Patent Application
20220404366
The present invention relates to monitoring expression levels of one or more cell surface marker in circulating T cell populations in relation to anti-Clever-1 treatment and choosing the best combination agent to initiate treatment together with anti-Clever-1 therapy after the observed changes in the expression of one or more cell surface marker.
The vast number of genetic and epigenetic changes that are inherent to cancer cells provide plenty of tumor-associated antigens that the host immune system can recognize, thereby requiring tumors to develop specific immune resistance mechanisms. An important immune resistance mechanism involves immune-inhibitory pathways, termed immune checkpoints, which normally mediate immune tolerance and mitigate collateral tissue damage. A particularly important immune-checkpoint receptor is cytotoxic T-lymphocyte associated antigen 4 (CTLA-4), which downmodulates the amplitude 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 functions within tissues. By upregulating ligands for PD-1, tumor cells block antitumor immune responses in the tumor microenvironment. [1] Anti-PD-1, anti-PD-L1 and anti-CTLA-4 immune-checkpoint inhibitors are extensively used in clinical patient care. The second generation immune-checkpoint receptors including but not limited to ICOS (Inducible T-cell COStimulator), OX40, 41BB, TIM3 and LAG3, and antagonist antibodies against these checkpoint inhibitors (ICIs) are under clinical development as anti-cancer agents.
Currently, immune checkpoint modulators targeting CTLA-4 and the PD-1/PD-L1 axis are approved for clinical use, and while highly efficacious in about 10-20% of patients with melanoma and certain other tumors, several other important cancer types (such as prostate, breast and colorectal cancer) remain refractory to them, and there is no clear biomarker available that could differentiate responders from non-responders and guide treatment [2]. Patients responding favorably to checkpoint inhibition usually have a preexisting antitumor immune response, which is characterized by high density of interferon gamma (IFNg) producing CD8+ T cells, expression of PD-L1 in tumor-infiltrating immune cells, and high mutational load. Tumors that do not respond to immune checkpoint blockage show either a stromal T cell phenotype where infiltration of T cells (TIL) into tumors or activation of T cells in the tumor microenvironment (TME) is prevented by immunosuppressive stromal compartments, or a non-inflamed phenotype characterized by low T cell infiltration, low mutational load and high proliferation of tumor cells. The tumors can be classified immunologically (inflamed vs. non-inflamed) based on the presence of tumor infiltrating cytotoxic CD8 T cells [3]. The inflamed tumors show high mutational load, high IFNg and PD-L1 expression, and respond favorably to immune checkpoint blocking therapies. IFNg produced by T cells is considered necessary for ICIs to work as anti-cancer agents. IFNg secretion however, is known to increase PD-L1 expression on cells, which can be a source of immunotherapy resistance [3].
Innate immune cells such as macrophages, however, can dampen T cell activation and contribute to tumor progression despite high mutational load. The macrophages that contribute to tumor-related immunosuppression and provide tumor growth supporting signals may be highly eligible candidates for targeted therapies, since these cells are abundantly present in various tumors, they are very plastic and can be converted into pro-inflammatory macrophages supporting T cell activation and tumor rejection [4, 5]. To date, macrophage targeted strategies under clinical development utilize macrophage colony-stimulating factor receptor inhibition to deplete macrophage populations in tumors [6]. However, resistances to these approaches have already been reported [7]. Thus, there is a need to find novel ways to utilize these cells to induce tumor cell killing by the immune system.
In recent years, increasing attention has been paid to the contribution of scavenger receptors in regulating macrophage responses to different stimuli. Clever-1 (also known as Stabilin-1) is a multifunctional molecule conferring scavenging ability on a subset of anti-inflammatory macrophages [8, 9]. In these cells, it is involved in receptor-mediated endocytosis and recycling, intracellular sorting, and transcytosis of altered and normal self-components. More recently, it has been found that the growth and metastases are attenuated in Stab1−/− (Clever-1 knock out) mice in several tumor models, and in mice treated with anti-Clever-1 therapy [10, 11]. In addition, combination treatments with an anti-Clever-1 agent together with an anti-PD-1 agent has shown to produce anti-tumor responses in mouse models of triple negative breast cancer and colorectal cancer [11].
Now, it has been surprisingly found out that anti-Clever-1 treatment in cancer patients decreases PD-1, PD-L1, CTLA-4, OX40, 41BB, LAG3, TIM3 and CD28 expression on leucocytes, especially in circulating T cell populations, and increases the expression of CD25 (IL-2RA), CXCR3 and CD69, and also affecting the expression level of ICOS. In addition, anti-tumor responses with anti-Clever-1 treatment was found to associate with an increase in circulating interferon gamma (IFNg). In line with current knowledge, this means that anti-Clever-1 treatment could later build resistance as the IFNg response could lead to an increase in PD-1 and/or PD-L1 expression. Especially, it has been found that anti-Clever-1 treatment removes 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 the change in these commonly known checkpoint molecules during anti-Clever-1 treatment, we can pick a checkpoint inhibitor that should be given in combination with the on-going anti-Clever-1 treatment. If a certain checkpoint molecule does not react to anti-Clever-1 treatment in the wanted way, then a specific checkpoint inhibitor targeting the wanted checkpoint molecule is administered in combination with to the on-going anti-Clever-1 treatment. The expression of cell surface markers can be used to guide anti-Clever-1 treatment or the best possible checkpoint inhibitor(s) combination treatment with anti-Clever-1 therapy. More detailed, it has been found that 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 patient's response to anti-Clever-1 therapy and to evaluate the need for combination therapy with anti-Clever-1 therapy. The present finding provides a method for choosing the best combination agent(s) to initiate treatment together with anti-Clever-1 therapy after the observed changes in the expression of one, two, three or more cell surface marker.
Hence, the present invention provides a method for monitoring a patient's response to anti-Clever-1 monotherapy and estimating the need for combination therapy based on the expression levels of one, two, three or more cell surface marker selected from PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25, CXCR3 and CD69 on leucocytes, especially in circulating T cell populations in relation to anti-Clever-1 treatment and choosing the best combination agent(s) to initiate treatment together with anti-Clever-1 therapy after the observed changes in the expression of one or more cell surface marker.
A typical method for monitoring a patient's response to anti-Clever-1 therapy and estimating the need for combination therapy, when an agent capable of binding to Clever-1 has been administered in a patient, the method comprising
The present invention also provides a novel biomarker-based combination treatment for cancer patients and thus reduce or even eliminate the abovementioned problems in defining non-responders from responding patients. The present invention gives the possibility of probing the molecular landscape of solid tumors via a blood draw to define changes in the patient during anti-Clever-1 therapy and subsequently gives the opportunity to select the best possible check-point inhibition combination treatment with anti-Clever-1 therapy.
Therefore, the findings of the present invention also provide a combination of therapeutically effective amounts of:
The present invention is based on the findings that anti-Clever-1 treatment alternates the expression of several cell surface receptors on leucocytes and that the expression levels of these checkpoints may vary during the treatment course of anti-Clever-1 therapy. This enables the best possible biological rationale to choose which checkpoint inhibitor (ICI) treatment should be combined with anti-Clever-1 treatment and when to initiate that combination treatment during anti-Clever-1 therapy.
The present invention provides means to choose when to start anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-ICOS, anti-OX40, anti-41BB, anti-LAG3, anti-TIM3 and/or anti-CD28 treatment in combination with anti-Clever-1 treatment.
Anti-Clever-1 treatment has surprisingly been shown to downregulate exhaustion markers PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3 and CD28 in the beginning of the anti-Clever-1 treatment. Thus, for example, if anti-Clever-1 treatment would not downregulate the expression of CTLA-4, one would combine anti-CTLA-4 treatment with anti-Clever-1 treatment. Or if anti-Clever-1 treatment would not downregulate OX40, one would combine anti-OX40 treatment with anti-Clever-1 treatment etc. In similar fashion, despite a significant downregulation of exhaustion markers selected from PD-1, PD-L1, CTLA-4, OX40, 41BB, LAG3, TIM3 and CD28, when beginning anti-Clever-1 treatment, if said exhaustion markers would be upregulated during anti-Clever therapy, then one would combine the given ICI treatment with anti-Clever-1 treatment. Hence, the findings of the present invention can also be used to monitor patient's response to anti-Clever-1 treatment during the treatment. For example, despite an initially favorable response to single agent anti-Clever-1 treatment and an increase in serum IFNg levels, T cell activation and an anti-tumor response, the IFNg response could later increase PD-L1 expression, and then it would be best to combine anti-PD-1 and/or anti-PD-L1 treatment with anti-Clever-1 treatment. A method according to an embodiment of the present invention for monitoring patient's response to anti-Clever-1 therapy comprises measuring and monitoring IFNg levels during anti-Clever-1 treatment.
In a similar fashion, anti-Clever-1 treatment was surprisingly shown to upregulate CD25 (IL-2RA), CXCR3 and CD69 expression in the beginning of the anti-Clever-1 treatment. If this increase would not be seen when initiating anti-Clever-1 treatment, then anti-Clever-1 treatment should be combined with a CD25 (IL-2-RA) or a CXCR3 inducing therapy. In addition, agonists that stimulate CD25 (IL-2-RA) or CXCR3 could be used with anti-Clever-1 treatment to further enhance the proliferation, activation and/or migration of the cells with induced expression of the said molecules.
The embodiments and advantages mentioned in this text relate, where applicable, both to the combination of the said agents, the method as well as to the uses according to the invention, even though it is not always specifically mentioned.
CLEVER-1 is a protein disclosed in the patent publication WO 03/057130, Common Lymphatic Endothelial and Vascular Endothelial Receptor-1. It is a binding protein that mediates adhesion of lymphocytes (and malignant tumor cells) to endothelium in both the systemic vasculature and in the lymphatics. By blocking the interaction of Clever-1 and its lymphocyte substrate, it is possible to simultaneously control lymphocyte recirculation and lymphocyte migration, and related conditions such as inflammation, at the site of lymphocyte influx into, and efflux from, the tissues.
The terms “an agent capable of binding to Clever-1”, “Clever-1 inhibitor” and “anti-clever-1 agent” are interchangeable and refers to agents including antibodies and fragments thereof, peptides or the like, which are capable of binding to Clever-1 for blocking the interaction of Clever-1 and malignant tumor cells. The agent may also be any other inhibitor, such as small molecule inhibitor or macromolecule having an adequate affinity to bind to Clever-1 receptor and to inhibit the protein activity. The term “an antibody or a fragment thereof” is used in the broadest sense to cover an antibody or a fragment thereof which are capable to bind Clever-1 molecule in an individual. Especially, it shall be understood to include chimeric, humanized or primatized antibodies, as well as antibody fragments and single chain antibodies (e.g. Fab, Fv), so long they exhibit the desired biological activities. Particular useful agents are anti-Clever-1 antibodies and fragments thereof. Therefore, according to an embodiment of the present invention an agent capable of binding to Clever-1, i.e. Clever-1 inhibitor or anti-Clever-1 agent, is selected from the group consisting of an antibody or a 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 the treatment comprising administration of at least one agent capable of binding Clever-1. Anti-Clever-1 monotherapy refers in the present disclosure to the therapy including anti-Clever-1 agent(s) as a single agent.
According to an embodiment of the invention, an anti-Clever-1 antibody is a therapeutic humanized anti-Clever-1 antibody. According to an embodiment of the present invention, an anti-Clever-1 antibody is a humanized monoclonal Clever-1 antibody, previously presented in the patent publication WO2017/182705.
In an embodiment of the present invention, an anti-Clever-1 antibody is a humanized monoclonal immunoglobulin G4K antibody bexmarilimab (International Nonproprietary Name (INN)) as disclosed in WHO Drug Information, Vol. 33, No. 4, pages 814-815 (2019)), or bexmarilimab variant or the antibody in a bexmarilimab biosimilar. As used herein, “bexmarilimab” means the IgG4 monoclonal antibody with the structure described in WHO Drug Information, Vol. 33, No. 4, pages 814-815 (2019).
A bexmarilimab biosimilar means a biological product which is approved by a regulatory agency in any country for marketing as a bexmarilimab biosimilar. In an embodiment, a bexmarilimab biosimilar comprises a bexmarilimab variant as the drug substance. In an embodiment, a bexmarilimab biosimilar has substantially the same amino acid sequence of heavy and light chains as bexmarilimab. As used herein, a “bexmarilimab variant” means an antibody which comprises sequences of heavy chain and light chain that are identical to those in bexmarilimab, except for having one or more conservative amino acid substitutions at positions that are located outside of the light chain CDRs and/or one or more conservative amino acid substitutions that are located outside of the heavy chain CDRs, e.g. the variant positions are located in the framework regions or the constant region. In other words, bexmarilimab and a bexmarilimab variant comprise identical CDR sequences, but differ from each other due to having a conservative amino acid substitution at other positions in their full-length light and heavy chain sequences. A bexmarilimab variant is substantially the same as bexmarilimab with respect to binding affinity to CLEVER-1.
According to an embodiment of the present invention, a cell line producing the therapeutic anti-Clever-1 antibody bexmarilimab (FP-1305) has been deposited on 27 May 2020 under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure with the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, and has the accession number DSM ACC3361. The present invention is not to be limited in scope by the culture deposited, since the deposited embodiment is intended as a single illustration of one aspect of the invention and any culture that is functionally equivalent is within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustration that it represents.
It has been observed that anti-Clever-1 treatment has an ability to decrease expression of cell surface markers PD-1, PD-L1, CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3 and CD28 on leucocytes, especially on circulating T cell populations, and an ability to increase the expression of cell surface markers CD25 (IL-2RA), CXCR3 and CD69. However, the effect may vary from patient to other and/or from cancer type to other. For example, cell surface marker ICOS may be upregulate or downregulate in relation to anti-Clever-1 therapy. Therefore for providing most efficient treatment with anti-Clever-1 therapy, the expression of one or more said cell surface marker is monitored when initiating anti-Clever-1 therapy and/or during anti-Clever-1 therapy for providing required information to start the best combination agent(s) treatment together with anti-Clever-1 therapy after the observed changes in the expression of one or more cell surface marker.
The present invention provides a method for monitoring cancer patient's response to anti-Clever-1 therapy and for evaluating the need for combination therapy, when an agent capable of binding to Clever-1 has been administered in a patient. According to an embodiment of the present invention, one or more cell surface markers are monitored when initiating anti-Clever-1 therapy. 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.
A method according to an embodiment of the present invention for monitoring a patient's response to anti-Clever-1 therapy and evaluating the need for combination therapy, when an agent capable of binding to Clever-1 has been administered in a patient, the method comprising
According to an embodiment of the invention, a sample obtained at a first point in time prior to the administration of an agent capable of binding to Clever-1 to a patient refers to a sample obtained prior to the first administration of an agent capable of binding to Clever-1 (i.e. the first sample). A sample obtained at a later point in time after the administration of an agent capable of binding to Clever-1 to a patient refers to a sample obtained at any time point during the 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.
According to an embodiment of the invention, the method comprising
According to another embodiment of the present invention, the method comprising
In an embodiment according to the present invention, the expression of one, two, three or more cell surface marker selected from CTLA-4, ICOS, OX40, 41BB, LAG3, TIM3, CD28, CD25 and CXCR3 is measured from the obtained samples and the expression level of said cell surface marker from the sample obtained at a later point of time is compared to the expression level of said cell surface marker measured from the sample obtained at a first point of time for evaluating for initiation the concomitant administration of an agent that affects said cell surface marker. In an embodiment according to the present invention, an expression of PD-1 and/or PD-L1 can be analyzed in addition to above mentioned cell surface markers for evaluating for initiation the concomitant administration of PD-1/PD-1 inhibitor(s).
According to an embodiment of the present invention, a method further comprises measuring interferon gamma (IFNg) from the obtained samples and comparing the IFNg level measured from the sample at a later point of time to the IFNg level measured from the sample obtained at a first point of time, wherein an increase in the IFNg level is an indication for initiation the concomitant administration of PD-1 and/or PD-L1 inhibitor.
According to an embodiment of the invention sample is a blood sample drawn from a patient. In a typical embodiment of the invention, an expression of one or more cell surface markers are analyzed from leucocytes, especially from T cell populations, obtained from a blood sample drawn from the patient.
According to the present invention, the expression levels of the cell surface markers may be measured any suitable method known in the art.
According to an embodiment of the present invention a combination of therapeutically effective amounts of:
According to the present invention, it has been noticed that the combination treatment with anti-Clever-1 therapy can be selected on the basis of the measured expressions of the cell surface marker(s). If there is not desired change in the expression level by anti-Clever-1 therapy alone as a single agent, it is an indication to start the administration of an agent affecting said cell surface marker.
According to an embodiment of the present invention, also other cell surface markers can be defined for monitoring the treatment response and evaluating the need for initiating the combination treatment(s) to induce for example CD69, CD95, CD45RO and/or HLA DR expression.
According to an embodiment of the present invention a combination for use in a treatment of cancer in an individual may further comprise a PD-1 and/or PD-L1 inhibitor, when anti-Clever-1 monotherapy does not show a downregulation of PD-1 and/or PD-L1. A combination according to the present invention for use in a treatment of cancer in an individual may comprise one, two, three, four or more agent 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, a cytokine IL-2 including modified versions of it or CD25 (IL-2RA) agonist and a CXCR3 inducer or CXCR3 agonist in addition to an agent capable of binding to Clever-1 and PD-1 and/or PD-L1.
According to an embodiment of the present invention, the combination for use in a treatment of cancer in an individual further comprises a PD-1 and/or PD-L1 inhibitor, when an upregulation of interferon gamma (IFNg) and/or an upregulation of PD-1 and/or PD-L1 is observed during anti-Clever-1 therapy in combination with one or more said agents affecting cell surface marker(s). It has been observed that anti-tumor responses with anti-Clever-1 treatment associates with an increase in circulating interferon gamma (IFNg), which would later lead to an increase in PD-1 and/or PD-L1 expression level. Hence, when increased expression level of IFNg and/or increased expression level of PD-1 and/or PD-L1 is observed during anti-Clever-1 therapy alone or in combination with one or more said agents affecting cell surface marker(s), it is an indication to initiate anti-PD-1 and/or PD-L1 therapy with anti-Clever-1 treatment.
In an embodiment, a combination for use in a treatment of cancer in an individual may comprise a combination of a PD-1 and/or PD-L1 inhibitor and an agent capable of binding to Clever-1, when anti-Clever-1 monotherapy does not show a downregulation of PD-1 and/or PD-L1 on lymphocytes and/or an upregulation of IFNg and/or an upregulation of PD-1 and/or PD-L1 is observed during anti-Clever-1 therapy.
According to the present invention, CTLA-4 inhibitor, ICOS inhibitor, ICOS inducer, OX40 inhibitor, 41BB inhibitor, LAG3 inhibitor, TIM3 inhibitor, CD28 inhibitor, PD-1 inhibitor and PD-L1 inhibitor comprises an antibody or fragment thereof capable of blocking said cell surface receptors. A combination treatment according to the present invention can comprise any known agent(s) capable of blocking said cell surface receptors.
According to the present invention, a cytokine IL-2 including modified versions of it, CD25 (IL-2RA) agonist, a CXCR3 inducer and a CXCR3 agonist comprises an agent capable of activating said cell surface receptor. IL-2 is pro-inflammatory cytokine known to stimulate the immune system (mainly T cells) to kill tumor cells. IL-2 therapies are, however, rather toxic and many developmental technologies are being explored to induce IL-2 in the tumor microenvironment without toxic side effect. Such technologies include IL-2 receptor agonist, cancer vaccines introducing IL-2 producing viruses etc. A combination treatment according to the present invention can comprise any known agent(s) capable of activating said cell surface receptors.
According to an embodiment of the invention, a combination for use in a treatment of cancer in an individual comprises therapeutically effective amounts of
According to an embodiment of the present invention a combination for use in a treatment of cancer in an individual further comprises therapeutically effective amounts of a PD-1 and/or PD-L1 inhibitor, such as anti-PD-1 and/or anti-PD-L1 antibody or fragment thereof that binds specifically to a Programmed Death-1 (PD-1) receptor and inhibits PD-1 activity, when anti-Clever-1 single agent activity does not lead to decreased PD-1/PD-L1 expression and/or an IFNg response lead to increased levels of PD-1/PD-L1 expression.
A method according to an embodiment of the present invention for treating a cancer patient comprises administering to said cancer patient a combination of
According to an embodiment of the present invention a method for treating a cancer patient comprises administering to said cancer patient a combination of
In an embodiment, a method for treating a cancer patient comprises administering to said cancer patient a combination of
The term “treatment” or “treating” shall be understood to include complete curing of a disease or disorder, as well as amelioration or alleviation of said disease or disorder. The term “therapeutically effective amount” is meant to include any amount of an agent according to the present invention that is sufficient to bring about a desired therapeutic result.
The present invention provides a method and a composition for treating cancer by reducing malignant tumor growth and/or by inhibiting metastasis formation is applicable to all forms of cancers. Thus, any malignant tumor or metastasis can be treated.
“Administering” refers to the physical introduction of a composition comprising said therapeutic agents to an individual, using any of the various methods and delivery systems known to those skilled in the art. The agents to be used in the present invention may be administered by any means that achieve their intended purpose. For example, administration may be intravenous, intramuscular, intraperitoneal, intra-tumoral, subcutaneous or other parenteral routes of administration, for example by injection. In addition to the pharmacologically active compounds, the pharmaceutical preparations of said agents preferably contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active agents into preparations that can be used pharmaceutically. The dose chosen should be sufficient to reduce or inhibit malignant tumor growth and/or inhibit metastasis formation.
A method according to an embodiment of the present invention for treating a cancer patient comprising
The following examples are merely illustrative of the principles of the present invention and are not intended to limit the scope of the invention.
An anti-Clever-1 antibody FP-1305 (DSM ACC3361) used in the following Examples is a humanized antibody currently being developed by Faron Pharmaceuticals for cancer treatment and it is disclosed more detailed in the patent publication WO2017/182705 and known as bexmarilimab.
Clever-1 inhibiting agent, an anti-Clever-1 antibody FP-1305 is currently being tested for safety and preliminary efficacy in a Phase I/II study in patient with advanced solid tumors (clinicaltrials.gov NCT03733990: A Study to Evaluate Safety, Tolerability and Preliminary Efficacy of FP-1305 in Cancer Patients (MATINS)). First (pre-dose) sample taken prior to initiating FP-1305. Second sample (post-dose) taken 8 days after beginning FP-1305 treatment. Cell surface markers are analyzed from the samples as described below.
Protocol for Mass Cytometry (CyTOF) Staining and Data Analysis
PBMCs were isolated from the samples with Ficoll-Paque density gradient centrifugation and frozen with 10% DMSO in RPM11640 (Sigma-Aldrich; RPM11640 supplemented with 10% FCS, 2 mmol/L L-glutamine) medium.
Prior to CyTOF staining frozen PBMCs were thawn and re-suspended in PBS counted. 1-3×106 cells were taken for stainings. First cells were stained with 2.5 μM Cell-ID cisplatin (Fluidigm; cat. 201064) viability reagent for 5 min at room temperature (RT). After washings, cells were barcoded with heavy-metal isotope-labelled anti-human CD45 (clone H130) antibodies (CD45_89Y, CD45_141Pr and CD45_147Sm, 1/200) for 30 min at RT, washed carefully and differentially barcoded samples were combined. Next, samples were blocked with human Kiovig solution (0.2 mg/ml) for 15 min at RT and stained with heavy-metal isotope-labelled anti-human antibody cocktail (Table 1) including cell surface markers for 30 min at RT, followed with washings.
Stained samples were incubated with DNA intercalation reagent (1/1000, Cell ID Intercalator-103Rh in MaxPar® Fix and Perm Buffer; cat. 201067; Fluidigm) for 1 h at RT, washed and fixed with 4% PFA solution overnight (o/n) at +4° C. Next day samples were washed, resuspended to MaxPar Water (cat. 201069; Fluidigm) containing a 1/10 dilution of EQ 4 Element Beads (Fluidigm) and immediately acquired to a CyTOF mass cytometer (Helios, Fluidigm). After the bead normalization of the samples, viable singlet cells were debarcoded by using FlowJo. CD45+CD3+ cells were gated and exported for further analysis.
The data analysis was performed similarly as in 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 Web site and Cytokit package was downloaded from Bioconductor and opened in R. Manually gated events (gated as explained above) were imported into Cytokit and subjected to Phenograph analysis. Clustering was performed by using 9 out of 23 markers (CD4, CD8, CD45RA, CCR7, CD45RO, CD127, CD25, CCR6, CXCR3) with additional settings: merge method; minimum, transformation; CytofAsinh, cluster method; Rphenograph, visualization method; tSNE and cellular progression NULL.
22 clusters were defined by Phenograph and these clusters were displayed on tSNE blots by using R package “shiny” to visualize different patients before and after treatment. Cluster colours, identification numbers, dot and label size was customised in the “shiny” app. The phenograph analysis produced several csv-files which were used to calculate mean expression values per sample for each marker and additionally the changes between pre and post samples per patient was calculated. Heatmap was generated with ComplexHeatmap package (“Gu, Z. (2016) Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics.”) downloaded from Bioconductor and statistical analysis was done with 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/.”) by using T-test.
The results are shown in
The anti-Clever-1 antibody FP-1305 has begun clinical development in the setting explained above. In this first-in human trial (clinicaltrials.gov NCT03733990) metastatic colorectal cancer patients that have not been responsive to any available therapy have shown anti-tumor responses. These so far have all been associated with an increase in serum IFNg levels during treatment (
| Number | Date | Country | Kind |
|---|---|---|---|
| 20195959 | Nov 2019 | FI | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FI2020/050741 | 11/10/2020 | WO |
