Anti-Clever-1 agents for controlling the expression of cell surface markers on leukocytes and their use to guide anti-Clever-1 based cancer therapy

Abstract

A method for monitoring a patient's response to anti-Clever-1 therapy, estimating the need for combination therapy based on the expression levels of one or more cell surface markers selected from PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25, and CXCR3 on leukocytes in association with the anti-Clever-1 therapy, and selecting the best combination agent to initiate treatment with anti-Clever-1 therapy after observed changes in the expression of one or more cell surface markers.

Description

[Technical Field]

[0001] The present invention relates to monitoring the expression levels of one or more cell surface markers in circulating T cell populations in association with anti-Clever-1 therapy, and selecting the best combination drug to initiate treatment with anti-Clever-1 therapy following observed changes in the expression of one or more cell surface markers. [Background technology]

[0002] The vast number of genetic and epigenetic alterations inherent in cancer cells provides numerous tumor-associated antigens that can be recognized by the host immune system, thereby requiring tumors to develop specific immune resistance mechanisms. Important immune resistance mechanisms involve immune inhibitory pathways called immune checkpoints, which typically mediate immune tolerance and mitigate collateral tissue damage. A particularly important immune checkpoint receptor is cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), which downregulates the magnitude of T cell activation. Antibody blockade of CTLA-4 in mouse models of cancer induces antitumor immunity. Some immune checkpoint receptors, such as programmed cell death protein 1 (PD-1), limit T cell effector function in tissues. By upregulating PD-1 ligands, tumor cells block antitumor immune responses in the tumor microenvironment. Anti-PD-1, anti-PD-L1, and anti-CTLA-4 immune checkpoint inhibitors are widely used in clinical patient care. Antagonistic antibodies against second-generation immune checkpoint receptors, including but not limited to ICOS (inducible T-cell costimulator), OX40, 41BB, TIM3, and LAG3, as well as checkpoint inhibitors (ICIs), are in clinical development as anticancer drugs (Non-Patent Document 1).

[0003] Currently, immune checkpoint modulators targeting the CTLA-4 and PD-1 / PD-L1 axis are approved for clinical use and are highly effective in approximately 10–20% of patients with melanoma and certain other tumors. However, several other important cancer types (e.g., prostate, breast, and colorectal cancer) remain refractory to these modulators, and clear biomarkers that can distinguish responders from non-responders and guide treatment are not available (Non-Patent Document 2). Patients who respond well to checkpoint blockade typically have high densities of interferon-gamma (IFNg)-producing CD8+ cells. + Tumors that do not respond to immune checkpoint blockade have a pre-existing antitumor immune response characterized by PD-L1 expression on T cells and tumor-infiltrating immune cells and a high mutational load. Tumors that do not respond to immune checkpoint blockade exhibit either a stromal T cell phenotype, in which T cell infiltration into the tumor (TIL) or T cell activation in the tumor microenvironment (TME) is prevented by immunosuppressive stromal compartments, or a non-inflammatory phenotype, characterized by low T cell infiltration, low mutational load, and high tumor cell proliferation. Tumors can be immunologically classified as inflammatory or non-inflammatory based on the presence of tumor-infiltrating cytotoxic CD8 T cells (NPL 3). Inflammatory tumors exhibit a high mutational load, high IFNg and PD-L1 expression, and a favorable response to immune checkpoint blockade therapy. IFNg produced by T cells is thought to be necessary for ICIs to function as anticancer agents. However, IFNg secretion is known to increase PD-L1 expression on cells, which may contribute to immunotherapy resistance (NPL 3).

[0004] However, innate immune cells such as macrophages can suppress T cell activation and contribute to tumor progression despite their high mutational burden. Macrophages, which contribute to tumor-associated immunosuppression and provide signals that support tumor growth, may be highly suitable candidates for targeted therapy because these cells are abundant in various tumors, highly plastic, and can be transformed into pro-inflammatory macrophages that support T cell activation and tumor rejection (Non-Patent Documents 4 and 5). To date, macrophage-targeting strategies under clinical development utilize inhibition of macrophage colony-stimulating factor receptors to dramatically reduce the number of macrophages in tumors (Non-Patent Document 6). However, resistance to these approaches has already been reported (Non-Patent Document 7). Therefore, there is a need to find novel ways to utilize these cells to induce tumor cell death by the immune system.

[0005] In recent years, the contribution of scavenger receptors in regulating macrophage responses to different stimuli has been receiving increasing attention. Clever-1 (also known as Stabilin-1) is a multifunctional molecule that confers scavenging ability to a subset of anti-inflammatory macrophages (Non-Patent Documents 8, 9). In these cells, Clever-1 is involved in receptor-mediated endocytosis and recycling, intracellular sorting, and transcytosis of altered and normal self-components. More recently, the role of Stab1 in several tumor models has been reported. - / - Growth and metastasis were found to be attenuated in Clever-1 knockout mice and in mice treated with anti-Clever-1 therapy (Non-Patent Documents 10 and 11). In addition, combined treatment with anti-Clever-1 agents together with anti-PD-1 agents has been shown to produce anti-tumor responses in mouse models of triple-negative breast cancer and colorectal cancer (Non-Patent Document 11). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy, Nature reviews cancer, 2012, 12(4): 252-264. [Non-patent document 2] Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature 2017; 541(7637): 321-30. [Non-patent document 3] Hegde PS, Karanikas V, Evers S. The Where, the When, and the How of Immune Monitoring for Cancer Immunotherapies in the Era of Checkpoint Inhibition. Clin Cancer Res 2016;22(8):1865-74 doi 10.1158 / 1078-0432.CCR-15-1507. [Non-patent document 4] Guerriero JL, Sotayo A, Ponichtera HE, Castrillon JA, Pourzia AL, Schad S, et al. Class IIa HDAC inhibition reduces breast tumours and metastases through anti-tumour macrophages. Nature 2017; 543(7645):428-32. [Non-Patent Document 5] Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell 2010; 141(1):39-51. [Non-patent document 6] Ries CH, Cannarile MA, Hoves S, Benz J, Wartha K, Runza V, et al. Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer Cell 2014; 25(6):846-59.

Direct Environment 7

Outdoor Track 8

Outdoor Tools9

Outdoor Tools 10

Non-Patent Document 11

Summary of the Invention

[0007] Surprisingly, we found that anti-Clever-1 therapy in cancer patients reduced the expression of PD-1, PD-L1, CTLA-4, OX40, 41BB, LAG3, TIM3, and CD28 on leukocytes, particularly in circulating T cell populations, and increased the expression of CD25 (IL-2RA), CXCR3, and CD69, as well as affecting the expression level of ICOS. Additionally, we found that anti-Clever-1 therapy-induced antitumor responses were associated with increased circulating interferon gamma (IFNg). In line with current findings, this suggests that anti-Clever-1 therapy may subsequently lead to resistance, as IFNg responses may increase PD-1 and / or PD-L1 expression. In particular, anti-Clever-1 therapy has been shown to relieve T cell exhaustion by downregulating the expression of cell surface markers known as exhaustion markers or checkpoint molecules, such as PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 (IL-2RA), CXCR3, and CD69. By monitoring changes in these commonly known checkpoint molecules during anti-Clever-1 therapy, it is possible to select a checkpoint inhibitor to be administered in combination with ongoing anti-Clever-1 therapy. If a particular checkpoint molecule does not respond to anti-Clever-1 therapy in the desired manner, a specific checkpoint inhibitor targeting the desired checkpoint molecule is administered in combination with ongoing anti-Clever-1 therapy. Expression of cell surface markers can be used to guide anti-Clever-1 therapy or the best possible combination treatment with checkpoint inhibitor(s) along with anti-Clever-1 therapy. More particularly, it has been found that the cell surface markers PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 (IL-2RA), CXCR3, and CD69 can be used to monitor a patient's response to anti-Clever-1 therapy and to assess the need for combination therapy with anti-Clever-1 therapy.The present findings provide a method for selecting the best combination drug(s) to initiate treatment with anti-Clever-1 therapy following observed changes in expression of one, two, three, or more cell surface markers.

[0008] Thus, the present invention provides methods for monitoring a patient's response to anti-Clever-1 monotherapy, assessing the need for combination therapy based on the expression levels of one, two, three or more cell surface markers selected from PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25, CXCR3, and CD69 on leukocytes, particularly in circulating T cell populations, in association with the anti-Clever-1 therapy, and selecting the best combination agent(s) to initiate treatment with anti-Clever-1 therapy following observed changes in one or more cell surface marker expression.

[0009] 1. An exemplary method for monitoring a patient's response to anti-Clever-1 therapy and predicting the need for combination therapy when an agent capable of binding to Clever-1 is administered to the patient, the method comprising: - Obtaining a sample from a patient at an initial time point prior to administering to the patient an agent capable of binding to Clever-1; - Obtaining a sample from the patient at a later time point after administering to the patient an agent capable of binding to Clever-1; - measuring the expression of one or more cell surface markers selected from PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 and CXCR3, and / or interferon gamma (IFNg) from the obtained sample; - comparing the expression level of the cell surface marker and / or IFNg measured from a sample obtained at a subsequent time point with the expression level of the cell surface marker and / or IFNg measured from a sample obtained at the initial time point, wherein the absence of a desired change in the expression level of the cell surface marker or an increase in IFNg levels is an indication to initiate co-administration of an agent that affects the cell surface marker.

[0010] The present invention also provides novel biomarker-based combination treatments for cancer patients, thus reducing or even eliminating the above-mentioned problems in defining non-responders from responding patients. The present invention offers the possibility to explore the molecular landscape of solid tumors by blood sampling to define changes in patients during anti-Clever-1 therapy, and then provides the opportunity to select the best possible checkpoint inhibitor combination treatment with anti-Clever-1 therapy.

[0011] Therefore, the present findings also support the idea that a therapeutically effective amount of a) an agent capable of binding to Clever-1; and b) in combination with one or more agents selected from the group comprising CTLA-4 inhibitors, ICOS inhibitors, ICOS inducers, OX40 inhibitors, 41BB inhibitors, LAG3 inhibitors, TIM3 inhibitors, CD28 inhibitors, cytokine IL-2, CD25 (IL-2RA) agonists, CXCR3 inducers, and CXCR3 agonists, Also provided are combinations for use in treating cancer in an individual treated with anti-Clever-1 monotherapy, where the monotherapy does not exhibit the desired change in expression of cell surface markers associated with the agent: CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 and / or CXCR3.

[0012] The present invention is based on the finding that anti-Clever-1 therapy sequentially alters the expression of several cell surface receptors on leukocytes, and the expression levels of these checkpoints may change during the course of anti-Clever-1 therapy, providing the best possible biological explanation for selecting which checkpoint inhibitor (ICI) treatment should be combined with anti-Clever-1 therapy and when to initiate the combined anti-Clever-1 therapy.

[0013] The present invention provides a means for selecting when to initiate anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-ICOS, anti-OX40, anti-41BB, anti-LAG3, anti-TIM3, and / or anti-CD28 therapy in combination with anti-Clever-1 therapy. Anti-Clever-1 therapy has surprisingly been shown to downregulate exhaustion markers PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, and CD28 at the initiation of anti-Clever-1 therapy. Thus, for example, if anti-Clever-1 therapy does not downregulate CTLA-4 expression, anti-CTLA-4 therapy will be combined with anti-Clever-1 therapy. Alternatively, if anti-Clever-1 therapy does not downregulate OX40, anti-OX40 therapy will be combined with anti-Clever-1 therapy, etc. In a similar manner, when anti-Clever-1 therapy is initiated, despite significant downregulation of exhaustion markers selected from PD-1, PD-L1, CTLA-4, OX40, 41BB, LAG3, TIM3, and CD28, if the exhaustion marker is upregulated during anti-Clever-1 therapy, a given ICI treatment will be combined with anti-Clever-1 therapy. Thus, the findings of the present invention can also be used to monitor a patient's response to anti-Clever-1 therapy during that treatment. For example, despite an initial favorable response to single-agent anti-Clever-1 therapy, as well as increased serum IFNg levels, T cell activation, and anti-tumor response, the IFNg response may subsequently increase PD-L1 expression, and it would then be best to combine anti-PD-1 and / or anti-PD-L1 therapy with anti-Clever-1 therapy. A method according to one embodiment of the present invention for monitoring a patient's response to anti-Clever-1 therapy includes measuring and monitoring IFNg levels during anti-Clever-1 therapy.

[0014] In a similar manner, anti-Clever-1 therapy has surprisingly been shown to upregulate CD25 (IL-2RA), CXCR3 and CD69 expression at the start of anti-Clever-1 therapy.If this increase is not observed when anti-Clever-1 therapy is started, anti-Clever-1 therapy should be combined with CD25 (IL-2-RA) or CXCR3 induction therapy.In addition, agonists that stimulate CD25 (IL-2-RA) or CXCR3 can be used together with anti-Clever-1 therapy to further enhance the proliferation, activation and / or migration of cells with the induced expression of these molecules.

[0015] The embodiments and advantages mentioned in this document, where applicable, but not always specifically mentioned, relate to both the drug combinations and uses according to the present invention as well as the methods. [Brief explanation of the drawings]

[0016] [Figure 1] Heatmap of cell surface marker (i.e., checkpoint) changes in seven cancer patients treated with anti-Clever-1 antibody (FP-1305) therapy. The first sample was collected immediately before FP-1305 administration, and the second sample was collected 8 days after FP-1305 infusion. Color changes indicate the mean logarithmic change (up- or down-regulation) in the detection level of the cell surface marker from these seven patients, and asterisks indicate the statistical significance of the change between pre- and post-dose from these seven patients (*p<0.05, **p<0.01, ***p<0.001). [Figure 2] Changes in serum IFNg levels during anti-Clever-1 antibody (FP-1305) treatment. Increases in serum IFNg during FP-1305 treatment are associated with anti-tumor responses in metastatic colorectal cancer that has previously failed to respond to any available therapy. DETAILED DESCRIPTION OF THE INVENTION

[0017] CLEVER-1 is a protein disclosed in International Publication No. 03 / 057130, "Common Lymphatic Endothelial and Vascular Endothelial Receptor-1." CLEVER-1 is a binding protein that mediates the adhesion of lymphocytes (and malignant tumor cells) to endothelium in both the systemic vascular and lymphatic systems. Blocking the interaction of Clever-1 with its lymphocyte substrate can simultaneously regulate lymphocyte recirculation and migration at sites of lymphocyte inflow and outflow into tissues, as well as related conditions such as inflammation.

[0018] The terms "agent capable of binding to Clever-1," "Clever-1 inhibitor," and "anti-Clever-1 agent" are interchangeable and refer to agents including antibodies and fragments thereof, peptides, etc., capable of binding to Clever-1 to block the interaction of Clever-1 with malignant tumor cells. The agent may also be any other inhibitor, such as a small molecule inhibitor or a macromolecule with suitable affinity to bind to the Clever-1 receptor and inhibit protein activity. The term "antibody or fragment thereof" is used in the broadest sense to encompass antibodies or fragments thereof capable of binding to Clever-1 molecules in individuals. In particular, it should be 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. Therefore, according to one embodiment of the present invention, the agent capable of binding to Clever-1 (i.e., Clever-1 inhibitor or anti-Clever-1 agent) is selected from the group consisting of antibodies or fragments thereof, peptide(s), macromolecules, and any combination thereof. According to the present invention, "anti-Clever-1 therapy" or "anti-Clever-1 therapy" refers to a therapy comprising the administration of at least one agent capable of binding to Clever-1. Anti-Clever-1 monotherapy, in the present disclosure, refers to a therapy comprising an anti-Clever-1 agent(s) as a single agent.

[0019] According to an embodiment of the invention, the anti-Clever-1 antibody is a therapeutic humanized anti-Clever-1 antibody. According to an embodiment of the invention, the anti-Clever-1 antibody is a humanized monoclonal Clever-1 antibody previously shown in WO 2017 / 182705.

[0020] In one embodiment of the present invention, the anti-Clever-1 antibody is the humanized monoclonal immunoglobulin G4κ antibody bexmarilimab (International Nonproprietary Name (INN) as disclosed in WHO Drug Information, Vol. 33, No. 4, pages 814-815 (2019)), or a bexmarilimab variant or antibody in a bexmarilimab biosimilar. As used herein, "bexmarilimab" refers to an IgG4 monoclonal antibody having the structure described in WHO Drug Information, Vol. 33, No. 4, pages 814-815 (2019).

[0021] A bexmalilimab biosimilar refers to a biological product approved by a regulatory authority in any country for marketing as a bexmalilimab biosimilar. In one embodiment, a bexmalilimab biosimilar contains a bexmalilimab variant as the drug substance. In one embodiment, a bexmalilimab biosimilar has heavy and light chains with substantially the same amino acid sequences as bexmalilimab. As used herein, a "bexmalilimab variant" refers to an antibody that contains the same heavy and light chain sequences as bexmalilimab, except that it has 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 are located in the framework or constant regions. In other words, bexmalilimab and bexmalilimab variants contain the same CDR sequences but differ from each other due to conservative amino acid substitutions at other positions in the full-length light and heavy chain sequences. The bexmarilimab variants are substantially identical to bexmarilimab in terms of binding affinity to CLEVER-1.

[0022] According to one embodiment of the present invention, a cell line producing the therapeutic anti-Clever-1 antibody bexmarilimab (FP-1305) has been deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, on May 27, 2020, and has accession number DSM ACC3361. The deposited embodiment is intended as a single illustration of one aspect of the invention, and the present invention should not be limited in scope by the deposited culture, as any culture that is functionally equivalent is within the scope of the invention. The deposit of material herein is not an admission that the written description contained herein is insufficient to enable the practice of any aspect of the invention, including its best mode, nor should it be construed as limiting the scope of the claims to the particular examples it represents.

[0023] It has been observed that anti-Clever-1 therapy has the ability to reduce the expression of cell surface markers PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, and CD28 on leukocytes, particularly in circulating T cell populations, and to increase the expression of cell surface markers CD25 (IL-2RA), CXCR3, and CD69. However, its effect may vary depending on the patient and / or cancer type. For example, the cell surface marker ICOS may be up-regulated or down-regulated in association with anti-Clever-1 therapy. Therefore, in order to provide the most effective treatment with anti-Clever-1 therapy, the expression of one or more cell surface markers is monitored when anti-Clever-1 therapy is initiated and / or during anti-Clever-1 therapy to provide the necessary information for starting treatment with the best combination drug(s) together with anti-Clever-1 therapy after the observed changes in the expression of one or more cell surface markers.

[0024] The present invention provides a method for monitoring a cancer patient's response to anti-Clever-1 therapy and assessing the need for combination therapy when an agent capable of binding to Clever-1 is administered to the patient. According to an embodiment of the present invention, one or more cell surface markers are monitored when anti-Clever-1 therapy is initiated. According to another embodiment of the present invention, one, two, three, or more cell surface markers are monitored during anti-Clever-1 therapy. The cell surface markers can be monitored as a single marker or as a panel of markers.

[0025] 1. A method according to one embodiment of the present invention for monitoring a patient's response to anti-Clever-1 therapy and assessing the need for combination therapy when an agent capable of binding to Clever-1 is administered to the patient, the method comprising: - Obtaining a sample from a patient at an initial time point prior to administering to the patient an agent capable of binding to Clever-1; - Obtaining a sample from the patient at a later time point after administering to the patient an agent capable of binding to Clever-1; - measuring the expression of one, two, three or more cell surface markers selected from PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 and CXCR3 from the obtained sample; - comparing the expression level of the cell surface marker measured from a sample obtained at a subsequent time point with the expression level of the cell surface marker measured from a sample obtained at the initial time point, wherein the absence of a desired change in the expression level of the cell surface marker is an indication to initiate co-administration of an agent that affects the cell surface marker.

[0026] According to one embodiment of the present invention, a sample obtained at an initial time point before administering an agent capable of binding to Clever-1 to a patient refers to a sample obtained before the first administration of an agent capable of binding to Clever-1 (i.e., the initial sample). A sample obtained at a later time point after administering an agent capable of binding to Clever-1 to a patient refers to a sample obtained at any time point during therapy, i.e., after the first administration of an agent capable of binding to Clever-1 to a patient and before the last administration of an agent capable of binding to Clever-1 to a patient.

[0027] According to an embodiment of the present invention, a method comprises: Obtaining a sample from a patient at an initial time point before starting anti-Clever-1 therapy; - obtaining a sample from the patient at a later time during said therapy, i.e. after the first administration of an agent capable of binding to Clever-1 to the patient and before the last administration of an agent capable of binding to Clever-1 to the patient; - comparing the expression level of the cell surface marker measured from the sample obtained at the subsequent time point with the expression level of the cell surface marker measured from the sample obtained at the initial time point, - the absence of downregulation of PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3 and / or CD28 is an indication to initiate co-administration of an agent that affects said cell surface marker; and / or - the absence of upregulation of ICOS, CD25, and / or CXCR3 is an indication to initiate co-administration of an agent that affects said cell surface markers; and / or - An increase in CD25 and / or CXCR3 expression is an indication to initiate co-administration of agents that affect said cell surface markers.

[0028] According to another embodiment of the present invention, the method comprises: - Obtaining a sample from the patient at a first time point, which is any time point during anti-Clever-1 therapy prior to further administration of an agent capable of binding to Clever-1 to the patient; - obtaining a sample from the patient at a later time point during anti-Clever-1 therapy after administering to the patient an agent capable of binding to Clever-1; - comparing the expression level of the cell surface marker measured from the sample obtained at the subsequent time point with the expression level of the cell surface marker measured from the sample obtained at the initial time point, - upregulation of PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, and / or CD28 relative to the expression levels of the cell surface markers measured in a sample obtained at the initial time point is an indication to initiate co-administration of an agent that affects the cell surface marker; and / or - Downregulation of ICOS, CD25, and / or CXCR3, relative to the expression levels of said cell surface markers measured from a sample obtained at an initial time point, is an indication to initiate co-administration of an agent that affects said cell surface markers; and / or - An increase in CD25 and / or CXCR3 is an indication to start the co-administration of drugs that affect said cell surface markers.

[0029] In one embodiment of the invention, the expression of one, two, three, or more cell surface markers selected from CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25, and CXCR3 is measured from the obtained sample, and the expression level of the cell surface marker from the sample obtained at a later time point is compared to the expression level of the cell surface marker measured from the sample obtained at the initial time point to assess the initiation of co-administration of an agent that affects the cell surface marker. In one embodiment of the invention, in addition to the cell surface markers mentioned above, the expression of PD-1 and / or PD-L1 can be analyzed to assess the initiation of co-administration of PD-1 / PD-1 inhibitor(s).

[0030] According to one embodiment of the invention, the method further comprises measuring interferon gamma (IFNg) from the obtained sample and comparing the IFNg level measured from the sample at the subsequent time point with the IFNg level measured from the sample obtained at the initial time point, wherein an increase in IFNg level is an indication to initiate co-administration of a PD-1 and / or PD-L1 inhibitor.

[0031] According to one embodiment of the invention, the sample is a blood sample drawn from a patient. In a typical embodiment of the invention, the expression of one or more cell surface markers is analyzed from leukocytes, particularly from the T cell population, obtained from the blood sample drawn from the patient.

[0032] According to the present invention, the expression level of a cell surface marker may be measured by any suitable method known in the art.

[0033] According to one embodiment of the present invention, a therapeutically effective amount of a) an agent capable of binding to Clever-1; and b) with one or more agents selected from the group comprising CTLA-4 inhibitors, ICOS inhibitors, ICOS inducers, OX40 inhibitors, 41BB inhibitors, LAG3 inhibitors, TIM3 inhibitors, CD28 inhibitors, cytokines IL-2 including modified versions thereof, CD25 (IL-2RA) agonists, CXCR3 inducers, and CXCR3 agonists, A combination for use in treating cancer in an individual who has been treated with anti-Clever-1 monotherapy, and the monotherapy does not show downregulation of the cell surface markers CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, and / or CD28, and / or upregulation of the cell surface markers ICOS, CD25, and / or CXCR3. The combination of the present invention for use in treating cancer in an individual may comprise, in addition to an agent capable of binding to Clever-1, one, two, three, four, or more agents selected from the group including a CTLA-4 inhibitor, an ICOS inhibitor, an ICOS inducer, an OX40 inhibitor, a 41BB inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a CD28 inhibitor, the cytokine IL-2 including modified versions thereof, a CD25 (IL-2RA) agonist, a CXCR3 inducer, and a CXCR3 agonist.

[0034] It is noted that, according to the present invention, combination treatment with anti-Clever-1 therapy can be selected based on the measured expression of cell surface marker(s). The absence of the desired change in expression level with anti-Clever-1 therapy alone as a single agent is an indication to start administering an agent that affects said cell surface marker.

[0035] According to one embodiment of the present invention, other cell surface markers can also be defined to monitor treatment response and assess the need to initiate combination therapy(ies), for example, to induce CD69, CD95, CD45RO, and / or HLA DR expression.

[0036] According to one embodiment of the present invention, the combination for use in treating cancer in an individual may further comprise a PD-1 and / or PD-L1 inhibitor when anti-Clever-1 monotherapy does not show downregulation of PD-1 and / or PD-L1. The combination according to the present invention for use in treating cancer in an individual may comprise, in addition to an agent capable of binding to Clever-1 and PD-1 and / or PD-L1, one, two, three, four or more agents selected from the group comprising a CTLA-4 inhibitor, an ICOS inhibitor or inducer, an OX40 inhibitor, a 41BB inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a CD28 inhibitor, the cytokine IL-2 including modified versions thereof, or a CD25 (IL-2RA) agonist and a CXCR3 inducer, or a CXCR3 agonist.

[0037] According to one embodiment of the present invention, the combination for use in treating cancer in an individual further comprises a PD-1 and / or PD-L1 inhibitor if upregulation of interferon gamma (IFNg) and / or upregulation of PD-1 and / or PD-L1 is observed during anti-Clever-1 therapy in combination with one or more of the agents that affect cell surface marker(s). It has been observed that an anti-tumor response with anti-Clever-1 therapy is associated with an increase in circulating interferon gamma (IFNg), which subsequently leads to an increase in PD-1 and / or PD-L1 expression levels. Therefore, if an increase in IFNg expression levels and / or an increase in PD-1 and / or PD-L1 expression levels is observed during anti-Clever-1 therapy alone or in combination with one or more of the agents that affect cell surface markers, this is an indication to start anti-PD-1 and / or anti-PD-L1 therapy together with anti-Clever-1 therapy.

[0038] In one embodiment, a combination for use in treating cancer in an individual may include a combination of a PD-1 and / or PD-L1 inhibitor with an agent capable of binding to Clever-1, where anti-Clever-1 monotherapy does not show downregulation of PD-1 and / or PD-L1 on leukocytes and / or upregulation of IFNg and / or upregulation of PD-1 and / or PD-L1 is observed during anti-Clever-1 therapy.

[0039] According to the present invention, CTLA-4 inhibitors, ICOS inhibitors, ICOS inducers, OX40 inhibitors, 41BB inhibitors, LAG3 inhibitors, TIM3 inhibitors, CD28 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors include antibodies or fragments thereof capable of blocking said cell surface receptors. Combination treatments according to the present invention can include any known agent(s) capable of blocking said cell surface receptors.

[0040] According to the present invention, the cytokine IL-2, including its modified forms, CD25 (IL-2RA) agonists, CXCR3 inducers, and CXCR3 agonists include agents capable of activating the cell surface receptor. IL-2 is a pro-inflammatory cytokine known to stimulate the immune system (mainly T cells) to kill tumor cells. However, IL-2 therapy is rather toxic, and many developmental technologies are being explored to induce IL-2 in the tumor microenvironment without toxic side effects. Such technologies include IL-2 receptor agonists, cancer vaccines that introduce IL-2-producing viruses, and the like. The combination therapy of the present invention can include any known agent(s) capable of activating the cell surface receptor.

[0041] According to one embodiment of the present invention, the combination for use in treating cancer in an individual comprises a therapeutically effective amount of a) an agent capable of binding to Clever-1, such as an anti-Clever-1 antibody or a fragment thereof; b) The following drugs: - CTLA-4 inhibitors, such as anti-CTLA-4 antibodies or fragments thereof, which specifically bind to the CTLA-4 receptor and inhibit CTLA-4 activity, if anti-Clever-1 single agent activity does not lead to a reduction in CTLA-4 expression; - if anti-Clever-1 single agent activity does not lead to changes in ICOS expression, ICOS inhibitors and / or ICOS inducers, e.g., anti-ICOS antibodies or fragments thereof that specifically bind to the ICOS receptor; - if anti-Clever-1 single agent activity does not lead to a decrease in OX40 expression, an OX40 inhibitor, such as an anti-OX40 antibody or a fragment thereof that specifically binds to the OX40 receptor and inhibits OX40 activity; - If anti-Clever-1 single agent activity does not lead to a decrease in 41BB expression, a 41BB inhibitor, such as an anti-41BB antibody or a fragment thereof that specifically binds to the 41BB receptor and inhibits 41BB activity; - if anti-Clever-1 single agent activity does not lead to a decrease in LAG3 expression, a LAG3 inhibitor, such as an anti-LAG3 antibody or fragment thereof that specifically binds to the LAG3 receptor and inhibits LAG3 activity; - if anti-Clever-1 single agent activity does not lead to a decrease in TIM3 expression, a TIM3 inhibitor, such as an anti-TIM3 antibody or a fragment thereof that specifically binds to the TIM3 receptor and inhibits TIM3 activity; - if anti-Clever-1 single agent activity does not lead to a decrease in CD28 expression, a CD28 inhibitor, such as an anti-CD28 antibody or a fragment thereof that specifically binds to the CD28 receptor and inhibits CD28 activity; - when the molecule is used to stimulate the immune system in combination with the cytokine IL-2, anti-Clever-1, including modified versions thereof, - CD25 agonists, if anti-Clever-1 single agent activity does not lead to increased CD25 expression; - CXCR3 inducers, if anti-Clever-1 single agent activity does not lead to increased CXCR3 expression; - when the molecule is used to stimulate the immune system in combination with a CXCR3 agonist, anti-Clever-1, and one or more of:

[0042] According to one embodiment of the present invention, the combination for use in treating cancer in an individual further comprises a therapeutically effective amount of a PD-1 and / or PD-L1 inhibitor, e.g., an anti-PD-1 and / or anti-PD-L1 antibody or fragment thereof that specifically binds to the programmed cell death protein 1 (PD-1) receptor and inhibits PD-1 activity, when the anti-Clever-1 single agent activity does not lead to a decrease in PD-1 / PD-L1 expression and / or the IFNg response leads to an increase in the level of PD-1 / PD-L1 expression.

[0043] According to one embodiment of the present invention, a method for treating a cancer patient comprises administering to the cancer patient: a) an agent capable of binding to Clever-1; and b) administering a combination of one or more agents selected from the group including a CTLA-4 inhibitor, an ICOS inhibitor, an ICOS inducer, an OX40 inhibitor, a 41BB inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a CD28 inhibitor, the cytokine IL-2 including modified versions thereof, a CD25 (IL-2RA) agonist, a CXCR3 inducer, and a CXCR3 agonist.

[0044] According to one embodiment of the present invention, a method for treating a cancer patient comprises administering to the cancer patient: a) an agent capable of binding to Clever-1; and b) one or more agents selected from the group comprising CTLA-4 inhibitors, ICOS inhibitors, ICOS inducers, OX40 inhibitors, 41BB inhibitors, LAG3 inhibitors, TIM3 inhibitors, CD28 inhibitors, cytokines IL-2 including modified versions thereof, CD25 (IL-2RA) agonists, CXCR3 inducers, and CXCR3 agonists; c) administering a combination of a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0045] In one embodiment, the method for treating a cancer patient comprises administering to the cancer patient: a) an agent capable of binding to Clever-1; and b) in combination with a PD-1 inhibitor and / or a PD-L1 inhibitor, This includes administering when anti-Clever-1 monotherapy does not show downregulation of PD-1 and / or PD-L1 on leukocytes and / or upregulation of IFNg and / or PD-1 and / or PD-L1 is observed during anti-Clever-1 therapy.

[0046] The term "treatment" or "treating" should be understood to include complete cure of a disease or disorder, as well as amelioration or alleviation of the disease or disorder. The term "therapeutically effective amount" is meant to include any amount of an agent of the present invention sufficient to produce the desired therapeutic result.

[0047] The present invention provides methods and compositions for treating cancer by reducing malignant tumor growth and / or inhibiting metastasis formation, which are applicable to all forms of cancer, and thus can treat any malignant tumor or metastasis.

[0048] "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 agents used in the present invention can be administered by any means that achieve their intended purpose. For example, administration may be by injection, intravenous, intramuscular, intraperitoneal, intratumoral, subcutaneous, or other parenteral routes. In addition to the pharmacologically active compound, the pharmaceutical formulation of the agent preferably contains a suitable pharmaceutically acceptable carrier, including excipients and adjuvants that facilitate processing of the active agent into a pharmaceutically usable formulation. The selected dose should be sufficient to reduce or inhibit malignant tumor growth and / or inhibit metastasis formation.

[0049] A method according to one embodiment of the present invention for treating a cancer patient includes: - measuring the expression of one or more cell surface markers selected from PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 and CXCR3, and / or IFNg levels from a sample obtained from the patient; administering to the patient an agent capable of binding to Clever-1; - measuring the expression of one or more cell surface markers selected from PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 and CXCR3, and / or IFNg levels from a sample obtained from the patient after administering to the patient an agent capable of binding to Clever-1; - comparing the expression levels of said cell surface markers and / or IFNg levels measured from a sample obtained at a later time point with the expression levels of said cell surface markers and / or IFNg levels measured from a sample obtained at an earlier time point, wherein the absence of a desired change in the expression levels of the cell surface markers and / or an increase in IFNg levels is an indication to initiate co-administration of an agent that affects said cell surface marker; - administering one or more agents that affect said cell surface markers, where no desired change in expression levels is observed. [Example]

[0050] The following examples are merely illustrative of the principles of the present invention and are not intended to limit the scope of the invention.

[0051] The anti-Clever-1 antibody FP-1305 (DSM ACC3361) used in the following examples is a humanized antibody currently being developed by Fallon Pharmaceuticals for the treatment of cancer, and is disclosed in more detail in WO 2017 / 182705, where it is known as bexmarilimab.

[0052] Example 1. Cell surface / exhaustion markers are altered in leukocyte populations in cancer patients initiating anti-Clever-1 (FP-1305) treatment The Clever-1 inhibitor, anti-Clever-1 antibody FP-1305, 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)). The first (pre-dose) sample was collected before initiating FP-1305. The second (post-dose) sample was collected 8 days after initiating FP-1305 treatment. Cell surface markers were analyzed from the samples as described below.

[0053] Protocol for Mass Cytometry (CyTOF) Staining and Data Analysis PBMCs were isolated from the samples using Ficoll-Paque™ density gradient centrifugation and frozen in RPMI 1640 (Sigma-Aldrich, RPMI 1640 supplemented with 10% FCS, 2 mmol / L L-glutamine) medium with 10% DMSO.

[0054] Before CyTOF staining, frozen PBMCs were thawed, resuspended in PBS, and counted. For staining, 1–3 × 10 6 Cells were harvested. First, cells were stained with 2.5 μM Cell-ID cisplatin (Fluidigm, catalog no. 201064) viability reagent for 5 minutes at room temperature (RT). After washing, cells were barcoded with heavy metal isotope-labeled anti-human CD45 (clone H130) antibodies (CD45_89Y, CD45_141Pr, and CD45_147Sm, 1 / 200) for 30 minutes at RT, carefully washed, and the different barcoded samples were combined. Next, samples were blocked with human Kiovig solution (0.2 mg / ml) for 15 minutes at RT and stained with a heavy metal isotope-labeled anti-human antibody cocktail containing cell surface markers (Table 1) for 30 minutes at RT, followed by washing.

[0055] [Table 1]

[0056] The stained samples were incubated with DNA intercalation reagent (1 / 1000, Cell ID Intercalator-103Rh, Fluidigm, in MaxPar® Fix and Perm Buffer, catalog number 201067) for 1 hour at room temperature, washed, and fixed overnight (o / n) with 4% PFA solution at +4°C. The following day, samples were washed, resuspended in MaxPar Water (catalog number 201069, Fluidigm) containing a 1 / 10 dilution of EQ 4 Element Beads (Fludigm), and immediately acquired on a CyTOF mass cytometer (Helios, Fluidigm). After bead normalization of the samples, viable singlet cells were debarcoded by using FlowJo. CD45+CD3+ cells were gated and exported for further analysis.

[0057] Data analysis was performed similarly to Kimball et al. (2019) J Immunol (A Beginner's Guide to Analyzing and Visualizing Mass Cytometry Data). R studio version 1.2.1335 was downloaded from the official R website, and the Cytokit package was downloaded from Bioconductor and opened in R. Manually gated events (gated as described above) were imported into Cytokit and subjected to Phenograph analysis. Clustering was performed using 9 of the 23 markers (CD4, CD8, CD45RA, CCR7, CD45RO, CD127, CD25, CCR6, and CXCR3) with the following additional settings: merge; minimal; transform; CytofAsinh; cluster; Rphenograph; visualization; tSNE with cell progression null.

[0058] Twenty-two clusters were defined by Phenograph, and these clusters were displayed on the tSNE blot using the R package "shiny" to visualize different patients before and after treatment. Cluster colors, identification numbers, dot and label sizes were customized in the "shiny" application. Phenograph analysis generated several CSV files, which were used to calculate the average expression value per sample for each marker and the change between pre- and post-treatment samples per patient. Heatmaps were generated using the ComplexHeatmap package downloaded from Bioconductor (Gu, Z. (2016) Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics.). Statistical analysis was performed using R version 3.6.1 (R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org / ) using t-tests.

[0059] The results are shown in Figure 1: Differences in marker expression between pre- and post- (day 8) samples in each cluster.

[0060] Example 2. Increased serum IFNg is associated with anti-tumor responses in cancer patients treated with anti-Clever-1 (FP-1305) The anti-Clever-1 antibody FP-1305 has begun clinical development in the setting described above. In this first-in-human trial (clinicaltrials.gov NCT03733990), patients with metastatic colorectal cancer who had not responded to any available therapy demonstrated antitumor responses. These responses, so far, have all been associated with increased serum IFNg levels during treatment (Figure 2). IFNg serum levels were measured using a multiplex cytokine panel at the start of every treatment cycle. Treatment cycles (i.e., the interval between every FP-1305 infusion) were 3 weeks apart. It is now known that elevated IFNg levels can lead to elevated PD-L1 levels and resistance to T cell-targeted tumor killing. If patients receiving anti-Clever-1 therapy subsequently develop resistance due to increased IFNg and subsequent PD-L1 levels, these patients will require anti-PD-1 / L1 therapy in addition to anti-Clever-1 therapy.

Claims

1. 1. A method for monitoring the response of a cancer patient to anti-Clever-1 therapy and assessing the need for combination therapy when an agent capable of binding to Clever-1 is administered to said cancer patient, said method comprising: - providing a sample obtained from a patient at a first time point before administering to said patient an agent capable of binding to Clever-1; - providing a sample obtained from said patient at a later time point after administering to said patient an agent capable of binding to Clever-1; - measuring the expression of two, three or more cell surface markers selected from PD-1 or PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 and CXCR3 from the obtained sample; - comparing the expression level of said cell surface marker measured from said sample obtained at a subsequent time point with the expression level of said cell surface marker measured from said sample obtained at an initial time point, wherein the absence of a desired change in the expression level of said cell surface marker is an indication to initiate co-administration of an agent that affects said cell surface marker; the cell surface marker is measured on circulating T cells; and the agent capable of binding to Clever-1 comprises an anti-Clever-1 antibody or a fragment thereof; The method, wherein the agent that affects the cell surface marker is selected from a CTLA-4 inhibitor, an ICOS inhibitor, an ICOS inducer, an OX40 inhibitor, a 41BB inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a CD28 inhibitor, a CD25 (IL-2RA) agonist, a CXCR3 inducer, a CXCR3 agonist, and a PD-1 inhibitor and / or a PD-L1 inhibitor.

2. The method according to claim 1, wherein the anti-Clever-1 antibody is bexmarilimab.

3. The method according to claim 1 or 2, wherein the anti-Clever-1 antibody is anti-Clever-1 antibody FP-1305 (DSM ACC3361).

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