Method for identifying cancer patients who benefit from anti-CLEVER-1 therapy

Abstract

A method for identifying, prior to treatment, cancer patients who respond to anti-CLEVER-1 therapy, which includes administration of an agent capable of binding to common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) in a patient. In this method, the presence of PD-L1-expressing cells and CLEVER-1-expressing cells is detected in a tumor sample obtained from a cancer patient by immunohistochemical staining with a PD-L1-specific antibody and a mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), and the ratio of PD-L1-expressing cells from the total amount of viable cells present in the stained sample and the ratio of CLEVER-1-expressing cells within the tumor from the total amount of viable tumor cells present in the stained sample are calculated. A tumor sample with a low ratio of PD-L1-expressing cells or no PD-L1-expressing cells and a significant proportion of CLEVER-1-expressing cells within the tumor indicates that the cancer patient is responsive to anti-CLEVER-1 therapy.

Description

Technical Field

[0001] The present invention relates to the pre-treatment identification of cancer patients who respond to treatment including administration of an agent capable of binding to common lymphatic and vascular endothelial receptor-1 (CLEVER-1) using immunohistochemical staining of pre-treatment tumor biopsies.

Background Art

[0002] Currently, immune checkpoint inhibitors targeting the CTLA-4 and PD-1 / PD-L1 axes have been approved for clinical use, and high efficacy is observed in about 10-20% of patients, while the majority of cancer patients do not respond to checkpoint inhibitors or other novel immunotherapies. The key to success in immunotherapy lies in identifying responsive patients [1]. Patients who respond well to checkpoint inhibition typically exhibit a pre-existing anti-cancer immune response characterized by a high density of IFNγ-producing CD8 + T cells, expression of PD-L1 in tumor-infiltrating immune cells, and a high mutation burden. PD-L1 expression in pre-treatment biopsies is often used to select patients for anti-PD-1 or anti-PD-L1 treatment.

[0003] Anti-CLEVER-1 is a novel immunotherapy targeting tumor-associated macrophages and has shown promising monotherapy efficacy in a first-in-human (FiH) clinical trial named MATINS (ClinicalTrials.gov NCT03733990: A Study to Evaluate Safety, Tolerability and Preliminary Efficacy of FP-1305 in Cancer Patients (MATINS)) [2].

[0004] Natural immune cells such as macrophages can suppress T cell activation and contribute to tumor progression despite a high mutation load. Macrophages that contribute to tumor-associated immunosuppression and provide signals to support tumor growth are very suitable candidates for targeted therapy because these cells are abundant in various tumors, they can deform very freely, and they can be converted into inflammatory macrophages that support T cell activation and tumor killing [3, 4]. CLEVER-1 (also known as STABILIN-1) is a multifunctional molecule that confers scavenging ability to a subset of anti-inflammatory macrophages [5, 6]. However, there is still a need to identify patients who respond to anti-CLEVER-1 therapy and to find methods for pretreatment identification and patient selection to reduce or eliminate unnecessary treatment.

Summary of the Invention

[0005] It has been found that cancer patients showing low PD-L1 expression in tumor biopsies respond to CLEVER-1 inhibition, which is completely opposite to the prior art regarding checkpoint inhibitors. Also, the best way to predict the response to treatment based on CLEVER-1 inhibition is to use the combination of the expression of PD-L1 and CLEVER-1 and / or the ratio of PD-L1 to CLEVER-1 in immunohistochemical (IHC) staining of tumor biopsies before the start of anti-CLEVER-1 treatment. The clinical benefit of anti-CLEVER-1 treatment was seen in patients with low PD-L1 expression and high CLEVER-1 expression within the tumor in pretreatment tumor biopsies. The present invention discloses a method for detailed evaluation of the ratio of PD-L1 expression to CLEVER-1 expression and provides a tool for patient selection.

[0006] Accordingly, an object of the present invention is to provide a novel method for pre-treatment identification and patient selection of cancer patients responsive to anti-CLEVER-1 therapy using immunohistochemical staining of tumor biopsies before the initiation of anti-CLEVER-1 therapy, thus reducing or eliminating unnecessary treatments. More particularly, the present invention provides a method for pre-treatment identification and patient selection of cancer patients responsive to anti-CLEVER-1 therapy, which comprises administration of an anti-CLEVER-1 antibody, preferably the anti-CLEVER-1 antibody bemcentinib. By the method of the present invention, cancer patients to be treated with anti-CLEVER-1 therapy can be selected, and a low ratio of PD-L1 expression to CLEVER-1 intratumoral expression in immunohistochemical staining of tumor biopsies before the initiation of anti-CLEVER-1 therapy is an indication that the cancer patient is responsive to anti-CLEVER-1 therapy.

[0007] An object of the present invention is also to provide a method for treating a specific patient group having an improved treatment response, and this patient is selected by the method according to the present invention.

[0008] In particular, to achieve the object indicated above, the present invention is characterized by what is shown in the characterizing part of the appended independent claims. Some preferred embodiments of the present invention are described in the other claims. The embodiments and advantages described in this disclosure are applicable to the method and use of the present invention even if not specifically described.

[0009] A typical method of the present invention for pre-treatment identification of cancer patients responsive to anti-CLEVER-1 therapy, which comprises administration of an agent capable of binding to common lymphatic and vascular endothelial receptor-1 (CLEVER-1) in a patient, preferably administration of an anti-CLEVER-1 antibody, is - providing a tumor sample obtained from a cancer patient, - detecting the presence of PD-L1-expressing cells and CLEVER-1-expressing cells in the tumor sample by immunohistochemical staining with a PD-L1-specific antibody and a mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), and Calculating the proportion of PD-L1-expressing cells from the total amount of viable cells present in the stained sample and calculating the proportion of intratumoral CLEVER-1-expressing cells from the total amount of intratumoral viable cells present in the stained sample comprising Tumor samples showing a low proportion of PD-L1-expressing cells or no PD-L1-expressing cells and, together, a substantial proportion of Clever-1-expressing intratumoral cells indicate that the cancer patient is responsive to anti-Clever-1 therapy. More particularly, stained tumor samples showing from 0 to 2% PD-L1-expressing cells, calculated from the total amount of intratumoral viable cells present in the stained sample, and, together, at least 1% CLEVER-1-expressing cells, calculated from the total amount of intratumoral viable cells present in the stained sample, indicate that the cancer patient is responsive to anti-Clever-1 therapy, typically anti-CLEVER-1 antibody therapy.

[0010] The present invention also relates to a method of treating a cancer patient, comprising administering a therapeutically effective amount of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably anti-CLEVER-1 antibody vecsumarlimab, for use in the treatment of cancer in a patient diagnosed with a tumor showing no or low expression of PD-L1 (proportion of positive cells from all viable cells) and, together, substantial intratumoral expression of CLEVER-1 (proportion of positive cells from all intratumoral viable cells). More particularly, the present invention relates to an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably anti-CLEVER-1 antibody vecsumarlimab, for use in the treatment of cancer in a patient diagnosed with a tumor showing a low ratio of PD-L1-expressing cells to intratumoral CLEVER-1-expressing cells in a sample obtained from the tumor. According to the present invention, an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably anti-CLEVER-1 antibody vecsumarlimab, is used in the treatment of cancer by reducing the growth of malignant tumors in the patient and / or inhibiting metastasis formation.

[0011] Furthermore, the present invention is a method for treating cancer patients, comprising: - obtaining a tumor sample from a cancer patient; - detecting the presence of PD-L1-expressing cells and CLEVER-1-expressing cells in the tumor sample by immunohistochemical staining with a PD-L1-specific antibody and a mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9); - calculating the proportion of PD-L1-expressing cells from the total amount of viable cells present in the stained sample and calculating the proportion of CLEVER-1-expressing cells within the tumor from the total amount of viable intratumoral cells present in the stained sample; and - when the tumor sample shows a low proportion of PD-L1-expressing cells or does not contain PD-L1-expressing cells and, in addition, shows a substantial proportion of CLEVER-1-expressing intratumoral cells, in a patient, initiating administration of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody bemcentinib; The present invention provides a method comprising at least one of the steps of:

[0012] The present invention is based on the finding that the selection of patients who will benefit from anti-CLEVER-1 therapy is more accurate and effective by using the combination of PD-L1 and CLEVER-1 expression and / or the ratio of PD-L1 / intratumoral CLEVER-1 expression measured from tumor samples. Based on the results, the intratumoral CLEVER-1 expression level is statistically significant on its own to identify patients who will benefit from anti-CLEVER-1 therapy from those who will not, but the combination of PD-L1 and CLEVER-1 expression and / or the ratio of PD-L1 / intratumoral CLEVER-1 expression is even better than CLEVER-1 expression alone and allows for a more accurate method for pre-treatment identification of patients. According to the present invention, tumor samples that do not express or have low expression of PD-L1 and show moderate to high intratumoral CLEVER-1 expression indicate that cancer patients are responsive to anti-CLEVER-1 therapy.

[0013] Based on the present invention, an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody bemcentinib, can be effectively used for the treatment of cancer in patients.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

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Figure 4

Mode for Carrying Out the Invention

[0015] CLEVER-1 is a protein disclosed in International Publication No. 03 / 057130 as Common Lymphatic Endothelial and Vascular Endothelial Receptor-1. It is a binding protein that mediates the adhesion of lymphocytes (and malignant tumor cells) to endothelium in both the systemic vascular and lymphatic systems. CLEVER-1 (also known as STABILIN-1) is a multifunctional molecule that confers the ability to capture on a subset of anti-inflammatory macrophages [5, 6]. In these cells, it is involved in receptor-mediated endocytosis and recycling, intracellular sorting, and transcytosis of altered and normal self-components. More recently, in Stab1 - / - (Clever-1 knockout) mice and in mice treated with anti-CLEVER-1 therapy, it has been found that the progression of melanoma tumor growth and metastasis is attenuated [7]. More recently, similar results have been achieved using a novel humanized anti-CLEVER-1 antibody (FP-1305) named bexmarilimab disclosed in International Publication No. 2017 / 182705 in melanoma patients as well as in other cancers [2]. By blocking the interaction between CLEVER-1 and its lymphocyte substrate, it is possible to simultaneously control lymphocyte recirculation and lymphocyte migration, as well as related conditions such as inflammation, at the sites of lymphocyte influx into and efflux from tissues.

[0016] The terms "agent capable of binding to CLEVER-1" and "anti-CLEVER-1 agent" refer to agents including antibodies and fragments thereof, peptides or equivalents capable of binding to CLEVER-1 to block the interaction between CLEVER-1 and malignant tumor cells. An agent capable of binding to CLEVER-1 can also be any other inhibitor such as a small molecule inhibitor or a macromolecule that binds to the CLEVER-1 receptor and has sufficient affinity to inhibit protein activity. The term "antibody or fragment thereof" is used in the broadest sense to include antibodies or fragments thereof capable of binding to CLEVER-1 molecules in an individual. In particular, this is understood to include chimeric antibodies, humanized antibodies or primatized antibodies, as well as antibody fragments and single-chain antibodies (e.g., Fab, Fv), as long as they exhibit the desired biological activity. Particularly useful agents are anti-CLEVER-1 antibodies and fragments thereof. Accordingly, according to one embodiment of the present invention, an agent capable of binding to CLEVER-1 is selected from the group consisting of an antibody or fragment thereof, peptide(s), macromolecule, and any combination thereof. According to the present invention, "anti-CLEVER-1 treatment" or "anti-CLEVER-1 therapy" refers to a treatment including administration of at least one agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody.

[0017] According to one embodiment of the present invention, the anti-CLEVER-1 antibody is a therapeutic humanized anti-CLEVER-1 antibody. According to one embodiment of the present invention, the anti-CLEVER-1 antibody is the humanized monoclonal anti-CLEVER-1 antibody previously presented in International Publication No. WO 2017 / 182705.

[0018] In one embodiment of the present invention, the anti-CLEVER-1 antibody is the humanized monoclonal immunoglobulin G4 kappa antibody vectibiximab (International Nonproprietary Name (INN) disclosed in WHO Drug Information, Vol. 34, No. 3 (2020), pages 699-700), or an antibody in a vectibiximab variant or vectibiximab biosimilar. As used herein, "vectibiximab" means a humanized monoclonal IgG4 antibody having the structure described in WHO Drug Information, Vol. 34, No. 3 (2020).

[0019] A vectibiximab biosimilar means a biological product approved by a regulatory authority in any country for marketing as a vectibiximab biosimilar. In one embodiment, the vectibiximab biosimilar contains a vectibiximab variant as the drug substance. In one embodiment, the vectibiximab biosimilar has heavy and light chains with substantially the same amino acid sequence as vectibiximab. As used herein, "vectibiximab variant" means an antibody having one or more conservative amino acid substitutions at positions located outside the light chain CDRs and / or one or more conservative amino acid substitutions at positions located outside the heavy chain CDRs, e.g., the variant positions contain the same heavy and light chain sequences as vectibiximab except that they are located in the framework region or the constant region. In other words, vectibiximab and vectibiximab variants contain the same CDR sequences but differ from each other due to having conservative amino acid substitutions at other positions in the sequences of their full-length light and heavy chains. The vectibiximab variant is substantially the same as vectibiximab with respect to binding affinity for CLEVER-1.

[0020] According to one embodiment of the present invention, the cell line producing the therapeutic anti-CLEVER-1 antibody vexolimab (FP-1305) has been deposited on May 27, 2020, with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, Federal Republic of Germany, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and has the accession number DSM ACC3361. Since the deposited embodiment is intended as a single illustration of one aspect of the present invention and any culture that is functionally equivalent is within the scope of the present invention, the present invention is not limited in scope by the deposited culture. The deposit of the materials herein is not intended to constitute an admission that the description contained in the documents herein is insufficient to enable the practice of any aspect of the present invention, including its best mode, nor should it be construed as limiting the scope of the claims to the specific exemplifications it represents.

[0021] In the present invention, an effective method has been found for selecting cancer patients who respond to anti-CLEVER-1 therapy, including the administration of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody vexolimab.

[0022] In the method of the present invention, a tumor sample is obtained from a cancer patient in the patient before initiation of anti-CLEVER-1 treatment, which includes administration of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody vecsumab. Based on the tumor sample, the amount of cells expressing PD-L1 (the ratio of positive cells from all viable cells) and the amount of intratumoral cells expressing CLEVER-1 (the ratio of positive cells from all viable intratumoral cells) are evaluated for pretreatment identification and patient selection of cancer patients responsive to anti-CLEVER-1 treatment.

[0023] According to one embodiment of the present invention, the tumor sample is a tumor biopsy sample. There are many different types of biopsy techniques for obtaining a tumor sample. The biopsy may be the removal of a small piece of tissue or a sample of cells.

[0024] In the method of the present invention, both PD-L1 expression and CLEVER-1 expression are detected in the cells of the tumor sample by immunohistochemical staining with antibodies. In the method of the present invention, PD-L1-expressing cells are detected by staining the cells of the sample with a PD-L1-specific antibody, and CLEVER-1-expressing cells are detected by staining the cells of the sample with a mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9). Further, the method of the present invention for pretreatment identification of a cancer patient includes calculating the ratio of PD-L1-expressing cells from the total amount of viable cells present in the stained tumor sample, and calculating the ratio of CLEVER-1-expressing cells from the total amount of viable intratumoral cells present in the stained tumor sample. A stained tumor sample showing a low ratio of PD-L1-expressing cells or no PD-L1-expressing cells and a moderate to high ratio of CLEVER-1-expressing intratumoral cells indicates that the cancer patient is responsive to anti-CLEVER-1 treatment. The method may further include calculating the ratio of PD-L1 expression to intratumoral CLEVER-1 expression, and a low ratio of PD-L1 expression to CLEVER-1 intratumoral expression in the immunohistochemical staining of a tumor biopsy before initiation of anti-CLEVER-1 treatment indicates that the cancer patient is responsive to anti-CLEVER-1 treatment.

[0025] More particularly, PD-L1 expression in the sample is calculated using the combined positive score (CPS), which is obtained by dividing the number of PD-L1 stained cells (tumor cells, lymphocytes and macrophages) by the total number of viable tumor cells in the sample and multiplying by 100. In the method according to the invention, CLEVER-1 expression is obtained by dividing only CLEVER-1 expressing tumor cells by the total number of viable intratumoral cells in the sample and multiplying by 100. According to one embodiment of the invention, the intratumoral cells include macrophages and / or tumor endothelial cells.

[0026] According to the invention, PD-1 expression can be evaluated or detected, for example, by using different staining platforms and antibodies. The evaluation of PD-L1 expression can be performed by any suitable method using any suitable antibody specific for PD-L1. Several commercially available diagnostic assays for identifying PD-L1 expression are available, some of which are specific for certain cancer types and are intended to be used in combination with specific anti-PD-L1 inhibitors.

[0027] In the pre-treatment screening method according to the present invention, for CLEVER-1 staining, a highly specific anti-CLEVER-1 antibody, more particularly the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), is required for proper staining of CLEVER-1 positive cells. The monoclonal IgG2a kappa STAB-1 antibody (clone 4G9) is produced in the mouse clone 4G9. The mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9) is made against amino acids 1804-1902 (SEQ ID NO: 1) of human-derived STABILIN-1 (CLEVER-1). In an exemplary embodiment, the anti-CLEVER-1 antibody used in the method of the present invention is the STAB1 monoclonal antibody (M05), clone 4G9 from Abnova (Taiwan) (Product details: http: / / www.abnova.com / products / products_detail.asp?catalog_id=H00023166-M05). The amount of CLEVER-1 expressing cells from a sample can be calculated by a pathologist using a microscope, but for more accurate results, the sample is stained and machine read by an automated system. Therefore, the staining needs to be done with an antibody that is suitable for machine reading and does not produce a background color that may interfere with machine reading of the stained sample. In the present invention, it has been found that the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9) provides accurate staining without interfering with the background color, and thus appropriate staining for pre-treatment evaluation is achieved by the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), and the calculation of CLEVER-1 expressing cells can be made more accurately by machine reading.

[0028] Generally, PD-L1 expression in cells from a tumor biopsy is used to predict patient eligibility for treatment with a PD-L1 inhibitor. The level of PD-L1 expression can vary depending on the type of cells in which PD-L1 is evaluated (tumor vs. immune cells), or the source and timing of sample collection. PD-L1 expression (CPS) in more than 50% of the viable cells (tumor cells, lymphocytes, and macrophages) present in the sample is usually regarded as high expression for PPD-L1 and predicts responsiveness to a PD-1 / PD-L1 inhibitor. PD-L1 expression (CPS) in less than 1% of the viable cells present in the sample is regarded as not expressed, and PD-L1 expression (CPS) in 1-49% of the viable cells present in the sample is regarded as PD-L1 positive, but responsiveness can be treatment-dependent.

[0029] First human data using the humanized anti-CLEVER-1 antibody (bexmarilimab) from the clinical trial MATINS show that patients who experience clinical benefit (tumor regression or disease stabilization) from anti-CLEVER-1 therapy, including administration of the anti-CLEVER-1 antibody bexmarilimab, show low PD-L1 expression of 0-2% (median 1%) calculated from the total amount of viable cells present in the stained sample, while patients who do not respond to anti-CLEVER-1 therapy can show PD-L1 expression of 0-100% (median 5%) calculated from the total amount of viable cells present in the stained sample (Figure 2). According to one embodiment of the present invention, the low percentage of PD-L1-expressing cells is 0-2% calculated from the total amount of viable cells present in the stained sample, which, together with moderate to high CLEVER-1 expression levels, indicates that cancer patients are responsive to anti-Clever-1 treatment.

[0030] According to one embodiment of the present invention, the amount of CLEVER-1-expressing tumor cells is at least 1% calculated from the total amount of viable tumor cells present in the stained sample.

[0031] The present invention, including the administration of the anti-CLEVER-1 antibody vecsumalimab, is most valuable for patients diagnosed with tumors that do not express or show low expression of PD-L1 and, together, show a significant amount of CLEVER-1 positive intratumoral cells.

[0032] Accordingly, according to one embodiment of the present invention, the expression of PD-L1 and the intratumoral CLEVER-1 expression can be used as pretreatment predictive biomarkers to identify the responsiveness of patients to anti-CLEVER-1 therapy. Typically, patients who respond well to anti-PD-1 therapy have PD-L1 positive and T lymphocyte-rich tumor specimens. According to one embodiment of the present invention, the expression of PD-L1 can be tested by staining tumor cells with an anti-PD-L1 antibody. The expression of the PD-L1 protein is usually determined by calculating the proportion of tumor live cells showing partial or complete membrane staining at any intensity from the total amount of live cells present in the sample, while for CLEVER-1 staining, only the positive cells within the tumor are meaningful. In the method of the present invention, the proportion of the PD-L1 expression level is calculated from the total amount of live cells present in the stained sample, and the proportion of the CLEVER-1 intratumoral expression level is calculated from the total amount of cells within the tumor.

[0033] According to one embodiment of the present invention, the method includes calculating the ratio of PD-L1-expressing cells to CLEVER-1-expressing cells within a tumor in a stained sample, and a low PD-L1 / intratumoral CLEVER-1 ratio indicates that a cancer patient is responsive to anti-CLEVER-1 therapy. The PD-L1 / intratumoral CLEVER-1 ratio is calculated from the proportion of PD-L1-expressing cells and the proportion of CLEVER-1 intratumoral cells. According to one embodiment of the present invention, the stained tumor sample shows 0 to 2% PD-L1-expressing cells, calculated from the total amount of viable cells present in the stained sample, and together at least 1% of CLEVER-1-expressing intratumoral cells, calculated from the total amount of viable intratumoral cells present in the stained sample, indicates that a cancer patient is responsive to anti-CLEVER-1 therapy. Thus, according to one embodiment of the present invention, the ratio of PD-L1 expression to intratumoral CLEVER-1 expression is ≦2, preferably <2, calculated based on the ratio values, and indicates that a cancer patient is responsive to anti-CLEVER-1 therapy.

[0034] In the method of the present invention, the decision to initiate anti-CLEVER-1 therapy is made based on a pre-treatment diagnosis. Absence or low PD-L1 expression and moderate to high CLEVER-1 expression indicate that a patient is responsive to anti-CLEVER-1 therapy. According to the present invention, the anti-CLEVER-1 therapy or treatment relates to a treatment comprising administration of an anti-CLEVER-1 antibody, preferably the anti-CLEVER-1 antibody vecsmarlimab. In a typical method for treating a cancer patient, the anti-CLVER-1 antibody is administered in a therapeutically effective amount.

[0035] The terms "therapy", "treatment", or "treating" are to be understood as including the complete cure of a disease or disorder, as well as the amelioration or alleviation of the disease or disorder. In one embodiment according to the present invention, the anti-CLEVER-1 therapy refers to an anti-CLEVER-1 antibody therapy comprising the administration of a therapeutically effective amount of an anti-CLEVER-1 antibody, preferably the anti-CLEVER-1 antibody vectocumab. The term "therapeutically effective amount" means any amount of a drug according to the present invention that is sufficient to bring about the desired therapeutic result. "Administering" refers to the physical introduction of a composition containing the therapeutic agent into an individual, using any of a variety of methods and delivery systems known to those skilled in the art. The drugs used in the present invention can be administered by any means that achieves their intended purpose. For example, administration can be, for example, by injection, intravenous, intramuscular, intraperitoneal, intratumoral, subcutaneous, or other parenteral routes of administration. In addition to the pharmacologically active compound, the pharmaceutical preparation of the drug preferably contains a suitable pharmaceutically acceptable carrier, including excipients and adjuvants that facilitate the processing of the active agent into a pharmaceutically usable preparation. The selected dosage must be sufficient to reduce or inhibit malignant tumor growth and / or inhibit metastasis formation.

[0036] The present invention for treating cancer by reducing malignant tumor proliferation and / or inhibiting metastasis formation is applicable to all forms of cancer. Thus, any benign or malignant tumor or metastasis of a malignant tumor can be treated.

[0037] Experimental Section First-in-Human Clinical Trial of Anti-CLEVER-1 Antibody The anti-CLEVER-1 antibody FP-1305, a CLEVER-1 inhibitor, is currently being tested for safety and preliminary efficacy in a Phase I / II trial in patients with advanced solid tumors (ClinicalTrials.gov NCT03733990: A study to evaluate the safety, tolerability and preliminary efficacy of FP-1305 in cancer patients (MATINS)).

[0038] The anti-CLEVER-1 antibody FP-1305 is a humanized monoclonal CLEVER-1 antibody previously presented in International Publication No. WO 2017 / 182705. More precisely, FP-1305 (DSM ACC3361) is a humanized monoclonal immunoglobulin G4 kappa antibody vectiximab (International Nonproprietary Name (INN) as disclosed on pages 699 - 700 of WHO Drug Information, Vol. 34, No.3 (2020)) produced in CHO cells.

[0039] In this study, pre-treatment tumor biopsies were obtained before initiating anti-CLEVER-1 antibody FP-1305 treatment, fixed in formalin (FFPE) for subsequent immunohistochemical (IHC) staining, and analyzed by an independent centralized pathologist. Tumor progression and regression were repeatedly evaluated by CT scans compared to existing scans taken before initiating anti-CLEVER-1 treatment. Progressive disease (PD) means the cancer is growing. In cancers that are highly invasive and untreatable, such as in the MATINS trial, no significant change in tumor size, which is a positive effect, is designated as stable disease (SD) and considered a good response. Tumor regression is called partial response (PR) according to the RECIST criteria used to evaluate treatment response. Patients with DCR (DCR = disease control rate) are those who achieved complete response, partial response, and stable disease with the therapeutic intervention of anti-Clever-1 antibody FP-1305.

[0040] IHC staining of tumor biopsies before anti-Clever-1 therapy FFPE (formalin-fixed paraffin-embedded) tumor samples obtained from patients in the MATINS clinical trial of 4-5 μm sections were stained with Ventana Benchmark Ultra (Roche Diagnostics, Basel, Switzerland). For Clever-1 staining, the UltraView Universal DAB Detection Kit (Roche Diagnostics) was used in combination with the CLEVER-1 primary antibody (monoclonal IgG2a kappa STAB-1 (clone 4G9), Abnova (Taiwan)) at a 1:100 dilution. PD-L1 staining was performed using the 22C3 pharmDx assay (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Clever-1 and PD-L1 staining of DCR patients are shown in Figure 1. Interpretation and scoring were performed by a board-certified pathologist using a brightfield microscope. The percentage of Clever-1 positive viable cells (calculated by dividing the number of all Clever-1 positive cells by the total number of cells and multiplying by 100) was scored regardless of site, and the percentage of Clever-1 positive viable cells was scored in both tumor and stroma. PD-L1 was scored as the combined positive score (CPS) by calculating the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by the number of total viable tumor cells and multiplying by 100. The results are shown in Figure 2.

[0041] Figure 2 shows the relationship between the percentage of viable cells (VC) positive for Clever-1 and PD-L1 staining and clinical benefit (DCR patients) from the total cell amount in biopsies obtained from patients prior to anti-Clever-1 therapy in the MATINS clinical trial. Also shown are the percentage of Clever-1 expressing cells in the stroma and cells within viable tumor. It was observed that DCR patients had higher intratumoral Clever-1 staining and lower PD-L1 staining.

[0042] Based on the results, the CLEVER-1 expression level in tumors was statistically significant (p = 0.038) (Figure 3) when used alone to identify patients who would benefit from anti-CLEVER-1 therapy from those who would not. However, the ratio of PD-L1 / CLEVER-1 expression in tumors was even better than CLEVER-1 expression alone (p < 0.01) (Figure 4). Therefore, according to the present invention, tumors that do not show PD-L1 expression or show low PD-L1 expression and show moderate to high CLEVER-1 expression levels among tumor-infiltrating cells in biopsy samples are indicators of responsiveness to treatment. The percentage of expression levels is calculated from the total amount of living cells present in the stained samples for PD-L1 and from tumor cells only for CLEVER-1. The PD-L1 / CLEVER-1 ratio in tumors was highly significant in separating non-DCR patients from DCR patients (p < 0.01 in Figure 4) and can thus predict patients who would benefit from anti-CLEVER-1 therapy.

[0043] TIFF2025516877000002.tif166143

Claims

1. A method for identifying cancer patients who will respond to anti-CLEVER-1 therapy, including the administration of a drug capable of binding to common lymphatic endothelium and vascular endothelial receptor-1 (CLEVER-1), before treatment, - To provide tumor samples obtained from cancer patients, - To detect the presence of PD-L1-expressing cells and CLEVER-1-expressing cells in the tumor sample by immunohistochemical staining with a PD-L1-specific antibody and a mouse monoclonal IgG2a copper STAB-1 antibody (clone 4G9), and - Calculate the percentage of PD-L1-expressing cells from the total amount of viable cells present in the stained sample, and calculate the percentage of intratumoral CLEVER-1-expressing cells from the total amount of viable intratumoral cells present in the stained sample. Includes, A method for demonstrating that a tumor sample showing a low percentage of PD-L1-expressing cells or no PD-L1-expressing cells, along with a significant percentage of Clever-1-expressing intratumor cells, indicates that the cancer patient is responsive to anti-Clever-1 therapy.

2. The method according to claim 1, comprising calculating the ratio of PD-L1-expressing cells to intratumoral CLEVER-1-expressing cells in a stained sample, wherein a low PD-L1 / intratumoral CLEVER-1 ratio indicates that the cancer patient is responsive to anti-CLEVER-1 therapy.

3. The method according to claim 1, wherein the agent capable of binding to CLEVER-1 comprises an anti-CLEVER-1 antibody, preferably the anti-CLEVER-1 antibody bexmarilimab.

4. The method according to claim 1, wherein the tumor sample is a tumor biopsy sample.

5. The method according to claim 1, wherein the intratumor cells include macrophages and / or tumor endothelial cells.

6. The method according to claim 1, wherein the low percentage of PD-L1-expressing cells, calculated from the total amount of living tumor cells present in the stained sample, is 0-2%.

7. The method according to claim 1, wherein a substantial proportion of the CLEVER-1 expressing cells is at least 1%, and is calculated from the total amount of living tumor cells present in the stained sample.

8. The method according to claim 2, wherein the ratio of PD-L1-expressing cells to tumor-derived CLEVER-1-expressing cells in the stained sample is ≤2, preferably less than 2.

9. A drug capable of binding to CLEVER-1 for use in the treatment of cancer in an individual identified by any one of claims 1 to 8.

10. A drug capable of binding to CLEVER-1 for use in the treatment of cancer in an individual according to claim 9, wherein the drug capable of binding to CLEVER-1 comprises an anti-Clever-1 antibody, preferably the anti-Clever-1 antibody bexmarilimab.

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