Method for predicting in vitro responsiveness of subject to anti PD-1 antibody

Abstract

To provide a method for predicting treatment responsiveness in a cancer patient who receives treatment with an anti PD-1 antibody (especially, Nivolumab).SOLUTION: Provided is a method for predicting in vitro the responsiveness of a subject to an anti PD-1 antibody, and the method includes: a measurement step of measuring the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject; and a prediction step of predicting the responsiveness of the subject to treatment with the anti PD-1 antibody, on the basis of a ratio calculated from the measured amounts of two types of protein.SELECTED DRAWING: Figure 7D

Description

Technical Field

[0001] The present invention provides a method for predicting, in vitro, the therapeutic responsiveness of a subject undergoing cancer treatment with an anti-PD-1 antibody (particularly nivolumab) to the anti-PD-1 antibody. The present invention further provides a kit and an apparatus for use in the above method.

Background Art

[0002] With the recent progress in anti-cancer drug development, in addition to conventional anti-cancer drugs, the development of molecular target drugs and the progress of cancer immunotherapy have improved the treatment outcomes in many cancers. Immune checkpoint inhibitors (ICIs), that is, inhibitors of the programmed cell death protein pathway (PD-1 / PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), have greatly changed the treatment of many cancers.

[0003] Currently, the standard treatment for unresectable and recurrent gastric cancer is chemotherapy. Among them, nivolumab (an anti-PD-1 antibody), an immune checkpoint inhibitor, has brought about a clear improvement in prognosis compared with existing chemotherapy and is evaluated as a key drug for gastric cancer treatment (Non-Patent Document 1). Therefore, it is currently desired to find a biomarker that can predict the efficacy of treatment with ICIs.

[0004] In the treatment of unresectable and recurrent gastric cancer, nivolumab is used in combination with other drugs in the first-line chemotherapy, and monotherapy with nivolumab is used after the third-line treatment. At that time, the timing of introducing nivolumab therapy in the first-line treatment and the timing of switching from the previous treatment when used after the third-line treatment are important points that affect the prognosis.

[0005] To assist in determining the treatment strategy with nivolumab, many studies have been conducted on biomarkers that can be used to predict the therapeutic effect of nivolumab. However, currently, only microsatellite instability (MSI) testing (gene testing) and Combined Positive Score (CPS) testing (tissue testing) have been reported to be useful (Non-Patent Document 1). However, since both require the collection of tumor tissue, there are problems such as a large burden on patients and difficulty in clinical handling. Furthermore, there is also a problem that these tests cannot be performed on patients who have had their primary tumors resected, and there is a need for biomarkers that are more convenient for sample collection and measurement and are useful for predicting the therapeutic effect of nivolumab.

[0006] By the way, VEGF (vascular endothelial growth factor) is a protein involved in angiogenesis of blood vessels, lymphatic vessels, etc. In the cancer-bearing state, VEGF is known to have effects such as promoting tumor growth and progression associated with tumor angiogenesis, promoting lymph node metastasis associated with lymphangiogenesis, and suppressing tumor immunity. It has been reported that an increase in VEGF reflects tumor aggravation in many solid cancers (Non-Patent Document 2).

[0007] The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E, which are encoded by different genes. Furthermore, there are two other family proteins (a total of seven types) in the VEGF family. Additionally, VEGF-A and -B have isoforms due to differences in splicing (Non-Patent Document 2). These isoforms will be described in detail later. In multiple studies, it has been reported that mainly VEGF-A, -C, and -D are related to the pathogenesis of gastric cancer (Non-Patent Document 3). In fact, ramucirumab, an inhibitor against VEGF-R2, which is a receptor common to VEGF-A, -C, and -D, has been developed and is used as a standard therapeutic agent for the treatment of gastric cancer (Non-Patent Document 4).

[0008] VEGF-A, -C, and -D, along with PD-1, PD-L1, etc., which are target molecules of immune checkpoint inhibitors, have been previously verified for their usefulness as biomarkers in clinical trials such as the AVAGAST trial (Non-Patent Document 5), the RAINBOW trial (Non-Patent Documents 4 and 6), the ATTRACTION-2 trial (Non-Patent Document 7), and the ATTRACTION-4 trial (Non-Patent Document 1). However, except for PD-L1 in tumor tissues, no association with treatment efficacy has been shown.

[0009] In recent years, it has been reported that VEGF-A, particularly in its isoforms VEGF-A121 and VEGF-A165, has different effects in the tumor environment (Non-Patent Document 8), and it is presumed that the same is true in humans. Until now, it has been difficult to accurately quantify each isoform of the VEGF-A protein. However, for example, with the ELISA kit developed by Sinotest Co., Ltd., it has become possible to measure the respective protein amounts of VEGF-A121 protein and VEGF-A165 protein.

[0010] Previously, Patent Document 1 is an existing technology that used isoforms of VEGF-121, VEGF-165, and VEGF-A in the diagnosis and treatment of cancer. Patent Document 1 disclosed a method for predicting the therapeutic responsiveness of cancer to an anti-VEGF-A antibody. However, Patent Document 1 does not disclose anything about predicting the therapeutic responsiveness of cancer to an anti-PD-1 antibody.

[0011] Patent Document 2 discloses the use of VEGF-165 as a biomarker for the therapeutic effects of severe ischemic limb, renal function, and heart disease, but does not disclose its use as a biomarker for cancer.

[0012] Patent Document 3 discloses the use of VEGF-121 as a biomarker for predicting the efficacy of cancer treatment with bevacizumab, an anti-VEGF-A antibody, but does not disclose its use as a biomarker for predicting the efficacy of nivolumab (an anti-PD-1 antibody).

[0013] Patent Document 4 discloses the use of VEGF-165 as a biomarker for cancer treatment. However, Patent Document 4 does not disclose the use of the ratio of the amounts of VEGF-121 and VEGF-165 as a biomarker.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0016] The problem of the present invention is to develop and provide a method for predicting the therapeutic effect of nivolumab using biomarkers in cancer patients.

Means for Solving the Problems

[0017] As a result of intensive research by the present inventors to solve the above problems, it has been newly found that the protein quantitative ratio of VEGF-A121 and VEGF-A165 contained in the serum of a subject who is a cancer patient can predict the responsiveness of the subject to cancer treatment with an anti-PD-1 antibody. That is, it has been clarified that the two proteins are useful as biomarkers for predicting the cancer responsiveness of anti-PD-1 antibodies.

[0018] The present invention is based on the above new findings and is as follows. (1) A method for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody, a prediction step of predicting the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the ratio calculated from the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject, the method comprising the above. (2) The method according to (1), further comprising a measurement step of measuring the amounts of VEGF-A121 protein and VEGF-A165 protein in a sample derived from the subject before the prediction step. (3) The ratio is the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein, when the ratio is high, it is predicted that the subject has poor responsiveness to the anti-PD-1 antibody, when the ratio is low, it is predicted that the subject has good responsiveness to the anti-PD-1 antibody, the method according to (1) or (2). (4) The method according to (3), wherein the high or low level of the ratio is determined based on a cut-off value. (5) The method according to (4), wherein the cut-off value is in the range of 6 to 10. (6) The high or low level of the ratio is determined based on a control value, wherein the control value is a ratio calculated from the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a control subject with low responsiveness to the anti-PD-1 antibody, the method according to (1). (7) When the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in the sample derived from the subject is higher than or equal to the control value, it is predicted that the subject has poor responsiveness to the anti-PD-1 antibody, when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in the sample derived from the subject is lower than the control value, it is predicted that the subject has good responsiveness to the anti-PD-1 antibody, the method according to (6). (8) In the measurement step, the amounts of the respective proteins are measured by an immunological assay method, the method according to any one of (2) to (7). (9) The method according to any one of (1) to (8), wherein the subject has cancer. (10) The method according to (9), wherein the anti-PD-1 antibody is nivolumab. (11) The method according to (9) or (10), wherein the cancer is selected from the group consisting of gastric cancer, malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, cancer of unknown primary origin, and urothelial carcinoma. (12) A method for determining the administration timing of nivolumab based on the prediction result obtained by the method according to (10) or (11). (13) A kit for use in the method according to any one of (1) to (12), the kit comprising an anti-VEGF-A121 antibody and an anti-VEGF-A165 antibody. (14) The anti-VEGF-A121 antibody is As the sequences of the complementarity determining regions (CDRs) of the heavy chain variable region, the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3), and As the CDR sequences of the light chain variable region, respectively include the amino acid sequence of SEQ ID NO: 4 (CDR1), the amino acid sequence consisting of lysine (Lys)-valine (Val)-serine (Ser) (CDR2), and the amino acid sequence of SEQ ID NO: 6 (CDR3). (13) The kit according to (13). (15) The kit according to (13), wherein the heavy chain variable region of the anti-VEGF-A121 antibody comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of the anti-VEGF-A121 antibody comprises the amino acid sequence of SEQ ID NO: 8. (16) The anti-VEGF-A165 antibody is As the sequences of the complementarity determining regions (CDRs) of the heavy chain variable region, the amino acid sequences of SEQ ID NO: 11 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 13 (CDR3), and As the CDR sequences of the light chain variable region, respectively include the amino acid sequence of SEQ ID NO: 4 (CDR1), the amino acid sequence consisting of lysine (Lys)-valine (Val)-serine (Ser) (CDR2), and the amino acid sequence of SEQ ID NO: 14 (CDR3). (13) The kit according to (13). (17) The kit according to (13), wherein the heavy chain variable region of the anti-VEGF-A165 antibody contains the amino acid sequence of SEQ ID NO: 15, and the light chain variable region of the anti-VEGF-A165 antibody contains the amino acid sequence of SEQ ID NO: 16. (18) The kit according to any one of (13) to (17), wherein the anti-VEGF-A121 antibody and the anti-VEGF-A165 antibody are labeled. (19) An apparatus for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody, a prediction unit that predicts the responsiveness of the subject to treatment with the anti-PD-1 antibody based on a ratio calculated from the amounts of VEGF-A121 protein and VEGF-A165 protein in a sample derived from the subject, The apparatus comprising the same. (20) The apparatus according to (19), comprising a measurement unit that measures the amounts of VEGF-A121 protein and VEGF-A165 protein in a sample derived from the subject. (21) The apparatus according to (19) or (20), comprising a measured value input unit that inputs the amounts of VEGF-A121 protein and VEGF-A165 protein in a sample derived from the subject. (22) The prediction unit a control recording means for preliminarily recording a cut-off value or a control value, a prediction means for predicting the presence or degree of responsiveness of the subject to treatment with the anti-PD-1 antibody from the ratio of the input amounts of VEGF-A121 protein and VEGF-A165 protein and the cut-off value or the control value, an output means for outputting the result of predicting the responsiveness of the subject to treatment with the anti-PD-1 antibody based on the result of the analysis, The apparatus according to any one of (19) to (21), comprising the same. (23) A program for controlling an apparatus for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody, wherein in the apparatus, the ratio is calculated from the amounts of VEGF-A121 protein and VEGF-A165 protein in a sample derived from the subject, Comparatively analyze the calculated ratio based on known results, a prediction unit that predicts the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the result of the analysis, The computer program configured to execute. (24) In the device, a measurement unit that measures the amounts of the two proteins, The computer program according to (23), further configured to execute.

Advantages of the Invention

[0019] According to the method of the present invention, the responsiveness of a subject to an anti-PD-1 antibody can be predicted in vitro based on a sample derived from the subject.

Brief Description of the Drawings

[0020]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0021] The present invention will be described in more detail. 1. Method for Predicting Responsiveness to Anti-PD-1 Antibody 1-1. Overview The first aspect of the present invention is a method for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody (method for predicting anti-PD-1 antibody responsiveness). The method of the present invention includes a measurement step and a prediction step. According to the method of the present invention, it is possible to predict the responsiveness of a subject to treatment with nivolumab.

[0022] 1-2. Definitions The main terms used in this specification are defined below. As used herein, the term "subject" refers to a person to whom the method, kit, and apparatus of the present invention are applied. Without limitation, the subjects in this specification mainly correspond to those who are scheduled to receive treatment with an anti-PD-1 antibody, preferably treatment with nivolumab.

[0023] As used herein, the term "sample" refers to a biological component derived from a subject. Without limitation, examples include blood (including whole blood, serum, and plasma), lymph fluid, extracts of various tissues or cells, pleural effusion, sputum, tears, nasal discharge, saliva, urine, cerebrospinal fluid, interstitial fluid, etc., collected from a subject, preferably serum or plasma prepared from blood, and particularly preferably serum prepared from blood.

[0024] As used herein, "PD-1" (Programmed death receptor-1) refers to an immune checkpoint protein that functions as a factor suppressing T cell responses, and is an immunosuppressive receptor belonging to the CD28 family expressed on the surface of activated T cells.

[0025] As used herein, "PD-L1" refers to a protein expressed on the surface of cancer cells that binds to PD-1 and suppresses activation on T cells. The binding of PD-1 and PD-L1 causes immune escape of cancer cells.

[0026] As used herein, "anti-PD-1 antibody" refers to an antibody that binds to the above PD-1 receptor. By binding to PD-1 on T cells, the anti-PD-1 antibody inhibits the binding of PD-1 and PD-L1. Thereby, the anti-PD-1 antibody inhibits the transmission of inhibitory signals of T cells, maintains the activation of T cells, and restores the antitumor effect. Specific anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab, etc., but are not limited thereto.

[0027] As used herein, "responsiveness" refers to the responsiveness of a subject to an anti-PD-1 antibody, particularly nivolumab, in the treatment of cancer. Examples include the characteristics of the response occurring in the subject, such as whether cancer reduction or remission occurs upon administration of the anti-PD-1 antibody to the subject.

[0028] As used herein, "prediction" or "predict" means assisting or supplementing in vitro determination of a subject's "responsiveness to anti-PD-1 antibody treatment" by implementing the method of the present invention without relying on a direct diagnosis by a physician.

[0029] As used herein, "VEGF protein" (vascular endothelial growth factor; also referred to as "vascular endothelial growth factor") is one of the growth factors, binds to the VEGF receptor, and is involved in biological functions such as proliferation of vascular endothelial cells, angiogenesis and vasculogenesis, survival of endothelial cells, vascular permeability and vasodilation, monocyte chemotaxis and calcium influx, etc. VEGF forms a family, and as shown in Figure 1, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E are known. In the present specification, "VEGF protein" may be abbreviated as "VEGF", and for example, "VEGF-A protein" may be abbreviated as "VEGF-A", and they are synonymous with each other. In addition, the VEGF protein in the present specification means human VEGF protein unless otherwise specified.

[0030] As used herein, "measurement value" refers to the quantified amount of protein obtained in the measurement step described below. The measurement value may be an absolute value such as a quantity, or a relative value with respect to a specific reference value.

[0031] As used herein, "amount of protein" refers to the measured amount of a specific protein contained in a sample derived from a subject, preferably the amount of VEGF-A121 protein and the amount of VEGF-A165.

[0032] As used herein, "cutoff value" refers to a boundary value or threshold value determined to distinguish a group in a specific disease or state (from a group that is not). In the present invention, the "cutoff value" serves as a criterion for predicting whether a subject has good or poor responsiveness to an anti-PD-1 antibody.

[0033] As used herein, the "control value" refers to a boundary value or threshold value determined based on the measured value of a specific protein or the ratio of the measured values of a specific plurality of proteins in a subject known to have good responsiveness to an anti-PD-1 antibody or a subject known to have poor responsiveness to an anti-PD-1 antibody.

[0034] As used herein, "cancer" refers to a cell population that grows disorderly beyond the normal range without receiving normal control due to gene mutations or the like in cells. However, the "cancer" described in this specification refers to cancer that can be suppressed or inhibited in the growth of cancer cells by an anti-PD-1 antibody (for example, nivolumab) unless otherwise specified. Such cancers include, for example, gastric cancer, malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, cancer of unknown primary origin, urothelial cancer, etc., but are not limited thereto.

[0035] As used herein, "inoperable gastric cancer" refers to gastric cancer that cannot be surgically resected due to reasons such as high invasion degree of cancer or distant metastasis to other organs without staying in the primary focus or the vicinity of the primary focus.

[0036] As used herein, "recurrent gastric cancer" refers to gastric cancer that occurs again after the cancer confirmed by examination has disappeared due to surgery or drug therapy. Recurrent gastric cancer may have an organ other than the stomach as the primary focus.

[0037] 1-3. Method FIG. 5 shows a flowchart of the anti-PD-1 antibody responsiveness prediction method (S0500) of the present invention. As shown in this figure, this method includes a prediction step (S0520) as an essential step and a measurement step (S0510) as an optional step. Hereinafter, each step will be specifically described.

[0038] 1-3-1. Measurement step In the method of the present invention, the "measurement step" is a step of measuring the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject. As described above, this step is an optional step in the method and may be performed before the next prediction step as necessary. By this step, the two proteins contained in the sample derived from the subject can be quantified, and their absolute values or relative values can be obtained as measurement values.

[0039] The method for measuring the amounts of VEGF-A121 protein and VEGF-A165 protein may be any measurement method as long as it can measure and quantify their absolute amounts or relative amounts, and is not particularly limited. For example, immunological assay methods can be mentioned. Immunological assay methods further include enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, radioimmunoassay (RIA), surface plasmon resonance method (SPR method), quartz crystal microbalance (QCM) method, immunoturbidimetry, latex agglutination immunoassay, latex turbidimetry, hemagglutination reaction, particle agglutination reaction method, gold colloid method, capillary electrophoresis method, Western blot method or immunohistochemistry (immunostaining method), etc. are known, and any of them may be used.

[0040] The "enzyme immunoassay" is a method of quantifying a target substance by causing a color-developing substance or a fluorescent substance to be generated by the action of an enzyme through a secondary antibody or the like, which is an enzyme-labeled intervening substance, for a primary antibody bound to the target molecule, and based on the color development concentration or fluorescence intensity thereof. Also, the "fluorescence immunoassay", "luminescence immunoassay", and "radioimmunoassay (RIA)" are methods of quantifying a target substance by labeling an antibody (primary antibody) against the target substance or a secondary antibody that binds to the antibody with a fluorescent substance, a chemiluminescent substance, and a radiolabeled substance, respectively, and based on the luminescence intensity, radiation intensity, etc. of these labeled substances. Specific examples of these measurement methods include ELISA method, sandwich ELISA method, or EIA method, etc.

[0041] The "Surface Plasmon Resonance (SPR) method" is a method for detecting and quantifying with high sensitivity adsorbates on the surface of a metal thin film by utilizing the surface plasmon resonance phenomenon, in which the reflected light intensity is significantly attenuated at a specific angle of incidence (resonance angle) when the angle of incidence of laser light irradiated onto a metal thin film is changed. Detection and quantification by the surface plasmon resonance method can be performed, for example, by using an SPR sensor commercially available from Biacore.

[0042] The "Quartz Crystal Microbalance (QCM) method" is a mass measurement method that utilizes the phenomenon in which the resonant frequency of a quartz crystal oscillator decreases in proportion to the mass of a substance adsorbed on the surface of an electrode attached to the quartz crystal oscillator. This method quantitatively captures extremely small amounts of adsorbed substances based on the change in resonant frequency.

[0043] The "enzyme" includes, for example, peroxidase, alkaline phosphatase, and the like.

[0044] The "fluorescent substance" refers to a substance that becomes excited by absorbing excitation light of a specific wavelength and emits fluorescence when returning to the original ground state. Examples of such substances include FITC, rhodamine, fluorescamine, fluorescein, Texas Red [registered trademark], Cy3, Cy5, Cy7, FAM, HEX, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar [registered trademark], CAL Fluor (registered trademark) Red610, SYBR Green [registered trademark], Eva Green [registered trademark], and SYTOX Green [registered trademark].

[0045] The term "chemiluminescent substance" refers to a substance that has the property of emitting the difference in energy as light when returning to the ground state after being excited by a chemical reaction, such as acridinium ester.

[0046] The term "radioisotope" refers to an element that emits radiation among isotopes with different mass numbers. For example, 35 S,3 H, 14 C, etc.

[0047] The antibodies used in the immunological assay method are antibodies that specifically recognize and bind to VEGF-A121 protein and VEGF-A165 protein, respectively. The type of each antibody is not particularly limited. For example, either monoclonal antibody or polyclonal antibody may be used. When the antibody is a polyclonal antibody or a monoclonal antibody, the immunoglobulin constituting it may be of any class (e.g., IgG, IgE, IgM, IgA, IgD, and IgY). Also, the antibody may be derived from any animal including mammals and birds. Examples of antibody-derived animals include rabbit, mouse, rat, guinea pig, goat, donkey, sheep, camel, llama, alpaca, horse, chicken, or human, etc.

[0048] In the immunological assay method, artificially prepared antibodies can also be used. Such antibodies include, for example, recombinant antibodies, synthetic antibodies, and antibody fragments.

[0049] "Recombinant antibody" is an antibody prepared using gene recombination technology, including chimeric antibodies, humanized antibodies, and multispecific antibodies, etc.

[0050] "Chimeric antibody" is an antibody in which the variable region (V region) of an antibody derived from one animal is replaced with the V region of an antibody derived from another different animal using molecular biological techniques.

[0051] "Humanized antibody" is a grafted antibody in which the complementarity-determining regions (CDRs; CDR1, CDR2, and CDR3) in the V region of a monoclonal antibody derived from a non-human mammal, for example, a mouse, are replaced with the corresponding CDRs of an appropriate human monoclonal antibody. For example, an anti-human VEGF-A121 humanized antibody obtained by replacing each CDR in a mouse-derived anti-human VEGF-A121 monoclonal antibody with the corresponding CDR of a human monoclonal antibody corresponds to this.

[0052] The term "multispecific antibody" refers to a multivalent antibody, that is, an antibody having a plurality of antigen-binding sites within a single molecule, and in which each antigen-binding site binds to a different epitope. For example, in the case of an antibody having two antigen-binding sites such as IgG, a bispecific antibody (Bispecific antibody) in which each antigen-binding site specifically binds to the same or different antigens can be mentioned.

[0053] The term "synthetic antibody" refers to an antibody synthesized by using a chemical method or a recombinant DNA method. For example, a monomeric polypeptide molecule in which one or more light chain variable regions (VL regions) and one or more heavy chain variable regions (VH regions) of a specific antibody are artificially linked via a linker peptide or the like having an appropriate length and sequence, or a multimeric polypeptide thereof corresponds. Specific examples of such polypeptides include single-chain Fv (scFv: single chain Fragment of variable region), diabody, triabody, or tetrabody. A diabody is a bivalent antibody fragment, but each antigen-binding site does not necessarily bind to the same epitope, and each may have bispecificity to recognize and specifically bind to different epitopes. Triabody and tetrabody have a trimeric and tetrameric structure based on the single-chain Fv structure, similar to diabody. They are trivalent and tetravalent antibody fragments, respectively, and may also be multispecific antibodies.

[0054] The term "antibody fragment" refers to, for example, Fab, F(ab’) 2 , Fv, etc.

[0055] 1-3-2. Prediction step The "prediction step" in the method of the present invention is a step of predicting the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the ratio calculated from the measured amounts of the two proteins, that is, the amounts of VEGF-A121 protein and VEGF-A165 protein.

[0056] The "ratio" calculated in this project may be either the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein, or the ratio of the amount of VEGF-A165 protein to the amount of VEGF-A121 protein. Preferably, it is the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein.

[0057] "Based on the ratio" means making a judgment based on the high or low of the ratio. "The high or low of the ratio" means the high or low relative to the reference value.

[0058] Examples of the "reference value" include a cut-off value or a control value.

[0059] In this specification, "poor responsiveness" means that the prognosis (i.e., the course of the disease after treatment) of the subject with an anti-PD-1 antibody, particularly nivolumab, is poor. For example, it means that the survival rate of the subject is predicted to be low. For example, the prognosis of the subject can be predicted by the Kaplan-Meier test.

[0060] In this specification, "good responsiveness" means that the prognosis (i.e., the course of the disease after treatment) of the subject with an anti-PD-1 antibody, particularly nivolumab, is good. For example, it means that the survival rate of the subject is predicted to be high.

[0061] When the ratio is the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein, when the ratio is high, it is predicted that the subject has poor responsiveness to the anti-PD-1 antibody. On the other hand, when the ratio is low, it is predicted that the subject has good responsiveness to the anti-PD-1 antibody.

[0062] When the ratio is the ratio of the amount of VEGF-A165 protein to the amount of VEGF-A121 protein, when the ratio is low, it is predicted that the subject has poor responsiveness to the anti-PD-1 antibody. On the other hand, when the ratio is high, it is predicted that the subject has good responsiveness to the anti-PD-1 antibody.

[0063] VEGF-A has splicing variants that occur during the generation of mRNA by transcription. As such variants, for example, VEGF-A111, VEGF-A121, VEGF-A145, VEGF-A148, VEGF-A165, VEGF-A183, VEGF-A189, VEGF-A206, etc. are known (Figure 2). Although the functions of these variants are not clear, it has been reported that the angiogenesis images in mice differ depending on the variant, and that the expression levels and functions differ depending on the tissue and the microenvironment in the living body.

[0064] The variants related to the present invention are VEGF-A121 and VEGF-A165. The gene encoding VEGF-A consists of 8 exons. VEGF-A121 is a protein encoded by exons 1 to 5 and exons 8a and 8b. In contrast, VEGF-A165 is a protein encoded by exons 1 to 5, exons 7a and 7b, and exons 8a and 8b (Figure 2).

[0065] The VEGF-A121 and VEGF-A165 proteins may be natural proteins or may be recombinant proteins. Recombinant human proteins can be produced by known genetic recombination techniques based on nucleotide sequences shown, for example, in NCBI (USA) accession numbers NM_001025366, NM_001025370, NM_001171626, AF486837, etc. (Green, M.R. and Sambrook, J., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press (2012)).

[0066] In a preferred embodiment of the present invention, the high or low of the ratio can be determined based on a cut-off value. For example, when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein (VEGF-A121 / VEGF-A165) in the serum of a subject is a value equal to or higher than the "cut-off value", it is determined that the subject has poor responsiveness to the anti-PD-1 antibody, and when it is a value lower than the "cut-off value", it is determined that the subject has good responsiveness to the anti-PD-1 antibody.

[0067] Also, when the ratio of the amount of VEGF-A165 protein to the amount of VEGF-A121 protein (VEGF-A165 / VEGF-A121) in the serum of a subject is a value equal to or higher than the "cut-off value", it is determined that the subject has good responsiveness to the anti-PD-1 antibody, and when it is a value lower than the "cut-off value", it is determined that the subject has poor responsiveness to the anti-PD-1 antibody.

[0068] The method for setting the cut-off value is not particularly limited, and it can be freely set in order to obtain sensitivity and specificity suitable for the purpose of the test. By changing the cut-off value set according to the purpose, the sensitivity and specificity of the predicted responsiveness to the anti-PD-1 antibody in the subject can be adjusted.

[0069] Incidentally, the "ROC curve (Receiver Operating Characteristic curve)" has the true positive rate (TPF: True Position Fraction), that is, sensitivity, on the vertical axis, and the false positive rate (FPF: False Position Fraction), that is, (1 - specificity), on the horizontal axis, and is created by plotting while changing the threshold value (for example, the cut-off value) for determining which value of the determination result is judged to have a finding as an intermediate variable. Here, sensitivity is the rate of correctly judging positive cases as positive, and specificity is the rate of correctly judging negative cases as negative.

[0070] "AUC (Area Under the Curve)" refers to the value of the area under the ROC curve, and is a numerical value representing the discriminative ability of the index related to that ROC curve. Generally, the closer the AUC is to 1, the higher the discriminative ability, and the closer it is to 0.5, the lower the discriminative ability.

[0071] However, the method for determining the cut-off value is not limited to this. For example, a method that does not use a direct comparison between the control group and the disease group to be compared, or a method that does not use an ROC curve, may be used to set the cut-off value. As a method for determining the cut-off value using an ROC curve, any known method can be used.

[0072] The cut-off value can be set to a value in the range of, for example, 2 to 30, 4 to 20, 6 to 10, or 7 to 9.

[0073] In the following examples, the median of the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein measured in 30 subjects is set as the cut-off value. The cut-off value set in this way is 8.65, and when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein is 8.65 or more, the prognosis of treatment with nivolumab, an anti-PD-1 antibody, is predicted to be poor.

[0074] Furthermore, in a preferred embodiment of the present invention, it can also be determined based on the level of the control value of the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein. The method for setting this "control value" is not particularly limited either.

[0075] For example, as a preferable "control value" in the present invention, a ratio (VEGF-A121 / VEGF-A165) calculated from the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a control subject known to have poor responsiveness to an anti-PD-1 antibody can be used. Based on such a control value, when the ratio of the amount of VEGF-A165 protein to the amount of VEGF-A121 protein in a sample derived from a subject is equal to or higher than the control value, it can be predicted that the subject has poor responsiveness to the anti-PD-1 antibody. Furthermore, when the ratio of the amount of VEGF-A165 protein to the amount of VEGF-A121 protein in a sample derived from a subject is lower than the control value, it can be predicted that the subject has good responsiveness to the anti-PD-1 antibody.

[0076] As a "control value" in the present invention, a ratio (VEGF-A165 / VEGF-A121) calculated from the amount of VEGF-A165 protein to the amount of VEGF-A121 protein in a sample derived from a control subject known to have poor responsiveness to an anti-PD-1 antibody can be used. Based on such a control value, when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a subject is equal to or lower than the control value, it can be predicted that the subject has poor responsiveness to the anti-PD-1 antibody. Furthermore, when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a subject is higher than the control value, it can be predicted that the subject has good responsiveness to the anti-PD-1 antibody.

[0077] Alternatively, as the "control value" in the present invention, a ratio (VEGF-A121 / VEGF-A165) calculated from the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a control subject known to have good responsiveness to an anti-PD-1 antibody can also be used. Based on such a control value, when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a subject is higher than the control value, it can be predicted that the subject has poor responsiveness to the anti-PD-1 antibody. Further, when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a subject is equal to or lower than the control value, it can be predicted that the subject has good responsiveness to the anti-PD-1 antibody.

[0078] As the "control value" in the present invention, a ratio (VEGF-A165 / VEGF-A121) calculated from the amount of VEGF-A165 protein to the amount of VEGF-A121 protein in a sample derived from a control subject known to have good responsiveness to an anti-PD-1 antibody can also be used. Based on such a control value, when the ratio of the amount of VEGF-A165 protein to the amount of VEGF-A121 protein in a sample derived from a subject is equal to or lower than the control value, it can be predicted that the subject has good responsiveness to the anti-PD-1 antibody. Further, when the ratio of the amount of VEGF-A165 protein to the amount of VEGF-A121 protein in a sample derived from a subject is lower than the control value, it can be predicted that the subject has poor responsiveness to the anti-PD-1 antibody.

[0079] Furthermore, based on the prediction results obtained by the method of the present invention, the administration timing of nivolumab to a subject can be determined. According to the method of the present invention, based on the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a subject, the responsiveness of the subject to nivolumab can be predicted. Therefore, in a subject predicted to have good responsiveness to nivolumab, it can be determined that it is appropriate to administer nivolumab in addition to other chemotherapeutic agents in primary treatment. The method of the present invention is particularly useful for making such a determination.

[0080] In this specification, primary treatment refers to the first anti-cancer agent treatment performed when anti-cancer agent treatment is carried out for inoperable cancer.

[0081] After primary treatment, the cancer drug therapy may be continued by switching to another anti-cancer agent. In this specification, the treatment performed after primary treatment is referred to as secondary treatment and tertiary treatment.

[0082] It is also possible to administer nivolumab at a stage after secondary treatment or tertiary treatment, etc. to a subject who did not receive nivolumab administration in primary treatment. In secondary treatment or tertiary treatment, since the chemotherapeutic agent used in combination with nivolumab changes, it can also be expected that the effect of nivolumab can be obtained at that stage.

[0083] Furthermore, according to the method of the present invention, a subject to whom nivolumab has no effect can be discriminated. Therefore, the method of the present invention is also effective for the purpose of selecting a subject who does not undergo treatment with nivolumab.

[0084] 2. Kit for use in a method for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody The present invention relates to a kit for use in a method for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody, the kit comprising an anti-VEGF-A121 antibody and an anti-VEGF-A165 antibody. By measuring the amount of VEGF-A121 protein and the amount of VEGF-A165 protein contained in a sample derived from a subject using the anti-VEGF-A121 antibody and the anti-VEGF-A165 antibody contained in the kit of the present invention, the method described in 1 above can be carried out.

[0085] When the above method uses an immunological assay (such as a sandwich assay, an ELISA assay, etc.), which is a kind of enzyme antibody method, an enzyme label such as peroxidase or alkaline phosphatase is preferred.

[0086] Regarding the anti-VEGF-A121 antibody and the anti-VEGF-A165 antibody, non-limitingly, the above anti-VEGF-A121 antibody includes, for example, the following antibodies. (a) An antibody comprising, as the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region, the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2) and SEQ ID NO: 3 (CDR3), and, as the CDR sequences of the light chain variable region, the amino acid sequences of SEQ ID NO: 4 (CDR1), the amino acid sequence consisting of lysine (Lys)-valine (Val)-serine (Ser) (CDR2) and SEQ ID NO: 6 (CDR3). (b) An antibody comprising the amino acid sequence of the heavy chain variable region of SEQ ID NO: 7 and the amino acid sequence of the light chain variable region of SEQ ID NO: 8. (c) An antibody comprising the heavy chain variable region encoded by the nucleotide sequence of SEQ ID NO: 9 and the light chain variable region encoded by the nucleotide sequence of SEQ ID NO: 10.

[0087] The above anti-VEGF-A165 antibody includes, for example, the following antibodies. (d) As the sequences of the complementarity-determining regions (CDRs) of the heavy-chain variable region, the amino acid sequences of SEQ ID NO: 11 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 13 (CDR3), and as the CDR sequences of the light-chain variable region, the amino acid sequences of SEQ ID NO: 4 (CDR1), the amino acid sequence consisting of lysine (Lys)-valine (Val)-serine (Ser) (CDR2), and SEQ ID NO: 14 (CDR3), respectively, an antibody. (e) An antibody comprising the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 15 and the amino acid sequence of the light-chain variable region of SEQ ID NO: 16. (f) An antibody comprising the heavy-chain variable region encoded by the nucleotide sequence of SEQ ID NO: 17 and the light-chain variable region encoded by the nucleotide sequence of SEQ ID NO: 18.

[0088] The kit may further include an instruction manual for the above method.

[0089] In the following examples, the amount of VEGF-A121 protein was measured by an ELISA assay using an anti-VEGF-A121 antibody (monoclonal antibody 5A6G4C8) comprising the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 7 and the amino acid sequence of the light-chain variable region of SEQ ID NO: 8. Further, in the following examples, the amount of VEGF-A165 protein was measured by an ELISA assay using an anti-VEGF-A165 antibody (monoclonal antibody 4F9A9G9) comprising the amino acid sequence of the heavy-chain variable region of SEQ ID NO: 15 and the amino acid sequence of the light-chain variable region of SEQ ID NO: 16. A kit containing such anti-VEGF-A121 antibody and anti-VEGF-A165 antibody is suitable in the present invention.

[0090] 3. Anti-PD-1 Antibody Responsiveness Predicting Device and Its Device Control Program 3-1. Overview The third aspect of the present invention is an anti-PD-1 antibody responsiveness predicting device and its device control program. A block diagram showing the configuration of this device is shown in FIG. 3. By executing the device control program, this device can implement the prediction method described in the first aspect.

[0091] 3-2. Configuration of the Device As shown in FIG. 3, the device (0300) of the present invention includes a prediction unit (0310) as an essential component, and a measurement unit (0320) and a measured value input unit (0330) as optional components. The prediction unit (0310) may further include a reference recording means (0311), a prediction means (0312), and a predicted value output unit (0340). Each component of the present device will be specifically described below.

[0092] 3-2-1. Measurement Unit The "measurement unit" (0320) is a unit that measures the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject.

[0093] The measurement unit (0320) is configured in the device (0300) of the present invention to be able to perform the measurement step in the "method for predicting anti-PD-1 antibody responsiveness" described in the first aspect. Therefore, any configuration that can quantify the two proteins contained in a sample derived from a subject and obtain their absolute value or relative value as a measured value is acceptable, and its specific configuration is not particularly limited. For example, when quantifying the two proteins using an immunological assay method in the measurement step, any unit that can implement the immunological assay method is acceptable. Examples include ELISA devices, AIA devices, SPR devices, QCM devices, absorptiometers, fluorescence spectrometers, luminometers, scintillation counters, etc. Specific configurations of these devices are all known in the art.

[0094] 3-2-2. Prediction Unit The "prediction unit" (0310) is a unit that predicts the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the ratio calculated from the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject.

[0095] The prediction unit (0310) in the device (0300) of the present invention is configured to be able to perform the prediction step in the "method for predicting anti-PD-1 antibody responsiveness" described in the first aspect. It is sufficient that the ratio of VEGF-A165 protein to VEGF-A121 protein or the ratio of VEGF-A121 protein to VEGF-A165 protein can be calculated from the measured value based on the amounts of the two proteins contained in the sample derived from the subject, and further the output ratio is compared with the control value, and the responsiveness to the anti-PD-1 antibody can be predicted based on the comparison result, and the specific configuration is not particularly limited. Usually, the prediction unit (0310) is implemented by a computer. The hardware configuration of the prediction unit (0310) will be described in detail in another section.

[0096] Specifically, the prediction unit (0310) includes a control recording means (0311), a prediction means (0312), and a predicted value output unit (0340) as necessary. Each of the following means will be described below.

[0097] (1) The control recording means (0311) is a means for pre-recording a cut-off value or a control value as a control value. It is sufficient that those numerical values can be electronically stored and read out to the following prediction means (0312) connected to this means as necessary, and there is no particular limitation. Usually, the control recording means (0311) is implemented by a non-volatile memory such as a hard disk, a flash memory, a magnetic storage device, or an optical disk. Since the control value recorded by this means is as described in the first aspect, the description here is omitted.

[0098] (2) The prediction means (0312) analyzes and predicts the presence or absence or degree of responsiveness of the subject to treatment with an anti-PD-1 antibody from the ratio of the amount of VEGF-A121 protein and the amount of VEGF-A165 protein input and the cut-off value or control value. It is a means for prediction. It is a main constituent means in the prediction unit (0310), and although not limited, it is usually implemented by a CPU, a volatile memory, and a device control program in a computer. The result obtained by this means is output to the outside as a prediction value through the following prediction value output unit.

[0099] 3-2-3. Prediction value output unit The prediction value output unit (0340) is a means for outputting the result of predicting the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the result of the analysis. The "output" mentioned here means extracting a prediction value from the prediction means (0312) and displaying the extracted prediction value in a state recognizable by a person. There is no particular limitation as long as it has such an output and display function. Usually, it is implemented by an interface (including a port) and a monitor or a printer, etc.

[0100] 3-2-4. Measured value input unit The "measured value input unit" (0330) is a means for inputting the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject.

[0101] When the device (0300) of the present invention includes a measurement unit (0320) inside the device, information on the amount of VEGF-A121 protein and the amount of VEGF-A165 protein can be obtained from the measurement unit (0320) and output to the prediction unit (0310). However, when the measurement unit (0320) is not provided, measurement values regarding the above two proteins can be input from the outside to the prediction unit via this measurement value input unit (0330). The measurement value input unit (0330) is not particularly limited as long as it can input the measurement value to the prediction unit (0310). For example, it may be configured to input the measurement value actively. As a specific configuration, usually, it is embodied by an interface (including a port), and a keyboard, a mouse, a touch panel, or the like.

[0102] 3-3. Configuration of the device control program In the present invention, there is provided a device control program for controlling the anti-PD-1 antibody responsiveness prediction device described in 3-1 and executing the prediction step in the "anti-PD-1 antibody responsiveness prediction method" described in the first aspect of the present invention.

[0103] In the program (0300) of the present invention, in the prediction unit (0310) or the prediction means (0312), based on the amounts of VEGF-A121 protein and VEGF-A165 protein in the sample derived from the subject obtained, that is, from the respective measurement values, a ratio is calculated according to the formula of VEGF-A121 / VEGF-A165 or VEGF-A165 / VEGF-A121.

[0104] Subsequently, the control value (cutoff value, or control value) stored in the control recording means (0311) is called and compared with the calculated ratio. Based on the comparison result and a predetermined judgment criterion, the responsiveness of the subject to cancer treatment with an anti-PD-1 antibody is predicted.

[0105] Specifically, for example, using the "cutoff value" as a criterion, it is determined whether the calculated ratio is higher than the cutoff value. In the case of the ratio of VEGF-A121 / VEGF-A165, if the ratio is higher than the cutoff value, a predictive value that the responsiveness of the subject to cancer treatment with an anti-PD-1 antibody is poor is obtained. On the other hand, when the ratio is lower than the cutoff value, a predictive value that the responsiveness of the subject to treatment with an anti-PD-1 antibody is good is obtained.

[0106] As the cutoff value, it is also possible to use the median of the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein measured in advance in a plurality of subjects.

[0107] In addition, as a criterion for predicting responsiveness, the "control value" can also be used as a criterion. Specifically, the ratio based on the formula of VEGF-A121 / VEGF-A165 or VEGF-A165 / VEGF-A121 in a sample from a control subject known to have low responsiveness to an anti-PD-1 antibody is used as the control value. Based on this control value, when the ratio of VEGF-A121 / VEGF-A165 in the sample from the subject is equal to or higher than the control value, the subject is processed so as to predict that the responsiveness to cancer treatment with an anti-PD-1 antibody is poor. On the other hand, when the ratio of VEGF-A121 / VEGF-A165 in the sample from the subject is lower than the control value, the subject is processed so as to predict that the responsiveness to cancer treatment with an anti-PD-1 antibody is good.

[0108] Conversely, as a control value, a ratio based on the formula of VEGF-A121 / VEGF-A165 or VEGF-A165 / VEGF-A121 in a sample derived from a control subject known to have high responsiveness to cancer treatment with an anti-PD-1 antibody can also be used. In this case, when the ratio of VEGF-A121 / VEGF-A165 in the sample derived from the subject is higher than the control value, the subject can be processed so as to be predicted to have poor responsiveness to cancer treatment with the anti-PD-1 antibody. Also, when the ratio is equal to or lower than the control value, the subject can be processed so as to be predicted to have good responsiveness to cancer treatment with the anti-PD-1 antibody. The obtained predicted value (prediction result) can be stored in a non-volatile memory as needed, and can also be output to an internal monitor or an external predicted value output unit (0340) and commanded to be displayed.

[0109] 3-4. Hardware Configuration of the Device An example of the hardware configuration in the device of the present invention is shown in FIG. 4 and will be described below. The device (0400) of the present invention includes a CPU (Central Processing Unit) (0410), a non-volatile memory (0420), a volatile memory (0430), and an interface (0440) as essential hardware configurations, and also includes a keyboard (0450) and an external monitor (0460) as optional hardware configurations.

[0110] For the measurement value input into the prediction unit from the measurement unit or the keyboard (0450) via the interface (0440), the CPU (0410) reads out the device control program stored in the non-volatile memory (0420), expands it on the volatile memory (0430), and performs processing according to the program. Control value data such as a cut-off value and a control value are also read out from the non-volatile memory (0420) storing them onto the volatile memory (0430) by the CPU (0410) according to the program. The predicted value obtained after the program execution is output via the interface (0440) and displayed and expanded on the external monitor (0460). Also, it is stored in the volatile memory (0430) as needed.

Example

[0111] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited by these examples.

[0112] 1. Clinical pathological background of the subjects In the Department of Gastroenterological Surgery, Kagoshima University Hospital, among 37 patients with unresectable or recurrent advanced gastric cancer who received anti-PD-1 antibody (nivolumab) from 2017 to 2022, 30 subjects with a performance status (PS) of 1 or less before the introduction of nivolumab were selected according to the patient selection criteria of the double-blind randomized phase III trial (ATTRACTION-2) of nivolumab.

[0113] Among the selected subjects, 22 were male (73.3%), and the median age was 65 years (range 35 - 78). There were 11 subjects with PS0 (36.7%) and 19 subjects with PS1 (63.3%). As a result of endoscopic diagnosis, 5 subjects (16.7%) were of Borrmann type 4, and 23 subjects (76.7%) were of undifferentiated histological type. Peritoneal dissemination was observed in 11 subjects (36.7%), liver metastasis was observed in 6 subjects (20%), and distant lymph node metastasis was observed in 11 subjects (36.7%).

[0114] The median number of nivolumab treatment courses was 5.5 (1 - 26), 7 subjects (23.3%) had received more than 3 courses of chemotherapy before nivolumab treatment, and 27 subjects had received treatment with ramucirumab.

[0115] 2. Analysis of the relationship between the amount of protein of each isoform of VEGF-A in the serum of the subjects and the prognosis of nivolumab administration 2-1. Method for measuring each protein in the serum of the subjects Serum samples were collected within 24 hours before the treatment with anti-PD-1 antibody and stored at -60°C until processing. The amounts of total VEGF-A protein (hereinafter referred to as total VEGF-A), VEGF-A121 protein (hereinafter referred to as VEGF-A121), VEGF-A165 protein (hereinafter referred to as VEGF-A165), VEGF-C protein (hereinafter referred to as VEGF-C), PD-1 protein (hereinafter referred to as PD-1), and PD-L1 protein (hereinafter referred to as PD-L1) in the serum were measured by enzyme-linked immunosorbent assay (ELISA).

[0116] Total VEGF-A, VEGF-C, PD-1, and PD-L1 were measured using commercially available ELISA kits (R&D Biosystems, Minneapolis, Minnesota).

[0117] For the measurement of VEGF-A121, ELISA using an anti-VEGF-A121 antibody (monoclonal antibody 5A6G4C8) that specifically recognizes VEGF-A121 protein described in JP-A-2022-006774 was used. Monoclonal antibody "5A6G4C8" contains a heavy-chain variable region including the amino acid sequence of SEQ ID NO: 7 and a light-chain variable region including the amino acid sequence of SEQ ID NO: 8.

[0118] For the measurement of VEGF-A165, ELISA using an anti-VEGF-A165 antibody (monoclonal antibody 4F9A9G9) that specifically recognizes VEGF-A165 protein described in JP-A-2022-006774 was used. Monoclonal antibody "4F9A9G9" contains a heavy-chain variable region including the amino acid sequence of SEQ ID NO: 15 and a light-chain variable region including the amino acid sequence of SEQ ID NO: 16.

[0119] The ELISAs for VEGF-A121 and VEGF-A165 were performed as follows. A polystyrene microtiter plate was coated and incubated overnight (4°C) with 100 μL of anti-human VEGF-A polyclonal antibody (R&D Biosystems, Minneapolis, Minnesota) (1 μg / mL - PBS). The plate was washed three times with PBS containing 0.05% Tween 20 and incubated for 2 hours with a blocking solution containing 1% BSA to block the binding sites remaining in the wells.

[0120] After washing the plate, 50 μL of a serum sample from the subject and 50 μL of a buffer (0.2 mol / L Tris pH 8.5 and 0.15 mol / L sodium chloride containing 1% casein) (total 100 μL) were added to each well. Then, the plate was incubated at 25°C for 15 hours and washed again.

[0121] After washing, 100 μL of the above anti-VEGF-A121 antibody or anti-VEGF-A165 antibody labeled with peroxidase was added to each well and incubated at 25°C for 2 hours.

[0122] After further washing, the chromogenic substrate TMBZ (3,3',5,5'-tetra-methylbenzidine) (Dojindo Laboratories, Kumamoto, Japan) was added to each well.

[0123] Sodium sulfate was added to terminate the reaction, and the absorbance at 450 nm was measured using a microplate reader (Model 680, Bio-Rad, Irvine, California).

[0124] 2-2. Analysis of the correlation between each protein in serum and prognosis 2-2-1. Analysis by the Kaplan-Meier method Analysis was performed using the Kaplan-Meier method on the amount of total VEGF-A (total VEGF-A), the amount of VEGF-A121 (VEGF-A121), the amount of VEGF-A165 (VEGF-A165), the ratio of VEGF-A121 / 165, the amount of VEGF-C (VEGF-C), the amount of PD-1 (PD-1), and the amount of PD-L1 (PD-L1) in the sera of the subjects. That is, regarding these factors, the prognosis was verified using the Kaplan-Meier method for the overall survival (OS) and progression-free survival (PFS) after the introduction of nivolumab. The appropriate cut-off values for the values of these parameters in the sera were set at the median of all the subjects. Based on the two-sided hypothesis, a statistically significant difference was considered when all P-values were less than 0.05.

[0125] The results of analyzing the relationship between total VEGF-A in the sera of the subjects and the prognosis after the introduction of nivolumab are shown in FIGS. 6A and 7A. For OS (log-rank test p = 0.29) and PFS (log-rank test p = 0.15), no significant difference was observed between the low-value group and the high-value group of total VEGF-A.

[0126] The results of analyzing the relationship between VEGF-A121 in the sera of the subjects and the prognosis after the introduction of nivolumab are shown in FIGS. 6B and 7B. For OS (log-rank test p = 0.32) and PFS (log-rank test p = 0.19), no significant difference was observed between the low-value group and the high-value group of VEGF-A121.

[0127] The results of analyzing the relationship between VEGF-A165 in the sera of the subjects and the prognosis after the introduction of nivolumab are shown in FIGS. 6C and 7C. For OS (log-rank test p = 0.42) and PFS (log-rank test p = 0.87), no difference was observed between the low-value group and the high-value group of VEGF-A165.

[0128] The results of analyzing the relationship between the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 in the serum of the subjects (VEGF-A121 / 165 ratio) and the prognosis after the introduction of nivolumab are shown in FIGS. 6D and 7D. There was no significant difference in OS (log-rank test p = 0.55) between the low and high VEGF-A121 / 165 ratio groups (FIG. 6D). On the other hand, a significant prolongation of PFS (log-rank test p = 0.0088) was observed in the low VEGF-A121 / 165 ratio group (FIG. 7D).

[0129] As described above, in the Kaplan-Meier curves of FIGS. 6D and 7D, the median value of all subjects was set as the cut-off value. That is, the cut-off value was calculated using the ROC curve (X-axis: ratio of the amount of 121 protein to the amount of 165 protein, Y-axis: deterioration within 12 weeks) created from the VEGF-A121 / 165 ratios of 30 subjects.

[0130] The AUC of the ROC curve was 0.77333, the sensitivity was 0.80, the specificity was 0.80, and the cut-off value was 7.90. This value means that when the VEGF-A121 / 165 ratio is 7.90 or more, the prognosis of nivolumab treatment is judged to be poor.

[0131] The relationships between serum VEGF-C (FIGS. 6E, 7E), PD-1 (FIGS. 6F, 7F), and PD-L1 (FIGS. 6G, 7G) of the subjects and the prognosis after the introduction of nivolumab were analyzed. There were no significant differences in OS (log-rank test p = 0.70, p = 0.42, p = 0.86 respectively) and PFS (log-rank test p = 0.19, p = 0.65, p = 0.71 respectively) between the groups with high and low amounts of these proteins.

[0132] 2-2-2. Univariate analysis by the Cox proportional hazards model For the total VEGF-A amount, VEGF-A121 amount, VEGF-A165 amount, VEGF-A121 / 165 ratio, VEGF-C amount, PD-1 amount, and PD-L1 amount in the sera of the subjects, the correlation between their amounts in the sera of the subjects and the prognosis was verified by univariate analysis using the Cox proportional hazards model. All P-values were based on two-sided hypotheses, and values less than 0.05 were considered statistically significant. All statistical analyses were performed using JMP Pro software, v13.0.0 (SAS Institute, Cary, North Carolina). The results are shown in Table 1.

[0133] As shown in Table 1, only the serum VEGF-A121 / 165 ratio of the subjects was significantly associated with a shorter PFS (hazard ratio (HR): 1.08, 95% confidence interval (CI): 1.00 - 1.17, P = 0.0454).

[0134]

Table 1

[0135] 2-2-3. Evaluation by RECIST For the response of tumors to treatment, an objective evaluation was performed using RECIST (Response Evaluation Criteria in Solid Tumors) v1.1. For the correlation with the treatment effect, univariate analysis was performed by logistic simple regression analysis. That is, the treatment effects were compared between the low-value group (n = 15) and the high-value group (n = 15) of the serum VEGF-A121 / 165 ratio (Table 2). When RECIST (Response Evaluation Criteria in Solid Tumors) evaluation was performed, the number of subjects with complete response (CR) was 3 vs. 0 (low-value group vs. high-value group, the same below), the number of subjects with partial response (PR) was 3 vs. 0, the number of subjects with stable disease (SD) was 3 vs. 2, and the number of subjects with progressive disease (PD) was 3 vs. 10. Subjects in whom PR or SD was not clinically determined were classified as non-CR / non-PD. The number of classified subjects with non-CR / non-PD was 4 and 3 in the low-value group and the high-value group of the serum VEGF-A121 / 165 ratio, respectively.

[0136] The disease control rate (DCR) obtained from these results was 80% in the low serum VEGF-A121 / 165 ratio group and 33% (p = 0.0253) in the high ratio group.

[0137] Furthermore, focusing on the subjects with target lesions, RECIST evaluations were performed in the low serum VEGF-A121 / 165 ratio group (n = 8) and the high ratio group (n = 11). The number of CRs was 2 in the low ratio group, while it was 0 in the high ratio group. The number of PRs was 2 in the low ratio group, while it was 0 in the high ratio group. The number of subjects with SD was 3 in the low ratio group, while it was 2 in the high ratio group. The number of subjects with PD was 1 in the low ratio group, while it was 9 in the high ratio group. The objective response rate was 50% in the low ratio group, while it was 0% (p = 0.0181) in the high ratio group. The DCR was 88% in the low ratio group, while it was 18% (p = 0.0055) in the high ratio group.

[0138]

Table 2

[0139] As described above, in the subjects who received nivolumab treatment, it was clarified that the ratio of VEGF-A121 protein to VEGF-A165 protein in the serum of the subjects before treatment was significantly correlated with the progression-free survival period. That is, when comparing the low and high VEGF-A121 / 165 ratio groups, it was shown that the progression-free survival period was shorter and the prognosis was poor in the high VEGF-A121 / 165 ratio group. Therefore, the ratio of serum VEGF-A121 to VEGF-A165 is useful as a new biomarker for predicting the therapeutic effect of nivolumab.

Industrial Applicability

[0140] The method of the present invention can predict the treatment responsiveness in cancer patients receiving treatment with an anti-PD-1 antibody (particularly nivolumab), enabling efficient and economical treatment of patients.

Claims

1. A method for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody, comprising: a predicting step of predicting the responsiveness of the subject to treatment with an anti-PD-1 antibody based on a ratio calculated from the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject; the method comprising the above.

2. The method according to claim 1, further comprising a measuring step of measuring the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in a sample derived from the subject before the predicting step.

3. The ratio is the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein, when the ratio is high, it is predicted that the subject has poor responsiveness to the anti-PD-1 antibody, when the ratio is low, it is predicted that the subject has good responsiveness to the anti-PD-1 antibody, according to the method of claim 1 or claim 2.

4. The method according to claim 3, wherein the high or low of the ratio is determined based on a cut-off value.

5. The method according to claim 4, wherein the cut-off value is in the range of 6 to 10.

6. The high or low of the ratio is determined based on a control value, the control value is a ratio calculated from the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in a sample derived from a control subject with low responsiveness to the anti-PD-1 antibody, according to the method of claim 1.

7. when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in the sample derived from the subject is higher than or equal to the control value, it is predicted that the subject has poor responsiveness to the anti-PD-1 antibody, when the ratio of the amount of VEGF-A121 protein to the amount of VEGF-A165 protein in the sample derived from the subject is lower than the control value, it is predicted that the subject has good responsiveness to the anti-PD-1 antibody, according to the method of claim 6.

8. In the measuring step, the amount of each protein is measured by an immunological assay method, according to the method of any one of claims 2 to 7.

9. The method according to any one of claims 1 to 8, wherein the subject has cancer.

10. The method according to claim 9, wherein the anti-PD-1 antibody is nivolumab.

11. The method according to claim 9 or claim 10, wherein the cancer is selected from the group consisting of gastric cancer, malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, cancer of unknown primary origin, and urothelial cancer.

12. A method for determining the administration timing of nivolumab based on the prediction result obtained by the method according to claim 10 or claim 11.

13. A kit for use in the method according to any one of claims 1 to 12, the kit comprising an anti-VEGF-A121 antibody and an anti-VEGF-A165 antibody.

14. The anti-VEGF-A121 antibody, as the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region, the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3), and as the CDR sequences of the light chain variable region, comprising the amino acid sequence consisting of SEQ ID NO: 4 (CDR1), the amino acid sequence consisting of lysine (Lys)-valine (Val)-serine (Ser) (CDR2), and the amino acid sequence of SEQ ID NO: 6 (CDR3), respectively. The kit according to claim 13.

15. The kit according to claim 13, wherein the heavy chain variable region of the anti-VEGF-A121 antibody comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of the anti-VEGF-A121 antibody comprises the amino acid sequence of SEQ ID NO:

8.

16. The anti-VEGF-A165 antibody, as the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region, the amino acid sequences of SEQ ID NO: 11 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 13 (CDR3), and as the CDR sequences of the light chain variable region, comprising the amino acid sequence consisting of SEQ ID NO: 4 (CDR1), the amino acid sequence consisting of lysine (Lys)-valine (Val)-serine (Ser) (CDR2), and the amino acid sequence of SEQ ID NO: 14 (CDR3), respectively. The kit according to claim 13.

17. The kit according to claim 13, wherein the heavy chain variable region of the anti-VEGF-A165 antibody comprises the amino acid sequence of SEQ ID NO: 15, and the light chain variable region of the anti-VEGF-A165 antibody comprises the amino acid sequence of SEQ ID NO:

16.

18. The kit according to any one of claims 13 to 17, wherein the anti-VEGF-A121 antibody and the anti-VEGF-A165 antibody are labeled.

19. An apparatus for predicting in vitro the responsiveness of a subject to an anti-PD-1 antibody, A prediction unit that predicts the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the ratio calculated from the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in the sample derived from the subject. The apparatus including the same.

20. The apparatus according to claim 19, including a measurement unit that measures the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in the sample derived from the subject.

21. The apparatus according to claim 19 or claim 20, including a measurement value input unit that inputs the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in the sample derived from the subject.

22. The prediction unit A control recording means for previously recording a cut-off value or a control value, A prediction means for predicting the presence or degree of responsiveness of the subject to treatment with an anti-PD-1 antibody from the ratio of the input amount of VEGF-A121 protein and the amount of VEGF-A165 protein and the cut-off value or the control value, An output means for outputting the result of predicting the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the result of the analysis. The apparatus according to any one of claims 19 to 21, including the same.

23. A program for controlling an apparatus that predicts in vitro the responsiveness of a subject to an anti-PD-1 antibody. In the apparatus, Calculate the ratio from the amount of VEGF-A121 protein and the amount of VEGF-A165 protein in the sample derived from the subject, Perform a comparative analysis of the calculated ratio based on known results, A prediction unit that predicts the responsiveness of the subject to treatment with an anti-PD-1 antibody based on the result of the analysis. The computer program configured to execute the same.

24. In the apparatus, a measurement unit that measures the amounts of the two proteins. The computer program according to claim 23, further configured to execute the same.

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