WO2014203918A1 - Method of predicting therapeutic effect of epidermal growth factor receptor tyrosine kinase inhibitors - Google Patents

Abstract

The present invention provides a method of predicting the therapeutic effect of EGFR tyrosine kinase inhibitors, said method including the following steps: (1) measuring the quantity of antibodies against a peptide comprising an amino acid sequence selected from SEQ ID NO: 2-5 in a blood sample taken from a patient before treatment with an EGFR tyrosine kinase inhibitor; and (2) comparing the measured quantity of antibodies with a reference value, whereby the EGFR tyrosine kinase inhibitors are predicted to have a high therapeutic effect in the patient if the quantity of antibodies is higher than the reference value.

Description

Methods for predicting therapeutic effects of epidermal growth factor receptor tyrosine kinase inhibitors

The present invention relates to a method for predicting the therapeutic effect of an epidermal growth factor receptor tyrosine kinase inhibitor, a peptide used in the method, a diagnostic composition, and a kit.

It has been found that activation of tyrosine kinases such as epidermal growth factor receptor (EGFR) plays an important role in cancer development and pathological progress, and research and development of molecular targeted therapeutic agents targeting them have progressed. Yes. In particular, the advent of EGFR tyrosine kinase inhibitors (EGFR-TKI) has dramatically improved the prognosis of patients with non-small cell lung cancer who are unresectable and resistant to conventional chemotherapy. However, the clinical effect is not constant among individuals, and there are not a few patients who show resistance to treatment from the beginning.

About 90,000 cases of non-small cell lung cancer patients in Japan (third place among malignant tumors) and about 65,000 deaths per year (first place among malignant tumors) And approximately 40% of advanced stage non-small cell lung cancer patients have an EGFR mutation that is subject to EGFR-TKI treatment. Currently, as a biomarker for predicting the therapeutic effect of EGFR-TKI gefitinib, analysis of EGFR gene mutation is mainly carried out generally (Non-patent Documents 1 and 2; Some). Analysis of such gene mutations is usually performed using tissue or cytological specimens collected from cancer patients, but in advanced cancer patients, only a small amount of specimens can be collected, and there are situations where analysis is difficult.

An object of the present invention is to provide a method for predicting the therapeutic effect of an EGFR tyrosine kinase inhibitor.

The present inventors measured the antibody (IgG) titer against 60 kinds of peptides consisting of 20 mer derived from the amino acid sequence of EGFR using the plasma of 42 non-small cell lung cancer patients before gefitinib treatment. When the correlation between the measured antibody titer and the patient prognosis after gefitinib treatment was examined, three types of peptides (EGFR41-60, EGFR61-80, EGFR481-500) significantly correlated with progression-free survival (PFS). In addition, three peptides (EGFR41-60, EGFR481-500, EGFR881-900) were significantly correlated with overall survival (OS). In multivariate analysis, these antibody titers were found to be independent prognostic factors regardless of other clinicopathological factors (age, smoking history, EGFR gene mutation). From the above results, the present invention was completed.

The present invention is a method for predicting the therapeutic effect of an EGFR tyrosine kinase inhibitor, which comprises the following steps:
(1) a step of measuring the amount of an antibody against a peptide comprising an amino acid sequence selected from SEQ ID NOs: 2 to 5 in a blood sample collected from a patient before treatment with an EGFR tyrosine kinase inhibitor; and (2) Comparing the amount of antibody with a reference value;
Here, when the amount of the antibody is higher than a reference value, the patient is predicted to have a high therapeutic effect by an EGFR tyrosine kinase inhibitor.
A method comprising:

In another aspect, the present invention provides a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof.

In another aspect, the present invention provides a bead or plate to which a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof is bound.

In another aspect, the present invention predicts the therapeutic effect of an EGFR tyrosine kinase inhibitor comprising a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof, or a bead to which the peptide or derivative is bound. A diagnostic composition is provided.

In another embodiment, the present invention provides a therapeutic effect of an EGFR tyrosine kinase inhibitor comprising a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof, or a bead or plate to which the peptide or derivative is bound. Providing kits for prediction.

The present invention makes it possible to select patients who can expect a therapeutic effect of an EGFR tyrosine kinase inhibitor, and contributes to improving the response rate of the therapeutic agent and preventing serious side effects due to unnecessary treatment. The present invention can further improve the accuracy of prediction of therapeutic effect by using in combination with conventional gene mutation analysis.

FIG. 1 shows Kaplan-Meier analysis for PFS and OS after initiation of gefitinib treatment. FIG. 2 shows the amino acid sequence of the EGFR protein. The wavy lines represent the peptides that correlate with the EGFR mutation eg egfr — 481 — 500, egfr — 721 — 740, egfr — 741 — 760, egfr — 841 — 860, and egfr — 1001 — 1020. The encircled lines represent egfr_41_60, egfr_61_80, egfr_481_500, and egfr_881_900, which are peptides that correlate with survival (PFS or OS). FIG. 3 shows Kaplan-Meier plots for PFS and OS in the high and low antibody titers for the selected peptides. High and low antibody titers were defined by median values. FIG. 4 shows ROC curves for PFS and OS using antibody amounts against peptides and clinicopathological features (solid line) or clinicopathological features only (dashed line). A and B peptides: EGFR481-500, EGFR41-60, and EGFR61-80; C and D peptides: EGFR481-500 and EGFR41-60. FIG. 5 shows the Cox regression solution path with lasso penalty for PFS (A) and OS (B).

In the present specification, the epidermal growth factor receptor (EGFR) -derived peptide means a peptide consisting of a part of the amino acid sequence of EGFR (SEQ ID NO: 1). In the present specification, “EGFRx-y” or “egfr_x_y” means a peptide consisting of the amino acid sequence from amino acid position x to y of SEQ ID NO: 1. For example, “EGFR41-60” or “egfr_41_60” means a peptide consisting of the 41st to 60th amino acid sequences of SEQ ID NO: 1. The methods of the present invention include EGFR41-60 (LGTFEDHFLSLQRMFNNCEV: SEQ ID NO: 2) EGFR61-80 (VLGNLEITYVQRNYDLSFLK: SEQ ID NO: 3), EGFR481-500 (FGTSGQKTKIISNRGENSCK: SEQ ID NO: 4), and EGFR881-900 (MALESILHRIYTHQSDVWS: SEQ ID NO: 5) Measuring the amount of an antibody against a selected EGFR-derived peptide (ie, a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5). In the method of the present invention, the amount of antibody against two or more kinds of peptides may be measured. In certain embodiments, the methods of the invention measure the amount of antibody to an EGFR-derived peptide selected from EGFR41-60 (SEQ ID NO: 2), EGFR61-80 (SEQ ID NO: 3), and EGFR481-500 (SEQ ID NO: 4). Including doing. In another embodiment, the method of the invention comprises an EGFR-derived peptide selected from EGFR41-60 (SEQ ID NO: 2), EGFR481-500 (SEQ ID NO: 4), and EGFR881-900 (SEQ ID NO: 5), preferably EGFR41- Measuring the amount of antibody against an EGFR-derived peptide selected from 60 (SEQ ID NO: 2) and EGFR481-500 (SEQ ID NO: 4).

EGFR tyrosine kinase inhibitors (hereinafter also referred to as EGFR-TKI) include gefitinib and erlotinib. Preferably, the EGFR tyrosine kinase inhibitor is gefitinib.

Patients in the method of the present invention include patients with cancer types that can be expected to have therapeutic effects by EGFR-TKI, such as non-small cell lung cancer, brain glioblastoma, squamous cell carcinoma of the head and neck, and pancreatic cancer. Preferably, the patient is a non-small cell lung cancer patient.

The patient's blood sample is collected prior to the treatment from the patient undergoing treatment with the EGFR tyrosine kinase inhibitor. Patients receiving treatment with an EGFR tyrosine kinase inhibitor include those patients who are scheduled to receive such treatment and patients who are considered an option for such treatment. Blood samples include whole blood, serum and plasma. Preferably, the blood sample is serum or plasma, more preferably plasma. The blood sample may be stored frozen after measurement from the patient until measurement, or may be treated with a reagent or the like as necessary. Blood samples can be prepared by routine methods known in the art.

The amount of antibody may be measured by any method known in the art. As a measuring method, ELISA method (Pedersen MK, et al., J Immunol Methods. 2006 Apr 20; 311 (1-2): 198-206. Epub 2006 Mar 6.), RIA method (Maruta T, et al. , Immunol Invest. 2006; 35 (2): 137-48.), A method using the Luminex (registered trademark) system (Komatsu N, et al., Scand J Clin Lab Invest 64, 535-546, 2004). The anti-peptide antibody to be measured is not particularly limited, but is usually IgG.

The method using the Luminex (registered trademark) system is suitable for the present invention because a high-sensitivity and high-throughput analysis is possible with a very small amount (several microliters) of blood sample. Briefly, an antibody in a patient blood sample that binds to a bead to which an EGFR-derived peptide is bound is measured by fluorescence or the like. For example, a blood sample collected from a patient is diluted as necessary, and incubated with beads to which an EGFR-derived peptide is bound, so that the anti-peptide antibody in the blood sample is bound to the EGFR-derived peptide on the beads. The beads are then incubated with biotinylated anti-human IgG to bind the anti-human IgG to the anti-peptide antibody bound to the beads. Furthermore, the amount of the anti-peptide antibody can be measured by incubating the beads with streptavidin-PE and measuring the fluorescence intensity of the PE bound to the beads. The fluorescence intensity can be measured by a multiplex bead suspension array (Luminex® system).

For measurement of the amount of antibody, a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof can be used. In the present specification, a peptide derivative means that its amino acid sequence has been altered by deletion, substitution, or addition of one or several, preferably one or two amino acid residues, but is present in a blood sample. It means a peptide that maintains reactivity with anti-peptide antibodies. The amino group and carboxyl group of the peptide and its derivative may be modified as long as recognition by the antibody is not impaired.

The peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof may be bound to beads or a plate. Peptides and derivatives may be bound directly to beads or plates, or indirectly via a linker or the like. Examples of the beads include polystyrene beads and magnetic beads. The size of the beads is not particularly limited, but for example, the diameter is 5 to 7 μm. The beads may be Luminex® beads that can be used in the Luminex® system. Examples of the plate include a polystyrene plate. The number of wells is, for example, 6, 24, 96, and 384, preferably 96. The plate may be a plate conventionally used for ELISA or RIA. The peptide or derivative can be bound to beads or plates as appropriate by those skilled in the art. There may be one kind or plural kinds of peptides or derivatives bound to one well of one bead or plate.

Measured antibody amount is compared with a reference value. The reference value is, for example, a value determined in advance based on the amount of antibody before the treatment of a patient group in which disease has progressed and / or a patient group in which disease has not progressed within a certain period after the start of treatment with an EGFR tyrosine kinase inhibitor. Alternatively, the reference value may be the median antibody amount in the patient group before the start of treatment with the EGFR tyrosine kinase inhibitor.

In the present invention, when the measured antibody amount is higher than the reference value, the patient is predicted to have a high therapeutic effect by the EGFR tyrosine kinase inhibitor. In addition, it can be predicted that the greater the amount of antibody, the higher the therapeutic effect. In the present specification, “therapeutic effect of an EGFR tyrosine kinase inhibitor is high” means that the overall survival or progression-free survival is greater when treated with an EGFR tyrosine kinase inhibitor than when the treatment is not performed. It means being extended.

In certain embodiments, the methods of the invention measure the amount of antibody to an EGFR-derived peptide selected from EGFR41-60 (SEQ ID NO: 2), EGFR61-80 (SEQ ID NO: 3), and EGFR481-500 (SEQ ID NO: 4). When the measured antibody amount is higher than the reference value, the patient is treated with an EGFR tyrosine kinase inhibitor to prolong progression-free survival compared with the case where the treatment is not performed. is expected.

In another embodiment, the method of the invention comprises an EGFR-derived peptide selected from EGFR41-60 (SEQ ID NO: 2), EGFR481-500 (SEQ ID NO: 4), and EGFR881-900 (SEQ ID NO: 5), preferably EGFR41- Measuring the amount of an antibody against an EGFR-derived peptide selected from 60 (SEQ ID NO: 2) and EGFR481-500 (SEQ ID NO: 4), and if the measured antibody amount is higher than a reference value, the patient is treated with EGFR tyrosine Treatment with a kinase inhibitor is expected to extend overall survival compared to the absence of such treatment.

The diagnostic composition of the present invention comprises a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5, or a derivative thereof, or beads to which the peptide or derivative is bound. The diagnostic composition of the present invention is used according to the method for predicting the therapeutic effect of an EGFR tyrosine kinase inhibitor described herein. The diagnostic composition of the present invention may further contain an appropriate carrier and additives such as a buffer and an isotonic agent, and may be provided in a lyophilized state and prepared at the time of use. The diagnostic composition of the present invention may be provided with instructions for use along the method of the present invention.

The kit of the present invention comprises a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof, or a bead or plate to which the peptide or derivative is bound. The kits of the invention are used according to the methods described herein for predicting the therapeutic effect of an EGFR tyrosine kinase inhibitor. A kit is a kit for performing the method by ELISA method, RIA method, or Luminex (trademark) system, for example. The kit may further include a blocking solution, a washing solution, a diluent, a detection antibody, a control sample, a plate, and instructions for use according to the method of the present invention.

The peptides and derivatives used in the present invention may be modified at their amino groups or carboxyl groups as long as recognition by the antibody is not impaired. The peptides and derivatives thereof used in the present invention can be produced by conventional peptide synthesis methods (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol2, Academic Press Inc., New York, 1976; Maruzen Co., Ltd., 1975; Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd., 1985;

In another aspect, the present invention is a method of collecting data for predicting the therapeutic effect of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor comprising the following steps:
(1) a step of measuring the amount of an antibody against a peptide comprising an amino acid sequence selected from SEQ ID NOs: 2 to 5 in a blood sample collected from a patient before treatment with an EGFR tyrosine kinase inhibitor; and (2) Comparing the amount of antibody with a reference value;
Here, when the amount of the antibody is higher than a reference value, the patient is predicted to have a high therapeutic effect by an EGFR tyrosine kinase inhibitor.
A method comprising:

In another embodiment, the present invention is a method of treating cancer comprising the following steps:
(1) a step of measuring the amount of an antibody against a peptide comprising an amino acid sequence selected from SEQ ID NOs: 2 to 5 in a blood sample collected from a patient before treatment with an EGFR tyrosine kinase inhibitor;
(2) comparing the measured antibody amount with a reference value; and (3) treating a patient whose antibody amount is higher than the reference value with an EGFR tyrosine kinase inhibitor;
A method comprising:

In another aspect, the present invention provides a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof, for the manufacture of a diagnostic composition for predicting the therapeutic effect of an EGFR tyrosine kinase inhibitor, or Use of beads to which the peptide or derivative is bound is provided.

In another aspect, the present invention relates to a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof for the manufacture of a kit for predicting the therapeutic effect of an EGFR tyrosine kinase inhibitor, or the peptide Alternatively, the use of a derivative or bound bead or plate is provided.

Hereinafter, the present invention will be further described with reference to examples. However, the present invention is not limited to the following examples in any way.

1. Materials and Methods (1) Patient, Treatment, and Sample Collection In this study, 42 non-small cell lung cancer (NSCLC) patients treated with gefitinib were treated as single patients from January 2006 to December 2008. Screening was conducted at institutions (Kurume City, Japan, Kurume University Hospital). Details of the patient's clinicopathological features, including age, gender, histology, smoking status, general condition (PS), stage, and treatment, are provided by an independent reviewer who is not informed about the clinical course. Obtained from the examination (Table 1). Because of advanced NSCLC, all patients received gefitinib (250 mg) orally once daily. Patient characteristics recorded included gender, age, general condition (PS) by the Eastern Cooperative Oncology Group (ECOG), tumor histology, smoking history, prior chemotherapy, and type of EGFR mutation. Tumor reduction effect (tumor response) was examined via computed tomography (CT) and evaluated according to RECIST (Response Evaluation Criteria in Solid Tumors). The reduction effect was confirmed at least 4 weeks (in case of complete or partial response) or 6 weeks (in case of disease stability) from the first recording. PFS (progression-free survival) was calculated from the date of initiation of EGFR-TKI treatment to the date of disease progression or the date of last treatment. The study also complies with the provisions of the Declaration of Helsinki. This study was approved by the Institutional Review Board of Kurume University.

Serum or plasma was collected from NSCLC patients treated with gefitinib before gefitinib treatment. Explanation consent documents were obtained from all subjects. Once serum or plasma was obtained, it was frozen at -80 degrees until use.

(2) EGFR mutation analysis Mutations in the EGFR gene were examined in exons 19 (E746-A750del) and 21 (L858R) by peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp (22) as previously reported. Briefly, genomic DNA was purified from paraffin-embedded tissue using the QIAamp DNA Micro kit (QIAGEN). The PCR primers used were from Invitrogen Inc. I was commissioned to synthesize. PNA clamp primers and LNA mutant probes were purchased from FASMEC (Kanagawa, Japan) and IDT (Coralville, IA), respectively. PNA-LNA PCR clamps were performed using an SDS-7500 System (Applied BioSystems).

(3) Measurement of EGFR-derived peptide and amount of antibody reactive to EGFR-derived peptide As shown in FIG. 2, 60 different 20-mer peptides designed from the sequence of EGFR protein were synthesized and purchased from Sigma Aldrich did. The peptide was dissolved in DMSO as previously reported (23). The amount of antibody specific for the EGFR-derived peptide was measured via a multiplex bead suspension array using the Luminex® system as previously reported (24). Briefly, 100 μl of diluted plasma is placed in 96-well filter plates (MABVN1250; Millipore Corp., Bedford, MA) on a plate shaker with xMAP beads (Luminex Corp., Austin, TX) conjugated with EGFR-derived peptides. And incubated at room temperature for 2 hours. After 2 hours, the plates were washed with T-PBS and incubated with 100 μl of biotinylated goat anti-human IgG (BA-3080; Vector Laboratories, Burlingame, Calif.) For 1 hour at room temperature on a plate shaker. After washing, 100 μl streptavidin-PE was added to the wells and the plates were incubated for 30 minutes at room temperature on a plate shaker. After washing the bound beads three times, 100 μl PBS was added to each well. Fluorescence on the beads was detected with the Luminex® system using 50 μl of each sample. The amount of antibody was expressed by fluorescence intensity, and the value was shown by fluorescence intensity unit (FIU) as previously reported (24). Since the FIU linear curve obtained from the sample dilution assay was 5-10,000 FIU (data not shown), the cut-off level was set to 10 FIU. The amount of antibody reactive to each of the 60 different peptides was measured.

(4) Statistical analysis It was investigated whether the amount of antibody reactive to each of 60 different peptides correlated with the mutation status. To this end, using the Wilcoxon rank sum test, the median amount of antibody reactive to each of the 60 peptides was determined for EGFR mutants (delE746-A750 and L858R) and wild type. Compared. PFS (progression free survival) was defined as the period from the start of gefitinib treatment to the day of disease progression. OS (overall survival) was defined as the period from the start of gefitinib treatment to the date of death regardless of the cause of death. Patients who could not determine PFS or OS were treated as observational censorship at the last visit date. Next, it was examined whether the amount of antibody reactive to each of 60 different peptides correlates with PFS or OS. Cox's proportional hazards model was applied with the amount of antibody reactive to each peptide as an explanatory variable, mutation status, smoking status, gender, and general status. In addition, whether or not the amount of antibody reactive to each of 60 different peptides is related to the tumor reduction effect was examined by applying logistic regression, and CR (complete response) and PR (partial response) were determined. Considered to be successful. Since 60 types of peptides were used, a difficult multiplicity problem occurred. We have identified peptides that significantly correlate with PFS (OS or tumor reduction effect), which controls the false discovery rate (FDR) to a level of 5%.

As an explanation of the analysis results, we attempted to identify peptides useful for predicting the prognosis of patients more accurately than using only clinicopathological features. Standard multivariate Cox regression (multiple regression) could not be applied due to the use of more peptides than the number of patients, which was a very difficult problem in this study. In order to avoid high-impact observations, we converted the amount of antibody reactive to each peptide by log (antibody amount to peptide + 1) and normalized to zero mean and unit standard deviation. We applied Cox regression (25, 26) with a lasso-type penalty. The lasso method is useful and is increasingly being used to analyze high-dimensional data. A notable feature of the Rasso method is sparseness. That is, a regression coefficient of a peptide that does not correlate with PFS (OS) can be evaluated as zero. Based on this feature, the inventors have identified several peptides that are expected to be useful in predicting patient prognosis. Cox regression analysis and time-dependent ROC analysis (27) were applied to determine if the amount of antibody reactive to the selected peptides is actually useful for predicting patient prognosis. The area under the ROC curve (AUC) was assessed for risk score through Cox regression by clinicopathologic features alone and Cox regression by both the amount of antibody reactive to each peptide and clinicopathological features. . AUCs were compared by calculating P values for 1000 iterations by the bootstrap method and testing for AUC equivalence. Statistical analysis was performed with R version 2.13 software and SAS version 9.3 software (SAS Institute, Cary, NC).

2. Results (1) Patient characteristics and survival analysis Table 1 shows the clinical characteristics of 42 patients. Twenty-five (59%) patients were women, 24 (57%) were non-smokers, and the median age of all patients was 63.5 years (range: 38-82 years). 38 (90%) patients have adenocarcinoma, 34 (80%) are in good general condition (Eastern Cooperative Oncology Group rating scale is 0), 15 patients (32%) EGFR-TKI treatment was given as first-line chemotherapy. To describe the types of EGFR mutations, 8 patients have a deletion in exon 19, 13 patients have a L858R missense mutation in exon 21, and 21 patients have the wild type. It was.

At the time of analysis, the median follow-up period was 418 days (range: 16-1532 days). The median PFS was 201 days (range: 11-1379 days) and the median OS was 418 days (range: 16-1532 days). A Kaplan-Meier analysis for PFS and OS after initiation of gefitinib treatment is shown in FIG. The log rank test significantly increased PFS in patients with EGFR mutations as a result of gefitinib treatment compared to PFS in patients without mutations (median 347 days versus 54 days, P = 0 .0029) (FIG. 1A) shows no significant difference between OS of these two patient groups (median 314 days vs. 128 days, P = 0.0.105, respectively) (FIG. 1B) Became clear. This PFS difference between patients with mutations and wild type patients was evident for both types of EGFR mutations (FIGS. 1C, D).

(2) Correlation between the amount of antibody to EGFR-derived peptide and EGFR mutation in NSCLC patients treated with gefitinib. We first have antibodies reactive to each of 60 different peptides. The plasma or serum from NSCLC patients was examined for quantification by the Luminex® system (Table 3, FIGS. 3A and 3B). Analyzing whether the amount of antibody reactive to each peptide correlates with the EGFR mutation, and in patients with exon 21 mutations, the amount of antibody against peptides egfr_481_500, egfr_721_740, egfr_741_760 is a mutation in exon 21 It was found to be significantly higher in patients without (P = 0.017 for egfr_481_500; P = 0.036 for egfr_721_740; P = 0.007 for egfr_741_760). In these three peptides, the median anti-peptide antibody amount in patients with the exon 21 mutation was approximately twice the median anti-peptide antibody amount in patients without the exon 21 mutation. (Table 3). On the other hand, the amount of antibody against egfr — 841 — 860 was significantly lower in patients with deletion in exon 19 than in patients without deletion (P = 0.047). The amount of antibody against egfr — 1001 — 1020 was significantly higher in patients with a deletion in exon 19. The amount of antibody reactive against the other peptides did not correlate with the EGFR mutation.

(3) Relationship between the amount of antibody against EGFR-derived peptide and survival in NSCLC patients treated with gefitinib Furthermore, whether the amount of anti-peptide antibody correlates well with the PFS and OS of NSCLC patients after treatment with gefitinib Examined. We have many p-values of less than 5% of all peptides, p-values of 38 and 32 peptides in Cox regression are less than 5%, and FDR to 5% level It was found that even with control, 35 peptides significant for PFS and 20 peptides significant for OS were identified (Table 4). We also examined whether the amount of antibody against each peptide correlates with the tumor reduction effect (CR or PR). Logistic regression analysis showed that the amount of antibody against any peptide did not correlate with the tumor reduction effect (data not shown).

(4) Identification of peptides useful for predicting patient prognosis As described above, the amount of antibody against many peptides significantly correlated with PFS and / or OS. Antibody levels for many peptides were moderately or highly correlated (data not shown). Thus, it was suggested that an antibody amount against a relatively small number of peptides may be sufficient to construct rules useful for predicting the prognosis of patients. From Cox regression with a lasso penalty, we found that the amount of antibody against egfr_41_60, egfr_61_80, and egfr_481_500 had a relatively large effect on PFS, and the amount of antibody against egfr_41_60, egfr_481_500, and egfr_881_900 compared to OS (Solution paths for PFS and OS are shown in FIGS. 5A and 5B, respectively). To construct the prediction rules for PFS, antibody amounts against egfr_41_60, egfr_61_80, and egfr_481_500 were used. In addition, since the antibody amount against egfr_41_60 and the antibody amount against egfr_881_900 were strongly correlated (Spearman's rank correlation coefficient was 0.71 and P <0.001), the present inventors Used antibody amounts against egfr_41_60 and egfr_481_500. Table 2A shows the results of Cox regression for PFS using antibody amounts to egfr_41_60, egfr_61_80, and egfr_481_500 adjusted for possible confounding PS, age, gender, and smoking status. All three peptides were found to be significant prognostic factors independent of clinicopathological features (P = 0.001 for egfr_41_60 and P = 0.020 for egfr_61_80 , Egfr_481_500, P = 0.028). Table 2B shows the results of Cox regression for OS using antibody amounts against egfr_41_60 and egfr_481_500. The amount of antibody against any peptide was found to be a significant prognostic factor independent of clinicopathological features (P = 0.018 for egfr_41_60 and P = 0.027 for egfr_481_500). Met). In order to grasp the marginal effect of the antibody amount against the selected peptide, Kaplan-Meier plots for PFS and OS in the high value group and the low value group of the antibody amount against the selected peptide are shown in FIGS. 3A and 3B, respectively. The effects of clinicopathological features have not been adjusted). In addition, using time-dependent ROC analysis, it was examined whether the prognosis prediction of patients can be improved by adding the amount of antibody against the peptide to the clinicopathological characteristics. 4A and 4B were evaluated by Cox regression as shown in Table 2A (for PFS) and Table 2B (for OS) using only the amount of antibody to the peptide and clinicopathological features, and clinicopathological features. Shown are ROC curves of annual and 2-year risk scores. The ROC curve shows that the diagnosis for 1-year and 2-year PFS is substantially improved (P <0.001 for the AUC comparison). For OS, the AUC of the 1-year and 2-year time-dependent ROC curves was significantly increased by adding the amount of antibody against the peptide (P <0.001). (FIGS. 4C and 4D). Therefore, time-dependent ROC analysis showed that the patient prognosis can be predicted more accurately for both PFS and OS by adding the amount of antibody against the peptide to the clinicopathological features.

References
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SEQ ID NO: 1 human EGFR
SEQ ID NO: 2: EGFR-derived peptide (EGFR41-60)
SEQ ID NO: 3: EGFR-derived peptide (EGFR61-80)
SEQ ID NO: 4: EGFR-derived peptide (EGFR481-500)
SEQ ID NO: 5: EGFR-derived peptide (EGFR 881-900)

Claims (10)

1. A method for predicting the therapeutic effect of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, comprising the following steps:
   (1) a step of measuring the amount of an antibody against a peptide comprising an amino acid sequence selected from SEQ ID NOs: 2 to 5 in a blood sample collected from a patient before treatment with an EGFR tyrosine kinase inhibitor; and (2) Comparing the amount of antibody with a reference value;
   Here, when the amount of the antibody is higher than the reference value, the patient is predicted to have a high therapeutic effect by the EGFR tyrosine kinase inhibitor.
2. The method according to claim 1, wherein the EGFR tyrosine kinase inhibitor is gefitinib.
3. The method according to claim 1 or 2, wherein the patient is a non-small cell lung cancer patient.
4. The method according to any one of claims 1 to 3, wherein the amount of the antibody is measured using beads or a plate to which a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof is bound.
5. A peptide consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 5 or a derivative thereof.
6. A bead to which the peptide or derivative according to claim 5 is bound.
7. A plate to which the peptide or derivative according to claim 5 is bound.
8. A diagnostic composition for predicting the therapeutic effect of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor comprising the peptide or derivative according to claim 5 or the bead according to claim 6.
9. A kit for predicting the therapeutic effect of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor comprising the peptide or derivative according to claim 5, the bead according to claim 6, or the plate according to claim 7. .
10. A method of treating cancer comprising the following steps:
    (1) A step of measuring the amount of an antibody against a peptide comprising an amino acid sequence selected from SEQ ID NOs: 2 to 5 in a blood sample collected from a patient before treatment with an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ;
    (2) comparing the measured antibody amount with a reference value; and (3) treating a patient whose antibody amount is higher than the reference value with an EGFR tyrosine kinase inhibitor.

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