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, including administration of an agent capable of binding to CLEVER-1, in a patient. In the method, the presence of CLEVER-1-expressing cells is detected in a tumor sample obtained from the cancer patient by immunohistochemical staining with the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), and the proportion of CLEVER-1-expressing cells within the tumor from the total amount of viable intratumoral cells present in the stained tumor sample is calculated. A tumor sample showing a substantial proportion of Clever-1-expressing intratumoral cells will indicate that the cancer patient is responsive to anti-Clever-1 therapy.

Description

Technical Field

[0001] The present invention relates to the pretreatment identification of cancer patients who respond to treatment comprising administration of an agent capable of binding to 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 mutational 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 the ability to capture on a subset of anti-inflammatory macrophages [5, 6]. However, there is still a need to identify patients who will 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] Cancer patients with CLEVER-1 positive macrophages are expected to benefit most from anti-CLEVER-1 treatment. Now, an accurate method has been found to identify cancer patients who respond to treatment involving the administration of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody vectalisimab. In particular, an accurate method for pretreatment identification of cancer patients has been found, which provides accurate results by machine reading from a sample stained with a specific staining antibody. The present invention is based on staining with a specific antibody that is suitable for machine reading and does not produce a background color that can interfere with machine reading of the stained sample. In addition, a specific composition of CLEVER-1 positive cells in tumor content / biopsy is required to identify patients who will benefit from anti-CLEVER-1 therapy.

[0006] Cancer patients having at least 1% CLEVER-1-expressing intratumoral cells from the total amount of viable intratumoral cells in a pre-treatment tumor sample such as a tumor biopsy according to the present invention are most responsive to a therapy comprising administration of an agent capable of binding to CLEVER-1, preferably a therapy comprising administration of an anti-CLEVER-1 antibody, more preferably a therapy comprising administration of the anti-CLEVER-1 antibody vecsumarlimab. Thus, it has been found that the threshold for selection of cancer patients to be treated with an anti-CLEVER-1 agent such as anti-CLEVER-1 antibody therapy is at least 1% calculated by the proportion of intratumoral CLEVER-1-expressing cells from the total amount of viable intratumoral cells. In the method of the present invention, CLEVER-1 expression is determined by immunohistochemical (IHC) staining of a tumor sample prior to the initiation of anti-CLEVER-1 therapy. The present invention discloses a detailed method for the evaluation of CLEVER-1 expression and provides a tool for patient selection.

[0007] The object of the present invention is to provide an accurate method for pre-treatment identification and patient selection of cancer patients responsive to anti-CLEVER-1 treatment using immunohistochemical staining of a tumor biopsy prior to the initiation of anti-CLEVER-1 treatment, thus reducing or eliminating unnecessary treatment. More particularly, the present invention provides a method for pre-treatment identification and patient selection of cancer patients responsive to anti-CLEVER-1 treatment comprising administration of an anti-CLEVER-1 antibody, preferably a treatment comprising administration of the anti-CLEVER-1 antibody vecsumarlimab. By the method of the present invention, cancer patients to be treated with anti-CLEVER-1 treatment can be selected, and the threshold of CLEVER-1-expressing intratumoral cells (proportion of CLEVER-1-expressing intratumoral cells from the total amount of viable intratumoral cells) is about 1% based on immunohistochemical staining of a tumor biopsy prior to the initiation of anti-CLEVER-1 therapy. When a tumor sample such as a tumor biopsy obtained from a cancer patient contains at least 1% CLEVER-1-expressing intratumoral cells from the total amount of viable intratumoral cells, it indicates that the cancer patient is responsive to anti-CLEVER-1 treatment, preferably anti-CLEVER-1 antibody therapy, more preferably anti-CLEVER-1 antibody vecsumarlimab therapy.

[0008] It is also an object of the present invention to provide a method for treating a specific group of patients having an improved treatment response, and this patient is selected by the method according to the present invention.

[0009] To achieve the object set forth above, the present invention is characterized by being present in 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.

[0010] The present invention provides an in vitro method for the pretreatment identification of cancer patients, which is a method for staining a tumor sample obtained from a cancer patient. A typical method of the present invention is a method for pretreating and identifying a cancer patient who responds to anti-CLEVER-1 therapy, including the administration of an agent capable of binding to common lymphatic and vascular endothelial receptor-1 (CLEVER-1) in a patient, - Detecting the presence of CLEVER-1-expressing cells in a tumor sample obtained from a cancer patient by immunohistochemical staining using the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), and - Calculating the proportion of CLEVER-1-expressing cells in the tumor from the total amount of viable tumor cells present in the stained tumor sample comprising said tumor sample containing at least 1% of Clever-1-expressing tumor cells from the total amount of viable tumor cells indicates that the cancer patient is responsive to said anti-Clever-1 therapy.

[0011] The present invention also relates to a method for treating a cancer patient, which comprises administering a therapeutically effective amount of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody bemcentinib, wherein the patient is diagnosed with a tumor that exhibits at least 1% CLEVER-1-expressing tumor cells from the total amount of cells in the primary tumor by immunohistochemical staining of the present invention. More particularly, the present invention relates to the use of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody bemcentinib, for the treatment of cancer in a patient diagnosed with a tumor containing at least 1% CLEVER-1-expressing tumor cells in the primary tumor from the total amount of cells in the primary tumor obtained from the tumor sample. According to 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, is used for the treatment of cancer by reducing the growth of malignant tumors in the patient and / or inhibiting metastasis formation.

[0012] The present invention relates to a method for treating a cancer patient, comprising - obtaining a tumor sample from the cancer patient, - detecting the presence of CLEVER-1-expressing cells in the tumor sample obtained from the cancer patient by immunohistochemical staining using the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), - calculating the proportion of CLEVER-1-expressing cells in the tumor from the total amount of primary tumor cells present in the stained tumor sample, and - starting the administration of an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody bemcentinib, to the patient if the stained tumor sample contains at least 1% Clever-1-expressing tumor cells from the total amount of primary tumor cells. The present invention provides a method comprising at least one of the steps.

[0013] Furthermore, the present invention relates to the use of a kit comprising at least one mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9) and a reagent for staining tumor samples for carrying out the method of the present invention for pre-treatment identification of cancer patients.

[0014] The present invention is based on the finding that the selection of patients who will benefit from anti-CLEVER-1 therapy, preferably anti-CLEVER-1 antibody therapy, more preferably anti-CLEVER-1 antibody bemcentinib therapy, is more accurate and effective when calculating CLEVER-1 expression in cells within a primary tumor using the disclosed immunohistochemical staining method. Based on the results, the CLEVER-1 expression level within the tumor is statistically significant on its own to identify patients who will benefit from anti-CLEVER-1 therapy from those who will not.

[0015] Based on the pre-treatment identification method of the present invention, an agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably anti-CLEVER-1 antibody bemcentinib, can be effectively used for the treatment of cancer in patients.

Brief Description of the Drawings

[0016]

Figure 1

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Mode for Carrying Out the Invention

[0017] CLEVER-1 is a protein disclosed in International Publication No. WO 2003 / 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 scavenging ability to 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 in melanoma patients as well as in other cancers [2] using a novel humanized anti-CLEVER-1 antibody (FP-1305) named vexamarlizumab disclosed in International Publication No. WO 2017 / 182705. By blocking the interaction of CLEVER-1 with its lymphocyte substrates, 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.

[0018] 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 that are 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 that are 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. Thus, 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 comprising administration of at least one agent capable of binding to CLEVER-1, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody vecsmarlimab.

[0019] According to one embodiment of the present invention, an agent capable of binding to CLEVER-1 includes an anti-CLEVER-1 antibody. Usually, 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.

[0020] In one embodiment of the present invention, the anti-CLEVER-1 antibody is the humanized monoclonal immunoglobulin G4κ antibody bevacizumab (International Nonproprietary Name (INN) disclosed in WHO Drug Information, Vol. 34, No. 3 (2020), pages 699-700), or an antibody in a bevacizumab variant or bevacizumab biosimilar. As used herein, "bevacizumab" means a humanized monoclonal IgG4 antibody having the structure described in WHO Drug Information, Vol. 34, No. 3 (2020).

[0021] A bevacizumab biosimilar means a biological product approved by a regulatory authority in any country for marketing as a bevacizumab biosimilar. In one embodiment, the bevacizumab biosimilar contains a bevacizumab variant as the drug substance. In one embodiment, the bevacizumab biosimilar has heavy and light chains with substantially the same amino acid sequence as bevacizumab. As used herein, "bevacizumab variant" means an antibody having one or more conservative amino acid substitutions at positions located outside the light chain CDR and / or one or more conservative amino acid substitutions at positions located outside the heavy chain CDR, e.g., the variant positions contain the same heavy and light chain sequences as bevacizumab except that they are located in the framework region or the constant region. In other words, bevacizumab and bevacizumab 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 bevacizumab variant is substantially the same as bevacizumab with respect to binding affinity for CLEVER-1.

[0022] According to one embodiment of the present invention, the cell line producing the therapeutic anti-CLEVER-1 antibody, vexolimab (FP-1305), was deposited on May 27, 2020, with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, 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 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 claims to the specific exemplifications it represents.

[0023] In the present invention, an effective and accurate method has been found for selecting cancer patients who respond to anti-CLEVER-1 treatment or therapy, including the administration of an agent capable of binding to CLEVER-1 in a patient, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody vexolimab. In the method of the present invention, a tumor sample is obtained from a cancer patient prior to the initiation of anti-CLEVER-1 treatment, including the administration of an agent capable of binding to CLEVER-1 in the patient, preferably an anti-CLEVER-1 antibody, more preferably the anti-CLEVER-1 antibody vexolimab, and based on that tumor sample, the amount of intratumoral cells expressing CLEVER-1 is evaluated for pretreatment identification and patient selection of cancer patients who respond to anti-CLEVER-1 treatment.

[0024] 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 tumor sample can also be obtained using any other suitable method. Biopsy may be the removal of a small piece of tissue or a sample of cells. The tumor sample may contain cells and / or small pieces of tissue. In one embodiment of the present invention, the tumor sample is a tumor tissue sample.

[0025] According to one embodiment of the present invention, the tumor tissue sample can be stained and examined for CLEVER-1 expressing cells as a fresh sample. According to other embodiments of the present invention, the tumor sample may be preserved by fixing and embedding in formalin, paraffin, etc. Thus, the tumor sample used in the method of the present invention may be a formalin or paraffin-fixed sample or a frozen sample. In a preferred embodiment, the tissue sample is fixed with formalin. In some embodiments, the fixed tissue sample is embedded in paraffin to prepare a formalin-fixed paraffin-embedded (FFPE) tissue sample. Cross-linking fixatives such as formalin are used to prepare samples for immunohistochemical staining because they help preserve the structural integrity of the tumor tissue and maintain the tissue architecture. The tissue sample can be fixed by conventional methods known in the art using a fixation length that depends on the size of the tissue sample and the fixative used. Another option is for use with frozen samples, and the tumor sample is cryopreserved, for example.

[0026] In the method of the present invention, CLEVER-1 expression is detected or identified in the cells of a tumor sample by immunohistochemical staining using a specific antibody. The immunohistochemical staining is performed according to a protocol known in the art and provided by the producer of the staining antibody. Two general methods of immunohistochemical staining can be used in direct and indirect assays. The immunohistochemical staining can be performed using an automated system. In the method of the present invention, CLEVER-1 expressing cells are detected by staining the cells of the tumor sample with the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9). Further, the method of the present invention for pre-treatment identification of cancer patients includes calculating the percentage of CLEVER-1 expressing cells within the tumor from the total amount of viable tumor cells present in the stained tumor sample, and a tumor sample containing at least 1% CLEVER-1 expressing tumor cells from the total amount of viable tumor cells indicates that the cancer patient is responsive to anti-CLEVER-1 treatment.

[0027] In the method according to the present invention, the CLEVER-1 expression is obtained by dividing the number of CLEVER-1 expressing tumor cells only by the total number of viable tumor cells in the sample and multiplying by 100. According to one embodiment of the present invention, the tumor cells include macrophages and / or tumor endothelial cells.

[0028] In the pre-treatment screening method according to the present invention, CLEVER-1 staining with a highly specific anti-CLEVER-1 antibody, more particularly the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9), is necessary for proper staining of CLEVER-1 positive tumor cells. The monoclonal IgG2a kappa STAB-1 antibody (clone 4G9) is produced in 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 STABILIN-1 (CLEVER-1) of human origin. 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). Immunohistochemical staining can be performed based on the protocol provided by the antibody producer. The amount of CLEVER-1 expressing cells in the tumor sample can be calculated by a pathologist using a microscope, but to obtain 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.

[0029] In the method of the present invention, the obtained tumor sample is stained in an immunohistochemistry (IHC) assay using an antibody that specifically binds to the CLEVER-1 protein, and the antibody is the mouse monoclonal IgG2a kappa STAB-1 antibody (clone 4G9).

[0030] First human data from the MATINS clinical trial (ClinicalTrials.gov NCT03733990) using the humanized anti-CLEVER-1 antibody (vexatumumab) showed that patients who benefited from anti-CLEVER-1 therapy (including administration of the anti-CLEVER-1 antibody vexatumumab) (tumor regression or disease stabilization) had at least 1% CLEVER-1-expressing tumor cells within the viable tumor, calculated from the total amount of viable tumor cells present in the stained samples obtained prior to initiation of anti-CLEVER-1 therapy. According to the ROC analysis performed, the threshold of the CLEVER-1 intratumoral score for reaching benefit from anti-CLEVER-1 therapy (vexatumumab) could be set in the range of 3 - 7%. The range of 1 - 10% of the intratumoral CLEVER-1 positive score is the best of the embodiments of the present invention. A 1% intratumoral CLEVER-1 positive score is required to benefit from anti-CLEVER-1 therapy.

[0031] The present invention, including administration of the anti-CLEVER-1 antibody vexatumumab, is most valuable for patients diagnosed with tumors containing a significant amount of CLEVER-1 positive tumor cells within the tumor. According to the present invention, it has been recognized that the anti-CLEVER-1 antibody vexatumumab is most valuable for cancer patients diagnosed with tumors containing at least 1% intratumoral CLEVER-1-expressing cells from the total amount of viable tumor cells in samples obtained from the tumor. In one embodiment of the present invention, tumor samples containing 1 - 10% or 3 - 7% CLEVER-1-expressing tumor cells from the total amount of viable tumor cells indicate that the cancer patient is responsive to anti-CLEVER-1 therapy.

[0032] Therefore, according to one embodiment of the present invention, CLEVER-1 expression within a tumor can be used as a pretreatment predictive biomarker to identify a patient's responsiveness to anti-CLEVER-1 therapy. In CLEVER-1 staining, it was found that only the amount of positive cells within the tumor was meaningful. In the method of the present invention, the percentage of CLEVER-1 expression level within the tumor is calculated from the total amount of cells within the viable tumor.

[0033] In the method of the present invention, the decision to initiate anti-CLEVER-1 therapy is made based on pretreatment in vitro diagnosis. Tumors containing specific CLEVER-1 expression indicate that the patient is responsive to anti-CLEVER-1 therapy. According to the present invention, anti-CLEVER-1 therapy or treatment relates to a treatment comprising administration of an anti-CLEVER-1 antibody, preferably the anti-CLEVER-1 antibody vecsumilimab. In a typical method for treating cancer patients, the anti-CLVER-1 antibody is administered in a therapeutically effective amount.

[0034] The terms "therapy", "treatment", or "treating" are understood to include 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, which includes the administration of a therapeutically effective amount of an anti-CLEVER-1 antibody, preferably the anti-CLEVER-1 antibody vexolizumab. 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 to an individual using any of a variety of methods and delivery systems known to those of skill in the art. The drugs used in the present invention can be administered by any means that achieve their intended purpose. For example, administration can be, for example, by injection, intravenous, intramuscular, intraperitoneal, intratumoral, subcutaneous, or other parenteral administration routes. 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 drug into a pharmaceutically usable preparation. The selected dosage must be sufficient to reduce or inhibit malignant tumor growth and / or inhibit metastasis formation.

[0035] 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.

[0036] 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 progressive solid tumors (ClinicalTrials.gov NCT03733990: A trial to evaluate the safety, tolerability, and preliminary efficacy of FP-1305 in cancer patients (MATINS)).

[0037] 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 vectoliximab (International Nonproprietary Name (INN) as disclosed on page 699 - 700 of WHO Drug Information, Vol. 34, No.3 (2020)) produced in CHO cells.

[0038] In this study, pre-treatment tumor biopsies were obtained before starting anti-CLEVER-1 antibody FP-1305 treatment. The obtained tumor samples were 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 starting anti-CLEVER-1 treatment. Progressive disease (PD) means the cancer is growing. In cancers that are neither aggressive nor treatable, 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 have achieved complete response, partial response, and stable disease with the treatment intervention of anti-Clever-1 antibody FP-1305.

[0039] 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. Clever-1 staining of DCR patients is 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 (obtained 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 also scored in the tumor and stroma. The results are shown in Figure 2.

[0040] Figure 2 shows the relationship between the percentage of viable cells (VC) positive for Clever-1 staining and clinical benefit (DCR patients) from the total cell mass 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 the viable tumor. It was observed that DCR patients had higher intratumoral Clever-1 staining.

[0041] Based on the results, the intratumoral Clever-1 expression level is statistically significant (p = 0.038) for identifying patients who benefit from anti-Clever-1 therapy from those who do not (Figure 3). The percentage of the expression level is calculated from the total amount of viable cells present in the stained sample, and for Clever-1, from the cells within the tumor.

[0042] Data was further analyzed based on receiver operating characteristic (ROC) curve analysis from pretreatment tumor biopsies from the MATINS (ClinicalTrials.gov NCT03733990), a first-in-human anti-CLEVER-1 clinical trial investigating vecsmarlimab (anti-CLEVER-1 antibody mAb) for the treatment of progressive solid tumors. The ROC curve is shown in Figure 4. The area under the ROC curve (AUC) indicates the accuracy of the method. In the most recent ROC using pretreatment tumor staining results, the AUC value was 0.72 (95% confidence interval 0.51 - 0.92), indicating that CLEVER-1 immunohistochemical staining according to the present invention can distinguish patients who will benefit from anti-CLEVER-1 treatment. The ROC analysis was performed on 8 patients who benefited from anti-CLEVER-1 treatment (tumor regression or stability on follow-up imaging) versus 75 patients who did not benefit from anti-CLEVER-1 treatment (tumor progression on follow-up imaging).

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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 detect the presence of CLEVER-1 expressing cells in tumor samples obtained from cancer patients by immunohistochemical staining with mouse monoclonal IgG2a copper STAB-1 antibody (clone 4G9), and - Calculate the percentage of intratumor CLEVER-1 expressing cells from the total amount of living intratumor cells present in the stained tumor sample. Includes, A method wherein a tumor sample containing at least 1% Clever-1 expressing tumor cells from the total amount of living tumor cells indicates that the cancer patient is responsive to the anti-Clever-1 therapy.

2. 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, a bexmarilimab biosimilar, or a bexmarilimab variant.

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

4. The method according to claim 1, wherein the tumor sample is a formalin-fixed sample, a formalin-fixed paraffin-embedded (FFPE) sample, or a frozen sample.

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

6. Use of a kit comprising at least one mouse monoclonal IgG2a copper STAB-1 antibody (clone 4G9) and a reagent for staining a tumor sample, for carrying out the method according to any one of claims 1 to 5.

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