WO2022049867A1 - がん免疫微小環境の調節物質およびそれによる予防・診断・治療的利用 - Google Patents
がん免疫微小環境の調節物質およびそれによる予防・診断・治療的利用 Download PDFInfo
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
Definitions
- the present invention relates to a regulator of a cancer immunomicroenvironment and its prophylactic / diagnostic / therapeutic use. More specifically, the present invention relates to a TCTP (translationally controlled tumor protein) function inhibitor, a therapeutic agent or therapeutic composition for cancer containing the substance, and the preventive / diagnostic / therapeutic use of the substance for cancer.
- TCTP translationally controlled tumor protein
- Non-Patent Document 1 a mechanism for suppressing an immune function that attacks cancer cells
- Non-Patent Document 2 a mechanism for suppressing an immune function that attacks cancer cells
- the cancer immune microenvironment is also called the "cancer cradle", where a mechanism that suppresses the immune response to cancer exists, creating an environment that is favorable for the growth of cancer cells.
- MDSC myeloid-derived suppressor cell
- PMN-MDSC polymorphonuclear myeloid-derived suppressor cell
- Non-Patent Document 3 A monocytic myeloid-derived suppressor cell (M-MDSC) that resembles monocytes and is known to differentiate into tumor-associated macrophage (TAM).
- TIME tumor-associated macrophage
- Non-Patent Document 3 Regarding the relationship between MDSC and cancer, in malignant tumors, the expression level of MDSC is increased and accumulated in TIME, and it is directly related to the clinical stage of cancer, the amount of metastatic tumor, and the prognosis of cancer. Since MDSC actually suppresses the activity and proliferation of lymphocytes (Non-Patent Document 4), it is considered to play a role in promoting it in the progression of tumors. However, the mechanism of MDSC accumulation in TIME has not yet been clarified.
- the present invention has a solution to identify a regulator of the cancer immunomicroenvironment and to provide a therapeutic utilization method by inhibiting the regulator.
- Non-Patent Document 12 Non-Patent Document 12
- TIME dead cells molecules released from dead tumor cells
- TIME dead cells As a result of diligent research after formulating a working hypothesis, the Translationally controlled tumor protein (TCTP) released from dead tumor cells is a novel immunomodulator that controls the function and dynamics of MDSC during TIME. was found for the first time.
- TCTP Translationally controlled tumor protein
- the present inventors have shown that extracellular TCTP induces the expression of CXCL1 family chemokines mainly by acting on M-MDSC.
- the CXCL1 family chemokines attract PMN-MDSC to TIME, making TIME highly immunosuppressive.
- administration of an anti-TCTP monoclonal antibody or TCTP function inhibitor inhibits the induction of PMN-MDSC to TIME and suppresses tumor growth.
- the present invention will be completed by clarifying the unsolved problem of how tumors construct and control TIME by identifying TCTP and elucidating its new function, and developing a method for inhibiting it. I arrived.
- the present invention is the following (1) to (13).
- suppressor cell (MDSC) accumulation inhibitor a substance that suppresses or inhibits the function of immunoregulatory factors released from dead tumor cells as an active ingredient.
- PMN-MDSC polymorphonuclear myeloid-derived suppressor cell
- TCTP translationally controlled tumor protein
- a therapeutic agent or composition for treating cancer which comprises the inhibitor according to any one of (1) to (5) above as an active ingredient.
- the therapeutic agent or therapeutic composition according to (6) above, wherein the cancer is colorectal cancer, malignant melanoma or fibrosarcoma.
- the diagnostic method or diagnostic assisting method for cancer which comprises measuring the amount of TCTP mRNA or TCTP protein present in a sample derived from a subject.
- the method according to (8) above, wherein the sample is blood or tissue.
- Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 2 Heavy chain CDR3, which comprises the amino acid sequence represented by SEQ ID NO: 3.
- Light chain CDR1, comprising the amino acid sequence represented by SEQ ID NO: 4. It has a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 5 and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 6.
- C Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 13.
- Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16. It has a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 17, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 18.
- the present invention provides a novel therapeutic agent for cancer and a novel therapeutic method for cancer.
- TCTP WT B16F10 cells TCTP-expressing B16F10 cells
- TCTP KO B16F10 cells TCTP-deficient B16F10 cells (1 ⁇ 10 5 cells, respectively
- Meth-A cells TCTP WT Meth-A-induced sarcoma cells
- Meth-A KO Meth-A cells TCTP KO Meth-A cells
- (A) Induced apoptosis (serum-free and adriamycin) or necrosis (freeze-thaw) in SL4 cells. The culture supernatant was collected and the amount of TCTP protein was measured by immunoblotting (n 3).
- C The protein expression of TCTP in wild-type (WT) or TCTP gene-inactive SL4 cells, B16F10 cells and Meth-A cells was investigated. Whole cytolysis of each cell line was prepared and immunoblotting analysis was performed for TCTP and ⁇ -actin.
- E TCTP WT SL4 cells and TCTP KO SL4 cells (2 ⁇ 10 5 cells) in standard state (20% O 2 , 10% FBS), low serum state (0% O 2 , 1% FBS) or low oxygen. The cells were cultured in the state (1% O 2 , 10% FBS).
- TCTP KO SL4 cells (Mock) or IL-2ss-TCTP SL4 cells (2 ⁇ 10 5 cells) were subcutaneously transplanted into C57BL / 6 mice. Twenty-one days after transplantation, the tumor was resected and a whole lysate of the tumor was prepared.
- TCTP KO SL4 cells left figure
- WT-TCTP cells TCTP KO cells expressing WT TCTP cDNA
- IL-2ss-TCTP SL4 cells IL-2ss-TCTP cDNA
- TCTP-secreting cells TCTP and nuclei (DAPI) in the right figure
- DAPI nuclei
- B PEC (2 ⁇ 10 5 cells) was stimulated with TCTP KO SL4 cell culture supernatant (Mock) or IL-2SS-TCTP SL4 cell culture supernatant (IL2ss-TCTP) for 2 hours.
- C WT SL4 cells or TCTP KO SL4 cells (2 ⁇ 10 5 cells) were subcutaneously transplanted into C57BL / 6 mice. Twenty-one days after transplantation, the tumor was resected and a whole lysate of the tumor was prepared. The protein levels of G-CSF (left) and GM-CSF (right) in the prepared lysates were measured using the cytometric bead assay (CBA).
- TCTP WT B16F10 cells (WT) or TCTP KO B16F10 cells (KO) were subcutaneously transplanted into C57BL / 6 mice.
- TCTP KO SL4 cells and IL-2ss-TCTP SL4 cells (2 ⁇ 10 5 cells) were subcutaneously transplanted into C57BL / 6 mice, respectively.
- Tumor volume was measured over time.
- the abundance ratio of PMN-MDSC in the CD45 + cell population is shown in%.
- the mean fluorescence intensity (MFI) of the markers (CD69, CD107a) that are indicators of NK cell activation was determined.
- MFI mean fluorescence intensity
- repeated measures one-way ANOVA with Tukey's multiple comparisons test. NS indicates no significant difference.
- Data are presented as mean ⁇ standard error (sem) of values. Examination of the immunosuppressive function of TCTP during TIME.
- A, b) WT SL4 cells or TCTP KO SL4 cells (2 ⁇ 10 5 cells) were subcutaneously transplanted into C57BL / 6 mice.
- SL4 cells (2 ⁇ 10 5 cells) were subcutaneously transplanted into C57BL / 6 mice.
- E WT SL4 cells or TCTP KO SL4 cells (2 ⁇ 10 5 cells) were administered to WT mice (2 ⁇ 10 5 cells).
- HEK293T cells that transiently express TLR2-YFP and Flag-TCTP were immunoprecipitated with an anti-GFP antibody and then analyzed with an anti-Flag antibody (upper figure) to detect TLR2 and TCTP in whole cytolysis. ( Figure below).
- F HEK293T cells were transfected with a luciferase reporter construct containing a multimerized NK ⁇ B binding motif and expression vectors for various proteins.
- HKE293T cells (2 ⁇ 10 4 cells) were seeded in 96-well plates and recombinant TCTP or agonists for each TLR (Pam3CSK4 300 ng / ml, poly I: C 100 ⁇ g / ml, poly U 10 ⁇ g). Stimulated with / ml, CpG-M 1 ⁇ M). Cytolysis was extracted 6 hours after stimulation and a luciferase assay was performed.
- IL-2ss-TCTP SL4 cells (2 ⁇ 10 5 cells) were subcutaneously transplanted into C57BL / 6 mice, and then the tumor volume was measured over time. O-vanillin (50 mg / kg) was orally administered every 2 days after tumor transplantation.
- (B) PEC (2 ⁇ 10 5 cells) was stimulated with the dead cell supernatant of SL4 cells containing 55F3 antibody or control IgG. After stimulation for 2 hours, the expressed Cxcl1 mRNA was quantified by RT-qPCR. n 3, Sup: Culture supernatant.
- (C) TCTP WT SL4 cells or TCTP KO SL4 cells (1 ⁇ 5 10 cells) were subcutaneously transplanted into C57BL / 6 mice. DHA (50 mg / kg) or DMSO was intraperitoneally administered daily after transplantation (n 6).
- D (Left figure) The expression levels of TCTP in the interstitial region of normal colon tissue and the interstitial region of colon cancer tissue were compared.
- the vertical axis shows the abundance ratio of PMN-MDSC to CD45 + cells.
- the first embodiment of the present invention is derived from bone marrow to a tumor immune microenvironment (TIME) containing a substance that suppresses or inhibits the function of an immunomodulatory factor released from dead tumor cells as an active ingredient. It is an accumulation inhibitor of immunosuppressive cells (myeloid-derived suppressor cell: MDSC) (hereinafter, also referred to as “inhibitor according to this embodiment”).
- TIME tumor immune microenvironment
- the cancer immunomicroenvironment refers to the field of interaction between cancer cells and non-cancer cells centered on immune cells inside the cancer, and is highly advanced.
- Non-Patent Document 1 Bone marrow-derived immunosuppressive cells (also referred to as “MDSC”) are immature myeloid cells that appear in chronic inflammation such as infectious diseases and cancer, and have strong immunosuppressive activity. Monocyte myeloid-derived immunosuppressive cells (also referred to as "M-MDSC”) and polymorphonuclear cell-derived myeloid-derived immunosuppressive cells (also referred to as "PMN-MDSC”) form major subpopulations, respectively. And neutrophils are similar in morphology and cell surface markers (Non-Patent Document 3). Accumulation of MDSC in TIME means that MDSC such as PMN-MDSC is localized in or near TIME.
- the immunomodulatory factor is a factor that promotes or suppresses an immune response and affects the function of the immune system.
- TCTP translationally controlled tumor protein
- CXCL1 and CXCL2 direct PMN-MDSC into or near the cancer immunomicroenvironment via the CXCR2 receptor on PMN-MDSC. It is known that PMN-MDSC induced in or near the cancer microimmune environment suppresses the attack of cancer cells by immune cells such as T cells and NK cells (Non-Patent Document 3).
- the above TCTP- (M-MDSC) -CXCL1 / 2- (PMN-MDSC) pathway suppresses the antitumor activity of immune cells by accumulating PMN-MDSC in TIME, and promotes tumor growth. This analysis newly revealed that. Therefore, by inhibiting the accumulation of MDSC (for example, PMN-MDSC) in TIME, the accumulation and activation of T cells and NK cells can be promoted, and as a result, the growth of tumor can be inhibited or suppressed.
- MDSC for example, PMN-MDSC
- TCTP- (M-MDSC) -CXCL1 / 2- (PMN-MDSC) pathway examples include the use of a substance that suppresses or inhibits the function of TCTP (NCBI accession number; cDNA sequence: CCDS9397.1 amino acid sequence: NP_003286.1 for human TCTP).
- the function of TCTP is, for example, the function of TCTP to bind to its receptor (eg, TLR2) and induce the release of cytokines (eg, CXCL1 / 2) from cells (eg, M-MDSC).
- TLR2 its receptor
- cytokines eg, CXCL1 / 2
- the substance that suppresses or inhibits the function of TCTP is not particularly limited, but is an antibody that suppresses or inhibits the function of TCTP, a peptide aptamer, or a substance that degrades or induces the degradation of TCTP, for example, dihydroartemisinin:
- substances such as DHA) and sertraline that suppress or inhibit the expression of TCTP, such as siRNA and miRNA.
- a substance that suppresses or inhibits the function of TCTP a substance that inhibits the binding between TCTP and its receptor (TLR2), for example, a TLR2 antagonist is also included.
- the inhibitor may be a substance that interacts with TCTP or the receptor (TLR2), or decomposes either of them.
- the inhibitor according to this embodiment may contain a substance that suppresses or inhibits the above-mentioned function of TCTP.
- a second embodiment of the present invention is an antibody that suppresses or inhibits the function of TCTP (hereinafter, also referred to as “anti-TCTP antibody according to the present embodiment”).
- the "antibody” in the present specification is not particularly limited in its preparation method and its structure, and is an “antibody” that binds to a desired antigen with desired properties such as a monoclonal antibody, polyclonal antibody or nanoantibody. All are included.
- the anti-TCTP antibody according to this embodiment is a polyclonal antibody, for example, an antigen and an adjuvant against an immune animal (for example, but not limited to, rabbit, goat, sheep, chicken, guinea pig, mouse, rat or pig). Can be prepared by injecting a mixture of.
- the antigen and / or adjuvant is injected multiple times subcutaneously or intraperitoneally in the immune animal.
- the adjuvant includes, but is not limited to, complete Freund and monophosphoryl lipid A synthesis-trehalose dicorinomicolet (MPL-TMD).
- MPL-TMD monophosphoryl lipid A synthesis-trehalose dicorinomicolet
- the anti-TCTP antibody can be purified from the serum derived from the immunized animal by a conventional method (for example, a method using Sepharose carrying Protein A).
- the anti-TCTP antibody according to this embodiment is a monoclonal antibody, it can be produced, for example, as follows.
- “monoclonal” suggests the characteristic of an antibody obtained from a substantially uniform group of antibodies (an antibody group having the same amino acid sequences of heavy chains and light chains constituting the antibody).
- the antibody is not limitedly interpreted as being produced by a specific method (for example, a hybridoma method).
- Examples of the method for producing a monoclonal antibody include a hybridoma method (Kohler and Milstein, Nature 256 495-497 1975) or a recombinant method (US Pat. No. 4,816,567).
- the anti-TCTP antibody according to this embodiment may be isolated from a phage antibody library (eg, Clackson et al., Nature 352 624-628 1991; Marks et al., J. Mol.Biol. 222 581-597 1991, etc.). good. More specifically, when prepared using the hybridoma method, the preparation method comprises, for example, the following four steps: (i) immunizing an antigen against an immune animal, (ii) a monoclonal antibody. Select cells that collect secretory (or potentially secretory) lymphocytes, (iii) fuse the lymphocytes to immortalized cells, and (iv) secrete the desired monoclonal antibody.
- a phage antibody library eg, Clackson et al., Nature 352 624-628 1991; Marks et al., J. Mol.Biol. 222 581-597 1991, etc.
- the preparation method comprises, for example, the following four steps: (i) immunizing an antigen against an immune
- lymphocytes obtained from the host animal fuse with the immortalized cell line using a fusion agent such as polyethylene glycol or an electrical fusion method to establish hybridoma cells.
- a fusion cell for example, a rat or mouse myeloma cell line is used.
- the cells are grown in a suitable medium containing a substrate that inhibits the growth or survival of unfused lymphocytes and immortalized cell lines.
- HGPRT hypoxanthine-guanine phosphoribosyltransferase
- aminopterin inhibits the growth of HGPRT-deficient cells and is added to a medium (HAT medium) that allows the growth of hybridomas. From the hybridomas thus obtained, a hybridoma that produces a desired antibody can be selected, and the desired monoclonal antibody can be obtained from the medium in which the selected hybridoma grows according to a conventional method.
- the hybridoma thus prepared can be cultured in vitro or in vivo in ascites such as a mouse, rat, guinea pig, or hamster, and the antibody of interest can be prepared from the culture supernatant or ascites.
- a nanoantibody is a polypeptide consisting of a variable domain of the heavy chain of heavy chain antibody (VHH).
- VHH heavy chain antibody
- Antibodies such as humans are usually composed of heavy chains and light chains, but camelid animals such as llamas, alpaca and camels produce single-chain antibodies (heavy chain antibodies) consisting only of heavy chains.
- a heavy chain antibody can recognize a target antigen and bind to the antigen in the same manner as an antibody consisting of a normal heavy chain and a light chain.
- the variable region of a heavy chain antibody is the smallest unit having an affinity for binding to an antigen, and this variable region fragment is called a "nanoantibody”.
- Nanoantibodies have high heat resistance, digestion resistance, and room temperature stability, and can be easily prepared in large quantities by genetic engineering techniques.
- the nanoantibody can be produced, for example, as follows. An antigen is immunized on a camel family animal, the presence or absence of the desired antibody is detected from the collected serum, and cDNA is prepared from RNA derived from peripheral blood lymphocytes of the immune animal in which the desired antibody titer is detected. A DNA fragment encoding VHH is amplified from the obtained cDNA and inserted into a phagemid to prepare a VHH phagemid library. The desired Nanoantibody can be prepared from the prepared VHH phagemid library through several screenings.
- the anti-TCTP antibody according to this embodiment may be a recombinant antibody.
- the recombinant antibody include, but are not limited to, a humanized antibody and a chimeric antibody with a human antibody.
- the chimeric antibody is, for example, an antibody in which a variable region derived from a different animal species and a constant region are linked (for example, an antibody in which a variable region of a rat-derived antibody is bound to a constant region derived from human) (for example, Morrison).
- Et al. Proc. Natl. Acad. Sci. USA 81, 6851-6855 1984.
- It can be easily constructed by gene recombination technology.
- a humanized antibody is an antibody having a human-derived sequence in the framework region (FR) and a complementarity determining region (CDR) consisting of a sequence derived from another animal species (for example, mouse).
- CDR complementarity determining region
- Humanized antibodies first described in other animal species, here mice, were transplanted from the variable region of a mouse-derived antibody into the human antibody variable region to reconstitute the heavy and light chain variable regions. Later, these humanized reconstituted human antibody variable regions can be produced by linking them to the human antibody constant region. Methods for producing such humanized antibodies are well known in the art (eg, Queen et al., Proc. Natl. Acad. Sci. USA, 86, 10029-10033 1989.).
- the antigen-binding fragment of the anti-TCTP antibody according to the present embodiment is a region of a part of the anti-TCTP antibody according to the present embodiment and is an antibody fragment that binds to TCTP.
- the fragment include Fab. Fab', F (ab') 2 , Fv (variable fragment of antibody), single chain antibody (heavy chain, light chain, heavy chain variable region, light chain variable region and nanoantibody, etc.), scFv (single chain Fv) , Diabody (scFv dimer), dsFv (disulfide-stabilized Fv), and peptides containing at least a part of the CDR of the anti-TCTP antibody according to the present embodiment.
- Fab is an antibody fragment having antigen-binding activity in which about half of the N-terminal side of a heavy chain and the entire light chain are bound by a disulfide bond among fragments obtained by treating an antibody molecule with the proteolytic enzyme papain.
- Fabs are prepared by treating antibody molecules with papain to obtain fragments, for example, constructing an appropriate expression vector into which a DNA encoding Fab is inserted, and using this as an appropriate host cell (for example, CHO cell). It can be carried out by expressing Fab in the cells after introduction into mammalian cells, yeast cells, insect cells, etc.).
- F (ab') 2 is an antibody fragment having antigen-binding activity, which is slightly larger than the fragment obtained by treating an antibody molecule with the proteolytic enzyme pepsin, in which Fab is bound via a disulfide bond in the hinge region.
- Is. F (ab') 2 can be produced by treating an antibody molecule with pepsin to obtain a fragment, or by thioether-bonding or disulfide-bonding Fab, and also by genetic engineering methods similar to Fab. Can be made.
- Fab' is an antibody fragment having antigen-binding activity in which the disulfide bond in the hinge region of F (ab') 2 is cleaved.
- scFv is a VH-linker-VL or VL-linker-VH polypeptide in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker. It is an antibody fragment having an antigen-binding activity.
- ScFv can be produced by genetic engineering techniques by obtaining cDNA encoding the heavy and light chain variable regions of an antibody.
- Diabody is an antibody fragment in which scFv is dimerized, and is an antibody fragment having divalent antigen-binding activity.
- the divalent antigen-binding activity may be the same antigen-binding activity, or one of them may be a different antigen-binding activity.
- diabody obtains the cDNA encoding the heavy chain variable region and the light chain variable region of the antibody, and constructs the cDNA encoding scFv in which the heavy chain variable region and the light chain variable region are bound by a peptide linker, and is genetically engineered. It can be produced by a method.
- DsFv refers to a polypeptide in which one amino acid residue in each of the heavy chain variable region and the light chain variable region is replaced with a cysteine residue, which is bound via a disulfide bond between the cysteine residues.
- the amino acid residue to be replaced with the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody.
- the cDNA encoding the heavy chain variable region and the light chain variable region of the antibody can be obtained, and the DNA encoding dsFv can be constructed and produced by a genetic engineering method.
- Peptides containing CDRs are configured to contain at least one region of the CDRs (CDR1-3) of the heavy or light chain. Peptides containing multiple CDRs can be linked either directly or via a suitable peptide linker.
- the peptide containing the CDR constructs the DNA encoding the CDR of the heavy or light chain of the antibody and inserts it into an expression vector.
- the type of vector is not particularly limited, and may be appropriately selected depending on the type of host cell to be introduced thereafter. These can be introduced into suitable host cells (for example, mammalian cells such as CHO cells, yeast cells, insect cells, etc.) for expression as antibodies and produced.
- the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
- a human antibody (fully human antibody) generally has the same structure as a human antibody in that the hypervariable region, which is the antigen binding site of the V region, the other part of the V region, and the constant region have the same structure. ..
- a human antibody can be easily produced by a person skilled in the art by a known technique.
- the human antibody is, for example, a method using a human antibody-producing mouse having a human chromosome fragment containing the H chain and L chain genes of the human antibody (for example, Tomizuka et al., Proc. Natl. Acad. Sci. USA, 97, 722.
- a multispecific antibody can be constructed by using the antigen-binding fragment of the anti-TCTP antibody according to the present embodiment.
- Multispecificity means having binding specificity for two or more antigens, for example, the form of a protein containing a monoclonal antibody or antigen binding fragment having binding specificity for two or more antigens. Can be mentioned. This is performed by one of ordinary skill in the art by known techniques.
- a method for constructing multispecificity a technique for constructing an asymmetric IgG in which two different types of antibody heavy chain molecules are subjected to protein engineering operations so as to form a heterodimer, and a low molecular weight antigen binding obtained from the antibody are used.
- the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy any of the following (A), (B) or (C).
- the characteristic antibodies and antigen-binding fragments thereof can be mentioned.
- (A) CDR of 55F3 antibody The heavy chain CDR1 amino acid sequence is GYSIASDYAWN (SEQ ID NO: 1), The heavy chain CDR2 amino acid sequence is YINYSGSTGYNPSLKS (SEQ ID NO: 2), The heavy chain CDR3 amino acid sequence is FEAGY (SEQ ID NO: 3), The light chain CDR1 amino acid sequence is KASQDINRYLS (SEQ ID NO: 4).
- the light chain CDR2 amino acid sequence has RANRLVD (SEQ ID NO: 5), and the light chain CDR3 amino acid sequence has LQYNEFPLT (SEQ ID NO: 6).
- (B) CDR of 44E1 antibody The heavy chain CDR1 amino acid sequence is GYTFTDHAIH (SEQ ID NO: 7), The heavy chain CDR2 amino acid sequence is YISPGNGDLKYNEKFKG (SEQ ID NO: 8), The heavy chain CDR3 amino acid sequence is GWTL (SEQ ID NO: 9), The light chain CDR1 amino acid sequence is KSSQSLLYRSNQKNYLV (SEQ ID NO: 10).
- the light chain CDR2 amino acid sequence has WAFTRES (SEQ ID NO: 11), and the light chain CDR3 amino acid sequence has QQHYSYPWT (SEQ ID NO: 12).
- C CDR of 51A9 antibody
- the heavy chain CDR1 amino acid sequence is GYSITSDYAWN (SEQ ID NO: 13),
- the heavy chain CDR2 amino acid sequence is YINYSGSTGYNPSLKS (SEQ ID NO: 14)
- the heavy chain CDR3 amino acid sequence is FEAGY (SEQ ID NO: 15)
- the light chain CDR1 amino acid sequence is KASQDINSYLS (SEQ ID NO: 16).
- the light chain CDR2 amino acid sequence has RANRLVD (SEQ ID NO: 17), and the light chain CDR3 amino acid sequence has LQYYEFPLT (SEQ ID NO: 18).
- an antibody containing any of a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and SEQ ID NO: 22 Antibodies containing any of the light chain variable regions comprising the amino acid sequence represented by SEQ ID NO: 23 or SEQ ID NO: 24 and antigen-binding fragments thereof, and heavy chain variable regions and / or light chains constituting these antibodies.
- An antibody consisting of an amino acid sequence having an amino acid sequence identity of% or more, about 97% or more, about 98% or more, and most preferably about 99% or more, and an antibody that inhibits the binding between TCTP and its receptor and those thereof. Contains antigen-binding fragments.
- Heavy chain variable region of 55F3 antibody MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTATGYSIASDYAWNWIRQFPGNKLEWMGYINYSGSTGYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARFEAGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLV Heavy chain variable region of 44E1 antibody MEWSWVFLFFLSVTTGVHSEVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWAKQKPEQGLEWIGYISPGNGDLKYNEKFKGKATLTTDKSSSTAYMQLNSLTSEDSAVYFCKSGWTLWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGC Heavy chain variable region of 51A9 antibody MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTATGYSITSDYAWNWIRQFPGNKLE
- the anti-TCTP antibody according to the present embodiment includes an antibody characterized in that the amino acid sequences of CDRs (complementarity determining regions) 1 to 3 satisfy any of the above (A), (B) or (C).
- An antibody that competitively inhibits binding to TCTP and suppresses or inhibits the function of TCTP (hereinafter, also referred to as “competitive antibody according to the present embodiment”) is also included.
- Competitive antibodies according to this embodiment can be prepared and obtained by competitive experiments and the like well known to those skilled in the art.
- the binding between the first anti-TCTP antibody (anti-TCTP antibody according to the present embodiment) and TCTP is competitively inhibited by the second anti-TCTP antibody, the first anti-TCTP antibody and the second It is judged that the anti-TCTP antibody of the above is bound to the antigen site substantially the same or in the immediate vicinity.
- the second anti-TCTP antibody suppresses or inhibits the function of TCTP
- the second anti-TCTP antibody is a competing antibody according to the present embodiment and is included in the anti-TCTP antibody according to the present embodiment.
- a method using a Fab fragment or the like is usually performed in the art.
- the anti-TCTP antibody according to the present embodiment also includes an antibody that binds to EDGVTPYMIFFKDGLEMEKC (SEQ ID NO: 25), which is a partial peptide of TCTP, and suppresses or inhibits the function of TCTP.
- a third embodiment of the present invention is a therapeutic agent or a therapeutic composition for cancer, wherein the therapeutic agent or the therapeutic composition containing the inhibitor of the present invention as an active ingredient (hereinafter, "therapeutic agent or the like").
- therapeutic agent or the like an active ingredient
- the active ingredient for example, a substance that suppresses or inhibits the function of TCTP
- the active ingredient 1 itself may be administered, but in general, the active ingredient 1 or It is desirable to administer in the form of a therapeutic composition containing one or more pharmaceutical additives in addition to the plurality of substances.
- the active ingredient of the therapeutic agent of the present invention may contain a plurality of different inhibitors of the present invention.
- the therapeutic agent of the present invention may contain known components such as other anticancer agents and immune checkpoint inhibitors.
- Dosage forms of therapeutic agents and the like according to the embodiment of the present invention include tablets, capsules, granules, powders, syrups, suspending agents, suppositories, ointments, creams, gels, patches, inhalants, etc.
- Examples include injections. These preparations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or another suitable solvent at the time of use. Further, tablets and granules may be coated by a well-known method. In the case of an injection, the active ingredient is prepared by dissolving it in water, but it may be dissolved in physiological saline or glucose solution as needed, and a buffering agent or a preservative may be added. ..
- the product for oral administration or parenteral administration is provided in any form.
- the pharmaceutical forms include, for example, therapeutic agents or compositions for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or liquids, intravenously.
- the type of pharmaceutical additive used for the production of the therapeutic agent or the like according to the embodiment of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing a pharmaceutical product or a pharmaceutical composition is the present invention according to the embodiment. It is possible for a person skilled in the art to select as appropriate.
- the additive for the preparation an inorganic or organic substance, or a solid or liquid substance can be used, and generally, it can be blended in the range of 1% by weight to 90% by weight based on the weight of the active ingredient. ..
- additives for preparation lactose, glucose, mannitt, dextrin, cyclodextrin, starch, fatty acid, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calciumcarboxymethylcellulose , Ion exchange resin, methyl cellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragant, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, Examples thereof include sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white vaseline, fluorocarbon, nonionic surfactant, prop
- the active ingredient and the excipient ingredient for example, lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid, etc. are mixed to form a powder, or if necessary, the powder is prepared.
- a binder such as sucrose, hydroxypropyl cellulose or polyvinylpyrrolidone, a disintegrant such as carboxymethyl cellulose or carboxymethyl cellulose calcium is added, and wet or dry granulation is performed to obtain granules.
- these powders and granules may be used as they are, or they may be tableted by adding a lubricant such as magnesium stearate or talc.
- These granules or tablets are coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methylmethacrylic acid polymer to form an enteric solvent preparation, or are coated with ethyl cellulose, carnauba wax, cured oil or the like to form a long-acting preparation. You can also do it.
- an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methylmethacrylic acid polymer to form an enteric solvent preparation
- ethyl cellulose, carnauba wax, cured oil or the like to form a long-acting preparation. You can also do it.
- To manufacture capsules hard capsules are filled with powders or granules, or the active ingredient is used as it is, or it is dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and then coated with a gelatin film to form soft capsules. can do.
- the active ingredient may be used as a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, glucose, etc. It may be dissolved in distilled water for injection together with an isotonic agent, filtered aseptically and filled in an ampol, or it may be vacuum-freeze-dried by adding mannitol, dextrin, cyclodextrin, gelatin, etc. to make a solution-type injection. .. Further, reticine, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like can be added to the active ingredient and emulsified in water to obtain an injection emulsion.
- a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, glucose, etc. It may be dissolved in distilled water for injection together
- the active ingredient may be moistened with a suppository substrate such as cacao butter, fatty acid bird, di and monoglyceride, polyethylene glycol, dissolved and poured into a mold for cooling, or the active ingredient may be polyethylene. After dissolving in glycol, soybean oil, etc., it may be coated with a gelatin film.
- a suppository substrate such as cacao butter, fatty acid bird, di and monoglyceride, polyethylene glycol, dissolved and poured into a mold for cooling, or the active ingredient may be polyethylene. After dissolving in glycol, soybean oil, etc., it may be coated with a gelatin film.
- the dose and frequency of administration of the therapeutic agent and the like according to the embodiment of the present invention are not particularly limited, and conditions such as prevention of deterioration / progression of the disease to be treated and / or purpose of treatment, type of disease, weight and age of patient. It is possible to make an appropriate selection at the discretion of the doctor.
- the daily dose for an adult in oral administration is about 0.01 to 1000 mg (active ingredient weight), and can be administered once or divided into several times a day, or every few days. .. When used as an injection, it is desirable to administer a daily dose of 0.001 to 100 mg (active ingredient weight) continuously or intermittently to an adult.
- the therapeutic agent or the like according to the embodiment of the present invention is prepared as a sustained release preparation such as an implantable tablet and a delivery system encapsulated in microcapsules, using a carrier that can prevent immediate removal from the body. May be good.
- a carrier that can prevent immediate removal from the body. May be good.
- Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid can be used as such carriers. Such materials can be readily prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.
- Liposomes are prepared, but not limited to, as a lipid composition containing phosphatidylcholine, cholesterol and PEG-induced phosphatidylethanol (PEG-PE) through a suitable pore-sized filter to a size suitable for use and reverse phase evaporation. Can be purified by.
- PEG-PE PEG-induced phosphatidylethanol
- the therapeutic agent or the like according to the embodiment of the present invention may be provided in the form of a kit together with an instruction manual such as an administration method.
- the drug contained in the kit is made of a container made of a material that effectively maintains the activity of the constituents of the therapeutic drug for a long period of time, does not adsorb to the inside of the container, and does not deteriorate the constituents.
- the sealed glass ampoule may include a buffer encapsulated in the presence of a neutral, non-reactive gas such as nitrogen gas.
- an instruction manual may be attached to the kit.
- the usage instructions of the kit may be printed on paper or the like, or may be stored in an electromagnetically readable medium such as a CD-ROM or a DVD-ROM and supplied to the user.
- a third embodiment of the present invention is a method for preventing or treating cancer, which comprises administering a therapeutic agent or the like (the second embodiment of the present invention) according to the embodiment of the present invention to a patient.
- treatment means preventing or alleviating the progression and exacerbation of a pathological condition caused in a mammal suffering from cancer.
- prevention means to prevent the onset of cancer in advance in a mammalian patient who may develop the cancer.
- the "mammal” to be prevented or treated means any animal classified as a mammal, and is not particularly limited, for example, in addition to humans, pet animals such as dogs, cats, rabbits, ferrets, and cows. , Pigs, sheep, horses and other domestic animals.
- a particularly preferred "milk animal” is human.
- cancer malignant tumor / malignant neoplasm
- examples of the cancer (malignant tumor / malignant neoplasm) targeted by the preventive or therapeutic method of the present invention include hepatocellular carcinoma, bile duct cell carcinoma, renal cell carcinoma, squamous cell carcinoma, basal cell carcinoma, and transitional cell carcinoma.
- Adenocarcinoma malignant gastrinoma, malignant melanoma, fibrosarcoma, mucoid sarcoma, liposarcoma, smooth myoma, rhizome myoma, malignant malformation, hemangiosarcoma, capoes sarcoma, osteosarcoma, chondrosarcoma, lymphangiosarcoma, malignant spinal cord Gastrointestinal cancers such as membranous, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, brain tumor, epithelial cell-derived neoplasm (epithelial carcinoma), basal cell carcinoma, adenocarcinoma, lip cancer, oral cancer, esophageal cancer, small bowel cancer and gastric cancer, Includes skin cancers such as colon cancer, rectal cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, squam
- a fourth embodiment of the present invention is a method for diagnosing or assisting a diagnosis of cancer, which comprises measuring the amount of TCTP mRNA or TCTP protein present in a sample derived from a subject. It is an auxiliary method.
- the amount of TCTP protein or TCTP mRNA in tumor tissue and blood is significantly higher than that in healthy subjects (those who have been confirmed not to develop cancer). (See, for example, FIG. 2b). Therefore, if the amount of TCTP mRNA or TCTP protein in the sample derived from the subject is significantly higher than that in the sample of a healthy subject, it is possible that the subject has developed some kind of cancer. Can be judged.
- the sample derived from the subject for example, blood (including components derived from blood such as serum), tissues suspected to be malignant tumors, and the like can be used, although not limited.
- a method for measuring the amount of TCTP protein in a sample a person skilled in the art can easily select an appropriate method, for example, an ELISA (enzyme-linked immuno-sorbent assay) method, a Western blotting method, or the like. Immunological assay can be used.
- a method for measuring the amount of TCTP mRMA in a sample a person skilled in the art can easily select an appropriate method, and examples thereof include a real-time PCR (RT-qPCR) method.
- RT-qPCR real-time PCR
- mice C57BL / 6 mice and BALB / c mice were purchased from CLEA Japan. Apc ⁇ 716 mice were prepared according to previously reported (19) and used in the background of C57BL / 6 mice.
- TCTP flox mice were donated by Dr. Hsin-Fang Yang-Yen of Academia Sinica (Republic of China) and used in the background of C57BL / 6 mice.
- Villin-CreER T2 mice were donated by Dr. Sylvie Robine of the Curie Institute (France) and used in the background of C57BL / 6 mice.
- MyD88 and IPS-I-deficient mice were donated by Dr.
- TLR2 and TLR4 deficient mice were purchased from Oriental Bio Service Co., Ltd. and used in the background of C57BL / 6 mice.
- STING-deficient mice were donated by Dr. Glen N. Barber of the University of Miami (USA) and used in the background of C57BL / 6 mice.
- the RAG1 knockout mouse was donated by Dr. Shinsuke Taki of Shinshu University. Unless otherwise specified, males and females (6-12 weeks old) were used in this example. All animal experiments were conducted with the approval of the University of Tokyo Animal Experiment Committee.
- Cells RAW264.7 cells, mouse melanoma cell line B16F10 cells, mouse fibroblast line Meth-A cells and HEK293T cells were obtained from RIKEN BioResource Research Center (Japan).
- the mouse colorectal cancer cell line SL4 cell line was donated by Dr. T Irimura (Juntendo University).
- RAW264.7 cells, B16F10 cells and HEK293T cells were maintained in DMEM (Nacalai Tesque) supplemented with 10% FBS (HyClone).
- Meth-A cells were maintained in RPMI (Narakaitesk) supplemented with 10% FBS.
- SL4 cells were maintained in DMEM-F12 (Gibco) supplemented with 10% FBS.
- peritoneal exudate cells 2 ml of 4% thioglycolate (DIFCO solution) was administered intraperitoneally to mice, and 4 days later, the abdominal cavity was washed with PBS to recover PEC. .. Cells were incubated overnight on Petri dishes in RPMI supplemented with 10% FBS. After incubation, cells were washed with RPMI medium and used in subsequent experiments.
- DIFCO solution 4% thioglycolate
- Tumor-infiltrated cells were collected after subcutaneous transplantation of the tumor into mice and analyzed by flow cytometry.
- the resected tumor is cut into small pieces, collagenase (0.75 mg / ml, cat # 11088882001, Roche), DNase I ((40 ⁇ g / ml, cat # 11284932001, Roche)) and dispase (0.5 mg / ml, cat # 17105041, Thermo Fisher Scientific). ) And then vigorously stirred (180 rpm, 37 ° C., 1 hour).
- the resulting cell suspension was passed through a cell strainer (cat # 352340, Falcon) and an RBC lysis buffer (cat # 00-4333-). 57, treated with Invitrogen). After washing with PBS, the cells were first incubated with anti-CD16 / 32 antibodies (clone 93, cat # 101302, BioLegend) for 5 minutes on ice, after which the cells were incubated with PFE (2% FBS and BioLegend). Stained on ice for 20 minutes in 1 mM of EDTA-supplemented PBS) and analyzed with LSR Fortessa (BD Biosciences). Cell sorting of tumor infiltrating cells was performed using FACSAria II (BD Biosciences). The data obtained were analyzed with FlowJo software (BD BioSciences).
- Ly6C-AF488 (clone HK1.4, cat # 128022, BioLegend)
- NK1.1-FITC (clone PK136, cat # 553164, Pharmingen).
- CD11b-PE (clone M1 / 70, cat # 101208, BioLegend) CD3 ⁇ -PE (clone 145-2C11, cat # 12-0031-81, eBioscience), CD11c-APC (clone N418, cat # 17-0114-81) , EBioscience), B220-APC (clone RA3-6B2, cat # 20-0452-U025, TONBO biosciences), F4 / 80-PerCP / Cy5.5 (clone BM8, cat # 123128, BioLegend), CD8 ⁇ -PerCP / Cy5 .5 (clone 53-6.7, cat # 100734, BioLegend), Ly6G-PE / Cy7 (clone 1A8, 127618, BioLegend), CD4-PE / Cy7 (clone GK1.5, cat # 100422, BioLegend), CD45.2 - PacificBlue (clone104, cat # 109820, BioLegend), anti-CD16 / 32 (clon
- the culture supernatant was prepared according to the previously reported (Non-Patent Document 5). SL4 cells were incubated overnight with indomethacin (10 ⁇ M) to remove PGE2 (prostaglandin E2), suspended in PBS at a concentration of 108 cells / ml, and used in a 37 ° C. constant temperature bath and liquid nitrogen. Then, freezing-thawing was performed 5 times. The cells were then centrifuged at 8,000 xg for 5 minutes to remove necrotic cell debris. The culture supernatant was obtained by filtering through a 0.45 ⁇ m membrane filter (Millipore).
- the reagent LPS (O55: B5) was purchased from SIGMA-Aldrich (cat # L2637). Oligonucleotides were purchased from FASMAC. Indomethacin was purchased from WAKO (cat # 093-02473). Recombinant IL-1 ⁇ was purchased from Peprotech (cat # 211-11A). Recombinant TCTP was purchased from ORIGENE (cat # TP301664). TUNEL staining was performed using the in situ Apoptosis Detection Kit (cat # MK500, TaKaRa).
- the endotoxin level of recombinant TCTP was assayed with the LAL Endotoxin Assay Kit, Chromogenic, ToxinSensor (cat # L00350, Genscript) and confirmed to have an endotoxin level ⁇ 0.1 EU / ⁇ g.
- Hitrap Capto S column (cat # 17544105, GE healthcare) or Hitrap Capto Q column (cat # 11001302, GE healthcare).
- Hitrap Capto S column or Hitrap Capto Q column First, washed with 1 ml DDW and then equilibrated with 30 ml PBS. Then charged with 5 ml SL4 cell culture supernatant and washed with 5 ml PBS. Flow-through was collected and there. RT-qPCR analysis showed that the flow-through of the Hitrap Capto S column induces cytokine mRNA but not the flow-through of the Hitrap Capto Q column. It suggests binding to the Hitrap Capto Q column.
- the Hitrap Capto S column was washed with 1 ml of DDW and then equilibrated with 30 ml of PBS. The column was charged with 5 ml of SL4 cell culture supernatant and then washed with 5 ml of PBS. The flow-through was collected and charged into the equilibrated Hitrap Capto Q column in the same manner as the Hitrap Capto S column. Next, the column was washed with 20 ml of PBS and eluted with 5 ml of 0.4 M NaCl.
- the eluate was concentrated with Spin-XUF10kMWCO (cat # CLS431488, MERCK). Finally, size exclusion chromatography was performed using a Superdex 200 Increase 10/300 GL column connected to AKTA purifier (GE Healthcare). The column was equilibrated with twice the volume of PBS and the column was charged with the concentrated eluate. Using the AC-5700P MicroCollector (ATTO), the eluted droplets (18 droplets) were continuously collected in the wells of a 48-well plate. Each fraction was added to PEC or RAW264.7 cells and the amounts of Cxcl1, Cxcl2, Tnf and Il1b mRNA were determined by RT-qPCR.
- the 18th to 27th fractions were silver-stained using SilverQuest (cat # LC6070, invitrogen), and the darkest band consistent with the cytokine-inducing activity was subjected to LC-MS (liquid chromatography-mass spectrometry). ..
- RT-qPCR Total tissue or cell RNA was extracted using NucleoSpin RNA II (MACHEREY NAGEL) and reverse transcribed using PrimeScript RT Master Mix (TaKaRa Bio). The RT-qPCR reaction was performed using TB Green Premix Ex Taq TM II (TaKaRa Bio) and LightCycler 480 (Roche Life Science) or LightCycler 96 (Roche Life Science). The values obtained were normalized to the expression level of Gapdh mRNA. Primers with the sequences shown below were used.
- CRISPR / Cas9 TCTP KO SL4 cells, TCTP KO B16F10 cells and TCTP KO Meth-A cells were generated by CRISPR / Cas9 genome editing using the CRISPR design tool (http://www.genome-engineering.org, accessed May 2017). ..
- the genomic sequence of the TCTP gene was targeted (5'-CGGGCGGAAAAGGCCGACGC-3'(SEQ ID NO: 38) and 5'-AGGCCCCGCCATTTCCCGCGC-3' (SEQ ID NO: 39)).
- the first exon of the TCTP gene was sandwiched between two sequences, SEQ ID NO: 38 and SEQ ID NO: 39.
- Oligonucleotides corresponding to these guide sequences were cloned into the BbsI sites of pSpCas9 (BB) -2A-GFP (PX458) (Addgene) and pSpCas9 (BB) -2A-Puro (PX459) V2.0 (Addgene), respectively. did. Both expression vector constructs were introduced into SL4 cells. The construct-introduced cells were selected with puromycin and then GFP-expressing cells were subjected to single cell sorting with FACSAria II (BD Biosciences) or SH800S (SONY).
- Recombinant TCTP (cat # TP301664, OriGene) was used to measure the amount of TCTP.
- Antibodies Rabbit IgG-HRP (cat # NA934V, GE healthcare) or anti-mouse IgG-HRP (cat # NA931V, GE healthcare) were used as secondary antibodies.
- the immunoblotting signal was detected by FUSION Solo S (Vilber-Lourmat) and analyzed by FUSION Capt Advanced software (Vilber-Lourmat).
- GFP-positive cells were subjected to Cell Sorter SH800S. Sorted by (SONY). Mock cells or IL-2ss-TCTP-introduced cells were identified by performing the above-mentioned immunobrotting analysis on the culture supernatant collected from a 60 mm culture dish seeded on 2 ⁇ 10 6 cells.
- Enzyme-linked Immunosorbent assay Tumors were resected 21 days after transplantation and finely chopped in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM Na 3 VO 4 , 1 mM APMSF). It was then incubated on ice for 30 minutes, the lysate was centrifuged and the supernatant was collected for ELISA. The production of mouse CXCL1 and CXCL2 during TIME was quantified using an ELISA kit (R & D Systems) according to the attached instruction manual.
- ELISA Enzyme-linked Immunosorbent assay
- PMN-MDSC T cells were collected from tumor-free mouse splenocytes using the Pan T cell isolation kit II (Miltenyi Biotec). The prepared cells were stained with CFSE using CellTrace TM CFSE Cell Proliferation Kit (Thermo Fischer Scientific) according to the attached instruction manual. PMN-MDSC was collected from spleen cells of mice subcutaneously transplanted with SL cells (2 ⁇ 10 5 cells) using anti-Ly6-G microbeads (Miltenyi Biotec) 17-19 days after transplantation. Neutrophils were collected from tumor-free C57BL / 6 mice by a method similar to PMN-MDSC.
- CFSE-labeled 1 ⁇ 10 5 cells of T cells and 5 ⁇ 10 4 cells, 2.5 ⁇ 10 4 cells or 1.25 ⁇ 10 4 cells of PMN-MDSC were seeded on 96-well plates.
- T cell proliferation was induced using Dynabeads Mouse T-Activator CD3 / CD28 (Thermo Fischer Scientific) for 3 days, followed by flow cytometric analysis to assess CFSE dilution in T cells.
- RAG1 KO mice were intraperitoneally administered with anti-asialo GM1 (cat # 014-09801, WAKO) or rat control IgG (cat # 31933, Thermofisher) (200 ⁇ g / mouse).
- anti-anti-Ly6G antibody cat # BE0075-1, Bio X Cell
- rat control IgG was administered intraperitoneally 1, 3, 5, 7, 9 and 11 days after tumor cell transplantation. did.
- DHA Dihydroartemisinin
- Selleck o-vanillin-treated DHA
- O-vanillin cat # 120804-10G, Merck
- RT reverse transcription
- reaction solution (II) (cDNA synthesis reaction) 2.2 ⁇ L H 2 O 2 ⁇ L 5 ⁇ SMART Scribe buffer 1 ⁇ L 20 mM DTT 0.3 ⁇ L 100 ⁇ M template-switch oligo F. 0.5 ⁇ L 100 U / ⁇ L SMARTScribe Reverse Transcriptase
- the above was mixed in an 8-well strip tube.
- the reaction solution (I) was heat-treated in a thermal cycler at 72 ° C. for 3 minutes. After the heat treatment, 6 ⁇ L of the reaction solution (II) was added to the reaction solution (I), and then the mixture was incubated with a thermal cycler at 42 ° C. for 60 minutes. Then, it was heat-treated at 70 ° C. for 5 minutes to stop the reaction.
- Primer ISPCR F 5'-aagcagtggtatcaacgcagag-3' (SEQ ID NO: 45)
- mIGK PCR 5'-acattgatgtctttggggtagaag-3' (SEQ ID NO: 46)
- mIGL PCR 5'-atcgtacacaccagtgtggc-3'(SEQ ID NO: 47)
- mIGHG PCR 5'-gggatccagagttccaggtc-3' (SEQ ID NO: 48)
- Synthesized cDNA from the RT reaction 2.5 ⁇ L 10 ⁇ M universal forward primer ISPCR 2.5 ⁇ L 10 ⁇ M reverse PCR primer (mIGK PCR, mIGL PCR or mIGHG PCR) 6 ⁇ L H 2 O 1 ⁇ L KOD FX (1 U /
- amino acid sequences of the determined antibody heavy and light chain variable regions can be found in the antibody sequence database (http://www.abybank.org/kabat/ and http://www.bioinf.org.uk/abs/info.html). By collating with #cdrid etc.), the amino acid sequence corresponding to CDR as defined by Kabat was identified.
- TCTP (cat # 133568, abcam) or CD15 (cat # M363129, Dako) was used for DAB staining. Briefly, paraffin-embedded samples were incubated twice with xylene to deparaffinize, then dehydrated in ethanol series and rehydrated in PBS. The activation of the epitope by heat treatment was carried out at 121 ° C. for 10 minutes using the antigen activating reagent pH 9 (cat # 415211, NICHIREI BIOSCIENCES INC).
- REAL Peroxidase-Blocking solution (cat # S2023, DAKO) was used according to the attached instruction manual, followed by blocking with 1% BSA / TBST at room temperature for 30 minutes. .. Sections were incubated with TCTP antibody or CD15 antibody at room temperature for 1 hour. The detection of the primary antibody was carried out using Histofine simple stain MAX-PO (MULTI) (cat # 424151, NICHIREI BIOSCIENCES INC.) And DAB (cat # 415171, NICHIREI BIOSCIENCES INC.) As a substrate. Samples were counterstained with hematoxylin (at no.
- the signal intensity from TCTP was quantified using ImageJ Fiji (BioProtoc.2019Dec20; 9 (24): e3465.) According to the previous report.
- the average intensity in the normal colonic mucosa was set to 1. Quantification of the CD31 positive region was performed using a BZ-9000 microscope and BZ-H4C (Keyence) of cell counting software.
- Microarray PEC was stimulated with the culture supernatant of necrotic cells (2 x 10 6 SL4 cells). After 2 hours of incubation, total RNA was extracted and used for analysis by Clariom S Array (Thermo Fisher Scientific). Volcano plot was created with Transcriptome Analysis Console (TAC) software v4.0 (ThermoFisher Scientific). The microarray data was registered in the Gene Expression Omnibus (GEO) database (accession No. GSE150465).
- GEO Gene Expression Omnibus
- Immunostained cells were cultured overnight in a 35 mm glass bottom dish (MATSUNAMI), washed with PBS and fixed with 4% paraformaldehyde / PBS for 15 minutes. Then, after washing with PBS, membrane permeation treatment was performed with 0.5% Triton X-100 / PBS for 15 minutes, and blocking was performed with 3% BSA / PBS. Then, it was incubated with an anti-TCTP antibody (ab37506) for 2 hours, washed, treated with a secondary antibody (Alexa Fluor 594 Goat anti-rabbit IgG, cat # A-11012, Invitrogen), and immunostained. ..
- PMN-MDSC adopted transfer Bone marrow cells and splenocytes were collected from SL4 cells (2 ⁇ 10 5 cells) from mice (with tumors) 17 days after subcutaneous transplantation.
- Ly-6G + cells were isolated using anti-Ly6-G MicroBead Kit, mouse (Miltenyi Biotec). Separated Ly-6G + cells (4 ⁇ 10 6 cells) were intravenously injected into mice on days 1, 4, 7, 10 and 13 subcutaneously transplanted with TCTP KO SL4 cells (2 ⁇ 10 5 cells). ..
- Cell lysates were then prepared using a lysis buffer (20 mM Tris-HCL pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF). Immunoprecipitation was performed on 1 mg of cytolysate using 1 ⁇ g of anti-GFP antibody (598; MBL) and 30 ⁇ l of Dynabeads Protein G for immunoprecipitation (Thermo Fisher Scientific). Then, the immunoprecipitate was immunoblotting using an anti-GFP antibody or an anti-FLAG M2 antibody (Sigma Aldrich).
- Mouse TLR3, TLR7, TLR9 and human CD14 cDNAs were cloned into the pCXNII-HA vector.
- Human TLR2 cDNA construct pcDNA3.1-hTLR2-YFP
- mouse TLR3 cDNA construct pCXNII-HA-mTLR3
- mouse TLR7 cDNA construct pCXNII-HA-mTLR7
- mouse TLR9 cDNA construct pCXNII-HA-mTLR9
- NF ⁇ B luciferase reporter was transfected into HEK293T cells.
- human CD4 cDNA construct (pCXNII-HA-hCD14) was co-transfected. Twenty-four hours after transfection, 2 ⁇ 10 4 cells were seeded in 96 weld dishes. After 24 hours, cells were treated with recombinant TCTP or each TLR agonist. Then, the luciferase activity was measured using the Pikka Gene Dual Assay kit (cat # PD-11, TOYO B-Net) and MicroLumat Plus LB96V (Berthold Technologies) according to the attached instruction manual.
- Freezing and thawing was carried out as described in "1-4. Preparation of culture supernatant" above. As a control, a supernatant of SL4 cells incubated in PBS for the same amount of time as freeze-thaw was used. Hypoxic treatment (1% O 2 , 5% CO 2 ) was performed using a multi-gas incubator (MCO-5MUV-PJ, Panasonic).
- TCTP WT cells or TCTP KO cells administered to a subcutaneous tumor model TCTP WT cells or TCTP KO cells on a 10 cm dish were irradiated with a lethal dose of X-ray (100 Gy). The same amount of the above TCTP WT cells or TCTP KO cells was mixed with TCTP WT SL4 cells (2 ⁇ 10 5 cells) and subcutaneously transplanted into C57BL / 6 cells.
- mRNAs were analyzed by microarray (Fig. 1b) and RT-qPCR (Fig. 1c).
- Cxcl1 and Cxcl2 are known to regulate the migration of hematopoietic cells such as MDSC, so we focused on Cxcl1 and Cxcl2 .
- CXCR2 which is a common receptor for CXCL1 and CXCL2, is known to be important for the transfer of PMN-MDSC in mice (Non-Patent Document 6).
- chemokines derived from dead tumor cells that induce the expression of Cxcl1 and Cxcl2 were used to determine the molecule of interest. An attempt was made to isolate.
- the culture supernatant of SL4 cells was passed through an anion exchange column (Hitrap Q) and a cation exchange column (Hitrap S). Flow-throughs from the Hitrap S column were collected and charged into the Hitrap Q column. Fractions bound to Hitrap Q were collected and charged into a size exclusion column (Superdex 200).
- TCTP Tumor cell-derived translationally controlled tumor
- TCTP has been reported to be present in the cytoplasm of eukaryotic cells (Non-Patent Document 7, Non-Patent Document 8 and Non-Patent Document 9), the function of TCTP present outside the cell has not been known so far. ..
- TCTP has an immunostimulatory function.
- TCTP mRNA expression was reported to be elevated in tumor samples (Du et al., Oncotarget 8, 101922-101935, doi: 10.18632 / oncotarget.21747 (2017)). However, it was not clear whether TCTP was functioning in tumor progression and, if so, how it was functioning.
- TCTP was abundant in the culture supernatant of dead tumor cells (tumor dead cells), whereas live tumor cells (living tumor cells) released almost no TCTP (Fig. 3a). .. Furthermore, as shown in FIG. 2b, the amount of TCTP protein in serum increased over time in C57BL / 6 mice subcutaneously transplanted with SL4 cells. This result suggests that TCTP is released from dying tumor cells in vivo. Similarly, an increase in TCTP protein levels was observed in the sera of mice with B16F10 melanoma and Meth-1 fibrosarcoma tumors (Fig. 3b).
- TCTP gene of B16F10 cells and Meth-A cells was disrupted (Fig. 3c), and the same studies as above were performed.
- the growth rate of the wild-type strain and the TCTP gene-deficient strain was similar in vitro (Fig. 3g and h), but the growth of the tumor transplanted into the mouse body was TCTP gene-deficient. Tumors from the strain were significantly slower than tumors from the wild-type strain (FIGS. 2d and e).
- TCTP flox / flox , Apc + / ⁇ 716 , villin-Cre ERT2 mice whose expression of the TCTP gene can be regulated by tamoxifen were used (Fig. 2f).
- administration of tamoxifen causes a deficiency of the TCTP gene in an intestinal epithelial cell-specific manner.
- TCTP-deficient mice TCTP-deficient mice
- untreated mice mice carrying the TCTP gene
- TCTP released from dying tumor cells induces CXCL1 and CXCL2, which attract PMN-MDSC to TIME and suppress the antitumor immune response at TIME. It is possible that this may promote the growth of the tumor.
- CXCL1 expression level was significantly higher in TCTP WTSL4 tumor (Fig. 5d). Similar results were observed with CXCL2 (Fig. 5d).
- CXCL1 / 2 in the IL-2ss-TCTP tumor was also significantly higher than the expression level in the TCTP KO SL4 tumor (Fig. 5e). This result indicates that extracellular TCTP induces these chemokines within TIME.
- tumor-invasive immune cells were prepared from TCTP WT SL4 tumor and TCTP KO SL4 tumor and analyzed by flow cytometry.
- FIG. 7a it was confirmed that CD11b + Ly6C low Ly6G + cells representing PMN-MDSC in mice were dramatically reduced in the TCTO KO SL4 tumor.
- myeloid cells such as CD11b + Ly6C high Ly6G - cells and CD11b + Ly6C-Ly6G - F4 / 80 + tumor-associated macrophage (TAM) representing M-MDSC have such dramatic changes.
- TAM tumor-associated macrophage
- the amount of PMN-MDSC was elevated in the IL-2ss-TCTP tumor compared to the TCTP KO tumor, whereas there was little difference in the number of M-MDSC (Fig. 7c). It is noteworthy that there was no significant difference in the expression levels of G-CSF and GM-CSF that promote MDSC proliferation between WT and TCTP KO tumors (Fig. 6c). This result indicates that G-CSF and GM-CSF are not the major causes of the decrease in PMN-MDSC cell count in TCTP KO tumors. Similarly, a decrease in the number of PMN-MDSC cells was also observed in B16F10 and Meth-A tumors (FIGS. 6d and e).
- CD11b + Ly6C low Ly6G + cells actually represent PMN-MDSC
- CD11b + Ly6C low Ly6G + cells from mice with SL4 tumor and CD11b + Ly6C from mice without SL4 tumor Low Ly6G + cells were subjected to an in vitro T cell proliferation assay.
- T cells were stimulated with anti-CD3 / CD28 antibody and T cell proliferation was observed.
- CD11b + Ly6C low Ly6G + T cell proliferation was suppressed depending on the cell abundance.
- mice with TCTP KO tumors show that antitumor lymphocytes are still activated in mice with TCTP KO tumors.
- the number of CD8 + T cells during TIME was significantly increased in mice with TCTP KO SL4 tumors compared to mice with TCTP WT SL4 tumors.
- IL-2ss-TCTP tumors had fewer CD8 + cells than TCTP KO tumors.
- intratumoral NK cell activation markers (CD69 and CD107a) were also elevated in mice with TCTP KO SL4 tumors than in mice with TCTP WT SL4 tumors (FIG. 6h).
- PMN-MDSC which is taken up by TIME by TCTP released from tumor cells, suppresses the antitumor immune effect of CD8 + T cells and NK cells and promotes tumor growth in at least a part of them. Will be done.
- Cxcr2 mRNA which is a receptor gene for CXCL1 / 2 chemokines
- TCTP acts on M-MDSC to induce the expression of CXCL1 / 2 chemokines
- the stimulation of the CXCL1 / 2 chemokines causes CXCR2-expressing PMN-MDSC cells to migrate to TIME and the antitumor immune system. It is suggested that tumor growth is promoted by the suppression of chemokines.
- TCTP-induced chemokines activate unique receptors.
- FIG. 10c the induction of chemokines by TCTP disappeared in PECs lacking the MyD88 gene, but not in PECs lacking the IPSI / MAVS or STING genes.
- MyD88 is an adapter molecule common to Toll-like receptors (TLRs).
- TLR2 and TLR4 are known to be able to recognize multiple proteins released from dead cells (Non-Patent Document 10).
- TLR2 The interaction between TCTP and TLR2 was confirmed by epitope-tagged TCTP and TLR2 co-immunoprecipitation (Fig. 11e). This result supports the role of TLR2 as a receptor for signals that induce CXCL1 / 2 expression via TCTP. As shown by the luciferase reporter assay, none of TLR3, TLR7 and TLR9 signaling via the MyD88 pathway was activated by recombinant TCTP (FIG. 11f).
- SL4 tumors transplanted into Tlr2- deficient mice grow slower than SL4 tumors transplanted into WT mice (Fig. 11g).
- the IL-2-ss-TCTP tumor transplanted into Tlr2 -deficient mice also grew slowly (Fig. 11h).
- the selective TLR2 inhibitor O-vanillin suppressed the growth of IL-2ss-TCTP tumors (Fig. 11i).
- SL4 cells were transplanted into C57BL / 6 mice by subcutaneous injection, and then 55F3 or control IgG (200 ⁇ g / mouse) was intraperitoneally administered every 1 to 2 days after transplantation to measure the tumor volume.
- 55F3 reduced the growth rate of SL4 tumor cells (Fig. 13a) and decreased the amount of PMN-MDSC during TIME (Fig. 6b).
- DHA dihydroartemisinin
- PD-1 immune checkpoint antibody an antibody of clone RMP1-14 (BioLegend) was used. After transplanting SL4 cells into C57BL / 6 mice by subcutaneous injection, 55F3 or DHA was intraperitoneally administered daily from 1 day after transplantation. Ten days after transplantation, anti-PD-1 monoclonal antibody was administered and the tumor volume was measured. As a result, it was confirmed that the combined administration of 55F3 or DHA and the PD-1 antagonist antibody improved the tumor growth inhibitory effect as compared with the case of 55F3 and DHA alone (FIGS. 13d and e).
- the results of this analysis show that amplification of the TCTP gene is observed in about 5% of colorectal cancer patients, and that the expression level of TCTP mRNA correlates with the number of TCTP gene copies (Fig. 13j).
- TCTP expression levels were negatively correlated with cytotoxic T cell or NK cell markers (ie, CD8A, GZMB, PRF1 and CD69) (FIG. 12e). This negative correlation was also observed for cytotoxic T cell and NK cell cytolytic responses (defined as geometric mean of GAMA mRNA and PRF1 mRNA expression levels, respectively) (FIG. 12f). Furthermore, notably, the progression-free survival (PFS) of patients with amplified TCTP genes was significantly lower (Fig. 13k).
- PFS progression-free survival
- TCTP is released from dead tumor cells, binds to the TLR2 receptor on M-MDSC, and induces CXCL1 / 2 chemokines. It is concluded that these chemokines attract PMN-MDSC to TIME, slowing the antitumor immune response and further promoting tumor growth.
- the present invention provides a therapeutic agent for cancer, a therapeutic method, and the like. Therefore, the present invention is highly expected to be used in the medical field.
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Abstract
Description
現在、実際にがん治療に用いられている免疫療法の主なものとしては、「チェックポイント阻害抗体療法」、「CAR-T細胞療法」が挙げられる。しかしながら、いずれもその効果については、ある種のがんでは一定の効果が確かめられているものの、他の多くのがんの治療には充分な療法とまではいえないのが現状である。
しかしながら、TIMEにおけるMDSCの蓄積の仕組みについては、未だ明らかにされていない。
具体的には、本発明者らは、細胞外TCTPが、主にM-MDSCに働くことによって、CXCL1ファミリーケモカインの発現を誘導することを明らかにした。CXCL1ファミリーケモカインは、PMN-MDSCをTIMEに呼び寄せ、TIMEを高度に免疫抑制的な状態にする。本発明者らは、抗TCTPモノクローナル抗体またはTCTP機能阻害物質の投与により、TIMEへのPMN-MDSCの誘導が阻害され、腫瘍の増殖が抑制されることを明らかにした。本発明は、腫瘍がTIMEをどのように構築し、制御するのか、という未解決な問題をTCTPの同定とその新たな機能の解明によって明らかにし、その阻害法を開発することによって完成されるに至った。
(1)腫瘍死細胞から放出される免疫調節因子の機能を抑制または阻害する物質を有効成分として含む、がん免疫微小環境(tumor immune microenvironment :TIME)への骨髄由来免疫抑制細胞(myeloid-derived suppressor cell:MDSC)の蓄積阻害剤。
(2)前記骨髄由来免疫抑制細胞が、多型核細胞系骨髄由来免疫抑制細胞(polymorphonuclear myeloid-derived suppressor cell:PMN-MDSC)である、上記(1)に記載の阻害剤。
(3)前記免疫調節因子が、TCTP(translationally controlled tumor protein)である、上記(1)または(2)に記載の阻害剤。
(4)前記TCTPの機能を抑制または阻害する物質が抗体である、上記(3)に記載の阻害剤。
(5)前記TCTPの機能を抑制または阻害する物質がジヒドロアルテミシニン(dihydroartemisinin:DHA)である、上記(3)に記載の阻害剤。
(6)がんの治療薬または治療用組成物であって、上記(1)から(5)までのいずれかに記載の阻害剤を有効成分として含む、前記治療薬または治療用組成物。
(7)前記がんが、大腸癌、悪性黒色腫または線維肉腫である、上記(6)に記載の治療薬または治療用組成物。
(8)がんの診断方法または診断補助方法であって、被験者由来のサンプル中に存在するTCTP mRNAまたはTCTPタンパク質の量を測定することを含む、前記診断方法または診断補助方法。
(9)前記サンプルが血液または組織である、上記(8)に記載の方法。
(10)CDR(complementarity determining region)1~3のアミノ酸配列が下記(A)、(B)または(C)のいずれかを満たすことを特徴とする抗体またはその抗原結合断片。
(A)配列番号1で表されるアミノ酸配列を含む重鎖CDR1、
配列番号2で表されるアミノ酸配列を含む重鎖CDR2、
配列番号3で表されるアミノ酸配列を含む重鎖CDR3、
配列番号4で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号5で表されるアミノ酸配列を含む軽鎖CDR2、および
配列番号6で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(B)配列番号7で表されるアミノ酸配列を含む重鎖CDR1、
配列番号8で表されるアミノ酸配列を含む重鎖CDR2、
配列番号9で表されるアミノ酸配列を含む重鎖CDR3、
配列番号10で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号11で表されるアミノ酸配列を含む軽鎖CDR2、および
配列番号12で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(C)配列番号13で表されるアミノ酸配列を含む重鎖CDR1、
配列番号14で表されるアミノ酸配列を含む重鎖CDR2、
配列番号15で表されるアミノ酸配列を含む重鎖CDR3、
配列番号16で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号17で表されるアミノ酸配列を含む軽鎖CDR2、および
配列番号18で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(11)TCTPの機能を抑制または阻害する抗体であって、上記(10)に記載の抗体とTCTPとの結合を競合阻害する抗体またはその抗原結合断片。
(12)ヒト化抗体であることを特徴とする上記(10)または(11)に記載の抗体またはその抗原結合断片。
(13)Fab、Fab’、F(ab’)2、Fv、一本鎖抗体、scFv、scFv二量体またはdsFvであることを特徴とする上記(10)から(12)までのいずれかに記載の抗原結合断片。
なお、本明細書において「~」の符号は、その左右の値を含む数値範囲を示す。
本発明の第1の実施形態は、腫瘍死細胞から放出される免疫調節因子の機能を抑制または阻害する物質を有効成分として含む、がん免疫微小環境(tumor immune microenvironment :TIME)への骨髄由来免疫抑制細胞(myeloid-derived suppressor cell:MDSC)の蓄積阻害剤(以下「本実施形態にかかる阻害剤」とも記載する)である。
ここで、がん免疫微小環境(以下「TIME」とも記載する)とは、がん内部における、がん細胞と、免疫細胞を中心とした非がん細胞の相互作用の場を指し、高度な免疫抑制を始めとしたがんの生存に有利な環境を備えたがん内部の微小環境と定義される(非特許文献1)。骨髄由来免疫抑制細胞(「MDSC」とも記載する)とは、感染症やがんなどの慢性炎症において出現する、未熟なミエロイド系細胞であり、強力な免疫抑制活性を有する。単球系骨髄由来免疫抑制細胞(「M-MDSC」とも記載する)および多型核細胞系骨髄由来免疫抑制細胞(「PMN-MDSC」とも記載する)が主要な亜集団をなし、それぞれ単球および好中球に形態および細胞表面マーカーが類似している(非特許文献3)。TIMEへのMDSCの蓄積とは、TIME内またはTIME付近にPMN-MDSCなどのMDSCが局在することを意味する。
ここで、免疫調節因子とは、免疫応答を促進または抑制し、免疫系の機能に影響を与える因子のことである。
TCTPの機能を抑制または阻害する物質として、特に限定はしないが、TCTPの機能を抑制または阻害する抗体、ペプチドアプタマーなど、あるいはTCTPを分解するもしくは分解を誘導する物質、例えば、ジヒドロアルテミシニン(dihydroartemisinin:DHA)やセルトラリン(Sertraline)など、TCTPの発現を抑制もしくは阻害する物質、例えば、siRNA、miRNAなどがある。さらに、TCTPの機能を抑制または阻害する物質として、TCTPとその受容体(TLR2)との結合を阻害する物質、例えば、TLR2アンタゴニストなども含まれる。当該阻害物質は、TCTPまたは受容体(TLR2)と相互作用する、あるいは、いずれかを分解等する物質であってもよい。
本実施形態にかかる阻害剤は、上記のTCTPの機能を抑制または阻害する物質を含むものであってもよい。
本明細書における「抗体」は、その調製方法およびその構造は特に限定されるものではなく、例えば、モノクローナル抗体、ポリクローナル抗体またはナノ抗体など、所望の抗原と所望の特性で結合する「抗体」の全てが含まれる。
本実施形態にかかる抗TCTP抗体がポリクローナル抗体の場合、例えば、免疫動物(限定はしないが、例えば、ウサギ、ヤギ、ヒツジ、ニワトリ、モルモット、マウス、ラットまたはブタなど)に対して、抗原およびアジュバントの混合物をインジェクトすることにより調製することができる。通常は、抗原および/またはアジュバントを免疫動物の皮下または腹腔内へ複数回インジェクトする。アジュバントとして、限定はしないが、例えば、完全フロイントおよびモノホスホリル脂質A合成-トレハロースジコリノミコレート(MPL-TMD)が含まれる。抗原の免疫後、免疫動物由来の血清から、定法により(例えば、ProteinAを保持したセファロースなどを用いる方法など)抗TCTP抗体を精製することができる。
モノクローナル抗体の作製方法としては、例えば、ハイブリドーマ法(KohlerおよびMilstein, Nature 256 495-497 1975)、または、組換え法(米国特許第4,816,567号)などを挙げることができる。あるいは、本実施形態にかかる抗TCTP抗体は、ファージ抗体ライブラリ(例えば、Clacksonら, Nature 352 624-628 1991;Marksら, J.Mol.Biol. 222 581-597 1991など)などから単離してもよい。より具体的に説明すると、ハイブリドーマ法を用いて調製する場合、その調製方法には、例えば、以下に示す4つの工程が含まれる:(i)抗原を免疫動物に免疫する、(ii)モノクローナル抗体分泌性(または潜在的に分泌性)のリンパ球を回収する、(iii)リンパ球を不死化細胞に融合させる、(iv)所望のモノクローナル抗体を分泌する細胞を選択する。免疫動物としては、例えば、マウス、ラット、モルモット、ハムスター、ウサギなどが選択可能である。免疫後、宿主動物から得られたリンパ球はハイブリドーマ細胞を樹立するために、ポリエチレングリコールなどの融合剤や電気融合法を用いて不死化細胞株と融合する。融合細胞としては、例えば、ラットもしくはマウスのミエローマ細胞株が使用される。細胞融合を行った後、融合しなかったリンパ球および不死化細胞株の成長または生存を阻害する基質を含む適切な培地中で細胞を生育させる。通常の技術では、酵素のヒポキサンチン-グアニンホスホリボシルトランスフェラーゼ(HGPRTまたはHPRT)を欠く親細胞を使用する。この場合、アミノプテリンがHGPRT欠損細胞の成長を阻害し、ハイブリドーマの成長を許容する培地(HAT培地)に添加される。このようにして得られたハイブリドーマから、所望の抗体を産生するハイブリドーマを選択し、選択したハイブリドーマが生育する培地から、常法に従い、目的のモノクローナル抗体を取得することができる。
このようにして調製したハイブリドーマをインビトロ培養し、あるいは、マウス、ラット、モルモット、ハムスターなどの腹水中でインビボ培養し、目的の抗体を培養上清、あるいは、腹水から調製することができる。
ナノ抗体は、例えば、以下のようにして作製することができる。ラクダ科の動物に抗原を免疫し、採取した血清から目的の抗体の有無を検出し、所望の抗体価が検出された免疫動物の末梢血リンパ球由来のRNAからcDNAを作製する。得られたcDNAからVHHをコードするDNA断片を増幅して、これを、ファージミドに挿入して、VHHファージミドライブラリを調製する。作製したVHHファージミドライブラリから数回のスクリーニングを経て、所望のナノ抗体を作製することができる。
(A)55F3抗体のCDR
重鎖CDR1アミノ酸配列が、GYSIASDYAWN(配列番号1)、
重鎖CDR2アミノ酸配列が、YINYSGSTGYNPSLKS(配列番号2)、
重鎖CDR3アミノ酸配列が、FEAGY(配列番号3)、
軽鎖CDR1アミノ酸配列が、KASQDINRYLS(配列番号4)
軽鎖CDR2アミノ酸配列が、RANRLVD(配列番号5)、および
軽鎖CDR3アミノ酸配列が、LQYNEFPLT(配列番号6)を有する。
(B)44E1抗体のCDR
重鎖CDR1アミノ酸配列が、GYTFTDHAIH(配列番号7)、
重鎖CDR2アミノ酸配列が、YISPGNGDLKYNEKFKG(配列番号8)、
重鎖CDR3アミノ酸配列が、GWTL(配列番号9)、
軽鎖CDR1アミノ酸配列が、KSSQSLLYRSNQKNYLV(配列番号10)
軽鎖CDR2アミノ酸配列が、WAFTRES(配列番号11)、および
軽鎖CDR3アミノ酸配列が、QQHYSYPWT(配列番号12)を有する。
(C)51A9抗体のCDR
重鎖CDR1アミノ酸配列が、GYSITSDYAWN(配列番号13)、
重鎖CDR2アミノ酸配列が、YINYSGSTGYNPSLKS(配列番号14)、
重鎖CDR3アミノ酸配列が、FEAGY(配列番号15)、
軽鎖CDR1アミノ酸配列が、KASQDINSYLS(配列番号16)
軽鎖CDR2アミノ酸配列が、RANRLVD(配列番号17)、および
軽鎖CDR3アミノ酸配列が、LQYYEFPLT(配列番号18)を有する。
MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTATGYSIASDYAWNWIRQFPGNKLEWMGYINYSGSTGYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARFEAGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT(配列番号19)
44E1抗体の重鎖可変領域
MEWSWVFLFFLSVTTGVHSEVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWAKQKPEQGLEWIGYISPGNGDLKYNEKFKGKATLTTDKSSSTAYMQLNSLTSEDSAVYFCKSGWTLWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT(配列番号20)
51A9抗体の重鎖可変領域
MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTATGYSITSDYAWNWIRQFPGNKLEWMGYINYSGSTGYNPSLKSRISITRDTSKNKFFLQLNSVTTEDTATYYCARFEAGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT(配列番号21)
MDMRTPAQFLGILLLWFPGIKCDIKMTQSPSSMSASLGERVTITCKASQDINRYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYGDMGIYYCLQYNEFPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLN(配列番号22)
44E1抗体の軽鎖可変領域
MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSPVVSVGEKVTMSCKSSQSLLYRSNQKNYLVWYQLKPGQSPKLLIYWAFTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQHYSYPWTFGGGTKLEVKRADAAPTVSIFPPSSEQLTSGGASVVCFLN(配列番号23)
51A9抗体の軽鎖可変領域
MDMRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTFTCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCLQYYEFPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLN(配列番号24)。
本発明の実施形態にかかるがんの治療薬として、有効成分(例えば、TCTPの機能を抑制または阻害する物質など)自体を投与してもよいが、一般的には、有効成分である1または複数の物質の他、1または2以上の製剤用添加物を含む治療組成物の形態で投与することが望ましい。本発明の治療薬等の有効成分としては、異なる複数の本発明の阻害剤を含んでもよい。また、本発明の治療薬等には、他の抗がん剤や免疫チェックポイント阻害剤等の既知の成分が配合されていてもよい。
一般的には、経口投与における成人一日あたりの投与量は0.01~1000mg(有効成分重量)程度であり、一日1回または数回に分けて、あるいは、数日ごとに投与することができる。注射剤として用いる場合には、成人に対して一日量0.001~100mg(有効成分重量)を連続投与または間欠投与することが望ましい。
また、キットには使用説明書が添付されてもよい。当該キットの使用説明は、紙などに印刷されたものであっても、CD-ROM、DVD-ROMなどの電磁的に読み取り可能な媒体に保存されて使用者に供給されてもよい。
ここで「治療」とは、がんに罹患したほ乳動物において引き起こされる病態の進行および悪化を阻止または緩和することを意味する。また、「予防」とは、がんを発症するおそれがあるほ乳動物患者において、その発症を予め阻止することを意味する。予防または治療の対象となる「ほ乳動物」は、ほ乳類に分類される任意の動物を意味し、特に限定はしないが、例えば、ヒトの他、イヌ、ネコ、ウサギ、フェレットなどのペット動物、ウシ、ブタ、ヒツジ、ウマなどの家畜動物などのことである。特に好ましい「ほ乳動物」は、ヒトである。
がんを発症している被験者においては、腫瘍組織や血液中において、TCTPタンパク質またはTCTP mRNAの量が健常者(がんを発症していないことが確認されている者)よりも有意に増大している(例えば、図2bなどを参照のこと)。従って、被験者由来のサンプル中において、TCTP mRNAの量またはTCTPタンパク質の量が健常者のサンプルと比較して、有意に高い場合に、当該被験者が何らかのがんを発症している可能性があるとの判断をすることができる。
本実施形態において、被験者由来のサンプルとして、限定はしないが、例えば、血液(血清など、血液由来の成分も含む)、悪性腫瘍が疑われる組織などを使用できる。サンプル中のTCTPタンパク質の量を測定する方法として、当業者であれば適切な方法を容易に選択することが可能であり、例えば、ELISA(enzyme-linked immuno-sorbent assay)法やウェスタンブロッティング法などの免疫学的測定法が使用できる。また、サンプル中のTCTP mRMAの量を測定する方法として、当業者であれば適切な方法を容易に選択することが可能であり、例えば、リアルタイムPCR(RT-qPCR)法などが挙げられる。
以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。
1-1.マウス
C57BL/6マウスおよびBALB/cマウスはCLEA Japanから購入した。ApcΔ716マウスは既報に従い作製し(19)、C57BL/6マウスのバックグラウンドで使用した。TCTP floxマウスは、中央研究院(中華民国)のHsin-Fang Yang-Yen博士にご供与頂き、C57BL/6マウスのバックグラウンドで使用した。Villin-CreERT2マウスはキュリー研究所(仏国)のSylvie Robine博士にご供与頂き、C57BL/6マウスのバックグラウンドで使用した。MyD88およびIPS-I欠損マウスは大阪大学のShizuo Akira博士にご供与頂き、C57BL/6マウスのバックグラウンドで使用した。TLR2およびTLR4欠損マウスは、株式会社オリエンタルバイオサービスから購入し、C57BL/6マウスのバックグラウンドで使用した。STING欠損マウスは、マイアミ大学(米国)のGlen N. Barber博士にご供与頂き、C57BL/6マウスのバックグラウンドで使用した。RAG1ノックアウトマウスは、信州大学のShinsuke Taki博士にご供与頂いた。別段の定めがない限り、本実施例においてオスおよびメス(6-12週齢)を使用した。全ての動物実験は、東京大学動物実験委員会の承認を得て行った。
RAW264.7細胞、マウスメラノーマ細胞株B16F10細胞、マウス線維芽細胞株Meth-A細胞およびHEK293T細胞は、 理化学研究所バイオリソース研究センター(日本)から入手した。マウス大腸癌細胞株SL4細胞株は、T Irimura博士(順天堂大学)からご供与頂いた。RAW264.7細胞、B16F10細胞およびHEK293T細胞は、10% FBS (HyClone)を添加したDMEM(ナカライテスク社)中で維持した。Meth-A細胞は、10% FBS を添加したRPMI(ナラカイテスク社)中で維持した。SL4細胞は、10% FBS を添加したDMEM-F12(Gibco)中で維持した。腹腔浸出細胞(peritoneal exudate cells:PEC)の調製については、マウス腹腔内に4% チオグリコレート(DIFCO溶液)を2 mlを投与し、4日後、腹腔をPBSで洗浄して、PECを回収した。細胞を10% FBS を添加したRPMI中、ペトリ皿上で一晩インキュベーションした。インキュベーション後、細胞をRPMI培地で洗浄し、その後の実験に使用した。
腫瘍浸潤細胞は、マウスへの腫瘍の皮下移植後回収し、フローサイトメトリーにより解析した。切除した腫瘍を細かく切断し、コラゲナーゼ(0.75 mg/ml、cat# 11088882001、Roche)、DNase I((40μg/ml、cat# 11284932001、Roche)およびディスパーゼ(0.5 mg/ml、cat# 17105041、ThermoFisher Scientific)で処理した後、激しく撹拌(180 rpm、37℃、1時間)した。得られた細胞懸濁液をセルストレイナー(cat# 352340、Falcon)に通し、RBC溶解バッファー(cat# 00-4333-57、Invitrogen)で処理した。PBSで洗浄後、最初に細胞を抗CD16/32抗体(clone 93、cat# 101302、BioLegend)と共に氷上で5分間インキュベートした。その後、細胞をPFE(2 % FBSおよび1 mM of EDTA を添加したPBS)中、氷上にて20分間染色し、LSR Fortessa (BD Biosciences)で解析した。腫瘍浸潤細胞のセルソーティングはFACSAria II(BD Biosciences)を使用して実施した。得られたデータは、FlowJoソフトウェア(BD BioSciences)で解析した。
培養上清は既報(非特許文献5)に従って調製した。SL4細胞を、PGE2(プロスタグランジンE2)を除去するために、インドメタシン(10μM)と共に一晩インキュベートし、108細胞/mlの濃度でPBSに懸濁し、37℃の恒温槽と液体窒素を使用して、凍結-融解を5回行った。その後、細胞を8,000×gで5分間遠心して壊死を起こした細胞片を除去した。培養上清は、0.45μmのメンブレンフィルター(Millipore)をと通して濾過して得た。
LPS(O55:B5)は、SIGMA-Aldrich(cat# L2637)から購入した。オリゴヌクレオチドは、FASMACから購入した。インドメタシンは、WAKO(cat# 093-02473)から購入した。組換えIL-1αは、Peprotech(cat# 211-11A)から購入した。組換えTCTPはORIGENE(cat# TP301664)から購入した。TUNEL染色は、in situ Apoptosis Detection Kit(cat# MK500、TaKaRa)を使用して実施した。組換えTCTPのエンドトキシンレベルは、LAL Endotoxin Assay Kit, Chromogenic, ToxinSensor (cat# L00350、Genscript)でアッセイし、エンドトキシンレベル< 0.1 EU/μgであることを確認した。
壊死細胞の培養上清は、まずDNase I溶液(0.5 U/μl;TaKaRa Bio)、RNase A(0.25 mg/ml;MACHEREY-NAGEL)またはPBSで処理し、37℃で30分間インキュベートした。プロテイナーゼK処理については、培養上清をプロテイナーゼK(100μg/ml;TaKaRa Bio)で、37℃にて1時間処理した。その後、プロテイナーゼKで処理したサンプルは、氷上にて20分間APMSF(5 mM;ナカライテスク)で処理し、プロテイナーゼKを不活性化した。サイトカインのmRNAの誘導は、プロテイナーゼK処理のみで消失したことから、活性化因子はタンパク質であることが示唆された。次に、培養上清をイオン交換クロマトグラフィー(Hitrap Capto Sカラム(cat# 17544105、GE healthcare)または Hitrap Capto Qカラム(cat# 11001302、GE healthcare)にかけた。Hitrap Capto S columnまたは Hitrap Capto Q columnは、まず、1 mlのDDWで洗浄した後、30 mlのPBSで平衡化した。その後、5 mlのSL4細胞培養上清をチャージし、5 mlのPBSで洗浄した。フロースルーを回収し、そこにPECを添加した。RT-qPCR解析により、Hitrap Capto SカラムのフロースルーはサイトカインmRNAを誘導するが、Hitrap Capto Qカラムのフロースルーは誘導しないことが分かった。このことは、同定対象分子がHitrap Capto Qカラムに結合することを示唆している。
組織または細胞の総RNAはNucleoSpin RNA II(MACHEREY NAGEL)を用いて抽出し、PrimeScript RT Master Mix(TaKaRa Bio)を使用して逆転写した。RT-qPCR反応は、TB Green Premix Ex TaqTM II(TaKaRa Bio)を用いて、LightCycler 480(Roche Life Science)またはLightCycler 96(Roche Life Science) を使用して行った。得られた値はGapdh mRNAの発現量に対して正規化した。プライマーは以下に示す配列のものを使用した。
Gapdh
フォワード;5’-ctcatgaccacagtccatgc-3’(配列番号26)
リバース;5’-cacattgggggtaggaacac-3’ (配列番号27)
Tnf
フォワード;5’-tcataccaggagaaagtcaacctc-3’ (配列番号28)
リバース;5’-gtatatgggctcataccagggttt-3’(配列番号29)
Cxcl1
フォワード;5’-agaccatggctgggattcac-3’(配列番号30)
リバース;5’-agcttcagggtcaaggcaag-3’ (配列番号31)
Cxcl2
フォワード;5’-tccagagcttgagtgtgacg-3’ (配列番号32)
リバース;5’-tcagttagccttgcctttgttc-3’ (配列番号33)
Cxcr2
フォワード;5’- gacaccctcatgagaaccaagc-3’ (配列番号34)
リバース;5’- gttaaggcagctgtggaggaag-3’ (配列番号35)
Il1b
フォワード;5’-gtggaccttccaggatgagg-3’ (配列番号36)
リバース;5’-cggagcctgtagtgcagttg-3’ (配列番号37)
TCTP KO SL4細胞、TCTP KO B16F10細胞およびTCTP KO Meth-A細胞は、CRISPRデザインツール(http://www.genome-engineering.org, accessed May 2017)を使用し、CRISPR/Cas9ゲノム編集により作製した。TCTP遺伝子のゲノム配列を標的化した(5’- CGGGCGGAAAAGGCCGACGC-3’(配列番号38)および5’- AGGCCCGCCATTTCCCGCGC-3’(配列番号39))。TCTP遺伝子の第1エクソンは配列番号38および配列番号39の2つの配列によって挟まれていた。これらのガイド配列に対応するオリゴヌクレオチドは、各々、pSpCas9(BB)-2A-GFP (PX458) (Addgene)およびpSpCas9(BB)-2A-Puro (PX459) V2.0(Addgene)のBbsIサイトにクローニングした。両方の発現ベクターコンストラクトをSL4細胞に導入した。コンストラクトが導入された細胞は、ピューロマイシンで選択し、次いでGFP発現細胞をFACSAria II(BD Biosciences)またはSH800S(SONY)を用いた単一細胞ソーティングに供した。
イムノブロッティング解析は、既報に従って行った(Inoueら, Nature 434, 243-249, doi:10.1038/nature03308 (2005))。β-アクチン(cat# A5441)はSigma-Aldrichから購入した。TCTP抗体(cat# ab133568およびcat# ab37506)はabcamから購入した。TCTP抗体(cat# 5128S)はCell Signaling Technologyから購入した。β-アクチンはloadingコントロールとして使用した。血清中のTCTP量を決定するために、血清をTCTP抗体(ab133568)によるイムノブロッティング解析に供した。組換えTCTP(cat# TP301664、OriGene)はTCTP量の測定のために使用した。抗体ウサギIgG-HRP (cat# NA934V、GE healthcare)または抗マウスIgG-HRP(cat# NA931V、GE healthcare)は2次抗体として使用した。イムノブロッティングシグナルはFUSION Solo S(Vilber-Lourmat)で検出しFUSION Capt Advanced software(Vilber-Lourmat)で解析した。
WT SL4細胞およびTCTP KO SL4細胞(2.5×105 細胞)、B16F10細胞(1.2×105 細胞)またはMeth-A細胞(2.5×105 細胞)は、6ウェルプレートに各ウェルに播種した。3日毎に、細胞の8分の1を新しい6ウェルプレートに継代した。細胞数は3、6および9日目に算出した。
マウスの皮下に、2×105個のSL4細胞、1×105個のB16F10細胞または5×105個のMeth-A細胞を移植した。腫瘍体積は、平均体積=πab2/6(aおよびbは、各々、長径および短径)で算出した。
TCTPflox/flox, Apc+/Δ716マウスまたはTCTPflox/flox, Apc+/Δ716, Villin-CreERT2マウスは、4 mg/マウスのタモキシフェンで5週齢から1週間に1回処理した。このマウスの腸内の腫瘍をステレオスコープ(Leica S9 D、Leica)を用いて10週齢時に解析した。
ヒトIL-2ペプチド(MYRMQLLSCIALSLALVTNS:配列番号40)のコードDNAをマウスTCTP cDNAの5’末端に融合させた。IL-2ss-TCTPおよびWT TCTPの各 cDNAフラグメントは、pMXs-IRES-GFPレトロウイルス発現ベクターに挿入した。TCTP KO SL4細胞へのレトロウイルスによるベクターの導入は、既報に従って行った(Chibaら, Elife. 2014 Aug 22;3:e04177. doi: 10.7554/eLife.04177.)その後、GFP陽性細胞をCell Sorter SH800S(SONY)で選別した。Mock細胞またはIL-2ss-TCTP導入細胞は、2×106細胞上を播種した60 mm培養ディッシュから回収した培養上清に対して、上述のイムノブロッティング解析を実施することにより同定した。
腫瘍は、移植から21日目に切除し、溶解バッファー(20mM Tris-HCl、150mM NaCl、1mM EDTA、1 % Triton X-100、1mM Na3VO4、1mM APMSF)中で細かく刻んだ。その後、氷上にて30分間インキュベーションし、溶解物を遠心し、ELISAのために上清を回収した。TIME中のマウスCXCL1およびCXCL2の産生量は、ELISAキット(R&D Systems)を使用し、添付の使用説明書に従って定量した。
T細胞は、腫瘍を持たないマウスの脾細胞からPan T cell isolation kit II(Miltenyi Biotec)を用いて収集した。調製した細胞は、CellTraceTM CFSE Cell Proliferation Kit(Thermo Fischer Scientific)を使用し、添付の使用説明書に従ってCFSE染色を行った。PMN-MDSCは、SL細胞(2×105細胞)を皮下移植したマウスの脾細胞から、移植から17-19日目に、抗Ly6-Gマイクロビーズ(Miltenyi Biotec)を用いて収集した。好中球は、PMN-MDSCと同様の方法により、腫瘍を持たないC57BL/6マウスから収集した。CFSEで標識した1×105細胞のT細胞、および5×104細胞、2.5×104細胞もしくは1.25×104細胞のPMN-MDSCを96ウェルプレートに播種した。T細胞の増殖は、Dynabeads Mouse T-Activator CD3/CD28(Thermo Fischer Scientific)を使用して3日間誘導し、その後、T細胞におけるCFSE希釈率を評価するためにフローサイトメトリー解析を行った。
NK細胞の除去については、抗asialo GM1(cat# 014-09801、WAKO)またはラットコントロールIgG(cat# 31933、Thermofisher)を腫瘍細胞の移植後1、3、7、11および15日に腹腔内投与した(200μg/マウス)。CD8+T細胞の除去については、抗CD8α(clone 2.43、cat# BE0061、Bio X Cell)またはラットコントロールIgG(cat# 31933、Thermofisher)を腫瘍細胞の移植後1、3、7および11日に腹腔内投与した(100μg/マウス)。NK細胞およびCD8+T細胞の除去については、RAG1 KOマウスに抗asialo GM1(cat# 014-09801、WAKO)またはラットコントロールIgG(cat# 31933、Thermofisher)を腹腔内投与した(200μg/マウス)。 PMN-MDSCの除去については、抗anti-Ly6G抗体(cat# BE0075-1、Bio X Cell)またはラットコントロールIgGを腫瘍細胞の移植後1、3、5、7、9および11日に腹腔内投与した。
DHA(Selleck、TX、USA)はDMSOに溶解させ、腫瘍の移植後1日目から毎日腹腔内投与(50 mg/kg)した。抗PD-1抗体およびDHAの組み合わせ投与については、6日目にDHA投与を中止した。O-バニリン(cat# 120804-10G、Merck)は、まずDMSOに500 mg/mlとなるように溶解させ、その後PBSで最終濃度50 mg/mlとなるように希釈した。希釈した溶液を腫瘍の移植後1日目から2日毎に経口投与(50 mg/kg)を行った。
1-18-1.抗体の作製
TCTPに対するマウスモノクローナル抗体は、株式会社モノクローナル抗体研究所(http://www.monoclo.com;日本国、長野)に委託し、標準的なハイブリドーマ法により作製した。BALB/cマウスをヒトTCTPの一部の配列である合成ペプチド(EDGVTPYMIFFKDGLEMEKC:配列番号25)で免役した。抗体のスクリーニングは、TCTPに対するイムノブロッティング解析および免疫沈降解析に基づいて、スクリーニングを行った。その結果、抗体55F3、44E1および51A9を得た。抗体による処理については、55F3またはコントロールIgG(cat# 0107-01、SouthernBiotech)をマウスあたり200μg腹腔内投与した。
作製したモノクローナル抗体の重鎖および軽鎖のアミノ酸配列は、PLoS ONE e0218717,14: 2019を参考にして決定した。
ハイブリドーマ細胞より総RNAを抽出し、抗体重鎖および軽鎖の可変領域に特異的なRT(逆転写)プライマー(mIGK RT、mIGHG RT、Template-switch oligo F)を使用して、cDNAを合成した。次いで、作製したcDNAを鋳型とし、引き続いて特異的プライマー(ISPCR、mIGK PCR、mIGHG PCR)を用いてPCR反応を行った。得られたcDNAをTAクローニング(pTA2 vector、TOYOBO)し、配列決定を行った。
詳細な実験条件は以下の通りである。
逆転写反応に使用したプライマー
Template-switch oligo F:5’-aagcagtggtatcaacgcagagtacatGGG-3’ (Gはリボグアニン)(配列番号41)
mIGK RT:5’-ttgtcgttcactgccatcaatc-3’(配列番号42)
mIGL RT:6’-ggggtaccatctaccttccag-3’(配列番号43)
mIGHG RT:5’-agctgggaaggtgtgcacac-3’(配列番号44)
反応液(I)(cDNA合成反応)
2μL 50 ng/μL 総RNA
1μL 10μM 逆転写反応用プライマー(mIGK RT、mIGL RTまたはmIGHG)
1μL 10 mM dNTPs.
以上を8ウェルストリップチューブ中で混合した。
反応液(II)(cDNA合成反応)
2.2μL H2O
2μL 5×SMARTScribe buffer
1μL 20 mM DTT
0.3μL 100μM template-switch oligo F.
0.5μL 100 U/μL SMARTScribe Reverse Transcriptase
以上を8ウェルストリップチューブ中で混合した。
反応液(I)をサーマルサイクラーで、72℃、3分間熱処理した。熱処理後、6μLの反応液(II)を反応液(I)に加えた後、混合液をサーマルサイクラーで、42℃、60分間インキュベートした。その後、70℃、5分間熱処理し、反応を停止した。
合成したcDNAを鋳型とし、以下に示すプライマーを使用してPCR反応を行った。得られた増幅産物を用いて、抗体重鎖および軽鎖の可変領域のDNA配列を決定した後、それがコードするアミノ酸配列を決定した。
プライマー
ISPCR F:5’-aagcagtggtatcaacgcagag-3’(配列番号45)
mIGK PCR:5’-acattgatgtctttggggtagaag-3’(配列番号46)
mIGL PCR:5’-atcgtacacaccagtgtggc-3’(配列番号47)
mIGHG PCR:5’-gggatccagagttccaggtc-3’(配列番号48)
反応液
25μL 2×PCR buffer for KOD FX
10μL 2mM dNTPs
3μL Synthesized cDNA from the RT reaction
2.5μL 10μM universal forward primer ISPCR
2.5μL 10μM リバースPCR primer(mIGK PCR、mIGL PCRまたはmIGHG PCR)
6μL H2O
1μL KOD FX(1 U/μL)
PCRサイクル
98℃、30秒反応させた後、
98℃、15秒、
63~57.5℃、30秒(各サイクルにおいて温度を0.5℃ずつ低下させる)
72℃、30秒、
以上の反応を10サイクル行い、さらに、
98℃、15秒、
56℃、30秒、
72℃、30秒、
以上の反応を15サイクル行った後、
72℃で7分間反応させた後、4℃で静置した。
640人の大腸癌患者のTDGAデータセットをcBioPortal(https://www.cbioportal.org/, accessed December 2019)からダウンロードし、cBioportal platformにおいてGISTIC 2.0解析を行った。
SL4腫瘍およびApcΔ716マウス腫瘍由来の大腸は、4% パラホルムアルデヒドを含むPBS中に保存し、パラフィン包埋を行った。CD31(cat# ab28364、abcam)またはLy6G(cat# 127601、Biolegend)に対するヘマトキシリン/エオシン(H&E)染色、TUNEL染色および3,3’-ジアミノベンジジン(3,3’-diaminobenzidine:DAB)染色は、東京大学医科学研究所の病理コアラボラトリーにて実施した。大腸癌患者の大腸のホルマリン固定パラフィン包埋組織および同じ患者の正常大腸上皮のサンプル調製および解析は金沢大学にて行われた。同解析は金沢大学のHuman Genome/Gene Analysis Research Ethics Committee of Kanazawa University (2016-086-433)にて承認され、文書による同意が患者より得られている。
PECはネクローシスを起こした細胞(2×106個のSL4細胞)の培養上清で刺激した。2時間のインキュベーション後、総RNAを抽出して、Clariom S Array(Thermo Fisher Scientific)による解析に使用した。Volcano plotは、Transcriptome Analysis Console (TAC) software v4.0(ThermoFisher Scientific)で作成した。マイクロアレイデータは、Gene Expression Omnibus (GEO) database(accession No. GSE150465)に登録した。
細胞は、35 mmのガラスボトムディッシュ(MATSUNAMI)で一晩培養した後、PBSで洗浄し、4% パラホルムアルデヒド/PBSで15分間固定した。その後、PBSで洗浄した後、0.5% Triton X-100/PBSで15分間、膜透過処理を行い、3% BSA/PBSでブロッキングした。次いで、抗TCTP抗体(ab37506)と共に2時間インキューべーションし、洗浄後、2次抗体(Alexa Fluor 594 Goat anti-rabbit IgG、cat# A-11012、Invitrogen)で処理し、免疫染色を行った。PBSで洗浄した後、核の対比染色は、VECTASHIELD Hard Set Mounting Medium with DAPI(cat# H-1500、VECTOR LABORATORIES、INC)を使用して行い、すぐに、ECLIPSE Ti顕微鏡(NIKON)を備えたC2si共焦点顕微鏡システム(NIKON)で解析を行った。
骨髄細胞および脾細胞は、SL4細胞(2×105細胞)を皮下移植後17日目のマウス(腫瘍を持つ)から収集した。Ly-6G+細胞は、anti-Ly6-G MicroBead Kit, mouse(Miltenyi Biotec)を使用して分離した。TCTP KO SL4細胞(2×105細胞)を皮下移植した1、4、7、10および13日目のマウスに、分離したLy-6G+ 細胞(4×106細胞)を静脈内に注入した。
HEK293T細胞(5×106個)を播種した後、pCXNII-FLAG-hTCTP(2μg)のみ、または、pCXNII-FLAG-hTCTP(2μg)およびpcDNA3.1-hTLR2-YFP(2μg)を、X-tremeGENE9(Roche Life Science)を使用して、HEK293T細胞へ一過的に形質導入した。pcDNA3.1-hTLR2-YFPベクターはDouglas Golenbock博士にご供与頂いた。
その後、溶解バッファー(20 mM Tris-HCL pH7.5、150 mM NaCl、1 mM EDTA、1% Triton X-100、1 mM PMSF)を使用して細胞溶解物を調製した。1 mgの細胞溶解物に対し、1μgの抗GFP抗体(598;MBL)および30μlのDynabeads Protein G for immunoprecipitation(Thermo Fisher Scientific)を用いて免疫沈降を行った。その後、免疫沈降物に対して、抗GFP抗体または抗FLAG M2抗体(Sigma Aldrich)を使用してイムノブロッティングを行った。
マウスTLR3、TLR7、TLR9およびヒトCD14 cDNAは、pCXNII-HAベクターにクローニングした。ヒトTLR2 cDNAコンストラクト(pcDNA3.1-hTLR2-YFP)、マウスTLR3 cDNAコンストラクト(pCXNII-HA-mTLR3)、マウスTLR7 cDNAコンストラクト(pCXNII-HA-mTLR7)またはマウスTLR9 cDNAコンストラクト(pCXNII-HA-mTLR9)は、NFκBルシフェラーゼレポーター(pNFκB-Luc(Stratagene))と共に、HEK293T細胞に形質移入した。TLR2刺激に関しては、ヒトCD4 cDNAコンストラクト(pCXNII-HA-hCD14)を同時形質移入した。形質移入から24時間後、2×104細胞を96ウェルディッシュに播種した。24時間後、細胞を組換えTCTPまたは各TLRアゴニストで処理した。その後、ルシフェラーゼ活性を、Pikka Gene Dual Assay kit (cat# PD-11、TOYO B-Net)を用い、MicroLumat Plus LB96V(Berthold Technologies)を使用し、添付の使用説明書に従って測定した。
G-CSF(cat# 560152、BD Biosciences)およびGM-CSF(cat# 558347、BD Biosciences)の定量は、cytometric bead assay(CBA)を使用し、添付の使用説明書に従って行った。
アポトーシスを誘導するために、血清飢餓およびアドリアマイシン処理を行った。血清飢餓に関しては、SL4細胞(5×104細胞)を48ウェルディッシュに播種して、12時間後に、培地を血清フリー培地に交換した。血清飢餓から72時間後、培養上清を回収した。アドリアマイシン処理に関しては、SL4細胞を血清飢餓と同様に播種した。12時間後、アドリアマイシン(50μM)を含む培地で細胞を24時間処理し、培養上清を回収した。
ネクローシスを誘導するために、SL4細胞(1×107細胞)を1 mlのPBSに懸濁した。凍結融解を上記「1-4.培養上清の調製」に記載したように実施した。コントロールとして、PBS中で凍結融解と同じ時間だけインキュベートしたSL4細胞の上清を使用した。低酸素処理(1% O2、5% CO2)は、マルチガスインキュベーター(MCO-5MUV-PJ、Panasonic)を使用して行った。
大腸癌患者のおよび非癌患者の血清は、Bionbank Japanから入手した。本研究は、東京大学の倫理委員会によって承認された(20-239)。TCTPの定量はイムノブロッティングにより行った。
10 cmディッシュ上のTCTP WT細胞またはTCTP KO細胞に、致死量のX線(100 Gy)を照射した。TCTP WT SL4細胞(2×105細胞)に、同量の上記TCTP WT細胞またはTCTP KO細胞を混合して、C57BL/6細胞へ皮下移植した。
サンプルサイズおよび統計的検定は、図の説明に記載したように行った。データは、別段の定めが無い限り、値の平均値±標準誤差(s.e.m.)で表示した。One-way ANOVA後のDunnet multiple comparisonもしくはOne-way ANOVA後のTukey’s multiple comparson、Spearman correlation coefficients calculation、Log-rank testおよびtwo-dided Student’s t testは、Prism 8.0(GraphPad Software)を用いて行った。全てのα水準は、0.05、p<0.05を有意差有り、とした。
2-1.免疫調節因子としての腫瘍細胞由来TCTPの同定
相当数の腫瘍細胞は、腫瘍が増殖する間に死滅(ほとんどが壊死(ネクローシス))しているため、壊死細胞の環境が、がん免疫微小環境(tumor immune microenvironment :TIME)内のMDSC(myeloid-derived suppressor cell、骨髄由来免疫抑制細胞)の免疫調節因子として機能しているとの作業仮説を立てた。腫瘍死細胞由来の免疫調節因子を同定するためのアプローチとして、まず、壊死したSL4細胞(マウス大腸癌細胞株)の培養上清モデルを使用した(非特許文献5)(図1a)。SL4細胞培養上清をマウスのPECに暴露すると、複数のサイトカインmRNAの発現が誘導された。これらmRNAをマイクロアレイ(図1b)およびRT-qPCR(図1c)で解析した。mRNAの発現誘導が確認されたたサイトカインのうち、Cxcl1およびCxcl2は、MDSCなどの造血細胞の移動を制御することが知られているため、Cxcl1およびCxcl2に着目した。CXCL1およびCXCL2に共通のレセプターであるCXCR2は、マウスにおいてPMN-MDSCの移動に重要であることが知られている(非特許文献6)。
上記結果を踏まえて、TCTP発現SL4細胞およびTCTP欠損SL4細胞によって形成される腫瘍の増殖に対し、TCTPがどのような影響を与えるかについて検討を行った。TCTP発現SL4細胞(TCTP WT SL4:WT)とTCTP欠損SL4細胞(TCTP KO SL4:KO)の全細胞溶解物を調製し、TCTPおよびβ-アクチンに対しイムノブロッティングを行い、TCTP KO SL4においてTCTPが欠損していることを確認した(図3c)。TCTP WT SL4細胞およびTCTP KO SL4細胞の増殖速度は、インビトロでは、標準条件、低酸素条件および低血清条件において、同程度であった(図3dおよびe)。これに対し、TCTP WT SL4細胞とTCTP KO SL4細胞を、C57BL/6マウスに皮下注射により移植し、インビボで生じた腫瘍の体積を経時的に測定したところ、TCTP KO SL4細胞由来の腫瘍の増殖は、TCTP WT SL4細胞由来の腫瘍に比べて、著しく遅くなっていることが分かった(図2c)。また、TCTP KO SL4細胞にTCTP遺伝子を再移入して、再びTCTPタンパク質を発現させると、インビボにおける腫瘍の増殖速度が回復することが確認された(図3f)。さらに、B16F10細胞およびMeth-A細胞のTCTP遺伝子を破壊して(図3c)、上記と同様の検討を行った。その結果、SL4細胞と同様に、野生型株とTCTP遺伝子欠損株のインビトロにおける増殖速度は同程度であったが(図3gおよびh)、マウス生体内に移植した腫瘍の増殖は、TCTP遺伝子欠損株由来の腫瘍の方が、野生型株由来の腫瘍よりも著しく遅くなっていた(図2dおよびe)。
以上の結果は、インビボにおいて、TCTPが腫瘍の増殖を促進することを示している。
さらに、腫瘍進行におけるTCTPの役割を明らかにするために、腫瘍抑制因子であるApc 遺伝子の欠損変異(Apc Δ716)によって促進される大腸腫瘍のモデルを、TCTP遺伝子の存在下または非存在下において検討した(図2f)。タモキシフェンによりTCTP遺伝子の発現を調節することが可能なTCTPflox/flox, Apc+/Δ716, villin-Cre ERT2マウスを使用した(図2f)。このマウスではタモキシフェン投与によって腸管上皮細胞特異的にTCTP遺伝子の欠損が生じる。その結果、タモキシフェン処理したマウス(TCTP欠損マウス)と処理していないマウス(TCTP遺伝子を保持するマウス)の腸に発生した腫瘍の総数を比較したところ、両者に有意差は認められなかった(図4a、Total)。他方、腫瘍の直径が2.0 mmを超える腫瘍の数が、TCTP欠損マウスにおいて著しく低下していた(図4a、2.0 mm<)。この結果は、TCTPは腫瘍の発生率(発生頻度)には影響を及ぼさないが、腫瘍の増殖を促進していることを示しており、前出のがん細胞移植マウスモデルを用いた実験結果と一致している。すなわち、TCTPは、腫瘍の発生率に関与するのではなく、生じたがんの増殖を促進させる、ということを意味している。
次に、細胞外に放出されたTCTPが腫瘍の増殖を促進するかどうか検討した。細胞外にTCTPを分泌させるために、レトロウイルス遺伝子トランスファーベクターを用いて、ヒトIL-2シグナル配列を融合したキメラTCTPタンパク質をコードするcDNAをTCTP KO SL4細胞中で発現させた細胞(IL-2ss-TCTP SL4細胞)を細胞培地で増殖させた。インビトロにおいて、TCTPタンパク質は培養上清に検出され(図5a)、細胞内には検出されなかった(図6a)。次に、TCTP KO SL4細胞の培養上清およびIL-2SS-TCTP SL4細胞の培養上清でPECを刺激し、サイトカインの誘導が見られるか調べた。その結果、IL-2ss-TCTP SL4細胞の培養上清の刺激により、Cxcl1およびCxcl2 mRNAの発現量の上昇が確認された(図6b、IL2ss-TCTP)。インビトロにおけるIL-2ss-TCTP SL4細胞の増殖速度は、TCTP KO SL4細胞と同程度であったが(図5b)、インビボにおけるIL-2ss-TCTP SL4細胞由来の腫瘍の増殖速度は、TCTP KO SL4細胞由来の腫瘍よりも有意に速くなっていた(図5c)。
以上の結果から、細胞外に放出されたTCTPは、免疫調節因子として機能し、インビボにおける腫瘍増殖を促進させる因子として機能していることが示された。
実際に、TCTP KO SL4腫瘍内とTCTP WT SL4(TCTP発現SL4)腫瘍内におけるCXCL1の量を比較すると、TCTP WTSL4腫瘍において、CXCL1発現量が有意に高かった(図5d)。同様の結果がCXCL2においても認められた(図5d)。さらに、IL-2ss-TCTP腫瘍内のCXCL1/2の量もTCTP KO SL4腫瘍内における発現量よりも有意に高かった(図5e)。この結果は、細胞外TCTPがTIME内においてこれらのケモカインを誘導することを示している。
特に、TCTP KO SL4腫瘍の増殖は、CD8+T細胞およびNK細胞のいずれかを除去することにより一部回復し(図8cおよびd)、両方の細胞を除去することで、腫瘍の増殖が促進された(図8e)。以上の観察結果に対しては、MDSCのレパトアが欠損したことにより、CD8+T細胞およびNK細胞の両方がTIME中のTCTP KO腫瘍に対して活性を保持した状態で存在しているとの説明が可能である。他方、WT腫瘍においてCD8+T細胞およびNK細胞が消失させても、腫瘍増殖には影響が生じなかった。この結果は、これらのエジェクター細胞が、より強力なMDSCレパトアによって、すでに機能不全の状態に陥っていることを示唆する(図8c~e)。注目すべきは、TAM、MDSCおよび間質細胞上のPD-L1の発現量、およびTCTP WT腫瘍およびTCTP KO腫瘍中のCD8+T細胞上のPD-1の発現量には、基本的に差が見られなかったことである(図9aおよびb)。さらに、TCTP KO腫瘍中の内皮細胞(CD31+細胞)の密度は、TCTP WT腫瘍中の密度と同程度であった(図9c)。
以上の結果から、腫瘍細胞から放出されるTCTPによってTIMEに取り込まれるPMN-MDSCが、CD8+T細胞およびNK細胞による抗腫瘍免疫作用を抑制して少なくとも一部において腫瘍増殖を促進することを示唆される。
これまでの結果から、TIME(がん免疫微小環境)において、TCTP-CXCL1/2-PMN-MDSC経路が抗腫瘍免疫の抑制に関与していることが考えられる。そこで、次に、どのような細胞種がTIMEにおけるケモカインの誘導の要因になっているかを検討した。TCTP WT SL4腫瘍のTIMEおよびTCTP KO SL4腫瘍のTIMEから、免疫細胞のサブセットを選別し、ケモカインのmRNA発現レベルを調べたところ、D11b+Ly6ChighLy6G細胞(M-MDSC)が、最も多くの量のCxcl1 mRNAを発現していた(図10a)。ここで注目すべきは、TCTP KO SL4腫瘍から分離したM-MDSCのCxcl1 mRNAの発現レベルが著しく低下している点である(図10b)。この点、インビトロにおいて、SL4腫瘍細胞は、TCTPに応答せず、Cxcl1 mRNAの発現を誘導しなかった(図11a)。さらに、インビトロにおいて、TCTP WT SL細胞とTCTP KO SL4細胞におけるCxcl1 mRNAの発現レベルは同等であった(図11b)。従って、インビボにおいて、腫瘍細胞自体は、TCTPの刺激により、CXCL1をほとんど誘導しないと考えられる。また、CXCL1/2ケモカインのレセプター遺伝子であるCxcr2のmRNAの発現は、他のどの細胞よりもPMN-MDSCにおいて顕著に高かった(図11c)。これらの結果は、TCTPがM-MDSCに作用し、CXCL1/2ケモカインの発現を誘導し、そのCXCL1/2ケモカインの刺激により、CXCR2発現PMN-MDSC細胞がTIMEへの移動し、抗腫瘍免疫系の抑制が起きることによって腫瘍成長が促進されることを示唆する。
次に、TCTPの阻害抗体および阻害剤が、腫瘍の増殖へどのような影響をおよぼすか検討した。ヒトTCTPペプチド(配列番号25)に対するモノクローナル抗体(55F3、44E1、51A9)を作製した。配列番号25のペプチドは、ヒトTCTPタンパクのC末端側20残基である。
まず、55F3抗体(55F3)がマウスTCTPに対して種交差性があることを確認した(図12a)。55F3またはコントロールIgGを含むSL4細胞の培養上清でPECを刺激したところ、55F3を含むSL4細胞の培養上清で刺激したPECからのCxcl1 mRNAの発現が抑制された(図12b)。次に、SL4細胞をC57BL/6マウスに皮下注入により移植した後、55F3またはコントロールIgG(200μg/マウス)を、移植後1日から2日毎に腹腔内投与し、腫瘍体積を測定した。その結果、55F3を投与したことにより、SL4腫瘍細胞の増殖速度が低下し(図13a)、TIME中におけるPMN-MDSCの量が減少した(図6b)。この結果は、TCTP-CXCL1/2-MDSC経路を阻害すると、腫瘍の増殖が抑制されることを示している。
また、TCTPに結合し、TCTPのプロテアソームでの分解を促進することが知られているジヒドロアルテミシニン(dihydroartemisinin:DHA)の効果(非特許文献11)を調べた。図6cに示すように、DHAの腹腔内投与により、SL4腫瘍の増殖が阻害されことが分かった。他方、このDHAによる腫瘍増殖の阻害効果は、TCTP KO腫瘍では観察されなかったことから(図12c)、腫瘍の増殖阻害においてTCTPが実際にターゲットになっていることが示された。以上の結果は、TCTP阻害物質(TCTPアンタゴニスト)ががん治療剤として有効であることを示すものである。
ヒトのがんにおけるTCTPの役割について検討するために、ヒト大腸癌(human colorectal cancer :CRC)患者由来の血清中におけるTCTPタンパク質量を測定した。マウスモデル(図2bおよび図3g)と同様に、がん患者由来の血清サンプル中のTCTPタンパク質量の方が、コントロールサンプル中の量よりも多かった(図13f)。また、正常大腸組織と比較して、CRC組織中のTCTPタンパク質の量の方が多かった(図13g)。さらに、TCTPの発現量の上昇が、がんの進行と相関していた(図13h)。TCTPタンパク質量の上昇は、腫瘍病巣において確認されたが、間質領域では確認されなかった(図12d)。このことは、TCTPの発現は腫瘍細胞において選択的に上昇することを示している。また、CRC組織をヒトPMN-MDSCマーカーであるCD15に対する抗体で染色したところ、TCTPタンパク質の発現量とCD15+細胞の数が、正の相関を示した(図13i)。これらの結果は、TCTP-OMN-MDSC経路がヒトの癌においても機能していることを示唆している。
Claims (13)
- 腫瘍死細胞から放出される免疫調節因子の機能を抑制または阻害する物質を有効成分として含む、がん免疫微小環境(tumor immune microenvironment :TIME)への骨髄由来免疫抑制細胞(myeloid-derived suppressor cell:MDSC)の蓄積阻害剤。
- 前記骨髄由来免疫抑制細胞が、多型核細胞系骨髄由来免疫抑制細胞(polymorphonuclear myeloid-derived suppressor cell:PMN-MDSC)である、請求項1に記載の阻害剤。
- 前記免疫調節因子が、TCTP(translationally controlled tumor protein)である、請求項1または請求項2に記載の阻害剤。
- 前記TCTPの機能を抑制または阻害する物質が抗体である、請求項3に記載の阻害剤。
- 前記TCTPの機能を抑制または阻害する物質がジヒドロアルテミシニン(dihydroartemisinin:DHA)である、請求項3に記載の阻害剤。
- がんの治療薬または治療用組成物であって、請求項1から請求項5までのいずれか1項に記載の阻害剤を有効成分として含む、前記治療薬または治療用組成物。
- 前記がんが、大腸癌、悪性黒色腫または線維肉腫である、請求項6に記載の治療薬または治療用組成物。
- がんの診断方法または診断補助方法であって、被験者由来のサンプル中に存在するTCTP mRNAまたはTCTPタンパク質の量を測定することを含む、前記診断方法または診断補助方法。
- 前記サンプルが血液または組織である、請求項8に記載の方法。
- CDR(complementarity determining region)1~3のアミノ酸配列が下記(A)、(B)または(C)のいずれかを満たすことを特徴とする抗体またはその抗原結合断片。
(A)配列番号1で表されるアミノ酸配列を含む重鎖CDR1、
配列番号2で表されるアミノ酸配列を含む重鎖CDR2、
配列番号3で表されるアミノ酸配列を含む重鎖CDR3、
配列番号4で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号5で表されるアミノ酸配列を含む軽鎖CDR2、および
配列番号6で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(B)配列番号7で表されるアミノ酸配列を含む重鎖CDR1、
配列番号8で表されるアミノ酸配列を含む重鎖CDR2、
配列番号9で表されるアミノ酸配列を含む重鎖CDR3、
配列番号10で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号11で表されるアミノ酸配列を含む軽鎖CDR2、および
配列番号12で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(C)配列番号13で表されるアミノ酸配列を含む重鎖CDR1、
配列番号14で表されるアミノ酸配列を含む重鎖CDR2、
配列番号15で表されるアミノ酸配列を含む重鎖CDR3、
配列番号16で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号17で表されるアミノ酸配列を含む軽鎖CDR2、および
配列番号18で表されるアミノ酸配列を含む軽鎖CDR3を有する。 - TCTPの機能を抑制または阻害する抗体であって、請求項10に記載の抗体とTCTPとの結合を競合阻害する抗体またはその抗原結合断片。
- ヒト化抗体であることを特徴とする請求項10または請求項11に記載の抗体またはその抗原結合断片。
- Fab、Fab’、F(ab’)2、Fv、一本鎖抗体、scFv、scFv二量体またはdsFvであることを特徴とする請求項10から請求項12までのいずれか1項に記載の抗原結合断片。
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