な細胞傷害活性を有することが確認できた。
(4)抗CCR8-CARを発現する末梢血由来NK細胞によるヒト腫瘍内浸潤Treg細胞傷害活性評価
上記(1)で得られたヒト卵巣癌由来腫瘍内浸潤Treg細胞をターゲット細胞、実施例11で得られたαCCR8-2-CAR-2 mRNAまたは陰性コントロールmRNAを形質転換した末梢血由来NK細胞をエフェクター細胞として用いた。
形質転換20時間後に細胞を回収、Human TruStain FcX (BioLegend社)およびZombie NIR Fixable Viability Kit(Biolegend社)を添加したHBSS/2%FCS/10mM HEPESバッファーに懸濁し、4℃に30分間静置した。洗浄後、各種抗体を添加して4℃に30分間静置した(Bv785標識抗ヒトCD45抗体、AlexaFlour700標識抗ヒトCD3抗体、Bv605標識抗ヒトCD56抗体、PE標識抗FLAG抗体(全てBiolegend社))。洗浄後、フローサイトメーターを用いてαCCR8-2-CAR-2発現率を算定したところ、5%であった。細胞傷害活性評価は、上記(3)と同様に行った。
その結果、陰性コントロールmRNA導入末梢血由来NK細胞に比べ、αCCR8-2-CAR-2 mRNA導入末梢血由来NK細胞では腫瘍内浸潤Treg細胞の有意な減少が認められた。(図19、Student t-test、P<0.005)。また、その減少幅は、腫瘍内浸潤Treg細胞のCCR8陽性率とほぼ一致したことから、αCCR8-2-CAR-2を発現させた末梢血由来NK細胞は、CCR8を発現する腫瘍内浸潤Treg細胞に対して強力な細胞傷害活性を有することが確認できた。
実施例17(1)と同様の方法でヒト大腸癌患者由来腫瘍組織から得られた腫瘍内浸潤Treg細胞をターゲット細胞、実施例11で得られたαCCR8-2-CAR-2 mRNAまたは陰性コントロールmRNAを形質転換した末梢血由来NK細胞をエフェクター細胞として用いた。実施例17(3)と同様の方法で細胞を播種後、5日目にフローサイトメーターを用いて腫瘍内浸潤Treg細胞の存在比、および、CD8陽性T細胞と腫瘍内浸潤Treg細胞以外のCD4陽性T細胞のIFNγ産生を定量した。細胞を回収後、IFNγ検出試薬(IFN-γ Secretion Assay - Detection Kits, human、Miltenyi Biotech社)および実施例17(3)の各種標識抗体で細胞染色を行った。
フローサイトメーターを用いた測定の結果、陰性コントロールmRNA導入末梢血由来NK細胞に比べ、αCCR8-2-CAR-2 mRNA導入末梢血由来NK細胞では腫瘍内浸潤Treg細胞の有意な減少が認められた(図20、Student t-test、P<0.005)。また、CD8陽性T細胞および腫瘍内浸潤Treg細胞以外のCD4陽性T細胞のIFNγ陽性率が増加する傾向が認められた。
以上より、αCCR8-2-CAR-2を発現する末梢血由来NK細胞は、CCR8発現腫瘍内浸潤Treg細胞除去活性およびT細胞活性化効果を有することが確認できた。
ヒトIL-15をノックインしたNOGマウス(29週齢、メス)の腹腔内に、ルシフェラーゼ遺伝子を導入したATN-1細胞を1×107個(100μl)移植した。ATN-1細胞はヒトCCR8を自然発現している細胞株である。
腫瘍移植2、5日後に、αCCR8-2-CAR-12発現KHYG-1細胞投与群は実施例13で得られたαCCR8-2-CAR-12発現KHYG-1細胞を、コントロール群はKHYG-1細胞を2×106個(100μl)腹腔内に投与した(N=7)。腫瘍移植2、7、12、14日後にマウスにD-Luciferin(住商ファーマインターナショナル)PBS希釈液(15mg/ml)100μlを静脈投与し、IVIS Spectrum(PerkinElmer)を用いて、腫瘍の発光強度を測定した。発光強度は測定実数値からBack groundの測定値を差し引いて算出し、測定は、Exposure time:5秒、Binning:low、F/Stop:1の条件で行った。発光強度はROIの範囲内の総発光強度を定量化した数値であり、毎測定時ROI面積は同じになるよう設定した。発光画像の解析ソフトにはliving image○R3.2を用いた。有意差検定にはMann-Whitney U法を用いた(有意水準:P<0.05)。
その結果、腫瘍移植7、12、14日後において、αCCR8-2-CAR-12発現KHYG-1細胞投与群では、コントロール群と比較し、腫瘍の発光強度に有意な減少が見られた(図21及び22)。
以上より、αCCR8-2-CAR-12発現KHYG-1細胞は、ヒトCCR8発現腫瘍細胞移植マウスにおいて抗腫瘍活性を示すことが明らかとなった。
hCCR8-KI(KI/KI)マウス(13週齢、メス)の背部皮内にCT26細胞株を移植した。腫瘍移植7、9、10日後に、αCCR8-2-CAR-12発現KHYG-1細胞投与群には実施例13で得られたαCCR8-2-CAR-12発現KHYG-1細胞を、コントロール群にはKHYG-1細胞をそれぞれ2×106個(100μl)腹腔内に投与した(N=7)。腫瘍移植7日後より3~4日ごとに腫瘍体積を測定した。腫瘍体積(mm3)は、長径(mm)×短径(mm)×短径(mm)/2で計量し、Mann-Whitney U法による有意差検定を行った(有意水準:P<0.05)。
その結果、腫瘍移植10日後において、αCCR8-2-CAR-12発現KHYG-1細胞投与群では、コントロール群と比較し有意な腫瘍体積の減少が認められた。また腫瘍移植14日後においても減少傾向が認められた(図23、P=0.0973)。
以上より、αCCR8-2-CAR-12発現KHYG-1細胞は、大腸癌移植hCCR8-KI(KI/KI)マウスにおいて抗腫瘍活性を示すことが明らかとなった。
hCCR8-KI(KI/KI)マウス(10週齢、メス)の背部皮内にCT26細胞株を移植した。腫瘍移植8日後に、αCCR8-2-CAR-12発現KHYG-1細胞投与群には実施例13で得られたαCCR8-2-CAR-12発現KHYG-1細胞を、コントロール群にはKHYG-1細胞を2×106個(40μl)腫瘍内に投与した(N=5)。細胞投与1日後にマウスから腫瘍を回収した。腫瘍塊をハサミで細かく切断し、Tumor Dissociation Kit, mouse(Miltenyi)を用いてキットの添付プロトコルにしたがい腫瘍浸潤細胞懸濁液を調整した。細胞懸濁液をFixable Viability Dye eFluor 780試薬(eBioscience)を用いて、4℃、30分間染色した。2%FCS/10mM HEPES/HBSSで1回洗浄後、非蛍光標識抗ヒトCCR8抗体(又はisotype control抗体)4℃、30分間染色した。2%FCS/10mM HEPES/HBSSで2回洗浄後、AF647標識抗ラットIgG抗体で4℃、30分間染色した。Bv510標識抗マウスCD45、 Bv605標識抗マウスTCRβ、Bv785標識抗マウスCD4抗体で4℃、30分間染色した。2%FCS/HEPES/HBSSで洗浄後、細胞をFoxp3 / Transcription Factor Staining Buffer Set(eBioscience)を用いて添付プロトコルにしたがい固定し、PE標識抗マウスFoxP3抗体を用いて細胞内FoxP3を染色した。当該キット添付バッファーで洗浄後、フローサイトメーターを用いてT細胞(CD45+TCRβ+)をゲーティングし、腫瘍内浸潤Treg細胞(CD4+Foxp3+)中のCCR8陽性率を解析した。有意差検定にはStudent t-test法を用いた(有意水準:P<0.05)。以下に用いた染色抗体を示す。なお、抗ヒトCCR8抗体は、WO2020/138489号公報の実施例2記載の方法に基づき得られた抗体を用いた。
rat IgG2a isotype control抗体 (BioXcell)
AF647 AffiniPure F(ab') Fragment Donkey Anti-Rat IgG(H+L) (Jackson Immuno Research)
PE anti-mouse/rat FoxP3 (clone FJK-16s, eBiosciences)
Bv510 anti-mouse CD45 (clone 30-F11, Biolegend)
Bv605 anti-mouse TCRβ (clone PC61, BioLegend)
Bv421 anti-mouse CD25 (clone PC61, BioLegend)
Bv785 anti-mouse CD4 (clone RM4-5, BioLegend)
その結果、αCCR8-2-CAR-12発現KHYG-1細胞投与群では、コントロール群と比較し、腫瘍内浸潤Treg細胞中のCCR8陽性細胞の有意な減少が認められた(図24)。
以上より、αCCR8-2-CAR-12発現KHYG-1細胞は、マウス生体内においてヒトCCR8を発現する腫瘍内浸潤Treg細胞を除去する傾向を有することが明らかとなった。
Claims (44)
- CCR8に結合する細胞外領域、膜貫通領域及び細胞内領域を含み、細胞内領域にエフェクター細胞として機能するリンパ球に発現するシグナル伝達分子の細胞内シグナル領域を含むキメラ抗原受容体。
- CCR8に結合する細胞外領域に抗CCR8-scFv領域を含む、請求項1記載のキメラ抗原受容体。
- 抗CCR8-scFv領域が、
1)配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域;
2)配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域;
3)配列番号35のアミノ酸配列からなるCDR1、
配列番号36のアミノ酸配列からなるCDR2及び
配列番号37のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号38のアミノ酸配列からなるCDR1、
配列番号39のアミノ酸配列からなるCDR2及び
配列番号40のアミノ酸配列からなるCDR3を含む重鎖可変領域;
4)配列番号30のアミノ酸配列からなるCDR1、
配列番号41のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号42のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号43のアミノ酸配列からなるCDR3を含む重鎖可変領域;
5)配列番号44のアミノ酸配列からなるCDR1、
配列番号45のアミノ酸配列からなるCDR2及び
配列番号46のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号47のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号48のアミノ酸配列からなるCDR3を含む重鎖可変領域;又は
6)配列番号56のアミノ酸配列からなるCDR1、
配列番号57のアミノ酸配列からなるCDR2及び
配列番号58のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号59のアミノ酸配列からなるCDR1、
配列番号60のアミノ酸配列からなるCDR2及び
配列番号61のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、請求項2記載のキメラ抗原受容体。 - 抗CCR8-scFv領域が、
1)配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域;
2)配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域;又は
4)配列番号30のアミノ酸配列からなるCDR1、
配列番号41のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号42のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号43のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、請求項3記載のキメラ抗原受容体。 - 抗CCR8-scFv領域が、
配列番号24のアミノ酸配列からなるCDR1、
配列番号25のアミノ酸配列からなるCDR2及び
配列番号26のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号27のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号29のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、請求項4記載のキメラ抗原受容体。 - 抗CCR8-scFv領域が、
配列番号30のアミノ酸配列からなるCDR1、
配列番号31のアミノ酸配列からなるCDR2及び
配列番号32のアミノ酸配列からなるCDR3を含む軽鎖可変領域並びに
配列番号33のアミノ酸配列からなるCDR1、
配列番号28のアミノ酸配列からなるCDR2及び
配列番号34のアミノ酸配列からなるCDR3を含む重鎖可変領域
を含む、請求項4記載のキメラ抗原受容体。 - 抗CCR8-scFv領域が、
1)配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域;
2)配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域;又は
3)配列番号16のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号17のアミノ酸配列を有する重鎖可変領域;
を含む、請求項2又は3記載のキメラ抗原受容体。 - 抗CCR8-scFv領域が、
1)配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域;又は
2)配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域;
を含む、請求項7記載のキメラ抗原受容体。 - 抗CCR8-scFv領域が、
配列番号12のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号13のアミノ酸配列を有する重鎖可変領域
を含む、請求項8記載のキメラ抗原受容体。 - 抗CCR8-scFv領域が、
配列番号14のアミノ酸配列を有する軽鎖可変領域、及び、
配列番号15のアミノ酸配列を有する重鎖可変領域
を含む、請求項8記載のキメラ抗原受容体。 - 抗CCR8-scFv領域において、軽鎖可変領域と重鎖可変領域が配列番号2のリンカー配列を介して連結している、請求項2~10のいずれかに記載のキメラ抗原受容体。
- CCR8に結合する細胞外領域にCD8Aシグナル配列領域を含む、請求項2~11のいずれかに記載のキメラ抗原受容体。
- CD8Aシグナル配列領域が、抗CCR8-scFv領域のN末端側に存在する、請求項12記載のキメラ抗原受容体。
- CD8Aシグナル配列領域が、配列番号1のアミノ酸配列を有する、請求項12又は13記載のキメラ抗原受容体。
- 抗CCR8-scFv領域と膜貫通領域がスペーサー配列を介して連結している、請求項2~14のいずれかに記載のキメラ抗原受容体。
- スペーサー配列に、Flagエピトープ含有配列領域を含む、請求項15記載のキメラ抗原受容体。
- Flagエピトープ含有配列領域が、配列番号3のアミノ酸配列を有する、請求項16記載のキメラ抗原受容体。
- スペーサー配列に、CD8Aヒンジ領域を含む、請求項15~17のいずれかに記載のキメラ抗原受容体。
- CD8Aヒンジ領域が、配列番号4のアミノ酸配列を有する、請求項18記載のキメラ抗原受容体。
- 膜貫通領域にCD8A膜貫通領域を含む、請求項1~19のいずれかに記載のキメラ抗原受容体。
- CD8A膜貫通領域が、配列番号5のアミノ酸配列を有する、請求項20記載のキメラ抗原受容体。
- 膜貫通領域にCD28膜貫通領域を含み、細胞内領域にCD28細胞内シグナル領域を含む、請求項1~19のいずれかに記載のキメラ抗原受容体。
- CD28膜貫通領域が、配列番号6のアミノ酸配列を有し、CD28細胞内シグナル領域が、配列番号7のアミノ酸配列を有する、請求項22記載のキメラ抗原受容体。
- 細胞内領域に4-1BB、2B4、CD3ζ及びDAP10の細胞内シグナル領域のうち、少なくとも一つを含む、請求項1~23のいずれかに記載のキメラ抗原受容体。
- 細胞内領域に4-1BB、2B4、CD3ζ及びDAP10の細胞内シグナル領域のうち、少なくとも二つを含む、請求項24記載のキメラ抗原受容体。
- 細胞内領域に4-1BB及びCD3ζの細胞内シグナル領域を含む、請求項25記載のキメラ抗原受容体。
- 細胞内領域に2B4及びCD3ζの細胞内シグナル領域を含む、請求項25記載のキメラ抗原受容体。
- 細胞内領域に4-1BB細胞内シグナル領域を含み、当該4-1BB細胞内シグナル領域が、配列番号8のアミノ酸配列を有する、請求項24~27のいずれかに記載のキメラ抗原受容体。
- 細胞内領域に2B4細胞内シグナル領域を含み、当該2B4細胞内シグナル領域が、配列番号9のアミノ酸配列を有する、請求項24~27のいずれかに記載のキメラ抗原受容体。
- 細胞内領域にCD3ζ細胞内シグナル領域を含み、当該CD3ζ細胞内シグナル領域が、配列番号10のアミノ酸配列を有する、請求項24~27のいずれかに記載のキメラ抗原受容体。
- 細胞内領域にDAP10細胞内シグナル領域を含み、当該DAP10細胞内シグナル領域が、配列番号11のアミノ酸配列を有する、請求項24~27記載のキメラ抗原受容体。
- 請求項1~31のいずれかに記載のキメラ抗原受容体を発現しているエフェクター細胞。
- エフェクター細胞がNK細胞である、請求項32記載の細胞。
- エフェクター細胞がT細胞である、請求項32記載の細胞。
- エフェクター細胞がマクロファージである、請求項32記載の細胞。
- エフェクター細胞が末梢血由来である、請求項32~35のいずれかに記載の細胞。
- エフェクター細胞が幹細胞から分化させたものである、請求項32~35のいずれか記載の細胞。
- 幹細胞がiPS細胞である、請求項36記載の細胞。
- 請求項32~38のいずれかに記載の細胞を含有する医薬組成物。
- 癌を治療するために用いる、請求項39記載の医薬組成物。
- CCR8を発現する腫瘍内浸潤Treg細胞の除去作用を有する、請求項40記載の医薬組成物。
- CCR8を発現する腫瘍細胞の除去作用を有する、請求項40記載の医薬組成物。
- 請求項1~31のいずれかに記載のキメラ抗原受容体のアミノ酸配列をコードするポリヌクレオチド。
- 請求項43記載のポリヌクレオチドを含む発現ベクター。
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