WO2020218322A1

WO2020218322A1 - Method for predicting therapeutic effect of immune checkpoint inhibitor using blood chemokine

Info

Publication number: WO2020218322A1
Application number: PCT/JP2020/017287
Authority: WO
Inventors: 卓藤村
Original assignee: 国立大学法人東北大学
Priority date: 2019-04-23
Filing date: 2020-04-22
Publication date: 2020-10-29

Abstract

This method for predicting the therapeutic effect of anti-PD-1 antibody or antigen-bound fragment thereof in a subject comprises determining the level of CXCL5 in a biological sample collected from the subject.

Description

Prediction of therapeutic effects of immune checkpoint inhibitors using blood chemokines

The present invention relates to a method, a biomarker, a diagnostic agent, and a kit for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof.

Cancer cells block the attack of immune cells by reducing the activity of the immune cells that attack the cancer cells. This mechanism is called an "immune checkpoint". Therefore, by inhibiting this "immune checkpoint", the function of immune cells can be activated again to attack cancer cells. Immune checkpoint inhibitors are drugs that activate immune cells whose activity has been reduced by cancer cells and attack them.

Specific examples of the immune checkpoint inhibitor include anti-PD-1 antibody and anti-CTLA4 antibody. The anti-PD-1 antibodies nivolumab and pembrolizumab are known to be useful in the treatment of malignant melanoma, and their therapeutic effects on non-small cell lung cancer and renal cell carcinoma have also been reported (Patent Document 1). , Non-Patent Document 1).

Since anti-PD-1 antibodies such as nivolumab and pembrolizumab are very expensive, biomarkers are needed to evaluate the effectiveness of anti-PD-1 antibody therapy. The efficacy of nivolumab and pembrolizumab in Japan has been reported to be 34.1% and 24.1%, respectively (Non-Patent Documents 2 and 3), and another drug that enhances the antitumor immune response in malignant melanoma is required. It is suggested that.

Therefore, it would be beneficial if patients with high therapeutic effects of anti-PD-1 antibody could be selected in advance and the antibody could be administered.

Ipilimumab is a fully human IgG1 monoclonal antibody that blocks cellular T lymphocyte antigens (CTLA4) and is one of the promising agents that enhances the antitumor immune response of patients with advanced malignant melanoma in combination with nivolumab. It is one. The efficacy of the combination therapy of nivolumab and ipilimumab in advanced-stage melanoma has been reported to be 57.8%, and in addition to the simultaneous administration of nivolumab and ipilimumab, continuous administration of nivolumab and ipilimumab by planned switching is also advanced. It is said to be highly effective in the treatment of stage malignant melanoma. However, such simultaneous and continuous administration of nivolumab and ipilimumab frequently causes severe immune-related side effects (irAE) such as hepatitis, colitis, and polyneuropathy in advanced malignant melanoma (Non-Patent Documents). 4).

It is known that the appearance of autoimmune-related side effects caused by the administration of immune checkpoint inhibitors is higher than that of conventional drugs. For example, the incidence of grade 3 or higher adverse events when anti-PD-1 antibody (nivolumab) or anti-CTLA4 antibody (ipilimumab) is administered to patients with unresectable malignant melanoma is 16.3% with nivolumab alone. It has been reported that ipilimumab alone accounts for 27.3%, and nivolumab in combination with ipilimumab accounts for 55.0% (Non-Patent Document 1).

Therefore, it is preferable to be able to predict the possibility of side effects caused by the administration of immune checkpoint inhibitors before the onset of side effects.

Patent Document 2 addresses the above-mentioned problems, and is collected from a subject to which at least one antibody drug selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody and an antigen-binding fragment thereof has been administered. Disclosed is a method for predicting the onset of side effects due to administration of the antibody drug by measuring the level of at least one marker selected from CD163 and CXCL5.

Japanese Unexamined Patent Publication No. 2016-064989 International Publication No. 2018/003995

In Patent Document 2, data before (0 weeks) and after (6 weeks) administration of anti-PD-1 antibody are measured, and side effects are obtained based on changes in serum CXCL5 concentration before and after administration of anti-PD-1 antibody. Predicts the possibility of developing.

However, the possibility of therapeutic effect of anti-PD-1 antibody cannot be evaluated based only on the data before administration of anti-PD-1 antibody.

It would be beneficial in selecting an appropriate drug if patients with high therapeutic effects could be selected in advance based on the data of patients before administration of anti-PD-1 antibody. Furthermore, if the therapeutic effect of the anti-PD-1 antibody can be predicted before the administration of the anti-PD-1 antibody, it is also useful in that the risk of side effects due to the administration of the anti-PD-1 antibody can be prevented.

An object of the present invention is to provide a method capable of accurately predicting the therapeutic effect of an anti-PD-1 antibody in a subject before administration of the anti-PD-1 antibody.

The present inventors diligently studied the relationship between various chemokines in a target biological sample and the therapeutic effect of an anti-PD-1 antibody, and found that the therapeutic effect of an anti-PD-1 antibody was obtained in a subject having a high level of CXCL5. We found that it was expensive and came to complete the present invention.

That is, the present invention includes, for example, the following embodiments.

Item 1. A method for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof in a subject.
A method comprising measuring the level of CXCL5 in a biological sample taken from a subject.

Item 2. If the level of CXCL5 in a biological sample taken from a subject is higher than a preset cutoff value, it involves predicting that the anti-PD-1 antibody is likely to be effective in treating the disease in the subject. Item 1. The method according to Item 1.

Item 3. A biomarker for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, including CXCL5.

Item 4. A diagnostic agent for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antibody-binding fragment thereof.

Item 5. A kit for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antigen-binding fragment thereof.

According to the present invention, a new method for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof in a subject is provided. That is, the prediction method of the present invention can predict a cancer patient who is significantly sensitive to an anti-PD-1 antibody or an antigen-binding fragment thereof based on the level of CXCL5. As a result, it is possible to select an appropriate drug for a subject in disease treatment or avoid unnecessary medication, and it is possible to formulate an appropriate administration plan and change to an appropriate administration plan.

The graph which shows the ROC curve (A) and the mean serum level (B) of CXCL5 in malignant melanoma. * Indicates p <0.05. The graph which shows the ROC curve (A) and the mean serum level (B) of CXCL10 in malignant melanoma. n. s. Indicates that there is no statistically significant difference. The graph which shows the ROC curve (A) and the mean serum level (B) of CCL22 in malignant melanoma. n. s. Indicates that there is no statistically significant difference.

According to the first aspect of the present invention, it is a method for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof in a subject, and includes measuring the level of CXCL5 in a biological sample collected from the subject. A method is provided. An antibody drug that is an anti-PD-1 antibody or an antigen-binding fragment thereof may be simply referred to as an antibody drug.

According to such a method, the therapeutic effect of an antibody drug consisting of an anti-PD-1 antibody or an antigen-binding fragment thereof in a subject can be predicted based on the level of CXCL5.

As used herein, "CXCL5" is a chemokine capable of recruiting not only neutrophils but also CXCR2 ⁺ bone marrow-derived immunosuppressive cells (MDSC) and CXCR2 ⁺ monocytes which can be progenitor cells of tumor-related macrophages (TAM). CXCL5 may include variants, isoforms, and species homologues of CXCL5. CXCL5 may also be referred to as LIX or GCP-2.

The level of CXCL5 is, for example, the concentration or amount of CXCL5 in the biological sample, and preferably the concentration of CXCL5 in the biological sample. Examples of the concentration of CXCL5 to be measured include a concentration in the range of 10 pg / mL to 10000 pg / mL, 10 pg / mL to 5000 pg / mL.

As used herein, an "antibody" is a full-length antibody comprising a glycoprotein containing at least two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (hereinafter, may be abbreviated as V _H ) and a heavy chain constant region. The heavy chain constant region is composed of three domains C _H1 , C _{H 2} and C _{H 3} . Each light chain is composed of a light chain variable region (hereinafter, may be abbreviated as _VL ) and a light chain constant region. The light chain constant region is comprised of one domain C _L. The V _H and _VL regions are further subdivided into highly mutagenic regions called complementarity determining regions (CDRs), which are referred to as framework regions (FRs) and are more conserved regions. Are scattered. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

Examples of such "antibodies" include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, low molecular weight antibodies, domain antibodies, synthetic antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody complexes, and single-stranded antibodies. , Antibody derivatives, antibody analogs, and their respective antigen-binding fragments.

As used herein, an "antigen-binding fragment" (or simply referred to as an "antibody fragment") of an antibody is one or more antibodies that retain the ability to specifically bind to an antigen (eg, PD-1). It shows a fragment. Examples of binding fragments contained in the "antigen binding fragment" of an antibody include (i) Fab fragment, which is a monovalent fragment composed of _VL , V _H , _CL and C _H1 domains, and (ii) in the hinge region. F (ab') ₂ fragments, which are divalent fragments containing 2 Fab fragments bonded by disulfide bridges, Fd fragments composed of (iii) V _H and C _H1 domains, and (iv) single arm of antibody. Examples thereof include an Fv fragment composed of _VL and V _H domains, a dAb fragment composed of (v) V _H domains, or (vi) isolated complementarity determining regions (CDRs). In addition, the two domains of the Fv fragment, _VL and _VH, are encoded by separate genes, which can be linked by synthetic linkers that can be made into single protein chains using recombination techniques. Within this chain, the _VL and _VH regions can be paired to form a monovalent molecule (single chain Fv (scFv)). Such single-stranded antibodies are also included in the "antigen-binding fragment" of the antibody.

In the present specification, "PD-1" is an immune receptor that mediates a signal for regulating the immune response, and is a type I membrane protein belonging to the CD28 / CTLA-4 family. "PD-1" in the present invention is used interchangeably with "ProgrammedDeath1", "ProgrammedCellDeath1", "Protein PD-1", "PD1", "PDCD1", and "hPD-1" to modify PD-1. It may include the body, isoforms, species homologues, and analogs that have at least one common epitope with PD-1.

In the present specification, the "anti-PD-1 antibody" is not particularly limited as long as it does not interfere with the effects of the present invention, and may be any antibody that specifically binds to PD-1. Such an antibody may be an antibody that specifically recognizes a part of the structure of the amino acid sequence, or may be an antibody that specifically recognizes the entire structure. The antibody is not particularly limited, and examples thereof include nivolumab, pembrolizumab, and lambbrolizumab, and nivolumab or pembrolizumab is preferable.

In the present specification, the subject (also referred to as a test subject) is a mammal such as a rodent, a dog, a cat, a cow, a primate, etc., preferably a human, and more preferably an anti-PD-1 antibody. A person who has or may have a disease that can be treated by administration of, and even more preferably a person who has cancer, sarcoma, or malignant mesothelioma. Here, "treatment" includes not only treating an established pathological condition but also preventing a pathological condition that may be established in the future. The subject is preferably a subject who has never been administered an anti-PD-1 antibody or an antigen-binding fragment thereof.

Specific examples of cancer, sarcoma, or malignant mesoderma include malignant melanoma (eg, metastatic malignant melanoma, unresectable malignant melanoma), cutaneous spinous cell carcinoma, and extramammary Paget's disease. , Or skin cancers such as Merkel cell carcinoma; renal cancers (eg, renal cell carcinomas, clear cell melanomas); prostate cancers (eg, hormone-refractory prostatic adenocalcinoma); breast cancers; colon cancers; lung cancers (eg, non-small cells) Lung cancer); Bone cancer; Pancreatic cancer; Head and neck cancer; Skin or intraorbital malignant melanoma; Uterine cancer; Ovarian cancer; Rectal cancer; Anal cancer; Gastric cancer; Testis cancer; Uterine cancer; Ovical carcinoma; Endometrial carcinoma; Cervical melanoma; Vaginal melanoma; genital melanoma; esophageal cancer; small intestine cancer; colon cancer; endocrine cancer; thyroid cancer; ad thyroid cancer; adrenal cancer; soft tissue sarcoma; multiple myeloma; urinary tract cancer; penis cancer; chronic Or acute leukemia (eg, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia); pediatric solid tumor; advanced solid cancer; bladder cancer; kidney or urinary tract cancer; renal pelvis Melanoma; Urinary tract epithelial cancer; Central nervous system (CNS) tumor; Lymphoma (eg, lymphocytic lymphoma, primary CNS lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma (Hodgkin's disease), T-cell lymphoma); pleural malignancies Pericardial malignant melanoma; Peritoneal malignant melanoma; Tumor neovascularization; Spinal tumor; Brain stem gliome; Hydrangea adenoma; Caposhi sarcoma; Squamous epithelial cancer; Squamous cell carcinoma; Environment-induced cancer including asbestos-induced cancer; Or a combination thereof, preferably skin cancer, more preferably malignant melanoma, cutaneous spinous cell cancer, extramammary Paget's disease, or Merkel cell cancer.

Examples of the biological sample include all body fluids such as serum, plasma, blood, lymph, tissue fluid, body cavity fluid, digestive juice, urine and the like, and serum is preferable.
The method for administering the anti-PD-1 antibody or the antigen-binding fragment thereof is not particularly limited, but intravenous administration is preferable.

The time for collecting the biological sample is before administration of the anti-PD-1 antibody or its antigen-binding fragment to the subject.

The measurement of the CXCL5 level is not particularly limited as long as the CXCL5 level can be measured quantitatively or semi-quantitatively, and any known method can be adopted. For example, an immunoassay method, an electrophoresis method, a Western blotting method, a mass spectrometry method and the like can be mentioned, and an immunoassay method is preferable.

Examples of immunoassays include immunoturbidimetric methods, enzyme immunoassays, and the like. The immunoassay is an immunoassay that measures a protein or the like as an antigen, and an anti-CXCL5 antibody or an antigen-binding fragment thereof can be used, preferably a polyclonal antibody or a monoclonal antibody thereof. The anti-CXCL5 antibody or an antigen-binding fragment thereof can be a commercially available antibody, or can be produced by a well-known method.

As an immunoturbidimetric measurement method, CXCL5 in a biological sample is reacted with an anti-CXCL5 antibody or an antigen-binding fragment thereof to cause an antigen-antibody reaction, and as a result, the level of CXCL5 is measured from the degree of turbidity generated. If so, it is not particularly limited. Examples of such a method include the TIA method, the latex immunoturbidimetric method, and the neferometry method. The TIA method is a method for measuring the degree of turbidity at a specific absorbance in an immunoturbidimetric measurement method. Further, the latex immunoturbidimetric method is a method for measuring an immunoturbidimetric method using an anti-CXCL5 antibody or an antigen-binding fragment thereof bound to latex particles as an antibody. Further, the neferometry method is a method in which the degree of turbidity is measured as scattered light by collecting light scattered to a size of a certain angle or more in the immunoturbidimetric measurement method.

Examples of the enzyme immunoassay method include an EIA method such as an ELISA method using a plate as a support. First, the anti-CXCL5 antibody or its antigen-binding fragment is directly or indirectly bound to the solid phase as a primary antibody. When CXCL5 is measured by an enzyme immunoassay, for example, a biological sample for measuring CXCL5 is added to a primary antibody bound to a solid phase and reacted. After reacting for a certain period of time, the solid phase is washed and a secondary labeled antibody is added for a secondary reaction. The solid phase is washed again and the labeled portion bound to the solid phase is measured.

In the immunoassay method using the above-mentioned secondary labeled antibody, an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase can be used as the labeling substance. For example, when an HRP-labeled antibody is used, known DAB, TMB, OPD and the like can be used as the substrate. In addition, the labeling substances include not only enzymes such as HRP, but also labeling metals such as gold colloid and europium, and various chemical and biological fluorescent substances such as FITC, rhodamine, Texas Red, Alexa, and GFP, ³² P, ^51. All labelable substances such as radioactive substances such as Cr can be mentioned. When a labeling substance is used in the present invention, an avidin-biotin system or a streptavidin-biotin system can also be used. In that case, for example, streptavidin or avidin labeled with an enzyme such as HRP can be used together with the secondary labeled antibody labeled with biotin. In addition, chemiluminescence immunoassay using a luciferase-labeled antibody, fluorescence immunoassay using a fluorescent dye-labeled antibody, flow cytometry method and the like can be mentioned.

As the electrophoresis method, generally, the SDS-PAGE method can be mentioned. In addition, there are those using cellulose acetate as a support. Protein staining includes Coomassie Brilliant Blue, Ponso S staining, amide black staining, and a method using direct enzyme activity.

In addition, detection by Western blotting is also effective. That is, the electrophoresed gel is transferred to a nitrocellulose membrane, PVDF membrane, etc., and then reacted with an anti-CXCL5 antibody, which is a primary antibody, or an antigen-binding fragment thereof, and an HRP-labeled anti-IgG, which is a secondary labeled antibody. Then, the color is developed with the HRP coloring reagent, and CXCL5 can be measured by the degree of color development of the band corresponding to CXCL5.

As a mass spectrometry method, for example, an analysis method using a mass spectrometer can be mentioned. For example, surface enhanced laser desorption ionization (Surface Enhanced Laser Deposition / Ionization) time-of-flight mass analyzer (SELDI-TOF MS method), matrix-assisted laser ionization (Matrix-Assisted Laser Deposition / Ionization) time-of-flight mass analyzer Examples of methods using the MALDI-TOF MS method) and the ESI method (Electrospray Ionization) can be exemplified. The SELDI-TOF MS method is preferable because impurities are removed while the target substance is uniformly captured by the functional groups on the chip surface and ionized by laser light, so that a reproducible ion spectrum with a high S / N ratio can be obtained. ..

Using the CXCL5 level data obtained from the above measurements, the therapeutic effect of the anti-PD-1 antibody or its antigen-binding fragment in the subject can be predicted.

As such a prediction, when the level of CXCL5 in a biological sample collected from a subject is higher than a preset cutoff value, an anti-PD-1 antibody or an antigen-binding fragment thereof may be effective for treating a disease in the subject. Predicting that the sex is high can be mentioned. Alternatively, if the level of CXCL5 in a biological sample taken from a subject is less than or equal to a preset cutoff value, it is unlikely that the anti-PD-1 antibody or antigen-binding fragment thereof is effective in treating the disease in the subject. Is to be predicted.

The cutoff value (threshold value) is a value that distinguishes between those who responded to the drug and those who did not, but since it varies depending on various conditions such as the measurement target and the type of measurement method, it is set in advance according to the conditions. There is a need to. Since the cutoff value varies depending on the measurement target (number of patients, age, gender, body weight, health condition, disease condition), measurement conditions (for example, type and sensitivity of antibody to be measured), statistical method, etc., the present invention. Broadly includes inventions using arbitrary cutoff values that may vary depending on these conditions, and is not limited to a specific value.

Those skilled in the art can obtain the cutoff value from the level of CXCL5 measured in advance by various statistical analysis methods. For example, the cutoff value is the average or median level of CXCL5 in the subject to which the anti-PD-1 antibody or the antigen-binding fragment thereof was administered; the subject to which the anti-PD-1 antibody or the antigen-binding fragment thereof was administered. ROC (Receiver Operating Characteristic) so that the sum of sensitivity and specificity is maximized from the relationship between the level of CXCL5 and the therapeutic effect of anti-PD-1 antibody or its antigen-binding fragment (tumor shrinkage effect, survival time prolonging effect, etc.) Values obtained based on analysis; CXCL5 levels in subjects administered with anti-PD-1 antibody or antigen-binding fragment thereof, and therapeutic effect of anti-PD-1 antibody or antigen-binding fragment thereof (tumor shrinkage effect, survival time prolonging effect) The value obtained based on the chi-square test (of which the P value is the minimum in the log rank test, and the P value is below a certain level (for example, the P value is 0.05 or less) from the relationship with the value. Value, P value is 0.01 or less), etc.));

The cutoff value may be one, or a plurality of cutoff values may be set according to the type of therapeutic effect, the type of antibody drug to be administered, the condition of the subject to which the antibody drug is administered, or a combination thereof.

According to the above prediction step, the level of CXCL5 in the biological sample collected from the subject is higher than the preset cutoff value, and it is predicted that the anti-PD-1 antibody is likely to be effective for the treatment of the disease in the subject. If so, it may be decided to administer the anti-PD-1 antibody to such subjects.

The effective amount of the anti-PD-1 antibody is not particularly limited, and depends on the type, purity, degree of side effects, target type, properties, gender, age, symptoms, presence / absence of concomitant drug, prior treatment history, etc. Determined appropriately by the vendor. For example, such an effective amount may be 0.1 to 20 mg / kg body weight / day, preferably 1.0 to 20 mg / kg body weight / day once or several times.

The treatment with the above-mentioned antibody drug may or may not have surgery to remove the tumor during or before or after the implementation period. Even if the treatment is not accompanied by removal of the tumor mainly for the purpose of prolonging the life, the tumor that has become smaller may be removed after the treatment with the above antibody drug, or the tumor for the purpose of suppressing recurrence / metastasis. After the removal of the tumor, treatment with the above antibody drug may be performed prophylactically.

In the present specification, "therapeutic effect" can be used interchangeably with "effectiveness", and can be evaluated by tumor shrinkage effect, recurrence / metastasis suppression effect, life extension effect, and the like. "Anti-PD-1 antibody is effective in treating a disease in a subject" means that the therapeutic effect in a patient whose CXCL5 level exceeds the cutoff value is the therapeutic effect in a patient whose CXCL5 level is below the cutoff value. It means that it is remarkably superior with a statistically significant difference in comparison.

According to the above prediction step, when the level of CXCL5 in the biological sample collected from the subject is equal to or less than the preset cutoff value, it can be decided not to administer the anti-PD-1 antibody to the subject. In this case, instead of the anti-PD-1 antibody, another drug such as an anti-CTLA4 antibody may be administered.

For example, in the case of treatment of patients with unresectable malignant melanoma, the level of CXCL5 in patients is measured before administration of anti-PD-1 antibody, and it is predicted that anti-PD-1 antibody is likely to be effective. Anti-PD-1 antibody (nivolumab or pembrolizumab) is administered to patients who have been treated, and antibody drugs other than anti-PD-1 antibody such as ipilimumab are administered to patients with negative judgment. Risk can be reduced.

Thus, according to the method of predicting the therapeutic effect of the anti-PD-1 antibody or its antigen-binding fragment in the subject of the first aspect of the present invention, the anti-PD-1 antibody or its antigen in the subject is based on the level of CXCL5. The therapeutic effect of the bound fragment can be predicted, an appropriate drug can be selected for each subject in the treatment of the disease, and the occurrence of side effects due to unnecessary medication can be avoided. Therefore, it is possible to formulate an appropriate administration plan and change to an appropriate administration plan.

The object of the first aspect of the present invention can also be seen as an auxiliary method for selecting a therapeutic agent in the treatment of a disease.

According to the second aspect of the present invention, a biomarker for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, including CXCL5, is provided.

According to the third aspect of the present invention, a diagnostic agent for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antibody-binding fragment thereof, is provided.

Since the above diagnostic agent can be used for measuring the level of CXCL5, various measuring methods described above can be performed. Examples of the above-mentioned measuring method include an immunoassay method, an electrophoresis method, a Western blotting method, a mass spectrometry method and the like, and an immunoassay method is preferable.

The aspect of the diagnostic agent can be carried out according to the description regarding the method for predicting the therapeutic effect of the first aspect.

According to the fourth aspect of the present invention, a kit for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antigen-binding fragment thereof, is provided.

Such a kit is suitably used for carrying out the prediction method of the first aspect of the present invention.

The anti-CXCL5 antibody or its antigen-binding fragment can measure the level of CXCL5 protein in a biological sample collected from a subject.

The kit may further contain a secondary antibody against the primary antibody in addition to the primary antibody consisting of the anti-CXCL5 antibody or an antigen-binding fragment thereof. The secondary antibody is preferably labeled with a luciferase label, a radioactive label, a fluorescent label, an enzyme label or the like.

The kit may further include an instruction manual that describes a procedure for carrying out the prediction method of the first embodiment of the present invention.

According to the fifth aspect of the present invention, the anti-PD-1 antibody or the anti-PD-1 antibody or an antigen-binding fragment thereof is predicted to be effective for a subject predicted to be effective by the prediction method of the first aspect. A method of treatment of a subject comprising administering the antigen-binding fragment is provided.

Subjects are mammals such as rodents, dogs, cats, cows, primates, etc., preferably humans, and more preferably can be treated by administration of an anti-PD-1 antibody or antigen-binding fragment thereof. A person who has or may have a disease, and even more preferably a person who has cancer, sarcoma, or malignant mesothelioma. Examples of the disease, the dose and the dosage form of the anti-PD-1 antibody or the antigen-binding fragment thereof are as described in the first embodiment.
The present invention can also adopt the following configurations.
<1> A method for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof in a subject.
A method comprising measuring the level of CXCL5 in a biological sample taken from a subject.
<2> When the level of CXCL5 in the biological sample collected from the subject is higher than the preset cutoff value, it is predicted that the anti-PD-1 antibody is likely to be effective for the treatment of the disease in the subject. The method according to <1>, which includes the above.
<3> A method for identifying a target having a high therapeutic effect on an anti-PD-1 antibody or an antigen-binding fragment thereof.
If the level of CXCL5 in a biological sample taken from a subject is measured and the level of CXCL5 in a biological sample taken from a subject is higher than a preset cutoff value, the subject is an anti-PD-1 antibody. Or a method comprising predicting that the therapeutic effect of the antigen-binding fragment thereof is high.
<4> The method according to any one of <1> to <3>, wherein the subject is a subject suffering from cancer, sarcoma, or malignant mesothelioma.
<5><3> Cancer, sarcoma, which comprises administering the anti-PD-1 antibody or its antigen-binding fragment to a subject predicted to have a high therapeutic effect on the anti-PD-1 antibody or its antigen-binding fragment. , Or a method of treating a subject suffering from malignant mesothelioma.
<6> A method for treating a subject suffering from cancer, sarcoma, or malignant mesothelioma.
When the level of CXCL5 in the biological sample collected from the subject is higher than the preset cutoff value, the subject is predicted to have a high therapeutic effect on the anti-PD-1 antibody or its antigen-binding fragment, and A method comprising administering an anti-PD-1 antibody or an antigen-binding fragment thereof to a subject predicted to have a high therapeutic effect on the anti-PD-1 antibody or the antigen-binding fragment thereof.
<7> The treatment method according to any one of <1> to <6>, wherein the subject is a mammal.
<8> The treatment method according to any one of <1> to <7>, wherein the subject is a human.
<9> The cutoff value is the average or median level of CXCL5 in a subject to which the anti-PD-1 antibody or an antigen-binding fragment thereof has been administered; a subject to which the anti-PD-1 antibody or an antigen-binding fragment thereof has been administered. ROC (Receivable Operating Chemical) so that the sum of sensitivity and specificity is maximized from the relationship between the level of CXCL5 and the therapeutic effect of anti-PD-1 antibody or its antigen-binding fragment (tumor shrinkage effect, survival time prolonging effect, etc.) ) Values determined based on analysis; or the level of CXCL5 in the subject to whom the anti-PD-1 antibody or antigen-binding fragment thereof was administered, and the therapeutic effect (tumor shrinkage effect or survival time) of the anti-PD-1 antibody or antigen-binding fragment thereof. From the relationship with the prolongation effect), the value obtained based on the chi-square test (of which the P value is the minimum in the log rank test, or the P value is below a certain level (for example, the P value is 0.05 or less). The method according to any one of <1> to <8>, wherein the value becomes 0.01 and the P value is 0.01 or less).
<10> A biomarker for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises CXCL5.
<11> A diagnostic agent for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antibody-binding fragment thereof.
<12> A kit for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antigen-binding fragment thereof.

Hereinafter, the present invention will be described in more detail based on Examples, but it goes without saying that the present invention is not limited to these Examples.

Example 1
1. 1. Esix Statement in Human Experiments This human research protocol was approved by the Ethics Committee of the Graduate School of Medicine, Tohoku University in Sendai, Japan (permission number: 2017-1-064). All methods were carried out in accordance with relevant guidelines and regulations. All patients provided written informed consent.
2. Patients Forty-six patients with advanced-stage melanoma were included. Patients had unresectable stage III malignant melanoma or stage IV malignant melanoma with distant metastases to the skin, subcutaneous tissue, and lymph nodes. Stage classification (staging) was performed according to AJCC 7th Edition, 2011.

Patients 1-46 were given 2 mg / kg of nivolumab for a 3-week rest period, or 3 mg / kg for a 2-week rest period. Blood was drawn from the patient prior to administration of nivolumab to obtain serum. Both are administration schedules approved in Japan. The response of nivolumab was assessed according to the response criteria (RECIST) in solid tumors.

3.3 Baseline serum levels of three chemokines Prior to administration of nivolumab, serum concentrations of CXCL5, CXCL10 and CCL22 obtained from patients were analyzed by ELISA according to the protocol of the manufacturer (R & D Systems, Minneapolis, Minnesota).

4. Statistical Methods The ROC curve was used to calculate the cutoff values for serum levels of CXCL5, CXCL10 and CCL22 and the area under the concentration curve (AUC). The cutoff value was determined using the Youden's index (sensitivity + specificity -1), and the point at which the index was maximized was determined. ROC curves were created to evaluate serum levels of CXCL5, CXCL10 and CCL22 in patients receiving nivolumab. The Mann-Whitney U-test was used to compare the two groups. The significance level was set to p <0.05. All statistical analyzes were performed using JMP version 14.1 software (SAS Institute, Tokyo, Japan).

5. Results Table 1 shows the data collected from patients with malignant melanoma who received nivolumab. The average age of the patients was 67 years (range 33-93 years). Of the patients with malignant melanoma, 58.7% were male and 41.3% were female.

In patients with advanced-stage malignant melanoma, complete response (CR) was seen in 3 patients (6.5%, 95% confidence interval (CI), 0-13.0%) and partial response (PR) was 11. It was found in 1 patient (23.9%, 95% CI, 0-47.8%) and disease stability (SD) was found in 13 patients (28.3%, 95% CI, 0-56.6). %), And disease progression (PD) was seen in 25 patients (41.3%, 95% CI, 0-82.6%). The objective response rate 3 months after the first dose was 30.4% (95% CI, 0-60.8%). The response of each person is shown in Table 1. The incidence of side effects was 41.3%.

46 suffering from advanced malignant melanoma treated with nivolumab to determine if baseline serum levels of CXCL5, CXCL10 and CCL22 are associated with the initial response of patients with malignant melanoma treated with nivolumab46 Their levels were evaluated in the first patients. Table 1 shows the baseline serum CXCL5 and increased efficacy 3 months after the first dose of nivolumab in each patient.

The baseline CXCL5 threshold (cutoff value) for distinguishing successful and non-successful individuals was 497.5 pg / ml. The sensitivity and specificity of baseline serum CXCL5 for advanced malignant melanoma were 70.6% and 69.0%, respectively (AUC = 0.732, threshold (cutoff value) 497.5, p = 0, respectively. .0016, FIG. 1A). High baseline serum levels of CXCL5 correlated with an objective response to nivolumab in patients with advanced-stage malignant melanoma (Fig. 1B).

On the other hand, no significant relationship was found between the serum levels of CXCL10 (Fig. 2A) and CCL22 (Fig. 3A) in patients with advanced malignant melanoma and the objective response to nivolumab in patients with advanced malignant melanoma (Fig. 3A). CXCL10: Sensitivity 28.4%, Specificity 93.1%, AUC = 0.523, Threshold (cutoff value) 336.8, p = 0.674, CCL22: Sensitivity 64.7%, Specificity 62.1 %, AUC = 0.582, threshold 619.5, p = 0.360).

The baseline CXCL10 and CCL22 thresholds that distinguish between successful and non-successful individuals were 336.8 and 619.5 pg / ml, respectively. There was no significant difference in serum CXCL10 and CCL22 levels between patients with objective response and those without response (FIGS. 2B and 3B).

Table 1 shows the baseline serum levels of CXCL5, CXCL10 and CCL22 in each patient. Significantly between the blood levels of CXCL5 (p = 0.0703), CXCL10 (p = 0.1748), CCL22 (p = 0.2207) and irAE in patients who received nivolumab. No relationship was found.

As described above, the baseline serum concentration of CXCL5 was significantly higher in the responding group for malignant melanoma than in the non-responding group. In contrast, no significant difference in baseline serum levels of CXCL10 and CCL22 was found between the responding and non-responding groups. The above experimental results suggest that baseline serum levels of CXCL5 are useful as biomarkers for identifying patients with malignant melanoma who can most benefit from immunotherapy for malignant melanoma.

Example 2
As a second cohort, serum samples from 19 new patients were analyzed and the CXCL5 concentration in the serum was measured (Table 2). The reaction evaluation criteria for PR, SD, and PD are the same as the reaction evaluation criteria for Example 1. PD and SD were hit below the cutoff, PR was above the cutoff, and the hit rate (distinguishing between successful and non-successful) at the cutoff value of 497.5 pg / ml for CXCL5 in Example 1 was 19 cases. It was a good result with 16 cases (84.2%).

Claims

A method for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof in a subject.
A method comprising measuring the level of CXCL5 in a biological sample taken from a subject.
If the level of CXCL5 in a biological sample taken from a subject is higher than a preset cutoff value, it includes predicting that the anti-PD-1 antibody is likely to be effective in treating the disease in the subject. The method according to claim 1.
A biomarker for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, including CXCL5.
A diagnostic agent for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antibody-binding fragment thereof.
A kit for predicting the therapeutic effect of an anti-PD-1 antibody or an antigen-binding fragment thereof, which comprises an anti-CXCL5 antibody or an antigen-binding fragment thereof.