WO2023002943A1 - がん患者の予後を予測するためのバイオマーカー、がん患者の予後を予測するための方法、がん患者における、がん治療薬の効果を予測するための方法、及び、がん患者の予後を予測するためのキット - Google Patents
がん患者の予後を予測するためのバイオマーカー、がん患者の予後を予測するための方法、がん患者における、がん治療薬の効果を予測するための方法、及び、がん患者の予後を予測するためのキット Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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Definitions
- Immune checkpoint inhibitors inhibit the transmission of immunosuppressive signals by binding to immune checkpoint molecules or their ligands that suppress immune responses to self and suppress excessive immune responses. It is a drug that releases the suppression of activation of T cells and other cells of the immune system by checkpoint molecules.
- immune checkpoint inhibitors suppress the transmission of immunosuppressive signals by binding to immune checkpoint molecules or their ligands that have the effect of suppressing immune responses to tumors, T by immune checkpoint molecules Releases suppression of activation of cells and other immune system cells, and exerts anti-tumor effects.
- LILRB4 Leukocyte Ig-like receptor B4, hereinafter also referred to as B4
- B4 is an immunosuppressive receptor for an unknown ligand
- Patent Document 2 reports that the physiological ligand of B4 is fibronectin, and that substances that inhibit the binding of B4 and fibronectin are effective in treating immune checkpoint-related diseases.
- B4 supports the infiltration of tumor cells into tissues, the onset of acute leukemia is suppressed in B4 knockout cell lines of tumor cells, and tumor-bearing mice with B4 knockout cell lines It has been reported that the survival rate is better than that of the parental tumor-bearing mouse (Non-Patent Document 1).
- TILs Tumor infiltrating lymphocytes
- Non-Patent Document 3 the prognosis of patients positive for TIL immunosuppressive receptor PD-1 expression is poor
- Non-Patent Document 4 the expression level of PD-1 on TIL did not affect the overall survival rate and recurrence-free survival rate
- the expression of immunosuppressive receptors on TIL is still uncertain.
- the present invention provides a biomarker for predicting the prognosis of cancer patients, a method for predicting the prognosis of cancer patients, and a method for predicting the effects of cancer therapeutic agents in cancer patients, comprising LILRB4. , and to provide a kit for predicting the prognosis of cancer patients.
- LILRB4 or the LILRB4 gene is useful as a biomarker for predicting the prognosis of cancer patients, and completed the present invention.
- the present invention includes the following aspects.
- a biomarker for predicting the prognosis of cancer patients comprising an immunosuppressive receptor LILRB4 or an immunosuppressive receptor LILRB4 gene.
- the biomarker of [1], wherein the cancer is lung cancer.
- a method for predicting the prognosis of a cancer patient comprising the step of measuring the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in a biological sample of the cancer patient.
- the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in the tumor-infiltrating immune system cells in the sample, and the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in the control is indicative of a poor prognosis for said cancer patient.
- the step of measuring the expression level of the immunosuppressive receptor LILRB4 is a step of measuring the expression level of the immunosuppressive receptor LILRB4 using an anti-immunosuppressive receptor LILRB4 monoclonal antibody or an antibody fragment thereof. , [3] or [4].
- the amino acid sequence of complementarity determining region (hereinafter also referred to as CDR) 1 of the heavy chain variable region (hereinafter also referred to as VH) of the anti-immunosuppressive receptor LILRB4 monoclonal antibody is SEQ ID NO: 3.
- the amino acid sequence of CDR2 of VH consists of the amino acid sequence of SEQ ID NO: 4
- the amino acid sequence of CDR3 of VH consists of the amino acid sequence of SEQ ID NO: 5
- the amino acid sequence of CDR1 of the chain variable region (hereinafter also referred to as VL) consists of the amino acid sequence represented by SEQ ID NO: 6
- the amino acid sequence of CDR2 of VL consists of the amino acid sequence represented by SEQ ID NO: 7
- a method for predicting the effect of a substance that inhibits the binding of fibronectin and the immunosuppressive receptor LILRB4 as a cancer therapeutic drug in a cancer patient comprising: A step of measuring the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene, wherein the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in the tumor-infiltrating immune system cells in the sample.
- the fact that the expression level is higher than the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in a control indicates that the cancer therapeutic agent has an effect on the cancer patient. show, how.
- the method of [10], wherein the cancer is lung cancer.
- the method of [10] or [11], wherein the substance that inhibits binding between fibronectin and immunosuppressive receptor LILRB4 is an anti-immunosuppressive receptor LILRB4 antibody.
- the step of measuring the expression level of the immunosuppressive receptor LILRB4 is a step of measuring the expression level of the immunosuppressive receptor LILRB4 using an anti-immunosuppressive receptor LILRB4 monoclonal antibody or an antibody fragment thereof. , the method according to any one of [10] to [12].
- the amino acid sequence of complementarity determining region (hereinafter also referred to as CDR) 1 of the heavy chain variable region (hereinafter also referred to as VH) of the anti-immunosuppressive receptor LILRB4 monoclonal antibody is SEQ ID NO: 3.
- the amino acid sequence of CDR2 of VH consists of the amino acid sequence of SEQ ID NO: 4
- the amino acid sequence of CDR3 of VH consists of the amino acid sequence of SEQ ID NO: 5
- the amino acid sequence of CDR1 of the chain variable region (hereinafter also referred to as VL) consists of the amino acid sequence represented by SEQ ID NO: 6
- the amino acid sequence of CDR2 of VL consists of the amino acid sequence represented by SEQ ID NO: 7
- the VL consists of the amino acid sequence represented by SEQ ID NO:8.
- the kit of [15], wherein the cancer is lung cancer.
- the method of [15] or [16], wherein the reagent for measuring the expression level of the biomarker comprises an anti-immunosuppressive receptor LILRB4 monoclonal antibody or an antibody fragment thereof.
- the amino acid sequence of complementarity determining region (hereinafter also referred to as CDR) 1 of the heavy chain variable region (hereinafter also referred to as VH) of the anti-immunosuppressive receptor LILRB4 monoclonal antibody is SEQ ID NO: 3.
- the amino acid sequence of CDR2 of VH consists of the amino acid sequence of SEQ ID NO: 4
- the amino acid sequence of CDR3 of VH consists of the amino acid sequence of SEQ ID NO: 5
- the amino acid sequence of CDR1 of the chain variable region (hereinafter also referred to as VL) consists of the amino acid sequence represented by SEQ ID NO: 6
- the amino acid sequence of CDR2 of VL consists of the amino acid sequence represented by SEQ ID NO: 7
- the VL The kit of [17], wherein the amino acid sequence of the CDR3 of A consists of the amino acid sequence represented by SEQ ID NO:8.
- a biomarker for predicting the prognosis of cancer patients a method for predicting the prognosis of cancer patients, comprising LILRB4 or the LILRB4 gene, a method for predicting the prognosis of cancer patients, and the effects of cancer therapeutic agents in cancer patients.
- a method for prediction and a kit for predicting the prognosis of cancer patients can be provided.
- FIG. 10 shows representative examples of LILRB4 immunohistochemical staining of TIL regions in lung adenocarcinoma patients in Experimental Example 4.
- FIG. The upper figure (No. 9605) shows a micrograph of immunohistochemical staining of a TIL region with low LILRB4 expression
- the lower figure (No. 0577) shows a micrograph of immunohistochemical staining of a TIL region with high LILRB4 expression.
- the portion enclosed by the dotted line is the tumor portion, and the portion except for that indicates the TIL region.
- the left figure shows a 10-fold enlarged photograph
- the right figure shows a 40-fold enlarged photograph.
- FIG. 10 is a diagram showing the overall survival rate of the LILRB4 high expression group and the LILRB4 low expression group in Experimental Example 5.
- FIG. 10 is a diagram showing the recurrence-free survival rates of the LILRB4 high expression group and the LILRB4 low expression group in Experimental Example 5.
- FIG. 10 shows representative examples of LILRB4 immunohistochemical staining of TIL regions in lung squamous cell carcinoma patients in Experimental Example 7.
- FIG. 10 is a diagram showing the overall survival rate of the LILRB4 high expression group and the LILRB4 low expression group in Experimental Example 9.
- FIG. 10 is a diagram showing the overall survival rate of the LILRB4 high expression group and the LILRB4 low expression group in Experimental Example 9.
- FIG. 10 is a diagram showing the recurrence-free survival rates of the LILRB4 high expression group and the LILRB4 low expression group in Experimental Example 9.
- FIG. 10 shows the overall survival rate of the LILRB4 high expression group and the LILRB4 low expression group in Experimental Example 10.
- FIG. 10 is a diagram showing the recurrence-free survival rates of the LILRB4 high expression group and the LILRB4 low expression group in Experimental Example 10.
- the present invention provides a biomarker for predicting the prognosis of cancer patients consisting of the immunosuppressive receptor LILRB4 or the LILRB4 gene.
- LILRB4 The base sequence and amino acid sequence of LILRB4 can be found in the database provided by the US Center for Biotechnology Information (NCBI).
- NCBI US Center for Biotechnology Information
- Entrez Gene ID is 11006 (as of June 17, 2019)
- RefSeq Protein ID is NP_001265355.2, NP_001265356.2, NP_001265357.2, NP_001265357.2, NP_001265356 (isoform NP_0012653502) to 5).
- Mouse (Mus musculus) LILRB4 includes GeneID 14728 (as of June 24, 2016) and NP_038560.1, and rat (Rattus norvegicus) LILRB4 has GeneID 292594 (as of April 18, 2019).
- RefSeq ProteinID is NP_001013916, and other animals are also known to have LILRB4.
- cancer patients with high levels of LILRB4 or LILRB4 gene expression in tumor-infiltrating immune system cells containing TIL have a poor prognosis.
- the marker for predicting the prognosis of cancer patients is useful for predicting the prognosis of cancer patients and as an appropriate selection index for cancer treatment strategies. For example, by measuring the expression of PD-1 or PD-L1 on the cancer tissue of cancer patients, it is possible to determine whether the patient is an application of an anti-PD-1 inhibitory antibody or an anti-PD-L1 inhibitory antibody.
- anti-PD-1 inhibitory antibody or PD-L1 inhibitory antibody is expected to be effective for patients with low expression levels of PD-1 and its ligand PD-L1 in cancer tissues. may not be possible.
- cancer patients with high expression levels of LILRB4 or LILRB4 gene in tumor-infiltrating immune system cells of cancer patients have a poor prognosis. Therefore, cancer patients with high LILRB4 or LILRB4 gene expression on tumor-infiltrating immune system cells are judged to have a poor prognosis.
- a substance that inhibits the binding of fibronectin to the immunosuppressive receptor LILRB4 such as an anti-LILRB4 inhibitory antibody.
- LILRB4 or the LILRB4 gene is preferably human for predicting the prognosis of cancer patients. That is, the biomarker for predicting the prognosis of cancer patients in this embodiment is preferably human LILRB4 or human LILRB4 gene.
- the LILRB4 gene may be mRNA of the LILRB4 gene, or cDNA obtained by reverse transcription of the mRNA of the LILRB4 gene.
- the cancer targeted by the biomarker for predicting the prognosis of cancer in cancer patients of the present embodiment is not particularly limited, but examples thereof include solid cancer, and more specifically, lung cancer. , breast cancer, liver cancer, colon cancer, malignant melanoma, etc., and is preferably used for lung cancer.
- Lung cancer includes non-small cell lung cancer and small cell lung cancer.
- Non-small cell lung cancer may be lung adenocarcinoma or lung squamous cell carcinoma.
- the present invention is a method for predicting the prognosis of a cancer patient, wherein the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in a biological sample of the cancer patient wherein the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in the tumor-infiltrating immune system cells in the sample is equal to the immunosuppressive receptor LILRB4 or the immunosuppressive receptor in the control
- a method is provided wherein a high relative expression level of the somatic LILRB4 gene is indicative of a poor prognosis for said cancer patient.
- the method of this embodiment can be used to predict the prognosis of cancer patients.
- the method of this embodiment can also be said to be a method for predicting the prognosis of cancer patients.
- a poor prognosis means a lower survival rate, a higher rate of cancer recurrence, and the like.
- a good prognosis means a higher survival rate, a lower rate of cancer recurrence, and the like. That is, the present invention is a method for predicting the prognosis of a cancer patient, comprising measuring the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in a biological sample of the cancer patient.
- the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in the tumor-infiltrating immune system cells in the sample is equal to the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in the control
- a method in which an expression level equal to or lower than the expression level of is indicative of a good prognosis for the cancer patient.
- the method of the present embodiment can be used to select patients for cancer therapy using a substance that inhibits the binding of LILRB4 to fibronectin. That is, in the method of the present embodiment, the application of a substance that inhibits the binding of LILRB4 and fibronectin to patients with high LILRB4 expression or LILRB4 gene expression and a poor prognosis, It can be judged to be effective for cancer treatment.
- the substance that inhibits the binding of fibronectin to the immunosuppressive receptor LILRB4 is not particularly limited as long as it has an activity to inhibit the binding of fibronectin to LILRB4. fibronectin analogs and the like.
- the method of the present embodiment can be used to predict the effects of cancer therapeutic drugs in cancer patients. That is, in the method of the present embodiment, LILRB4 expression or LILRB4 gene expression is high for patients shown to have a poor prognosis. It can be predicted that the application of substances that do Therefore, the method of the present embodiment can be used as a method for predicting the effect of a substance that inhibits the binding of fibronectin as a cancer therapeutic drug to the immunosuppressive receptor LILRB4 in cancer patients, which will be described later. can.
- the cancer targeted by the method of the present embodiment is not particularly limited, but examples include solid cancer, more specifically lung cancer, breast cancer, liver cancer, and the like. It is preferably used.
- Lung cancer includes non-small cell lung cancer and small cell lung cancer.
- Non-small cell lung cancer may be lung adenocarcinoma or lung squamous cell carcinoma.
- the biological samples of cancer patients include, in addition to tumor specimens collected by biopsy or surgery, blood such as serum, plasma or whole blood of cancer patients, lymph fluid, interstitial fluid, cerebrospinal fluid, body cavity fluids, digestive fluids, nasal mucus, tears, sweat, urine and the like.
- the biological sample may be a biological sample itself collected from a cancer patient, or may be a sample obtained by subjecting the collected biological sample to processing such as dilution and concentration that are usually performed.
- the biological sample may be one collected or prepared at the time of performing the measuring method, or one previously collected or prepared and stored.
- LILRB4 Specific binding substances for LILRB4 include antibodies, antibody fragments, aptamers, and the like. Antibodies that specifically bind to LILRB4 (hereinafter also referred to as anti-LILRB4 antibodies) can be produced, for example, by immunizing animals such as mice with LILRB4 or fragments thereof as antigens. Alternatively, it can be generated, for example, by screening a phage library.
- the expression level of LILRB4 reflects the expression level of LILRB4 to which fibronectin binds, so that the binding of LILRB4 and fibronectin is more accurately inhibited. This is because the patient to whom the substance is applied can be selected.
- Human LILRB4 containing a signal sequence consists of the amino acid sequence represented by SEQ ID NO:1.
- the human LILRB4 signal sequence is from the 1st methionine to the 23rd alanine in SEQ ID NO: 1, and the mature human LILRB4 is from the 24th glycine in the amino acid sequence represented by SEQ ID NO: 1 to the 258th tryptophan.
- consists of the amino acid sequence of Fibronectin, a ligand of LILRB4 can bind to human LILRB4 via the amino acid sequence represented by SEQ ID NO: 2 in fibronectin. That is, the amino acid sequence represented by SEQ ID NO: 2 is the target sequence of human LILRB4 in fibronectin.
- monoclonal antibodies For the production of monoclonal antibodies, known methods for producing monoclonal antibodies, for example, Kamei Nagamune and Hiroshi Terada, “Monoclonal Antibodies” Hirokawa Shoten (1990), James W. et al. Golding, “Monoclonal Antibody”, 3rd edition, Academic Press, 1996. Monoclonal antibodies can also be produced by the DNA immunization method, which is described in Nature 1992 Mar 12; 356 152-154 and J. Am. Immunol Methods Mar 1; 249 147-154.
- a LILRB4 protein, a partial fragment (peptide) thereof, or a vector incorporating a cDNA encoding the LILRB4 protein can be used.
- the full-length LILRB4 vector which is a construct containing the full-length LILRB4 gene, is the optimal antigen gene for immunization. Constructs can also be used as antigen genes for immunization.
- the partial region of the human LILRB4 sequence is preferably a region of LILRB4 where the amino acid sequence represented by SEQ ID NO: 2, which is the target sequence of human LILRB4 in fibronectin, binds to human LILRB4 (fibronectin binding site).
- various gene introduction methods e.g., intramuscular injection, electroporation, gene gun, etc.
- immunize animals mae, or It can be carried out by subcutaneously injecting into rats, etc.
- the antibody fragment is preferably an antibody fragment comprising an antigen-binding portion, such as F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, variants thereof, fusions comprising antibody portions Proteins, fusion peptides, and the like can be mentioned.
- the antibody fragment can be produced according to a known antibody fragment production method.
- an antibody “specifically binds” means that LILRB4 binds to LILRB4 without substantially binding to other different polypeptides.
- the amino acid sequence of complementarity determining region (hereinafter also referred to as CDR) 1 of the heavy chain variable region (hereinafter also referred to as VH) is represented by SEQ ID NO: 3.
- the amino acid sequence of CDR2 of VH consists of the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence of CDR3 of VH consists of the amino acid sequence represented by SEQ ID NO: 5, and the light chain variable region (hereinafter , also referred to as VL) consists of the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence of CDR2 of VL consists of the amino acid sequence represented by SEQ ID NO: 7, and the amino acid sequence of CDR3 of VL is an anti-LILRB4 monoclonal antibody consisting of the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence of VH consists of the amino acid sequence represented by SEQ ID NO: 9, and the amino acid sequence of VL is represented by SEQ ID NO: 10 An anti-LILRB4 monoclonal antibody, etc., comprising an amino acid sequence can be mentioned.
- the anti-LILRB4 monoclonal antibody can be used after further purification if necessary.
- Techniques for purifying and isolating the anti-LILRB4 monoclonal antibody include conventionally known methods, for example, salting-out such as ammonium sulfate precipitation, gel filtration using Sephadex or the like, ion-exchange chromatography, affinity purification using a protein A column, and the like. etc.
- An aptamer is a substance that has specific binding ability to a target substance.
- Aptamers include nucleic acid aptamers, peptide aptamers, and the like.
- Nucleic acid aptamers having specific binding ability to the target peptide can be selected by, for example, systematic evolution of ligand by exponential enrichment (SELEX) method.
- Peptide aptamers having specific binding ability to the target peptide can be selected by, for example, the two-hybrid method using yeast.
- Method for measuring expression level of LILRB4 or LILRB4 gene As a method for measuring the expression level of LILRB4, an immunological measuring method such as ELISA, Western blotting, or immunohistochemical staining using the aforementioned specific binding substance to LILRB4 can be used. As a method for measuring the expression level of the LILRB4 gene, microarray, RT-PCR, quantitative RT-PCR and the like can be used.
- an immunological measurement method using an anti-LILRB4 monoclonal antibody after reacting LILRB4 in a biological sample with the anti-LILRB4 monoclonal antibody or antibody fragment thereof, a label is bound to the anti-LILRB4 monoclonal antibody or antibody fragment thereof.
- the labeled antibody or labeled antibody fragment is added to generate an immune complex consisting of LILRB4, the anti-LILRB4 monoclonal antibody or antibody fragment thereof, and the labeled antibody or labeled antibody fragment, and the generated immune complex
- Examples thereof include an immunological measurement method for LILRB4 in a biological sample, which measures the amount of labeling in the sample.
- the immunological measurement method includes enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA), depending on the labeling of the labeled detection antibody. ), electrochemiluminescence immunoassay, immunohistochemical staining, etc., and any of these can be used in the immunological measurement method. Immunohistochemical staining is preferred because it allows easy and rapid measurement of the target to be detected.
- LILRB4 in the biological sample can be measured by measuring the label in the immune complex.
- LILRB4 in a biological sample can be measured by reacting a labeling enzyme with a substrate of the enzyme and measuring the absorbance of the colored product (sandwich method).
- an unlabeled anti-LILRB4 monoclonal antibody or antibody fragment thereof (primary antibody) is added, and this unlabeled LILRB4 in a biological sample can also be measured by further adding an enzyme-labeled secondary antibody to an antibody (secondary antibody) against the antibody or its antibody fragment and measuring the label of the secondary antibody.
- the secondary antibody is labeled with biotin, bound with enzyme-labeled avidin or streptavidin to biotin, the secondary antibody is labeled with an enzyme or the like, and the label of the secondary antibody is measured.
- LILRB4 can also be measured in biological samples.
- an unlabeled anti-LILRB4 monoclonal antibody or antibody fragment thereof is added to LILRB4 immobilized on a solid support or a conjugate of LILRB4 and a protein such as BSA, and LILRB4 and primary antibody are added to the solid support.
- a biological sample is added, a secondary antibody labeled with an antibody (secondary antibody) against this unlabeled antibody is added, and the labeled secondary antibody is labeled.
- LILRB4 in a biological sample can also be measured by measuring (competitive method).
- the step of measuring the expression level of the LILRB4 gene in the biological sample can be performed by RT-PCR, quantitative RT-PCR, etc. using a primer set capable of amplifying the LILRB4 gene.
- the expression level of the LILRB4 gene may be measured by DNA microarray analysis, northern blotting, or the like using a probe that specifically hybridizes to the mRNA of the LILRB4 gene.
- the mRNA of the LILRB4 gene has the nucleotide sequence described in the RefSeq database entry identified by the above ID.
- the mRNA of the LILRB4 gene is not limited to that specified by the above-mentioned RefSeq ID.
- the mRNA of the LILRB4 gene in the biological sample can be detected.
- the probe is not particularly limited as long as it specifically hybridizes to the mRNA of the LILRB4 gene.
- the probes may be immobilized on a carrier to constitute a DNA microarray or the like.
- the prognosis of cancer patients can be predicted by contacting the DNA microarray described above with mRNA extracted from cancer patient tissues and detecting LILRB4 gene mRNA hybridized to the probe.
- the expression level of LILRB4 or LILRB4 gene in the tumor-infiltrating immune system cells in the biological sample is higher than the expression level of LILRB4 or LILRB4 gene in the control, the prognosis of the cancer patient is bad. That is, when the expression level of the LILRB4 or LILRB4 gene in the tumor-infiltrating immune system cells in the biological sample is higher than the LILRB4 or LILRB4 gene expression level in the control, the prognosis of the cancer patient is poor. can be determined.
- the expression level of LILRB4 or LILRB4 gene in the tumor-infiltrating immune system cells in the cancer patient's biological sample is equal to or lower than the expression level of LILRB4 or LILRB4 gene in the control. It indicates that the prognosis of cancer patients is good. That is, when the expression level of the LILRB4 or LILRB4 gene in the tumor-infiltrating immune system cells in the cancer patient's biological sample is equal to or lower than the LILRB4 or LILRB4 gene expression level in the control, the prognosis of the cancer patient is improved. It can be determined to be good.
- Controls include, for example, biological samples derived from healthy individuals, blood such as serum, plasma or whole blood derived from healthy individuals, lymph, interstitial fluid, cerebrospinal fluid, body cavity fluid, digestive fluid, nasal mucus, tears, sweat and urine.
- blood such as serum, plasma or whole blood derived from healthy individuals
- lymph interstitial fluid
- cerebrospinal fluid cerebrospinal fluid
- body cavity fluid body cavity fluid
- digestive fluid nasal mucus, tears, sweat and urine.
- a normal tissue derived from a cancer patient and the like can be mentioned.
- the expression level of the LILRB4 or LILRB4 gene is higher in the tumor-infiltrating immune system cells in the cancer patient's biological sample than the expression level of the immunosuppressive receptor LILRB4 or immunosuppressive receptor LILRB4 gene in the control.
- an expression level of LILRB4 or the LILRB4 gene that is equal to or lower than that in the control indicates that the prognosis of the cancer patient is good.
- a reference value is set in advance based on the expression level of the LILRB4 or LILRB4 gene in a control, and a higher LILRB4 or LILRB4 gene expression level than the reference value indicates that the prognosis of the cancer patient is poor. , and being equal to or lower than the reference value may indicate that the prognosis of the cancer patient is good.
- the control may be LILRB4 or the average expression level of the LILRB4 gene in the TIL regions of multiple cancer patients.
- the average value of the expression level of the LILRB4 or LILRB4 gene in the TIL region of multiple cancer patients is obtained in advance, and this is set as a reference value, and the expression level of the LILRB4 or LILRB4 gene is higher than the reference value.
- the expression level of the LILRB4 or LILRB4 gene is higher than the reference value.
- Tumor-infiltrating immune system cells are immune system cells found in tumors, including tumor-infiltrating lymphocytes (TIL), bone marrow-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), Tumor-infiltrating dendritic cells and the like can be mentioned.
- TILs include intratumoral tumor-infiltrating lymphocytes (Intratumoral TIL) and intrastromal tumor-infiltrating lymphocytes (Stromal TIL).
- the TIL region means the tumor stroma where tumor-infiltrating immune system cells containing TIL are found.
- TIL regions in biological samples are evaluated qualitatively and quantitatively by microscopic examination of histopathological specimens of tumor specimens (hematoxylin and eosin (HE) stained specimens, immunohistochemically stained specimens, etc.) and cell analysis by flow cytometry. can do.
- the TIL region is preferably a stromal region that is not a tumor region, ie, a region where mononuclear cell infiltration is observed.
- TIL regions are also rich in stroma-associated granulocytes, tertiary lymphoid structures, i.e., follicular or distal lymphocyte aggregates with germinal centers, areas with necrosis, tumor borders. ie, outside the paratumor stromal area, other than areas such as TIL areas found in normal lung tissue.
- Non-small cell lung carcinoma (NSCLC, most of which are adenocarcinoma and squamous cell carcinoma to be resected) has also been reported to have a better prognosis as the number of stromal TILs increases. .
- NSCLC Non-small cell lung carcinoma
- the number of immune cells in the tumor site reflects the treatment response and prognosis of cancer-bearing patients.
- the prognosis of cancer patients is poor.
- the present invention provides a method for predicting the effect of a substance that inhibits the binding of fibronectin and immunosuppressive receptor LILRB4 as a cancer therapeutic drug in cancer patients, wherein the cancer measuring the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in a patient's biological sample, The expression level of the sex receptor LILRB4 gene is higher than the expression level of the immunosuppressive receptor LILRB4 or the immunosuppressive receptor LILRB4 gene in the control, and the cancer therapeutic drug is effective for the cancer patient A method is provided that shows that the
- the method of the present embodiment can be used to predict the effect of a substance that inhibits the binding of fibronectin and the immunosuppressive receptor LILRB4 as a cancer drug in cancer patients. That is, the method of the present embodiment can also be said to be a method of predicting the effect of a substance that inhibits the binding of fibronectin as a cancer therapeutic drug to the immunosuppressive receptor LILRB4 in cancer patients.
- a poor prognosis means a lower survival rate, a higher rate of cancer recurrence, and the like.
- the cancer targeted by the method of the present embodiment is not particularly limited, but examples include solid cancer, more specifically lung cancer, breast cancer, liver cancer, and the like. It is preferably used.
- Lung cancer includes non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer may be lung adenocarcinoma or lung squamous cell carcinoma.
- the cancer patient's biological sample and the control are the same as those used in the method for predicting the prognosis of the cancer patient, and the expression level of LILRB4 in the cancer patient's biological sample is
- the step of measuring and the step of measuring the expression level of the LILRB4 gene in the cancer patient's biological sample are the same as the steps in the method for predicting the prognosis of the cancer patient.
- the expression level of the LILRB4 or LILRB4 gene in the tumor-infiltrating immune system cells in the biological sample of the cancer patient is higher than the expression level of the LILRB4 or LILRB4 gene in the control.
- a substance that inhibits the binding of fibronectin and the immunosuppressive receptor LILRB4 as a cancer therapeutic agent administered to patients is shown to be effective for cancer patients. That is, when the expression level of LILRB4 or LILRB4 gene in tumor-infiltrating immune system cells in a biological sample of a cancer patient is higher than the expression level of LILRB4 or LILRB4 gene in a control, the cancer therapeutic agent is It can be expected to have an effect on the cancer patients.
- the substance that inhibits the binding of fibronectin to the immunosuppressive receptor LILRB4 is not particularly limited as long as it has an activity to inhibit the binding of fibronectin to LILRB4.
- Anti-fibronectin antibodies , anti-LILRB4 antibodies, fibronectin analogues, and the like are particularly limited as long as it has an activity to inhibit the binding of fibronectin to LILRB4.
- the amino acid sequence of complementarity determining region (hereinafter also referred to as CDR) 1 of the heavy chain variable region (hereinafter also referred to as VH) is the amino acid sequence represented by SEQ ID NO: 3.
- the amino acid sequence of CDR2 of VH consists of the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence of CDR3 of VH consists of the amino acid sequence represented by SEQ ID NO: 5, and the light chain variable region (hereinafter referred to as The amino acid sequence of CDR1 of VL) consists of the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence of CDR2 of VL consists of the amino acid sequence represented by SEQ ID NO: 7, and the amino acid sequence of CDR3 of VL consists of , an anti-human LILRB4 monoclonal antibody consisting of the amino acid sequence represented by SEQ ID NO: 8, the amino acid sequence of VH consisting of the amino acid sequence represented by SEQ ID NO: 9, and the amino acid sequence of VL being represented by SEQ ID NO: 10
- An anti-human LILRB4 monoclonal antibody which consists of an amino acid sequence, and the like can be mentioned.
- the fibronectin analogue includes any fibronectin analogue as long as it has an inhibitory effect on the binding of fibronectin to LILRB4. , any one of the following (a) to (c) peptides.
- a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 (b) contains an amino acid sequence in which one to several amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and against the fibronectin binding site of the immunosuppressive receptor LILRB4 a peptide capable of binding, (c) a peptide containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having the ability to bind to the fibronectin binding site of the immunosuppressive receptor LILRB4;
- the identity of the above amino acid sequences is 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.
- the number of deletions, substitutions or additions in the above amino acid sequence is preferably 1 to 5, more preferably 1 to 4, even more preferably 1 to 3, even more preferably 1 to 2.
- Amino acid sequence identity can be determined by BLAST searches provided on the GenBank database.
- the fibronectin analog may be a fibronectin analog in which any one of the peptides (a) to (c) and the Fc region of immunoglobulin G are fused.
- the cells were suspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 8.0), sonicated and centrifuged to collect inclusion bodies containing exB4.
- the resulting inclusion bodies were solubilized with a denaturing buffer (6 M guanidine-HCl, 50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 10 mM DTT, pH 8.0).
- denatured exB4 solution was added dropwise to refolding buffer (100 mM Tris-HCl, 2 mM EDTA, 0.4 M L-arginine-HCl, 0.5 mM oxidized glutathione) at 4°C.
- the refolded exB4 was dialyzed against PBS(-) and used as an immunogen.
- the value of SUVmax in the FDG-PET test is significantly higher in the LILRB4 high expression group, and the LILRB4 high expression group has a high degree of malignancy and is determined to have a poor cancer prognosis. be able to.
- the LILRB4 high-expressing group also had a significantly higher number of patients with positive vascular invasion, which is a factor of poor prognosis.
- the result was that multiple poor prognostic factors were observed in the LILRB4 high expression group.
- FN30-Fc mouse IgG2a Fc recombinant protein
- LILRB4 reporter cells were harvested from cultures and kept on ice until the start of the study. 5 ⁇ 10 4 B4 reporter cells were suspended in 50 ⁇ L of hybridoma culture supernatant containing anti-LILRB4 monoclonal antibody and incubated on ice for 30 minutes with gentle mixing every 10 minutes. LILRB4 reporter cells were then added directly to FN30-Fc coated or uncoated 96-well plates without washing. Next, the medium containing 60 ⁇ g/mL recombinant human ApoE (manufactured by Novoprotein, #CI02) (10% FBS, 50 ⁇ M 2-mercaptoethanol, 0.1 U/ml penicillin, RPMI- 1640) was added to the LILRB4 reporter cells.
- LILRB4 reporter cells 50 ⁇ L of medium without human ApoE was added to the LILRB4 reporter cells. Plates were then centrifuged at 400 xg for 3 minutes to attach the LILRB4 reporter cells to the bottom of the plate. LILRB4 reporter cells were incubated at 37° C. for 24 hours, GFP protein expression levels were measured by flow cytometry BDFACSAria TM III (BD Biosciences), and data were analyzed with FlowJo TM software (BD Biosciences). As a result, anti-human LILRB4 monoclonal antibody #10 was confirmed to have a good effect of inhibiting the binding of fibronectin and ApoE to human LILRB4.
- mRNA was extracted by a conventional method from the hybridoma strain producing anti-human LILRB4 monoclonal antibody #10, and the VH and VL nucleotide sequences of anti-human LILRB4 monoclonal antibody #10 were determined by the dideoxy method, and their nucleotide sequences were determined. determined the amino acid sequence encoded by Furthermore, amino acid sequences corresponding to the CDR regions were specified among the obtained amino acid sequences, and the base sequences and amino acid sequences of VH and VL were defined as CDR regions.
- the amino acid sequence of CDR1 of VH of anti-human LILRB4 monoclonal antibody #10 consists of the amino acid sequence represented by SEQ ID NO: 3
- the amino acid sequence of CDR2 of VH consists of the amino acid sequence represented by SEQ ID NO: 4
- the amino acid sequence of CDR3 of VH consists of the amino acid sequence represented by SEQ ID NO: 5
- the amino acid sequence of CDR1 of VL consists of the amino acid sequence represented by SEQ ID NO: 6
- the amino acid sequence of CDR2 of VL consists of SEQ ID NO: 7
- the amino acid sequence of CDR3 of VL was determined to consist of the amino acid sequence represented by SEQ ID NO:8.
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Abstract
Description
本願は、2021年7月20日に、日本に出願された特願2021-119757号に基づき優先権を主張し、その内容をここに援用する。
本発明は以下の態様を含む。
[1] 免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子からなる、がん患者の予後を予測するためのバイオマーカー。
[2] 前記がんが、肺がんである、[1]に記載のバイオマーカー。
[3] がん患者の予後を予測するための方法であって、前記がん患者の生体試料における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量を測定する工程を備え、前記試料中の腫瘍浸潤免疫系細胞における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量が、対照における免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量と比較して高いことが、前記がん患者の予後が不良であることを示す、方法。
[4] 前記がんが、肺がんである、[3]に記載の方法。
[5] 前記免疫抑制性受容体LILRB4の発現量を測定する工程が、抗免疫抑制性受容体LILRB4モノクローナル抗体又はその抗体断片を用いて免疫抑制性受容体LILRB4の発現量を測定する工程である、[3]又は[4]に記載の方法。
[6] 前記抗免疫抑制性受容体LILRB4モノクローナル抗体の重鎖可変領域(以下、VHとも称する)の相補性決定領域(Complementarity determining region;以下、CDRとも称する)1のアミノ酸配列が、配列番号3で表されるアミノ酸配列からなり、VHのCDR2のアミノ酸配列が、配列番号4で表されるアミノ酸配列からなり、VHのCDR3のアミノ酸配列が、配列番号5で表されるアミノ酸配列からなり、軽鎖可変領域(以下、VLとも称する)のCDR1のアミノ酸配列が、配列番号6で表されるアミノ酸配列からなり、VLのCDR2のアミノ酸配列が、配列番号7で表されるアミノ酸配列からなり、VLのCDR3のアミノ酸配列が、配列番号8で表されるアミノ酸配列からなる、[3]~[5]のいずれか一項に記載の方法。
[7] 前記がん患者が、がん治療薬を投与したがん患者である、[3]~[6]のいずれか一項に記載の方法。
[8] 前記がん治療薬が、フィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質である、[7]に記載の方法。
[9] 前記フィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質が、抗免疫抑制性受容体LILRB4抗体である、[8]に記載の方法。
[10] がん患者における、がん治療薬としてのフィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質の効果を予測するための方法であって、前記がん患者の生体試料における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量を測定する工程を備え、前記試料中の腫瘍浸潤免疫系細胞における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量が、対照における免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量と比較して高いことが、前記がん治療薬が、前記がん患者に対して効果を有することを示す、方法。
[11] 前記がんが、肺がんである、[10]に記載の方法。
[12] 前記フィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質が、抗免疫抑制性受容体LILRB4抗体である、[10]又は[11]に記載の方法。
[13] 前記免疫抑制性受容体LILRB4の発現量を測定する工程が、抗免疫抑制性受容体LILRB4モノクローナル抗体又はその抗体断片を用いて免疫抑制性受容体LILRB4の発現量を測定する工程である、[10]~[12]のいずれか一項に記載の方法。
[14] 前記抗免疫抑制性受容体LILRB4モノクローナル抗体の重鎖可変領域(以下、VHとも称する)の相補性決定領域(Complementarity determining region;以下、CDRとも称する)1のアミノ酸配列が、配列番号3で表されるアミノ酸配列からなり、VHのCDR2のアミノ酸配列が、配列番号4で表されるアミノ酸配列からなり、VHのCDR3のアミノ酸配列が、配列番号5で表されるアミノ酸配列からなり、軽鎖可変領域(以下、VLとも称する)のCDR1のアミノ酸配列が、配列番号6で表されるアミノ酸配列からなり、VLのCDR2のアミノ酸配列が、配列番号7で表されるアミノ酸配列からなり、VLのCDR3のアミノ酸配列が、配列番号8で表されるアミノ酸配列からなる、[13]に記載の方法。
[15] [1]又は[2]に記載のバイオマーカーの発現量を測定するための試薬を含む、がん患者の予後を予測するためのキット。
[16] 前記がんが、肺がんである、[15]に記載のキット。
[17] 前記バイオマーカーの発現量を測定するための試薬が、抗免疫抑制性受容体LILRB4モノクローナル抗体又はその抗体断片を含む、[15]又は[16]に記載の方法。
[18] 前記抗免疫抑制性受容体LILRB4モノクローナル抗体の重鎖可変領域(以下、VHとも称する)の相補性決定領域(Complementarity determining region;以下、CDRとも称する)1のアミノ酸配列が、配列番号3で表されるアミノ酸配列からなり、VHのCDR2のアミノ酸配列が、配列番号4で表されるアミノ酸配列からなり、VHのCDR3のアミノ酸配列が、配列番号5で表されるアミノ酸配列からなり、軽鎖可変領域(以下、VLとも称する)のCDR1のアミノ酸配列が、配列番号6で表されるアミノ酸配列からなり、VLのCDR2のアミノ酸配列が、配列番号7で表されるアミノ酸配列からなり、VLのCDR3のアミノ酸配列が、配列番号8で表されるアミノ酸配列からなる、[17]に記載のキット。
1実施形態において、本発明は、免疫抑制性受容体LILRB4又はLILRB4遺伝子からなる、がん患者の予後を予測するためのバイオマーカーを提供する。
1実施形態において、本発明は、がん患者の予後を予測するための方法であって、前記がん患者の生体試料における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量を測定する工程を備え、前記試料中の腫瘍浸潤免疫系細胞における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量が、対照における免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量と比較して高いことが、前記がん患者の予後が不良であることを示す、方法を提供する。
LILRB4に対する特異的結合物質としては、抗体、抗体断片、アプタマー等が挙げられる。LILRB4に特異的に結合する抗体(以下、抗LILRB4抗体とも称する)は、例えば、マウス等の動物にLILRB4又はその断片を抗原として免疫することによって作製することができる。あるいは、例えば、ファージライブラリーのスクリーニングにより作製することができる。
LILRB4の発現量の測定方法としては、上述のLILRB4に対する特異的結合物質を用いた、ELISA、ウエスタンブロット、免疫組織化学染色等の免疫学的測定方法により行うことができる。また、LILRB4遺伝子の発現量の測定方法としては、マイクロアレイ、RT-PCR、定量的RT-PCR等により行うことができる。
LILRB4遺伝子を増幅可能なプライマーセットとは、LILRB4遺伝子のmRNAをRT-PCR等により増幅することができるものであれば特に制限されない。
プローブとしては、LILRB4遺伝子のmRNAに特異的にハイブリダイズするものであれば特に限定されない。プローブは、担体上に固定されてDNAマイクロアレイ等を構成していてもよい。例えば、上記のDNAマイクロアレイに、がん患者の組織から抽出したmRNAを接触させ、プローブにハイブリダイズしたLILRB4遺伝子のmRNAを検出することにより、がん患者の予後を予測することができる。
このように、腫瘍局所の免疫細胞の数が、担がん患者の治療反応性や予後を反映することが明らかになっている。実施例で後述するように、がん患者の生体試料中のTILが多くても、TILを含む腫瘍浸潤免疫系細胞上のLILRB4又はLILRB4遺伝子の発現量が高ければ、がん患者の予後が不良となることが示される。すなわち、がん患者の生体試料中のTILが多くても、TILを含む腫瘍浸潤免疫系細胞上のLILRB4又はLILRB4遺伝子の発現量が高ければ、がん患者の予後が不良と判定することができる。
1実施形態において、本発明は、がん患者における、がん治療薬としてのフィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質の効果を予測するための方法であって、前記がん患者の生体試料における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量を測定する工程を備え、前記試料中の腫瘍浸潤免疫系細胞における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量が、対照における免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量と比較して高いことが、前記がん治療薬が、前記がん患者に対して効果を有することを示す、方法を提供する。
(a)配列番号2で表されるアミノ酸配列を含むペプチド、
(b)配列番号2で表されるアミノ酸配列において、1~数個のアミノ酸が欠失、挿入、置換若しくは付加されたアミノ酸配列を含み、且つ、免疫抑制性受容体LILRB4のフィブロネクチン結合部位に対し結合能を有するペプチド、
(c)配列番号2で表されるアミノ酸配列において、80%以上の同一性を有するアミノ酸配列を含み、且つ、免疫抑制性受容体LILRB4のフィブロネクチン結合部位に対し結合能を有するペプチド
1実施形態において、本発明は、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子からなる、がん患者の予後を予測するためのバイオマーカーの発現量を測定するための試薬を含む、がん患者の予後を予測するためのキットを提供する。本実施形態のキットは、本発明のがん患者の予後を予測するための方法に用いることができる。
免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子からなる、がん患者の予後を予測するためのバイオマーカーの発現量を測定するための試薬としては、上述したLILRB4に対する特異的結合物質、LILRB4遺伝子を増幅可能なプライマーセット又はLILRB4遺伝子のmRNAに特異的にハイブリダイズするプローブ等が挙げられる。LILRB4に対する特異的結合物質としては、前記抗LILRB4抗体又はその抗体断片、アプタマー等が挙げられる。LILRB4遺伝子を増幅可能なプライマーセット又はLILRB4遺伝子のmRNAに特異的にハイブリダイズするプローブとしては、上述した、プライマーセット、プローブ等が挙げられる。
非小細胞肺がん患者40例(年齢平均値68歳、35%が女性)の組織試料について、病理学的予後因子とされる、SUVmax、EGFR変異(EGFR mutation)、分化度(Degree of differenciation)、リンパ節転移(Lymph node differenciation)、脈管侵襲(Vascular invasion)、リンパ管侵襲(Lymphatic invasion)、胸膜浸潤(Pleural invasion)の有無等を調べた。その結果を表1に示す。
E.coliでの発現のために最適化されたコドンである、配列番号1で表されるアミノ酸配列を有するヒトLILRB4の156番目のイソロイシンがシステインに置換され、166番目のヒスチジンがシステインに置換されたヒトLILRB4の細胞外ドメイン(24番目のグリシンから219番目のグリシンまで;以下、exB4とも称する)のcDNAを、化学合成し、pUCIDT-KANベクター(Integrated DNA Technologies社製)に挿入し、プラスミドpUCIDT-KAN/exB4(I156C/H166C)を構築した。
このプラスミドをNdeIとXhoIで消化し、得られたLILRB4cDNA断片を、pET-26b(+)(Novagen社製)のNdeI-NcoI間をGly-×6 His tag-Alaに改変したpET-26MのNdeI/XhoI部位にライゲーションした。得られたプラスミドpET-26M/exB4(I156C/H166C)を大腸菌BL21(DE3)にトランスフェクトし、イソプロピル-β-D-チオガラクトピラノシド(IPTG)を培地に添加することによりLILRB4タンパク質の発現を誘導した。大腸菌を培養した後、菌体を溶解緩衝液(50mM Tris-HCl、300mM NaCl、pH8.0)に懸濁し、超音波処理し、遠心分離をして、exB4を含む封入体を採取した。得られた封入体を変性バッファー(6Mグアニジン-HCl、50mM Tris-HCl、100mM NaCl、10mM EDTA、10mM DTT、pH8.0)で可溶化した。exB4をリフォールディングするために、変性したexB4溶液をリフォールディングバッファー(100mM Tris-HCl、2mM EDTA、0.4M L-アルギニン-HCl、0.5mM 酸化型グルタチオン)に、4℃で滴下した。リフォールディングされたexB4をPBS(-)に透析し、免疫原とした。
ヒトLILRB4特異的モノクローナル抗体は、マウス腸骨リンパ節法(Y Sado et al.,Acta Histochem.Cytochem.,39:89-94、2006)を使用して、実験例2で得られたexB4でマウスを免疫することにより作製した。次に、以下の組換えタンパク質を用いたELISAで、モノクローナル抗体含有培養上清をスクリーニングすることにより、ヒトLILRB4特異的モノクローナル抗体産生ハイブリドーマを選択した。次に、選択されたヒトLILRB4特異的モノクローナル抗体産生ハイブリドーマを培養し、得られた培養液から、抗ヒトLILRB4モノクローナル抗体を得た。
組換えヒトLILRB2(Sino Biological社製、#14132-H08H)
組換えヒトLILRB3(Sino Biological社製、#11978-J08H)
組換えヒトLILRB4(Sino Biological社製、#16742-H08H)
組換えヒトLILRB5(Sino Biological社製、#17221-H08H)
組換えヒトLILRA1(R&D Systems社製、#9226-T4-050)
組換えヒトLILRA2(R&D Systems社製、#9040-T4-050)
組換えヒトLILRA3(Sino Biological社製、#13549-H08H)
組換えヒトLILRA4(Sino Biological社製、 #16058-H08H)
組換えヒトLILRA5(R&D Systems社製、#8956-T4-050)
組換えヒトLILRA6(Sino Biological社製、#9088-T4-050)
組換えマウスLILRB4(R&D Systems社製、#9095-T4-050)
ヤギ抗LILRB4ポリクローナル抗体(R&D Systems社製)を用いて、実験例1で用いた非小細胞肺がん患者40例のStromal TIL領域の免疫組織化学染色を行い、LILRB4の発現量を、2人の検者がダブルブラインドで評価した。乳がんで一般的に用いられている方法に倣い、LILRB4陽性細胞数は、腫瘍間質面積中に占めるLILRB4陽性細胞の面積の割合によって以下の0-5の陽性細胞スコアに分類して評価した。
0:1%未満
1:1%以上5%未満
2:5%以上10%未満
3:10%以上20%未満
4:20%以上50%未満
5:50%以上
表2に示したように、LILRB4高発現群において、肺扁平上皮がん患者の割合が77.8%と高かった。このことから、肺扁平上皮がんにおいては、TIL領域のLILRB4発現が高いことが明らかとなった。
実験例1の患者において、LILRB4高発現群と低発現群について、全生存率(全患者に対する、全患者から死亡患者を除く患者数の割合)、無再発生存率(全患者に対する、死亡患者及び再発患者を除く患者数の割合)を調べた。全生存率を図2Aに、無再発生存率を図2Bに示す。
図2A及び図2Bに示したように、全生存率及び無再発生存率ともに有意差は認められなかったが、全期間で全生存率、無再発生存率いずれもLILRB4低発現群の方が上回っていた。この結果からも、TIL領域においてLILRB4が高発現している場合は、がん患者の予後が不良となると考えられる。
実験例3で得られた抗ヒトLILRB4モノクローナル抗体について、フィブロネクチンがヒトLILRB4に結合することを阻害できるかについて、LILRB4キメラ受容体GFPレポーター細胞を用いて調べた。
マウスT細胞ハイブリドーマ細胞2B4に由来するレポーター細胞は、ヒト活性化ペア免疫グロブリン様受容体β(PILRβ)と活性化シグナルアダプタータンパク質DAP12とに融合されたヒトLILRB4の細胞外ドメインからなるキメラ受容体を発現し、活性化T細胞(NFAT)の核内転写因子の制御下で発現が誘導される緑色蛍光タンパク質(GFP)遺伝子を持つ(K Hirayasu et al.,Nat.Microbiol.1:16054、2016)。このLILRB4レポーター細胞を用いて、実験例3で得られた抗LILRB4モノクローナル抗体の、LILRB4とフィブロネクチンとの結合阻害効果を調べた。なお、試験を開始する前に、ヒトフィブロネクチンのN末端30kDa(Sigma社製、#9911、以下、FN30とも称する)をマウスIgG2aのFcと融合させた組換えタンパク質(以下、FN30-Fcとも称する)を、WO2021/029318に記載された方法で作製し、96ウェル平底プレートの各ウェルに50μLの10μg/mL FN30-Fcを含むPBS(-)を室温でプレコートした。
LILRB4レポーター細胞を加える直前に、1時間ウェルをPBS(-)で3回洗浄した。一部のウェルは、ネガティブコントロールのためにコーティングされていない状態に維持した。LILRB4レポーター細胞を培養物から回収し、試験開始まで氷上に静置した。5×104個のB4レポーター細胞を、抗LILRB4モノクローナル抗体を含む50μLのハイブリドーマ培養上清に懸濁し、10分ごとに穏やかに混合しながら氷上で30分間インキュベートした。次に、LILRB4レポーター細胞を洗浄せずに、FN30-Fcコーティングまたは非コーティングの96ウェルプレートに直接加えた。次に、60μg/mL組換えヒトApoE(Novoprotein社製、#CI02)を含む培地(10%FBS、50μM 2-メルカプトエタノール、0.1U/ml ペニシリン、0.1μg/mL ストレプトマイシンを添加したRPMI-1640)50μLをLILRB4レポーター細胞に添加した。コントロールとして、ヒトApoEを含まない50μLの培地をLILRB4レポーター細胞に添加した。次に、LILRB4レポーター細胞をプレートの底に付けるために、プレートを400×gで3分間遠心分離した。LILRB4レポーター細胞を37℃で24時間インキュベートし、GFPタンパク質の発現レベルをフローサイトメトリーBDFACSAriaTMIII(BD Biosciences社製)で測定し、データをFlowJoTMソフトウェア(BD Biosciences)で分析した。その結果、抗ヒトLILRB4モノクローナル抗体#10に良好なフィブロネクチン及びApoEとヒトLILRB4との結合を阻害する効果が確認された。
ヤギ抗LILRB4ポリクローナル抗体(R&D Systems社製)で予めLILRB4発現が強陽性(High),中陽性(Medium),弱陽性(Low)と判定された肺扁平上皮がん組織標本各1検体について、実験例6で得られた、フィブロネクチンとヒトLILRB4との結合を阻害する抗ヒトLILRB4モノクローナル抗体#10を用いて免疫組織化学染色を行った。その結果を図3に示す。図3に示したように、フィブロネクチンとヒトLILRB4との結合を阻害する抗ヒトLILRB4抗体を用いることにより、肺扁平上皮がん患者のTIL領域で、LILRB4が発現していることが確認された。フィブロネクチンとヒトLILRB4との結合を阻害する抗LILRB4抗体は、フィブロネクチンが結合するLILRB4の発現量をその生理的機能の観点で正確に反映するため、この結果は、フィブロネクチンとヒトLILRB4との結合を阻害する抗LILRB4抗体が、フィブロネクチンとヒトLILRB4との結合を阻害しない抗体よりも正確に抗LILRB4抗体が有効な患者を選択することに用いることができることを示している。
ヤギ抗LILRB4ポリクローナル抗体(R&D Systems社製)を用いて、肺腺がん患者142例、肺扁平上皮がん患者97例、合計239例を用い、実験例4と同様にして、SUVmax、EGFR変異(EGFR mutation)、分化度、リンパ節転移、脈管侵襲、リンパ管侵襲、胸膜浸潤の有無等を調べた。その結果を表3に示す。上記239例のうち、高発現群は75例、低発現群は164例で、それぞれの項目の患者数や割合は表3に記載した通りである。
また、実験例4と同様に、LILRB4高発現群において、肺扁平上皮がん患者の割合が65.3%と高かった。このことから、肺扁平上皮がんにおいては、TIL領域の腫瘍浸潤免疫系細胞上のLILRB4発現が高いことが明らかとなった。
実験例8の患者において、LILRB4高発現群と低発現群について、全生存率、無再発生存率を調べた。全生存率を図4Aに、無再発生存率を図4Bに示す。
図4A及び図4Bに示したように、全生存率及び無再発生存率ともに、LILRB4低発現群の方が有意に高かった。この結果からも、TIL領域においてLILRB4が高発現している場合は、がん患者の予後が不良となることが示された。
実験例8で用いたがん患者239例のうち、肺扁平上皮がん患者97例のみを用いて、LILRB4高発現群と低発現群について、全生存率、無再発生存率を調べた。全生存率を図5Aに、無再発生存率を図5Bに示す。
図5Aに示したように、全生存率は、有意差には至らないもののLILRB4低発現群の方が高い傾向にあった。また、図5Bに示したように、無再発生存率はLILRB4低発現群の方が有意に高かった。この結果から、肺扁平上皮がん患者においても、TIL領域においてLILRB4が高発現している場合は、予後が不良となることが示された。
Claims (18)
- 免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子からなる、がん患者の予後を予測するためのバイオマーカー。
- 前記がんが、肺がんである、請求項1に記載のバイオマーカー。
- がん患者の予後を予測するための方法であって、
前記がん患者の生体試料における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量を測定する工程を備え、
前記試料中の腫瘍浸潤免疫系細胞における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量が、対照における免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量と比較して高いことが、前記がん患者の予後が不良であることを示す、方法。 - 前記がんが、肺がんである、請求項3に記載の方法。
- 前記免疫抑制性受容体LILRB4の発現量を測定する工程が、抗免疫抑制性受容体LILRB4モノクローナル抗体又はその抗体断片を用いて免疫抑制性受容体LILRB4の発現量を測定する工程である、請求項3又は4に記載の方法。
- 前記抗免疫抑制性受容体LILRB4モノクローナル抗体の重鎖可変領域(以下、VHとも称する)の相補性決定領域(Complementarity determining region;以下、CDRとも称する)1のアミノ酸配列が、配列番号3で表されるアミノ酸配列からなり、VHのCDR2のアミノ酸配列が、配列番号4で表されるアミノ酸配列からなり、VHのCDR3のアミノ酸配列が、配列番号5で表されるアミノ酸配列からなり、軽鎖可変領域(以下、VLとも称する)のCDR1のアミノ酸配列が、配列番号6で表されるアミノ酸配列からなり、VLのCDR2のアミノ酸配列が、配列番号7で表されるアミノ酸配列からなり、VLのCDR3のアミノ酸配列が、配列番号8で表されるアミノ酸配列からなる、請求項5に記載の方法。
- 前記がん患者が、がん治療薬を投与したがん患者である、請求項3又は4に記載の方法。
- 前記がん治療薬が、フィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質である、請求項7に記載の方法。
- 前記フィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質が、抗ヒト免疫抑制性受容体LILRB4抗体である、請求項8に記載の方法。
- がん患者における、がん治療薬としてのフィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質の効果を予測するための方法であって、前記がん患者の生体試料における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量を測定する工程を備え、前記試料中の腫瘍浸潤免疫系細胞における、免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量が、対照における免疫抑制性受容体LILRB4又は免疫抑制性受容体LILRB4遺伝子の発現量と比較して高いことが、前記がん治療薬が、前記がん患者に対して効果を有することを示す、方法。
- 前記がんが、肺がんである、請求項10に記載の方法。
- 前記フィブロネクチンと免疫抑制性受容体LILRB4との結合を阻害する物質が、抗ヒト免疫抑制性受容体LILRB4抗体である、請求項10又は11に記載の方法。
- 前記免疫抑制性受容体LILRB4の発現量を測定する工程が、抗免疫抑制性受容体LILRB4モノクローナル抗体又はその抗体断片を用いて免疫抑制性受容体LILRB4の発現量を測定する工程である、請求項10又は11に記載の方法。
- 前記抗免疫抑制性受容体LILRB4モノクローナル抗体の重鎖可変領域(以下、VHとも称する)の相補性決定領域(Complementarity determining region;以下、CDRとも称する)1のアミノ酸配列が、配列番号3で表されるアミノ酸配列からなり、VHのCDR2のアミノ酸配列が、配列番号4で表されるアミノ酸配列からなり、VHのCDR3のアミノ酸配列が、配列番号5で表されるアミノ酸配列からなり、軽鎖可変領域(以下、VLとも称する)のCDR1のアミノ酸配列が、配列番号6で表されるアミノ酸配列からなり、VLのCDR2のアミノ酸配列が、配列番号7で表されるアミノ酸配列からなり、VLのCDR3のアミノ酸配列が、配列番号8で表されるアミノ酸配列からなる、請求項13に記載の方法。
- 請求項1又は2に記載のバイオマーカーの発現量を測定するための試薬を含む、がん患者の予後を予測するためのキット。
- 前記がんが、肺がんである、請求項15に記載のキット。
- 前記バイオマーカーの発現量を測定するための試薬が、抗免疫抑制性受容体LILRB4モノクローナル抗体又はその抗体断片を含む、請求項15に記載のキット。
- 前記抗免疫抑制性受容体LILRB4モノクローナル抗体の重鎖可変領域(以下、VHとも称する)の相補性決定領域(Complementarity determining region;以下、CDRとも称する)1のアミノ酸配列が、配列番号3で表されるアミノ酸配列からなり、VHのCDR2のアミノ酸配列が、配列番号4で表されるアミノ酸配列からなり、VHのCDR3のアミノ酸配列が、配列番号5で表されるアミノ酸配列からなり、軽鎖可変領域(以下、VLとも称する)のCDR1のアミノ酸配列が、配列番号6で表されるアミノ酸配列からなり、VLのCDR2のアミノ酸配列が、配列番号7で表されるアミノ酸配列からなり、VLのCDR3のアミノ酸配列が、配列番号8で表されるアミノ酸配列からなる、請求項17に記載のキット。
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