Small molecule targeting TLR4 overexpressing tumors and associated immunosuppressive, angiogenic and fibrotic complications

Abstract

The present disclosure relates to the use of compound (I) and any of its salts for the treatment of a neoplasm characterized by high expression of toll-like receptor 4 (TLR4) in the tumor or its microenvironment, associated with at least one of the pathophysiological complications including immunosuppression, angiogenesis, fibrosis, or metastasis.

Description

[0001] Small molecule targeting TLR4 overexpressing tumors and associated immunosuppressive, angiogenic and fibrotic complications

[0002] FIELD OF DISCLOSURE

[0003] The present disclosure relates to the use of compound (I) and any one of its salts for the treatment of a neoplasm characterized by high expression of toll-like receptor 4 (TLR4) in the tumor or its microenvironment, associated with at least one of the pathophysiological complications including immunosuppression, angiogenesis, fibrosis, or metastasis.

[0004] INTRODUCTION

[0005] Cancer is the second-leading cause of death worldwide which means that about 10 million people die from cancer every year. There are an estimated 20 million new cancer cases per year according to a WHO report dated February 1 , 2024 (WHO, https: / / www.who.int / news / item / 01-02-2024-global-cancer-burden-growing--amidst-mounting- need-for-services). The total annual economic cost of cancer is estimated at US$1.16 trillion. An ESMO study dated March 13, 2023, estimated that between 2020 and 2050, cancers will cost the world economy $25.2 trillion in international dollars (https: / / www.esmo.org / oncology- news / estimate-of-the-global-economic-cost-of-the-most-prevalent-cancers-in-204-countries- from-2020-to-2050). Significant advances in cancer treatment have been made in recent years. But most patients are poorly responsive due to factors including chronic non-resolved inflammation, angiogenesis and fibrosis.

[0006] Cancer-elicited inflammation can be activated by cancer-initiating mutations and contribute to tumor progression via the recruitment and activation of inflammatory cells resulting in immunosuppression and thereby providing a preferred condition for tumor development. Once an inflammatory tumor microenvironment (TME) is established, inflammatory factors derived from tumor cells and / or interstitial cells induce cell proliferation and prolong cell survival by initially activating oncogenes and subsequently inactivating tumor suppressor genes. On the other hand, when the inflammatory factors are persistent and so are not eliminated during the acute inflammation process, they likely lead to chronic non-resolved inflammation. In this context, tumors cannot use inflammation to change their microenvironment thereby allowing cancer cells to elude the natural immune response and to resist therapies especially in case of patients with advanced metastatic diseases. And those patients are also usually poorly responsive to immunotherapy or chemotherapy.

[0007] Toll Like Receptor 4 or TLR4 is recognized for inducing inflammation in response to endogenous molecules like pathogen-associated molecular patterns (PAMPs) and damage- associated molecular patterns (DAMPs), often released from injured or necrotic tissues following treatments like chemotherapy or radiotherapy. Thus, TLR4 activation has a beneficial role in tissue repair under normal conditions and has been considered as being beneficial for the treatment of cancer as well.

[0008] Based on that, considerable research has been performed over the past several years with the lipid A derivatives such as M LA (WO 93 / 12778, US 5,286,718; Mei et al., Am. J. Physiol. 1996, 271 , H2723-H2729; Elliott GT, J. Mol. Cell. Cardiol. 1998, 30, 3-17; Elliott et al., J. Cardiovasc. Pharmacol., 1998, 32(1), 49-56; Xi et al., Circulation, 1999, 99(16), 2157-2163; Xi et al., Cardiovascular Drug Reviews, 1999, 17(3), 265-280) and one synthetic analogue, RC-552 (WO 00 / 11010: phosphoglycolipid L34; Xi et al., American Journal of Physiology-Heart and Circulatory Physiology, 1999, 277(6), H2418-H2424; Xi and Kukreja, Toxicology, 2000, 155, 37-44, Pivotal role of nitric oxide in delayed pharmacological preconditioning against myocardial infarction; Elliot et al., Journal of Molecular and Cellular Cardiology. 2000, 32(7), 1327-39). Another lipid-A derivative, a triacyl named 2-deoxy-6-O-[2-deoxy-4-O- (dihydroxyphosphoryl)-2-[(R)-3-dodecanoyloxytetradecanoylamino]-p-D-glucopyranosyl]-2- [(R)-3-hydroxytetradecanoylamino]-a-D-glucopyranosyl dihydrogenophosphate or compound (I), has been supposed for many years to share a similar mechanism of action, especially mediated by the activation of TLR4 pathway, and compound (I) is still widely acknowledged in the scientific literature as a TLR4 agonist:

[0009] • Keshavarz A et al., Toll-like receptors (TLRs) in cancer; with an extensive focus on TLR agonists and antagonists. IUBMB Life. 2021 ; 73: 10-25: The review cites “3.1.2 TLR2 / 4 agonists: When it comes to TLR agonists in cancer treatment, ... Another chemically defined TLR2 / 4 agonist, ... [compound (I)], is a derivative of Escherichia coli lipid A that has been evaluated in a Phase I study in patients with solid tumors. The results of the investigations showed that probably compound (I) could increase the production of IFN-y and TNF-a and up-regulate iNOS.”

[0010] • Chakraborty S et al., Application of toll-like receptors (TLRs) and their agonists in cancer vaccines and immunotherapy. Frontiers in Immunology 14 (2023): 1227833. “Application of TLR agonists as adjuvants of cancer vaccine, TLR2 / TLR4: ... Similarly, anti-tumor effect generated through activation of TLR2 and 4, by ... [compound (I)], a lipid A (Escherichia coli origin) derivative, is currently being evaluated as a vaccine adjuvant for the treatment of melanoma in phase l / ll trials, in addition to a phase I trial against solid tumors. The observed results from a few preclinical studies depict the increase of TNF-a, IFN-y, and iNOS behind the therapeutic activity of ... [compound (I)] administration... .”

[0011] • Jiang Y et al., World J Gastroenterol, 2019, 25(25), 3151-3167, Hepatocellular carcinoma: Mechanisms of progression and immunotherapy “TLR agonists: The role of TLR agonists as a vaccine adjuvant and tumor immunotherapeutic agent has been recently noted. As a vaccine adjuvant, TLR agonists trigger antigen presentation by promoting the maturation of DCs. Multiple TLRs, such as TLR3 and TLR9, have been confirmed to be expressed on HCC cells, and the role of TLR agonists in tumor therapy has received much attention. The TLR2 / 4 agonist ... [compound (I)] has potential roles in the prevention of invasion and metastasis in HCC. ... TLR agonists can also work as adjuvants to stimulate the immune system during tumor treatment, but their effects on tumor cells cannot be ignored... .”

[0012] • Steeghs N et al., Manufacturing-dependent change in biological activity of the TLR4 agonist GSK1795091 and implications for lipid A analog development. Clin. Transl. Sci. 2022; 15: 2625-2639. “Considerable evidence supports the use of TLR agonists as immune adjuvants in combination with other anti-cancer treatments. The TLR4 agonists glucopyranosyl lipid A in a stable emulsion (GLASE) and ... [compound (I)] are being investigated in combination with other therapies in several cancer types.”

[0013] • Boucher, NG et al., TLR4 / IFNy pathways induce tumor regression via NOS Il-dependent NO and ROS production in murine breast cancer models. Oncoimmunolgy, 2016, 5, 5, 1- 9, e11233669. “Here, we report the investigation of the antitumor efficacy of an agonist of TLR4, ALA ... [compound (I)], in mouse models of mammary cancer.”

[0014] • D’Agostini C et al., Antitumour effect of ... [compound (I)] and cyclophosphamide on murine B16 melanoma in different experimental conditions. Int. Immunopharmacol. 2005; 5(7-8): 1205-1212.). “Nonetheless, data to be disclosed in this patent unexpectedly reveal a reversal of tumor-induced immunosuppression, along with anti-fibrotic and anti-angiogenic effects. These properties are surprisingly linked to potent TLR4 antagonism rather than agonism.”

[0015] • Only two papers contradictorily refer to compound (I) as a TLR4 antagonist: Dunn-Siegrist I et al., J. Biol. Chem. 2012, 287, 16121-16131 , Toll-like receptor activation of human cells by synthetic triacylated lipid A-like molecules. These results are cited by Peri F et al., Journal of Medicinal Chemistry, 2013, 57(9), 3612-3622, Toll-like Receptor 4 (TLR4) Modulation by Synthetic and Natural Compounds: An Update. However, the position expressed in those two references, which was based on in vitro experiments only, has not found any further acceptance in ensuing publications (see, for example: Boushehri, MAS and Lamprecht, A, TLR4-based immunotherapeutics in cancer: a review of the achievements and shortcomings. Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021 / acs.molpharmaceut.8b00691 • Publication Date (Web): 18 Sep 2018).

[0016] However, the role of TLR4 in cancer is far more complex and context-dependent, exhibiting both anti-tumoral and pro-tumoral effects influenced by various factors within the tumor microenvironment. Initially, TLR4 may in fact exert anti-tumoral effects, particularly in the early stages of cancer or in certain sterile cancer contexts. Nonetheless, a switch to pro-tumoral effects can occur in scenarios where TLR4 is overexpressed and activated on tumor cells, especially in the presence of chronic inflammation and Damage-Associated Molecular Patterns (DAMPs) released by dying tumor cells. This switch involves the hijacking of TLR4 by the tumor to promote immunosuppression and resistance to therapies.

[0017] Expressed in both immune and cancer cells, TLR4 activation leads to the overexpression of various cytokines, depending on the cell type. While immune cell activation by TLR4 can induce protective immune responses against infections, its activation in tumor cells often results in immunosuppression and facilitates tumor growth. Tumor cells exploit the inflammatory response driven by TLR4 for their benefit. In the context of chronic non-resolved immunosuppressive inflammation, where the overexpression of TLR4 in malignant cells amplifies pro-oncogenic effects, which is further enhanced by TLR4-positive tumor-associated cells, including endothelial cells. This collective activation fosters a TME favorable to chemoresistance and metastasis.

[0018] Moreover, TLR4 signaling upregulates inflammatory cytokines, pro-survival factors like vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-P), and proteases disrupting the extracellular matrix. These changes not only promote tumor growth but also enhance its metastatic potential by facilitating vascular invasion and resistance to cytotoxic therapies.

[0019] With respect to angiogenesis, it is the physiological process of new blood vessel formation from pre-existing ones, primarily mediated by factors like VEGF. In cancer, angiogenesis plays a critical role in tumor growth and progression by supplying nutrients and oxygen to the growing tumor mass. On the other side, the newly formed blood vessels could in principle facilitate the delivery of chemotherapeutic agents to the tumor site. However, the increased vascularization can also contribute to treatment resistance, since the complex network of angiogenic factors and cytokines can create a protective shield around the tumor, limiting drug penetration and promoting the survival of resistant tumor cells. Thus, angiogenesis not only fuels tumor growth but also poses a significant challenge in overcoming treatment resistance in cancer. With respect to fibrosis, also known as fibrotic scarring, this is a pathological wound healing in which connective tissue replaces normal parenchymal tissue to the extent that it goes unchecked, leading to considerable tissue remodeling and the formation of permanent scar tissue, which is mainly mediated by TGF-p. The TME drives fibrosis to stiffen the tumor cell stroma and promote malignancy. The stiffened stroma enhances tumor cell growth, survival and migration and drives a mesenchymal transition. A stiff microenvironment appears to promote angiogenesis, hypoxia and compromises anti-tumor immunity. Degree of tissue fibrosis and level of stromal stiffness seem to correlate with tumor aggressivity and poor patient survival prognosis.

[0020] With respect to tumor immunosuppression, TLR4 overexpression diminishes the cytotoxic activity of immune cells and promotes the growth and function of immunosuppressive cells like M2 polarized macrophages, or inhibits CD8-positive cytotoxic lymphocytes and natural killer (NK) cells. This immune modulation further aids in immune escape mechanisms through the expression of immunosuppressive cytokines and enhances processes like epithelial- mesenchymal transition (EMT), invasion, and metastasis by upregulating matrix metalloproteinases (MMPs) and integrins.

[0021] Importantly, many cancer treatments inadvertently contribute to the development of fibrosis, angiogenesis, and immunosuppression, posing significant medical challenges. The development of fibrosis, angiogenesis, and immunosuppression as a result of cancer treatments represents a substantial medical concern, impacting treatment efficacy and patient outcomes. The activation of the TGF-p pathway, a key regulator of fibrosis, by cancer treatments can further exacerbate fibrotic changes in the TME. Furthermore, anti-angiogenic therapies, while designed to inhibit tumor blood vessel formation, can lead to the recruitment of pro-angiogenic factors by cancer-associated fibroblasts (CAFs), promoting angiogenesis and contributing to resistance to anti-angiogenic agents. Moreover, the inflammatory tumor environment influenced by CAFs can drive immunosuppression, creating a favorable milieu for tumor progression and metastasis.

[0022] Given these complexities, TLR4 agonists may not be effective against tumors overexpressing TLR4 for the following reasons:

[0023] • Tumor-Promoting Effects: In tumors with high TLR4 expression, activation of TLR4 can enhance tumor growth and immunosuppression rather than fostering anti-tumor immunity. TLR4 signaling in these contexts may lead to increased production of immunosuppressive cytokines, enhanced tumor cell invasion and metastasis, and resistance to apoptosis. • Chronic Inflammation: Overexpression of TLR4 on tumor cells can induce chronic inflammation within the tumor microenvironment, contributing to tumor progression rather than regression.

[0024] • Immune Escape: High TLR4 expression on tumor cells may facilitate immune escape mechanisms, allowing cancer cells to evade detection and destruction by the immune system.

[0025] • Activation of Pro-Tumor Signaling Pathways: In TLR4-overexpressing tumors, agonists can activate downstream signaling molecules such as NF-KB, MAPK, and Akt, which are involved in promoting tumor cell proliferation and survival.

[0026] As a result of that dual role of TLR4, clinical and commercial success with TLR4 agonists has been limited, as indicated by Kaczanowska et al. (2013) in their study "TLR agonists: our best frenemy in cancer immunotherapy" (J Leukoc Biol, 93(6): 847-63). Moreover, multiple recent publications have demonstrated an association between high TLR4 expression and poor prognosis, especially in advanced cancer stages.

[0027] Therefore, the multifaceted impact of cancer treatments on fibrosis, angiogenesis, and immunosuppression necessitates a comprehensive understanding and there is a great and hitherto unmet need for the development of strategies to mitigate these adverse effects while optimizing therapeutic outcomes.

[0028] Summary of the invention

[0029] The factors discussed above suggest that in tumors with high TLR4 expression, TLR4 agonists may paradoxically promote tumor growth instead of inhibiting it. Consequently, the present invention is based on the fact that TLR4 antagonists might be more appropriate in these cases to counteract the pro-tumoral effects associated with TLR4 overexpression.

[0030] The disclosure relates to TLR4 antagonist pharmaceutical compositions and methods for treating neoplasm characterized by high expression of toll-like receptor 4 (TLR4) in the neoplasm or in the microenvironment of the neoplasm associated with at least one of the pathophysiological mechanisms including immunosuppression, angiogenesis, fibrosis, or metastasis. Surprisingly, the present inventors have found that compound (I) as defined below can act as a TLR4 antagonist, in contrast to what was known in the art, and the key to advancing new treatment options with compound (I) is by attacking TLR4 overexpressing tumors, which themselves are associated to TLR4-mediated pathophysiological mechanisms such as immunosuppressive inflammation, fibrosis, and angiogenesis. Compound (I) is a triacylated compound according to formula (I) or a salt thereof. The present invention provides methods for treating or alleviating a symptom of a neoplasm, mediated or promoted by increased TLR4 activity in a cell or subject in need thereof by contacting the cell or administering to the subject a therapeutically effective amount of compound (I) as a TLR4 antagonist.

[0031] Thus, in a first aspect, the present invention relates to the compound of formula (I) hereinafter referred to as “compound (I)”, or a pharmaceutical acceptable salt thereof for use in the treatment of patients with a neoplasm which have a high expression of toll-like receptor 4 in said neoplasm, wherein high expression means an RPKM (Reads Per Kilobase of Transcript, per Million mapped reads) of 1 or higher, particularly 2 or higher, 3 or higher 4 or higher, more particularly 4.5 or higher.

[0032] Furthermore, the disclosure relates to methods of treating a subject, preferably a human subject, having a neoplasm mediated or promoted by increased TLR4 expression or increased in TLR4 activity and associated to pretreatment abnormal increased level of immunosuppressive and / or angiogenic and / or fibrotic and / or metastatic markers in the blood and / or the TME.

[0033] Thus, in an aspect, the present invention relates to compound (I) or a pharmaceutically acceptable salt thereof for use in a treatment of patients with a neoplasm according to the present invention, wherein said treatment with compound (I) as a first compound is performed in combination with a second compound selected from the group consisting of immuno- oncologic agents, immunotherapeutic agents, adoptive cell therapies, targeted agents, chemotherapeutic agents, cancer vaccine and a combination of at least two of said agents, therapies and / or vaccines, or in combination with radiotherapy.

[0034] Thus, the disclosure provides a method for the administration of compound (I), which a first compound, in combination with a second compound selected from the group consisting of immuno-oncologic agents, immunotherapeutic agents, adoptive cell therapies, targeted agents, chemotherapeutic agents, cancer vaccine.

[0035] In an aspect, the present invention relates to compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) or said pharmaceutically acceptable salt thereof is comprised at a concentration of between 1 mg / ml and 10 mg / ml. In some embodiment, compound (I) is administered via intravenous route either as a bolus injection or as a slow infusion, i.e. given slowly over a long period, in saline.

[0036] Thus, the present disclosure relates to a treatment regimen for a neoplasm using compound (I) administered via an intravenous route.

[0037] Thus, in an aspect, the present invention relates to administration schemes, including a scheme where compound (I) or a salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein compound (I) or said salt thereof is for use in a treatment that comprising a weekly or biweekly administration of compound (I) or a pharmaceutically acceptable salt thereof for a period of 1 to 6 months.

[0038] In some embodiments, the disclosure relates to uses of compound (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a neoplasm.

[0039] In some embodiments, the disclosure relates to the sequential administration of compound (I), wherein the compound (I) is administered as a first therapy prior to the start of treatment with said second compound or start of said radiotherapy as a second therapy.

[0040] In some alternative embodiments, the disclosure relates to the sequential administration of compound (I), wherein the compound (I) is administered as a second therapy after the start of treatment of neoplasm with said second compound or after the start of said radiotherapy as a first therapy.

[0041] In the context of the present invention, the term “sequential administration”, with respect to a first compound or treatment regimen and a second compound or treatment regimen, means that in a first phase said first compound or treatment regimen is present at a therapeutically relevant level, whereas said second compound or treatment regimen is not present at a therapeutically relevant level, followed by a second phase, wherein said second compound or treatment regimen is present at a therapeutically relevant level, whereas said first compound or treatment regimen is not present at a therapeutically relevant level. One of ordinary skill in the art will understand that one or more additional phases with alternate presence of said first compound or treatment regimen and a second compound or treatment regimen may subsequently follow.

[0042] In some embodiments, the disclosure relates to the concomitant administration of compound (I) as a first compound and said second compound or said radiotherapy.

[0043] In the context of the present invention, the term “concomitant administration” with respect to a first compound and a second compound or treatment regimen, means that said first compound or treatment regimen and said second compound or treatment regimen are both present at a therapeutically relevant level at the same time. An alternative term is “simultaneous administration”.

[0044] One of ordinary skill in the art will understand that concomitant or simultaneous administration does not necessarily mean that both compounds or treatment regimens have to be administered at exactly the same time or as part of a single pharmaceutical composition, since the mode of administration for said first compound and said second compound or treatment regimen and their respective pharmacokinetic and pharmacodynamic properties may require different administration schemes in order to reach a therapeutically relevant level for both said first compound or treatment regimen and said second compound or treatment regimen.

[0045] In some embodiments, the disclosure relates to a combination of sequential and concomitant administration. In one embodiment, compound (I) is administered as a first therapy prior to the start of a second therapy which is treatment with a concomitant administration of said compound (I) and said second compound or start of said radiotherapy. In another embodiment, compound (I) is administered concomitantly with said second compound or said radiotherapy as a second therapy after the start of a first treatment with said second compound or said radiotherapy. One of ordinary skill in the art will understand that different combinations of sequential and concomitant administration are possible.

[0046] In another aspect, the present invention also provides pharmaceutical compositions comprising compound (I), or a pharmaceutically acceptable salt thereof, for the oral route and / or parenteral routes.

[0047] In some embodiments, the disclosure relates to compositions comprising compound (I), or a pharmaceutically acceptable salt thereof, for use in a method for the treatment of neoplasm, and a pharmaceutically acceptable carrier.

[0048] In another aspect, the invention relates to the use of compound (I) for the manufacture of a medicament for the treatment of neoplasia.

[0049] In other aspect, the invention provides the use of a pharmaceutical composition comprising compound (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of neoplasia.

[0050] In an aspect, the invention relates to kits comprising i) a compound (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound (I) or a pharmaceutically acceptable salt thereof; ii) optionally a pharmaceutical solution to dissolve or dilute i), and iii) instructions to administer said compound (I) or said pharmaceutically acceptable salt thereof, or said pharmaceutical composition comprising the compound (I) or a pharmaceutically acceptable salt thereof.

[0051] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0052] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 shows the Kibur Experimental Design: Figure lists the 16 treatment schemes using eight different anticancer drugs either alone (microwells 2 to 10) or in an in situ combination with an intravenous (IV) treatment with compound (I) (microwells 11 to 18) Microwells 1 and 2 were references (no compound, and compound (I) alone, respectively). Two separate cohorts of animals were used, one without IV compound (I) treatment and the second with IV compound (I) treatment to show the compound (I) IV priming effect on the combination treatments corresponding to a total of 36 treatment conditions.

[0055] Figure 2 shows the results of an in vivo efficacy combination study of compound (I) with anti PD-1 antibody Pembrolizumab against PBMC humanized model of metastatic melanoma A- 375: Figure shows the metastatic melanoma A-375 tumor volume measured as a function of time following a first treatment. Control and treated animal groups are shown with the drugs alone or in combination (compound (I) and / or Pembrolizumab). Treatment routes are indicated as IV (intravenous) for compound (I) and IP (intraperitoneal) for Pembrolizumab.

[0056] Figure 3 shows the results of an in vivo efficacy combination study of compound (I) with anti PD-1 antibody Pembrolizumab against PBMC humanized model of Small Cell Lung Cancer NCI-H69. Figure shows the Small Cell Lung Cancer NCI-H69 tumor volume measured as a function of time following the first treatment as in Figure 2. Control (vehicle) and treated animal groups are shown with the drugs alone (compound (I) and / or Pembrolizumab). Treatment routes are indicated as IV (intravenous) for compound (I) and IP (intraperitoneal) for Pembrolizumab.

[0057] Figure 4 illustrates the treatment protocol designed to investigate the effect of compound (I) on human macrophage polarization, specifically targeting the different polarization states: MO, M1 , and M2. Macrophages are generated from primary human peripheral blood monocytes, purified by anti-CD14 magnetic sorting and stimulated for 5 days with CSF-1 (100 ng / mL). The immature macrophages (M0) thus generated were then polarized for 2 or 4 days into pro- inflammatory macrophages (M1) by adding LPS (100 ng / mL) + IFNy (20 ng / mL) or into antiinflammatory macrophages (M2) by adding IL-4 / IL-13 (20 ng / mL). The efficiency of M1 and M2 polarization is quantified by flow cytometry by determining the expression level of pro- inflammatory membrane markers CD80 and CD86 and of anti-inflammatory membrane markers CD206, CD163, CD200R, CD209. The results are expressed as an arithmetic mean of fluorescence intensity (MFI). The effect of compound (I) on macrophage polarization was assessed on the polarization induction and on the reprogramming of already polarized macrophages (4 donors).

[0058] Figure 5 shows the evaluation of compound (I) toxicity on MO, M1 and M2 macrophages: the figure shows the toxicity of compound (I) compound assessed at 6 different concentrations (0.1 pM to 30 pM) versus vehicle alone (0 pM) on already polarized MO, M1 and M2 macrophages. Compound (I) was added at d7, and the evaluation of cell death was carried out by flow cytometry using DAPI labelling two days later (d9). These experiments were performed on two different donors.

[0059] Figure 6 shows the human macrophage polarization results (M0 / M1). Figure shows MFI (mean fluorescence intensity) of the different treatment conditions for the expression of CD80, CD86. The treatment with compound (I) strongly decreased the expression of pro-inflammatory markers CD80 and CD86 during M1 macrophage polarization.

[0060] Figure 7 shows the BioMAP profile of compound (I) in the non-small cell lung cancer stromal and vascular (NSCLC) panel: NSCLC tumor cells exposed to different concentrations of compound (I) 0.65 pM, 2 pM, 5.9 pM and 18 pM versus control vehicle (0 pM) in stromal conditions on the left part of the graph (StroNSCLC system is a co-culture of human primary fibroblasts, immune and HI 299 cells) and under vascular conditions on the right part of the graph (VascNSCLC system is a co-culture of human primary endothelial, immune and HI 299 cells). The biomarker readouts are shown as positive values which correspond to an upregulation of the selected marker or negative values which correspond to a downregulation of the selected marker. The dotted lines above and below zero are used for a comparison with the average upper and lower envelope of several thousands of drugs tested in this system.

[0061] Figure 8 shows the results of an in vivo efficacy combination study of compound (I) with anti PD-L1 antibody Atezolizumab against PBMC humanized model of small cell lung cancer NCI- H69: Figure shows the small cell lung cancer NCI-H69 tumor volume measured as a function of time following the first treatment as in Figure 2. Control and treated animal groups are shown with the drugs alone or in combination. Treatment routes are indicated as IV for compound (I) and IP for Atezolizumab.

[0062] Figure 9 shows the results of an in vivo efficacy (and pharmacodynamic) study of compound (I) combined with anti PD-L1 antibody Atezolizumab against PBMC humanized model of small cell lung cancer NCI-H69: Figure shows the small cell lung cancer NCI-H69 tumor volume measured as a function of time . Control and treated animal groups are shown with the drugs alone or in combination. Treatment routes are indicated as IV (intravenous) for compound (I) and IP (intraperitoneal) for Atezolizumab.

[0063] Figure 10 shows the human macrophage polarization results (M0 / M2): Figure shows MFI (mean fluorescence intensity in arbitrary units (A.U.) of the different treatment conditions for the expression of CD206, CD163, CD209, and CD200R. Compound (I) drastically decreased the CD163 expression level in MO and M2 conditions and partially dampened the expression of CD209, CD206 and CD200R during M2-macrophage polarization.

[0064] Figure 11 shows the BioMAP profile of compound (I) in the fibrosis panel: The figure shows the fibrosis panel stimulated to model TGFp- and TN Fa-driven myofibroblast differentiation during chronic inflammation and wound healing in different tissue settings. The biomarkers for the three panels are labelled (1) SAEMyoF for the interstitial lung diseases, including pulmonary fibrotic diseases such as idiopathic pulmonary fibrosis (IPF) which areis modeled by co-culturing small airway epithelial cells and adult fibroblasts; (2) REMyoF for fibrotic disease of the kidney associated with end-stage renal failure which are captured using a coculture of renal proximal tubule epithelial cells and adult fibroblasts; and (3) MyoF for fibroblasts alone as the monoculture. The three systems were exposed to different concentrations of compound (I) (0.65 pM, 2 pM, 5.9 pM and 18 pM) versus control vehicle (0 pM) The biomarker readouts at the different concentrations are shown as positive values which correspond to an upregulation of the selected marker or negative values which correspond to a downregulation of the selected marker. The dotted lines above and below zero are used for a comparison with the average upper and lower envelope of several thousands of drugs tested in this system.

[0065] Figure 12 shows the demonstration of compound (I) anti-fibrotic effect in Dog ischemia:

[0066] (A) Effect of compound (I) on area at risk / left ventricle in dogs: The area at risk as percent (%) of the left ventricular weight in the control and compound (l)-treated dogs. All values are the mean ± SEM of six dogs / group. There were no significant differences observed between groups.

[0067] (B) Effect of compound (I) on infarct size in grams in dogs: The effect of four different doses of (0.2 pg / kg, 1 pg / kg, 5 pg / kg and 50 pg / kg, IV administered) of compound (I) and saline vehicle given 24 hours prior to the acute experiment on myocardial infarct size in grams (g). Only the 5 pg / kg dose produced a significant reduction in infarct size. *P<0.05 versus the control group. All values are the mean ± SEM of six dogs / group.

[0068] (C) Effect of compound (I) on infarct size / area at risk in dogs: The effect of four different doses (0.2, 1 , 5 and 50 mg / kg, i.v.) of compound (I) and saline vehicle given 24 hours prior to the acute experiment on myocardial infarct size (IS) expressed as a percent of the area at risk (AAR). The three higher doses of compound (I) produced nearly equivalent reductions in IS / AAR. *P<0.05 versus the control group. All values are the mean ± SEM of six dogs / group.

[0069] (D) Effect of compound (I) on infarct size / left ventricle weight in dogs: the effect of four different doses (0.2, 1 , 5 and 50 mg / kg, i.v.) of compound (I) and saline vehicle given 24 hours prior to the acute experiment on myocardial infarct size (IS) expressed as a percent of the left ventricular weight (LV). The three higher doses of compound (I) produced nearly equivalent reductions in IS / LV. *P<0.05 versus the control group. All values are the mean ± SEM of six dogs / group.

[0070] (E) The effect of four different doses (0.2, 1 , 5 and 50 pg / kg, i.v.) of compound (I) and saline vehicle given 24 hours prior to the acute experiment, on transmural coronary collateral blood flow (TCCBF, ml / min / g) at 30 minutes of occlusion in the control and the compound (l)-treated groups. There were no significant differences between groups. All values are the mean ± SEM of six dogs / group.

[0071] Figure 13 shows that a single treatment of compound (I) demonstrates an increasing efficacy profile relative to the increasing TLR4 expression level of the tumor.

[0072] Figure 14 shows mean volume of NCI-N87 tumors in female NXG mice treated with PBMCs from two donors along with vehicle, trastuzumab or Compound (I). Values shown are mean ±SD; n=7 for all groups initially.

[0073] Figures 15 and 16 shows results from FACS experiment for antibody panel 1.

[0074] Figures 17 and 18 shows results from FACS experiment for antibody panel 2.

[0075] DETAILED DESCRIPTION

[0076] Terms and Definitions

[0077] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0078] Toll-like receptor 4 (TLR4) is a pattern recognition receptor (PRR) that plays a crucial role in the innate immune system by recognizing and responding to molecular patterns associated with microbial pathogens, like PAMPs (pathogen-associated molecular patterns) and endogenous signals from damaged cells, like DAMPs (damage-associated molecular patterns). TLR4 is expressed on various immune cells, including macrophages, dendritic cells, and certain epithelial cells. TLR4 recognizes PAMPs such as lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria. This recognition triggers an immune response aimed at eliminating the pathogen. TLR4 also binds to DAMPs released by stressed or damaged cells, such as high-mobility group box 1 (HMGB1), heat-shock proteins, and extracellular matrix components like hyaluronan fragments. These molecules signal tissue damage thereby initiating repair processes. In the context of cancer, TLR4 is often overexpressed and can be hijacked by tumor cells to promote a chronic inflammatory environment that supports tumor growth and metastasis. Kashani, B., Zandi, Z., Pourbagheri - Sigaroodi, A., Bashash, D., & Ghaffari, S. H. (2020). The role of toll - like receptor 4 (TLR4) in cancer progression: A possible therapeutic target? Journal of Cellular Physiology. doi:10.1002 / jcp.30166 The activation of TLR4 by DAMPs and PAMPs within the tumor microenvironment leads to the production of pro-inflammatory cytokines and chemokines, creating a pro-tumorigenic milieu. This persistent inflammation not only facilitates tumor progression and treatment resistance, but also contributes to immunosuppression, fibrosis, and angiogenesis, further complicating cancer treatment. By enhancing the expression of immunosuppressive factors and recruiting immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), TLR4 signaling helps tumors evade the immune system. Additionally, TLR4 activation can induce chemoresistance, making cancer cells less responsive to conventional therapies. Therefore, targeting TLR4 and its signaling pathways holds potential for improving cancer therapy outcomes by disrupting these pro- tumorigenic processes.

[0079] RPKM (Reads per kilo base of transcript per million mapped reads) is a normalized gene expression unit that measures the gene (transcript) abundance level in a sample. RPKM is normalized to correct the gene (transcript) lengths and library sizes (sequencing depth). Generally, the higher the RPKM of a gene, the higher the expression of that gene.

[0080] RPKM calculation formula

[0081] Number of reads mapped to gene x 103x 106

[0082] RPKM = -

[0083] Total number of mapped reads x gene length in base pairs

[0084] RPKM >1 reflects the extent to which the gene of interest is overexpressed vs basal healthy conditions.

[0085] DAMPs (damage-associated molecular patterns) are endogenous molecules released by stressed or damaged cells that can initiate and perpetuate an immune response. In cancer, DAMPs such as HMGB1 , ATP, S100A8, S100A9, S100A4, Annexin A1 , Calreticulin, IL-1 , IL- 33 and heat-shock proteins can activate immune cells and contribute to chronic inflammation, promoting tumor progression and influencing the tumor microenvironment.

[0086] PAMPs (pathogen-associated molecular patterns) are molecular structures found on pathogens, such as bacteria and viruses, that are recognized by the innate immune system's pattern recognition receptors (PRRs). In cancer, PAMPs can be involved in shaping the immune response, as some tumors exploit PAMP-induced signaling to create a pro- tumorigenic inflammatory environment.

[0087] Cancer is a neoplasm in which some of the body’s cells grow uncontrollably and spread to other parts of the body. It is caused by changes to genes that control the way our cells function, especially how they grow and divide. Normally, human cells grow and multiply, through a process called cell division, to form new cells as the body needs them. When cells grow old or become damaged, they die, and new cells take their place. Sometimes this orderly process breaks down, and abnormal or damaged cells grow and multiply when they shouldn’t. These cells may form tumors, which are lumps of tissue. Tumors can be cancerous or not cancerous i.e. benign. Cancerous tumors spread into, or invade, nearby tissues and can travel to distant places in the body to form new tumors (a process called metastasis). Cancerous tumors may also be called malignant tumors. Many cancers form solid tumors, but cancers of the blood, such as leukemias, generally do not.

[0088] Neoplasm and tumor have the same meaning and both refer to an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow after the stimuli that initiated proliferation is removed. Such abnormal tissue shows partial or complete lack of structural organization and functional coordination with the normal tissue which may be either benign (i.e., benign tumor) or malignant (i.e., malignant tumor). A neoplasm is an abnormal growth of cells, also known as a tumor. It is caused by changes to genes that control the way our cells function, especially how they grow and divide. Normally, human cells grow and multiply (through a process called cell division) to form new cells as the body needs them. When cells grow old or become damaged, they die, and new cells take their place. Sometimes this orderly process breaks down, and abnormal or damaged cells grow and multiply when they shouldn’t. These cells may form tumors, which are lumps of tissue. Tumors can be cancerous or not cancerous (benign) Neoplasms or neoplastic diseases are conditions that cause tumor growth. Neoplastic disease is the result of neoplasm and may alternatively be used.

[0089] The tumor microenvironment (TME) is a complex and dynamic network of cells, signaling molecules, extracellular matrix, and blood vessels surrounding and interacting with tumor cells. The TME plays a critical role in tumor progression by influencing tumor growth, metastasis, immune evasion, and response to therapies.

[0090] A pathobiological mechanism refers to the underlying biological processes that lead to the development and progression of a disease. In the context of cancer, it involves the molecular, cellular, and systemic interactions that result in tumor initiation, growth, metastasis, and the associated pathological changes in the tissue and organ systems.

[0091] Chronic non-resolved inflammation refers to a persistent inflammatory state that remains active over an extended period without proper resolution. Unlike acute inflammation, which helps the body heal and then switches off, chronic inflammation persists and can damage tissues, organs, and DNA. This prolonged inflammatory response is associated with various health problems.

[0092] Chronic non-resolved immunosuppressive inflammation in cancer refers to a prolonged and persistent inflammatory response within the tumor microenvironment that fails to resolve and, instead, promotes an immunosuppressive state. This type of inflammation is characterized by the continuous presence of immune cells, such as tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs), which secrete various cytokines and growth factors. A crucial element in this process is the over-solicitation and hijacking of Toll-like receptor 4 (TLR4), a key sentinel of the innate immune system, by the tumor. The persistent activation of TLR4 by tumor-derived signals leads to the sustained activation of signaling pathways such as NF-KB and STAT3, contributing to non-resolving inflammation. This, in turn, fosters tumorigenesis, angiogenesis, and tissue remodeling, while simultaneously hindering the immune system's ability to mount an effective attack against cancer cells. The exploitation of TLR4 by the tumor underscores the complexity of the tumor microenvironment and highlights the challenge of developing therapeutic strategies that can effectively target both the inflammatory and immunosuppressive aspects of cancer.

[0093] Tumor immunosuppression describes the process by which cancer cells evade the immune system by suppressing its ability to recognize and destroy tumor cells. This can occur through various mechanisms including secretion of immunosuppressive cytokines, recruitment of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and the expression of immune checkpoint molecules like PD-L1.

[0094] Angiogenesis refers to a process comprising the formation of new capillary blood vessels from pre- existing microvessels. Angiogenesis normally occurs during embryonic development, tissue regeneration, wound healing, and corpus luteum development that is a cyclical change in female reproductive system. Angiogenesis includes vascular angiogenesis and lymphatic angiogenesis.

[0095] Vascular angiogenesis refers to the formation of new blood vessels from pre-existing ones, driven primarily by endothelial cells in response to signals such as vascular endothelial growth factor (VEGF). This process supplies oxygen and nutrients to tissues, including tumors, thereby facilitating their growth and metastasis. Tumors exploit vascular angiogenesis to sustain their rapid growth and create pathways for cancer cells to enter the bloodstream, leading to the spread of cancer to distant organs. Anti-angiogenic therapies aim to inhibit this blood vessel formation, effectively starving the tumor of essential nutrients and slowing its growth, making vascular angiogenesis a critical target in cancer treatment.

[0096] Lymphatic angiogenesis involves the formation of new lymphatic vessels from existing ones, regulated by signals such as VEGF-C and VEGF-D. This process plays a crucial role in maintaining fluid balance and immune responses but also facilitates the metastasis of cancer cells through the lymphatic system. Newly formed lymphatic vessels provide routes for cancer cells to spread to regional lymph nodes and beyond, contributing to cancer dissemination. Additionally, these vessels can create environments that suppress the immune response, aiding tumor immune evasion. Targeting lymphatic angiogenesis can help prevent metastasis and improve immunotherapy efficacy, highlighting its importance in cancer progression and treatment strategies.

[0097] Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue, often as a reparative or reactive process. In cancer, fibrosis can result from chronic inflammation and the tumor's interaction with its microenvironment, leading to a stiffened, scar-like tissue that can support tumor growth and hinder effective drug delivery.

[0098] Desmoplasia is the growth of fibrous connective tissue around a neoplasm, causing dense fibrosis around the tumor, or scar tissue within the abdomen after abdominal surgery. It is characterized by low cellularity with hyalinized or sclerotic stroma and disorganized blood vessel infiltration. Desmoplasia is usually only associated with malignant neoplasms, which can evoke a fibrotic response invading healthy tissue. Desmoplasia and fibrosis are both characterized by the growth of fibrous connective tissue. However, they differ in their underlying causes and the types of tissue they affect. Desmoplasia is a response to an insult, such as a neoplasm, that causes the growth of fibrous connective tissue around the affected area. It is usually associated with malignant neoplasms and can cause dense fibrosis around the tumor or scar tissue within the abdomen after abdominal surgery. Fibrosis, on the other hand, is a pathological process that involves the accumulation of excess fibrous connective tissue in an organ or tissue. It can be caused by a variety of factors, including chronic inflammation, tissue injury, and exposure to toxins. Fibrosis can occur in many different organs, including the lungs, liver, and heart. In summary, desmoplasia is a response to an insult, such as a neoplasm, that causes the growth of fibrous connective tissue around the affected area, while fibrosis is a pathological process that involves the accumulation of excess fibrous connective tissue in an organ or tissue

[0099] Metastasis as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade tissues elsewhere in the body.

[0100] The hallmarks of cancer are a set of fundamental traits that cancer cells acquire during tumorigenesis, including sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, enabling of replicative immortality, induction of angiogenesis, and activation of invasion and metastasis. These characteristics collectively drive the progression and malignancy of cancer, providing a framework for understanding its biology and guiding therapeutic development.

[0101] Treatment, treat or treating refer to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of a disease. In certain embodiments, such terms refer to the amelioration or eradication of the disease, or symptoms associated with it. In other embodiments, this term refers to minimizing the spread or worsening of the disease, resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0102] Subject, individual or patient are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human.

[0103] A combination drug regimen involves the use of multiple medications together to treat a particular condition. Combination therapy is often employed to enhance efficacy as different drugs may work through distinct mechanisms, reducing the likelihood of drug-resistant cancer cells. Each drug can be used at its optimal dose, avoiding intolerable side effects. Combination treatment ? Combination therapy

[0104] Concomitant treatment refers to the administration of two or more drugs or therapies at the same time, either for the same condition or for different conditions. This term is commonly used in medicine and clinical trials. For example, chemotherapy for cancer often involves the use of multiple drugs that are given simultaneously. Concomitant treatment can also refer to the use of other pharmaceutical agents in addition to a primary therapy. Sequential treatment refers to a therapeutic strategy in which different treatments are administered in a specific order over a defined period, rather than simultaneously. This approach is designed to maximize therapeutic efficacy, manage side effects, and reduce the likelihood of resistance. Sequential treatment often involves using one modality (such as chemotherapy, or radiotherapy) initially to reduce tumor burden, followed by another modality (such as targeted therapy, immunotherapy, or additional chemotherapy) to eliminate remaining cancer cells and prevent recurrence. By tailoring the sequence of treatments based on the patient's response and the biological characteristics of the tumor, sequential treatment aims to improve overall survival and quality of life for cancer patients.

[0105] Pre-treatment with compound (I) in the example refers to the administration of compound (I) as a medication before the initiation of a primary therapy.

[0106] Treatment dose refers to a specific amount of medication taken at one specific time. It’s usually measured in metric mass units (e.g., milligrams or milliliters). The dosage is a broader term that specifies how much, when, and how long to take the medication. Dosage considers the overall amount of medication prescribed over a specific period (e.g., daily or weekly). So, a “treatment dose” typically refers to the specific amount of a drug administered during a single treatment,

[0107] Priming dose: The priming is performed before the therapeutic standard of care treatment. The interval between the priming dose and the treatment dose ranges from a few minutes up to several hours or several days. The goal of the priming dose is to enhance the efficacy of the primary therapy or to reduce the risk of adverse effects. In the example, a priming dose of compound (I) is the administration of compound (I) prior to any other treatment. Subsequent administrations of compound (I) can be pursued during the other treatment of neoplasm.

[0108] Priming effect: is the effect due to the administration of the priming dose. The priming effect is described in the examples as improving the anti-tumor immune response and inducing a significant reduction in tumor growth compared to the control without a priming dose. Another important aspect of the augmentation of anti-tumor activities using the priming effect of an inhibitor or antagonist (compound (I)) is the “priming effects” that sensitize cancer cells to the standard of care therapy. These effects result in the sensitization of cancer cells to subsequent standard of care therapy. The molecular mechanisms leading to chemo sensitization, immunomodulation, reversal of immunosuppression through these priming effects caused by compound (I), as a TLR4 antagonist and immune modulator, is dependent on treatment schedules.

[0109] Standard of care: standard of care is a treatment accepted by medical experts for a certain type of disease, in the example neoplasm, and that is widely used by healthcare professionals. It is also called best practice, standard medical care, and standard therapy. The standard of care is the medical treatment that is normally provided to people with a given condition. In many scientific studies, the control group receives the standard treatment rather than a placebo while a treatment group receives the experimental treatment, in the example with Compound (I). After the clinical trial, researchers compare the outcomes of the two groups to see if the experimental treatment is better than, as good as or not as beneficial as the standard treatment.

[0110] Systemic administration of a drug: the administration of a drug using the parenteral route into the circulatory system so that the entire body is exposed to the drug.

[0111] Intravenous bolus, intravenous infusion are the two ways in which a parenteral drug solution can be administered directly into a patient’s vein. A bolus is a rapid injection, typically within seconds or a few minutes, of a solution into a vein. An IV infusion, on the contrary, involves administering the drug solution for longer periods of time (slow infusion). While a short infusion can last 10-20 minutes, long infusions can take several hours, even days.

[0112] Radiotherapy: Radiotherapy is a cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumors. The dose of radiation used in radiotherapy depends on several factors, including the type of cancer, the size of the tumor, and the location of the tumor in the body and how close the tumor is to normal tissues that are sensitive to radiation, general health and medical history of the patient, other types of cancer treatment following radiotherapy, other factors, such as age and other medical conditions. The total dose is usually broken up into daily doses of five times a week for a total period of five to eight weeks. The aim of radiotherapy is to give a high dose of radiation to the cancer but as low a dose as possible to the surrounding healthy cells. This gives the highest chance of curing or shrinking the cancer, while reducing the risk of side effects.

[0113] Adoptive cell therapy (ACT) is an immunotherapy approach that involves isolating immune cells from a patient, expanding or genetically modifying them ex vivo to enhance their antitumor properties, and then reinfusing them into the patient. This therapy aims to boost the body's immune response against cancer, with strategies including T-cell receptor (TCR) transgenic T cells, chimeric antigen receptor -T (CAR-T) cells, and tumor-infiltrating lymphocytes (TILs).

[0114] An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria, viruses or cancer cells. The antibody recognizes a unique molecule of the foreign cell or of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly (for example, by blocking a part of a virus that is essential for its invasion). To allow the immune system to recognize millions of different antigens, the antigen-binding sites at both tips of the antibody come in an equally wide variety. In contrast, the remainder of the antibody is relatively constant. In mammals, antibodies occur in a few variants, which define the antibody's class or isotype: IgA, IgD, IgE, IgG, and IgM. The constant region at the trunk of the antibody includes sites involved in interactions with other components of the immune system. The class hence determines the function triggered by an antibody after binding to an antigen, in addition to some structural features. Antibodies from different classes also differ in where they are released in the body and at what stage of an immune response.

[0115] Therapeutic monoclonal antibody (mAb): A therapeutic monoclonal antibody is a type of medication that is designed to target specific cells or proteins in the body. These drugs are created by cloning a single type of immune cell, called a B cell, to produce large quantities of identical antibodies. The antibodies are then modified to enhance their therapeutic properties, such as their ability to bind to specific targets. Therapeutic monoclonal antibodies can be used to treat a variety of conditions, including cancer, autoimmune diseases, and infectious diseases. For example, rituximab is a monoclonal antibody that is used to treat certain types of cancer and autoimmune diseases. It works by binding to a protein called CD20, which is found on the surface of certain immune cells, and triggering their destruction. Another example is infliximab, which is used to treat inflammatory bowel disease and other autoimmune disorders. It works by binding to a protein called tumor necrosis factor-alpha (TNF-alpha), which is involved in inflammation, and blocking its activity. mAb: monoclonal antibody

[0116] Anti-cancer peptides: are short chains of amino acids that have been shown to have anticancer properties. They can be derived from a variety of sources, including plants, animals, and bacteria. Some anti-cancer peptides work by disrupting the cell membrane of cancer cells, while others target specific proteins involved in cancer growth and proliferation.

[0117] Fusion proteins: are proteins that are created when two or more genes fuse together.

[0118] Nucleic acid strands: are long chains of nucleotides that make up DNA and RNA. In cancer treatment, nucleic acid strands can be targeted using a variety of techniques, including gene therapy, RNA interference. Nucleic acid strands may comprise natural and non-natural nucleotides. Sensitize and sensitizing, as used herein, refer to making, through the administration of a first agent, an animal or a cell within a subject more susceptible, or more responsive, to the biological effects of a second agent. The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect observed upon the administration of a second agent with and without administration of the first agent. The response of the sensitized cell can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent. Biological effects include promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis.

[0119] Antibody drug conjugate (ADC, ADCs): Ado-trastuzumab emtansine (Kadcyla), Brentuximab vedotin (Adcetris), Gemtuzumab ozogamicin (Mylotarg), Enfortumab vedotin (Padcev), Sacituzumab govitecan (Trodelvy), Trastuzumab deruxtecan (Enhertu), Belantamab mafodotin-blmf (Blenrep) (withdrawn from FDA approval in November 2022), Loncastuximab tesirine-lpyl (ZYNLONTA), Tisotumab vedotin-tftv (Tivdak), Mirvetuximab soravtansine (ELAHERE).

[0120] Characteristics of the invention

[0121] The inventors have identified that compound (I) hereunder is of therapeutic interest for treating neoplasm as antagonist of TLR4 receptor. Such compound (I) is also useful for treating cancers associated mechanisms such as immunosuppression, angiogenesis, fibrosis or metastasis (major hallmarks of cancer). As demonstrated by the examples, such compound has multiple anti-cancer activity by exerting an effect against TLR4 and one of these mechanisms.

[0122] The present disclosure relates to a compound (I) or a pharmaceutically salt thereof as defined herein for use for treating a cancer.

[0123] Thus, the present invention relates to compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm characterized by high expression of toll-like receptor 4 in the neoplasm, wherein high expression means an RPKM of 1 or higher, particularly 2 or higher, 3 or higher, 4 or higher, more particularly 4.5 or higher. In particular embodiments, the neoplasm is further characterized byhigh expression of toll-like receptor 4 in the tumor microenvironment.

[0124] In the context of the present invention, overexpression of oncogenic markers is measured using Reads Per Kilobase of Transcript, per Million mapped reads (RPKM), a common metric in RNA sequencing analysis. RPKM normalizes the read counts by the length of the gene and the total number of reads, allowing for accurate comparisons of gene expression levels. An RPKM value above 1 indicates overexpression, while a value above 3, in particular above 4, more particularly above 4.5 denote very high overexpression, highlighting the potential impact of these markers in oncogenesis.

[0125] In some embodiments, “high expression” of TLR4 means an RPKM of 5 or higher, in particular 8 or higher.

[0126] As mentioned above, TLR4 agonism might have some beneficial effects in early stages of the development of a neoplasm, but become deleterious at later stages. Thus, TLR4 antagonism should be considered at specific switch points when high TLR4 expression is linked to worse prognosis compared to low TLR4 expression. In the context of the present invention, the term “poor prognosis” refers to a clinical prediction indicating that a patient is likely to have an unfavorable outcome from their cancer diagnosis. This is characterized by a lower likelihood of long-term survival, higher probability of disease progression or recurrence, and limited response to treatment. Factors contributing to a poor prognosis include advanced stage of cancer at diagnosis, aggressive tumor biology, poor patient health status, and lack of effective therapeutic options. Thus, in some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of a patient with a neoplasm according to the present invention, wherein a poor prognosis or a low chance of recovery from a neoplasm has been established for said patient.

[0127] For instance, in esophageal carcinoma, the switch point is identified around month 30, beyond which the use of TLR4 agonists in high TLR4-expressing tumors could be detrimental. Thus, in some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm has first been diagnosed at least 10 months prior to said treatment.

[0128] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by at least one of the following blood and / or tumor pathological features: patients exhibiting pretreatment an abnormally reduced level of activated CD8-positive cytotoxic T lymphocytes n the blood, wherein an abnormally reduced level means less than 800, in particular from 300 to 800, more particularly from 50 to 300, CD8-positive cytotoxic T lymphocytes per pL blood, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of exhausted CD8- positive cytotoxic T lymphocytes in the blood, wherein an abnormally increased level means more than 50, in particular from 50 to 200, more particularly from 200 to 600, exhausted CD8-positive cytotoxic T lymphocytes per pL blood, wherein said exhausted CD8-positive cytotoxic T lymphocytes are characterized by the expression of the immunological markers PD-1 , Tim-3, Lag 3 and / or RGMb, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of CD4-positive T regulatory lymphocytes in the blood, wherein an abnormally increased level means more than 50, in particular from 50 to 200, more particularly from 200 to 500, CD4- positive T regulatory lymphocytes per pL blood, wherein said CD4-positive T regulatory lymphocytes are characterized by the expression of the immunological marker FoxP3, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced concentration of activated CD8-positive cytotoxic T lymphocytes compared to activated CD4-positive T regulatory lymphocytes in the blood, wherein an abnormally reduced concentration means that the ratio of CD8-positive cytotoxic T lymphocytes to CD4-positive T regulatory lymphocytes is 1 or lower, in particular between 0.75 and 1 , more particularly lower than 0.75, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced level of activated dendritic cells in the blood, wherein an abnormally reduced level means less than 100, in particular from 50 to 100, more particularly from 10 to 50, CD11c-positive cells per pL blood, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced level of activated natural killer cells (NKs) in the blood, wherein an abnormally reduced level means less than 500, in particular from 200 to 500, more particularly from 50 to 200, CD56-positive cells per pL blood, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced level of polarized macrophages type 1 (M1) in the blood, wherein an abnormally reduced level means less than 300, in particular from 150 to 300, more particularly from 50 to 150, M1 cells per pL blood, wherein said M1 cells are characterized by the expression of the immunological markers CD80 and / or CD163, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of polarized macrophages type 2 (M2) in the blood, wherein an abnormally increased level means more than 100, in particular from 100 to 200, more particularly from 200 to 500, M2 cells per pL blood, wherein said M2 cells are characterized by the expression of the immunological markers CD206, CD163, CD200, CD200R and / or CD209, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally reduced concentration of polarized macrophages type 1 (M1) compared to polarized macrophages type 2 (M2) in the blood, wherein an abnormally reduced concentration means that the ratio of M1 / M2 is 1 or lower, in particular between 0.75 and 1 , more particularly below 0.75, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of immunosuppressive cells in the tumor, wherein an abnormally increased level means that the concentration of the immunological marker PD-L1 in the tumor is 2 ng / mL or higher, in particular between 2 and 5 ng / mL, more particularly between 5 and 20 ng / mL, and / or of the immunological marker PD-L2 in the tumor is 2 ng / mL or higher, in particular between 2 and 5 ng / pL, more particularly between 5 and 15 ng / mL, in particular wherein the concentration of PD-L1 and / or PD-L2 is deterimined by ELISA or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of TME angiogenesis in the tumor, wherein an abnormally increased level means that the concentration of the growth factors VEGFa and / or VEGFc is 50 pg / mL or higher, in particular between 50 and 100 pg / mL, more particularly between 100 and 200 pg / mL in particular as determined by ELISA; patients exhibiting pretreatment an abnormally increased level of TME fibrosis, wherein an abnormally increased level means that at least one of the following conditions is met: (i) in the case where said neoplasm is lung cancer, the Ashcroft score is 2 or higher, in particular between 2 and 5, more particularly between 5 and 8, in particular as determined by fibrotic histopathological assessment; (ii) the fibrotic stain score is 5% or higher, in particular between 5% and 15%, more particularly more than 15%, in particular as determined by fibrotic histopathological assessment using staining with Sirius Red, Fast Green, or via the Masson trichrome procedure; (iii) the concentration of the growth factor TGFbeta in the tumor is 5 ng / mL or higher, in particular between 5 and 10 ng / mL, more particularly between 10 and 20 ng / mL, in particular as determined by ELISA; (iv) the concentration of the growth factor CTGF in the tumor is 5 ng / mL or higher, in particular between 5 and 10 ng / mL, more particularly between 10 and 50 ng / mL, in particular as determined by ELISA, and (v) the concentration of the FGF growth factors in the tumor is 20 pg / mL or higher, in particular between 20 and 50 pg / mL, more particularly between 50 and 100 pg / mL, in particular as determined by ELISA; and patients exhibiting pretreatment an abnormally increased level of at least one of the following metastatic markers in the blood or tumor, respectively: (i) more than 1 circulating tumor cell (CTC) per mL of blood, in particular between 1 and 50 cells / mL, more particularly between 50 and 200 cells / mL, in particular as determined by liquid biopsy or flow cytometry; (iii) a concentration of the epithelial-mesenchymal transition (EMT) marker MMP9 of 50 ng / mL or more in the tumor, in particular between 50 and 100 ng / mL, more particularly between 100 and 300 ng / mL, in particular as determined by ELISA.

[0129] The term “FGFs” refers to a family of cell signaling proteins produced by macrophages. In humans, 23 FGFs are currently known. FGF1 through FGF10 bind to fibroblast growth factor receptors (FGFRs), while the remaining FGFs FGF11 to 23 are not binding to FGFRs, and their roles have not yet been fully elucidated. In the context of the present invention, when defining the concentration of FGFs to be present in neoplasms, the term “FGFs” refers to FGF1 and FGF2, also known as bFGF, only, and the given concentration is the total concentration for FGF1 and FGF2.

[0130] The following Table A and footnotes relating thereto provide a summary of the different blood and / or tumor pathological features:

[0131] Table A

[0132] Explanations / Footnotes for Table A:

[0133] - CD8+**: CD8+ cells are cytotoxic T lymphocytes involved in the direct killing of infected or cancerous cells.

[0134] - **CD8 exhausted**: CD8 exhausted cells are CD8+ T cells that have become dysfunctional due to chronic antigen exposure, characterized by reduced effector function.

[0135] - **CD4 Treg+**: CD4 Treg+ cells are regulatory T cells that maintain immune tolerance and prevent autoimmune reactions by suppressing other immune cells.

[0136] - **CD8 / CD4**: The CD8 / CD4 ratio compares the number of cytotoxic T cells (CD8+) to helper T cells (CD4+), indicating immune response balance.

[0137] - **aDCs**: Activated dendritic cells (aDCs) are antigen-presenting cells that initiate and regulate the adaptive immune response.

[0138] - **aNKs**: Activated natural killer cells (aNKs) are immune cells that can destroy virus-infected cells and tumor cells without prior sensitization.

[0139] - **M1**: M1 macrophages are a type of macrophage that promotes inflammation and has microbicidal and tumoricidal properties. - **M2**: M2 macrophages are anti-inflammatory macrophages involved in tissue repair and tumor progression.

[0140] - **M1 / M2**: The M1 / M2 ratio reflects the balance between pro-inflammatory (M1) and antiinflammatory (M2) macrophages.

[0141] - **PD-L1**: Programmed death-ligand 1 (PD-L1) is a protein that suppresses the immune response by interacting with PD-1 receptors on T cells.

[0142] - **PD-L2**: Programmed death-ligand 2 (PD-L2) is another ligand for PD-1 , involved in downregulating the immune response.

[0143] - **VEGFa**: Vascular endothelial growth factor A (VEGFa) is a signal protein that stimulates the formation of blood vessels.

[0144] - **VEGFc**: Vascular endothelial growth factor C (VEGFc) is involved in the formation of lymphatic vessels.

[0145] - ** Ashcroft score**: The Ashcroft score is a histological assessment used to quantify the extent of lung fibrosis. Score from 1 to 8 (max fibrosis)

[0146] - **TGFb**: Transforming growth factor beta (TGFb) is a cytokine involved in cell growth, differentiation, and immune regulation.

[0147] - **CTGF**: Connective tissue growth factor (CTGF) is a protein that plays a role in fibrosis and tissue repair.

[0148] - **FGFs**: Fibroblast growth factors (FGFs) are involved in wound healing, angiogenesis, and embryonic development.

[0149] - **CTCs**: Circulating tumor cells (CTCs) are cancer cells that have detached from the primary tumor and circulate in the bloodstream.

[0150] - **MMP9**: Matrix metallopeptidase 9 (MMP9) is an enzyme that breaks down extracellular matrix proteins and is involved in tissue remodelling and metastasis.

[0151] 1. Cytokines concentration calculation

[0152] The volume of tumor used for cytokine concentration measurement is typically standardized to ensure consistency and comparability across samples in accordance with the following general approach:

[0153] 1. 1. Weight or Volume of Tissue Sample:

[0154] A precise weight (e.g., 100 mg) of tumor tissue is used for the assay. This ensures that the amount of tissue being analyzed is consistent.

[0155] 1.2. Homogenization in Buffer:

[0156] The tumor tissue is homogenized in a specific volume of buffer (e.g., 1 mL of buffer per 100 mg of tissue). This creates a homogenate where the cytokines are extracted into the buffer solution.

[0157] 1.3. Normalization: The cytokine concentrations are often normalized to the amount of tissue used. For example, if 100 mg of tissue is used, the cytokine concentration might be expressed as pg / mg tissue.

[0158] Example Calculation:

[0159] - Sample Weight: 100 mg of tumor tissue.

[0160] - Buffer Volume: 1 mL of buffer added.

[0161] - Homogenization: The tissue is homogenized in the buffer.

[0162] - Centrifugation: The homogenate is centrifuged, and the supernatant is collected.

[0163] - Assay: The cytokine concentration is measured in the supernatant.

[0164] - Normalization: The measured cytokine concentration is then divided by the weight of the tissue to get the concentration per mg of tissue.

[0165] 1.4. Practical Steps:

[0166] 1. Weigh the tumor sample to ensure it matches the standard (e.g., 100 mg).

[0167] 2. Homogenize the tumor sample in a fixed volume of buffer (e.g., 1 mL for 100 mg).

[0168] 3. Centrifuge and collect the supernatant.

[0169] 4. Measure cytokine concentration in the supernatant using an appropriate assay.

[0170] 5. Normalize the concentration to the tissue weight.

[0171] Example Normalization:

[0172] - If the ELISA or Luminex assay measures 500 pg / mL of cytokine in the supernatant,

[0173] - And you used 100 mg of tumor tissue homogenized in 1 mL buffer,

[0174] - The cytokine concentration is 500 pg / mL per 100 mg of tissue,

[0175] - Normalized, this would be 5 pg / mg of tumor tissue. By using a consistent volume or weight of tumor tissue, researchers ensure that the cytokine concentrations are comparable across different samples and studies.

[0176] 2. Fibrotic scores

[0177] Fibrotic scores are clinical tools used to quantify the extent of fibrosis in tissues. Various scoring systems are used depending on the organ and context. Here are some commonly used fibrotic scoring systems:

[0178] 2. 1. Lung Fibrosis Scores

[0179] Ashcroft Score:

[0180] - 0: Normal lung

[0181] - 1 : Minimal fibrous thickening of alveolar or bronchiolar walls

[0182] - 2-3: Moderate thickening of walls without obvious damage to lung architecture

[0183] - 4-5: Increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses

[0184] - 6-7: Severe distortion of structure and large fibrous areas; "honeycomb lung" appearance

[0185] - 8: Total fibrous obliteration of the field

[0186] 2.2. Kidney Fibrosis Scores

[0187] Banff Classification (used for renal transplant biopsies):

[0188] - ciO: No interstitial fibrosis

[0189] - ci1 : Interstitial fibrosis involving up to 25% of cortical area

[0190] - ci2: Interstitial fibrosis involving 26-50% of cortical area

[0191] - ci3: Interstitial fibrosis involving more than 50% of cortical area

[0192] 2.3. Skin Fibrosis Scores

[0193] Modified Rodnan Skin Score (MRSS) (used in systemic sclerosis): - Skin thickness is scored in 17 body areas on a scale of 0-3:

[0194] - 0: Normal skin

[0195] - 1 : Mild thickness

[0196] - 2: Moderate thickness

[0197] - 3: Severe thickness

[0198] - Total score ranges from 0 to 51

[0199] 2.4. General Histopathological Scoring

[0200] Sirius Red / Fast Green Staining:

[0201] - Quantitative image analysis is used to measure the extent of fibrosis, often reported as a percentage of the stained area relative to the total tissue area.

[0202] Each scoring system is tailored to the specific tissue and context, enabling clinicians and researchers to assess and compare the severity of fibrosis accurately.

[0203] 3. Circulating tumor cell (CTC) detection:

[0204] Circulating tumor cells (CTCs) are cancer cells that have shed from a primary tumor and entered the bloodstream. They can provide valuable information about the tumor's biology and serve as a liquid biopsy for cancer diagnosis, prognosis, and treatment monitoring

[0001] [2][3][4],

[0205] Key points about CTC detection:

[0206] - CTCs are very rare, with only 1-100 CTCs per milliliter of blood along with billions of normal blood cells[1]

[0207] - Detection usually relies on capturing CTCs based on their expression of epithelial markers like EpCAM or cytokeratins[3][4]

[0208] - The FDA-approved CellSearch system is the most widely used method for CTC detection in breast, colorectal and prostate cancer[2][4] - Emerging technologies like microfluidics, nanoparticles, and physical filtration are improving CTC capture efficiency

[0001] [4][5]

[0209] - Single-cell analysis of captured CTCs can reveal their genomic and transcriptomic profiles[3]

[0210] - Monitoring CTC levels over time can assess treatment response and predict disease progression[2][4]

[0211] However, challenges remain in developing highly sensitive and specific CTC detection assays that can reliably capture the heterogeneous population of CTCs[1][3], Ongoing research aims to optimize CTC isolation and downstream analysis to fully realize their potential as a liquid biopsy for precision cancer management.

[0212] Citations:

[0213] [1] https: / / biomarkerres.biomedcentral.com / articles / 10.1186 / s40364-022-00403-2

[0214] [2] https: / / en.wikipedia.org / wiki / Circulating_tumor_cell

[0215] [3] https: / / www.nature.com / articles / s41392-021-00817-8

[0216] [4] https: / / www.frontiersin.org / journals / oncology / articles / 10.3389 / fonc.2021.652253 / full

[0217] [5] https: / / www.thelancet.com / journals / ebiom / article / PIIS2352-3964%2822%2900419- 4 / fulltext

[0218] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally reduced levels of activated CD8-positive cytotoxic T lymphocytes (CD8+ cytotox) in the blood, wherein an abnormally reduced level means less than 800 CD8-positive cytotoxic T lymphocytes per pL blood, in particular from 300 to 800 CD8-positive cytotoxic T lymphocytes per pL blood, more particularly from 50 to 300 CD8-positive cytotoxic T lymphocytes per pL blood. In some embodiments, level of activated CD8-positive cytotoxic T lymphocytes in the blood can be determined by flow cytometry or immunohistochemistry.

[0219] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally increased level of exhausted CD8-positive cytotoxic T lymphocytes in the blood, wherein an abnormally increased level means more than 50 exhausted CD8-positive cytotoxic T lymphocytes per pL blood, in particular from 50 to 200 exhausted CD8-positive cytotoxic T lymphocytes per pL blood, more particularly from 200 to 600 exhausted CD8-positive cytotoxic T lymphocytes per pL blood, wherein said exhausted CD8-positive cytotoxic T lymphocytes are characterized by the expression of the immunological markers PD-1 , Tim-3, Lag 3 and / or RGMb, in particular as determined by flow cytometry or immunohistochemistry.

[0220] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally increased level of CD4-positive T regulatory lymphocytes in the blood, wherein an abnormally increased level means more than 50 CD4-positive T regulatory lymphocytes per pL blood, in particular from 50 to 200 CD4-positive T regulatory lymphocytes per pL blood, more particularly from 200 to 500 CD4-positive T regulatory lymphocytes per pL blood, wherein said CD4-positive T regulatory lymphocytes are characterized by the expression of the immunological marker FoxP3, in particular as determined by flow cytometry.

[0221] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally reduced concentration of activated CD8-positive cytotoxic T lymphocytes compared to activated CD4- positive T regulatory lymphocytes in the blood, wherein an abnormally reduced concentration means that the ratio of CD8-positive cytotoxic T lymphocytes to CD4-positive T regulatory lymphocytes is 1 or lower, in particular wherein the ratio of CD8-positive cytotoxic T lymphocytes to CD4-positive T regulatory lymphocytes is between 0.75 and 1 , more particularly wherein the ratio of CD8-positive cytotoxic T lymphocytes to CD4-positive T regulatory lymphocytes is lower than 0.75, in particular wherein said ratio is determined by flow cytometry.

[0222] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally reduced level of activated dendritic cells in the blood, wherein an abnormally reduced level means less than 100 CD11c-positive cells per pL blood, in particular from 50 to 100 CD11c-positive cells per pL blood, more particularly from 10 to 50 CD11c-positive cells per pL blood, in particular as determined by flow cytometry. In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally reduced level of activated natural killer cells (NKs) in the blood, wherein an abnormally reduced level means less than 500 CD56-positive cells per pL blood, in particular from 200 to 500 CD56-positive cells per pL blood, more particularly from 50 to 200 CD56-positive cells per pL blood, in particular as determined by flow cytometry.

[0223] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally reduced level of polarized macrophages type 1 (M1) in the blood, wherein an abnormally reduced level means less than 300 M1 cells per pL of blood, in particular from 150 to 300 M1 cells per pL of blood, more particularly from 50 to 150 M1 cells per pL of blood, wherein said M1 cells are characterized by the expression of the immunological markers CD80 and / or CD163,

[0224] In some embodimentsin particular as determined by flow cytometry or immunohistochemistry.

[0225] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally increased level of polarized macrophages type 2 (M2) in the blood, wherein an abnormally increased level means more than 100 M2 cells per pL blood, in particular from 100 to 200 M2 cells per pL blood, more particularly from 200 to 500 M2 cells per pL blood, wherein said M2 cells are characterized by the expression of the immunological markers CD206, CD163, CD200, CD200R and / or CD209, in particular as determined by flow cytometry or immunohistochemistry.

[0226] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized abnormally reduced concentration of polarized macrophages type 1 (M1) compared to polarized macrophages type 2 (M2) in the blood, wherein an abnormally reduced concentration means that the ratio of M1 / M2 is 1 or lower, in particular wherein the ratio of M1 / M2 is between 0.75 and 1 , more particularly wherein the ratio of M1 / M2 is below 0.75, in particular as determined by flow cytometry or immunohistochemistry.

[0227] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally increased level of immunosuppressive cells in the tumor, wherein an abnormally increased level means that the concentration of the immunological marker PD-L1 in the tumor is 2 ng / mL or higher, in particular wherein the concentration of the immunological marker PD-L1 in the tumor is between 2 and 5 ng / mL, more particularly wherein the concentration of the immunological marker PD-L1 in the tumor is between 5 and 20 ng / mL, and / or of the immunological marker PD-L2 in the tumor is 2 ng / mL or higher, in particular wherein the immunological marker PD- L2 in the tumor is between 2 and 5 ng / mL, more particularly wherein the immunological marker PD-L2 in the tumor is between 5 and 15 ng / mL,

[0228] In some embodiments, the concentration of PD-L1 and / or PD-L2 is deterimined by ELISA or immunohistochemistry.

[0229] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally increased level of TME angiogenesis in the tumor, wherein an abnormally increased level means that the concentration of the growth factors VEGFa and / or VEGFc is 50 pg / mL or higher, in particular wherein the concentration of the growth factors VEGFa and / or VEGFc is between 50 and 100 pg / mL, more particularly wherein the concentration of the growth factors VEGFa and / or VEGFc is between 100 and 200 pg / mL,

[0230] In some embodiments, as determined by ELISA.

[0231] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally increased level of TME fibrosis, wherein an abnormally increased level means that at least one of the following conditions is met: (i) in the case where said neoplasm is lung cancer, the Ashcroft score is 2 or higher, in particular wherein the Ashcroft score is between 2 and 5, more particularly between 5 and 8,; (ii) the fibrotic stain score is 5% or higher, in particular between 5% and 15%, more particularly more than 15%, in particular; (iii) the concentration of the growth factor TGFbeta in the tumor is 5 ng / mL or higher, in particular between 5 and 10 ng / mL, more particularly between 10 and 20 ng / mL; (iv) the concentration of the growth factor CTGF in the tumor is 5 ng / mL or higher, in particular between 5 and 10 ng / mL, more particularly between 10 and 50 ng / mL, in particular as determined by ELISA, and (v) the concentration of the FGF growth factors in the tumor is 20 pg / mL or higher, in particular between 20 and 50 pg / mL, more particularly between 50 and 100 pg / mL.

[0232] In some embodiments, in particular as determined by fibrotic histopathological assessment In some embodiments, as determined by fibrotic histopathological assessment using staining with Sirius Red, Fast Green, or via the Masson trichrome procedure

[0233] In some embodiments, in particular as determined by ELISA

[0234] In some embodiments, in particular as determined by ELISA.

[0235] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said neoplasm is further characterized by abnormally increased level of at least one of the following metastatic markers in the blood or tumor, respectively: (i) more than 1 circulating tumor cell (CTC) per mL of blood, in particular between 1 and 50 circulating tumor cell (CTC) per mL of bloodL, more particularly between 50 and 200 circulating tumor cell (CTC) per mL of blood; (ii) a concentration of the epithelial-mesenchymal transition (EMT) marker MMP9 of 50 ng / mL or more in the tumor, in particular wherein the concentration of the epithelial-mesenchymal transition (EMT) marker MMP9 is between 50 and 100 ng / mL, more particularly wherein the concentration of the epithelial-mesenchymal transition (EMT) marker MMP9 is between 100 and 300 ng / mL.

[0236] In some embodiments, in particular as determined by liquid biopsy or flow cytometry

[0237] In some embodiments, in particular as determined by ELISA.

[0238] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein this neoplasm is caused and / or associated by one or more of the following: chronic non-resolved inflammation, chronic non-resolved infection, and fibrosis.

[0239] In some embodiments, the present invention relates to the compound (I) or a pharmaceuticall acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, for use in the treatment of a neoplasm according to the present invention, wherein the a neoplasm is selected from head & neck cancer, oesophageal cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, uterus cancer, ovarian cancer mesothelioma, non-small cell lung cancer, small cell lung cancer, triple negative breast cancer, soft tissue sarcoma, osteosarcoma, fibrosarcoma, myelofibrosis, and desmoid tumors.

[0240] In particular, the present inventors realized that the use of TLR4 antagonist might be a relevant therapeutic approach following the administration of a first therapeutic regimen. Thus, in some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said treatment is a second-line treatment after discontinuation of a first-line treatment of said neoplasm with a different anti-neoplasm drug. In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said treatment is in combination with a second compound selected from the group consisting of immuno-oncologic agents, immunotherapeutic agents, adoptive cell therapies, targeted agents, chemotherapeutic agents, cancer vaccine and a combination of at least two of said agents, therapies and / or vaccines, or in combination with radiotherapy.

[0241] In an other aspect, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least one second compound, in particular in combination with one second compound, wherein said at least one second compound is selected from an immuno-oncologic agent and an immunotherapeutic agent, in particular wherein said second compound targets CTLA-4, PD-1 , PD-L1 , PD-L2 and / or a combination thereof.

[0242] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is a monospecific or bispecific antibodybased drug.

[0243] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is selected from cemiplimab (REGN2810), nivolumab (BMS-936558, MDX-1106, ONO-4538), pembrolizumab (MK-3475, SCH 900475), SHR1210, sintilimab (IBI308), spartalizumab (PDR001), tislelizumab (BGB-A317), pidilizumab, BCD-100, toripalimab (JS001), PF-06801591 , AB122, AK105, AMG 404, BCD- 100, Bl 754091 , F520, HLX10, HX008, JTX-4014, LZM009, MEDI0680, MGA012, Sym021 , TSR-042, PSB205, MGD019, MGD013, AK104, XmAb20717, RO7121661 , CX-188, serplulimab, zimberelimab, dostarlimab, nivolumab, prolgolimab, Biocad, geptanolimab, spartalizumab, QL-1604, toripalimab with albumin-bound paclitaxel, nofazinlimab, pimivalimab, IBI-318, AZD-2936, AZD-7789, EMB-02, RG-6139, HX-009, MVR-T3011 , 609A, HLX-10 with cetuximab, RG-7769, HPV-E6-TCR-T cells, ezabenlimab, rosnilimab, LVGN- 3616, TQB-2868, SYN-125, Sofusa anti-PD-1 , Yinkang-001 , AMG-256, GNR-051 , BAT-1308, ONO-4685, LBL-015, LNL-005, zeluvalimab, F-520, CC-90006 and a combination of at least two of said drugs. In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second drug according to the present invention, wherein said second drug is selected from atezolizumab (MPDL3280A, RG7446, RO5541267), durvalumab (MEDI4736, MEDI-4736), avelumab (MSB0010718C), FS118, BCD- 135, BGB-A333, CBT-502, CK-301 , CS1001 , FAZ053, HLX20, KN035, MDX-1105, MSB2311 , SHR-1316, TG-1501 , ZKAB001 , INBRX-105, MCLA-145, KN046, M7824, LY3415244, sugemalimab, avelumab, socazolimab, adebrelimab, bintrafusp alfa, TQB-2450, KN-046, SHR-1701 , IBI-318, HB-0036, HLX-301 , PM-8002, PM-8001 , PD-L1 t-haNK, ADG- 104, LP-002, cosibelimab, MCLA-145, SAR-445710, PM-1022, SGN-PDL1V, dual anti- HER2 / anti-PD-L1 CAR-T cell therapy (peritoneal tumors), Sichuan University, GNC-035, PF- 07257876, CYTO-102, Y-101 D, SKB-337, TQB-2858, ATG-101 , IMM-2510, GS-19, Gensun Biopharma, LBL-024, QL-301 , Q-1802, TST-005, PM-1003, IBI-323, RC-98, HB-0025, IBI-322, MT-6402, BCD-135, CDX-527, ABL-503, anti-PD-L1 CSR T-cell therapy (cancer), Marino Biotechnology, HLX-20, ND-021 , lodapolimab.

[0244] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is selected from molecules inhibiting transcription PD-L1 genes, small molecules inhibiting translation of PD-L1 mRNA, and small molecules being antagonists of PD-L1 protein.

[0245] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is a molecule selected from peptides, fusion proteins and nucleic acid strands, particularly in their naked form or linked to a toxic payload, cytotoxic agent or radioisotope.

[0246] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I), which is a first compound, is for use in combination with at least a second compound according to the present invention, wherein said immuno-oncologic agents and / or the immunotherapeutic agents are adoptive cells.

[0247] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said adoptive cells are selected from the group consisting of CAR- T cells, NK cells, engineered T cells, tumor-infiltrating lymphocytes and a combination of at least two of said cells.

[0248] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said CAR-T cells are selected from the group consisting of tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, CAR-T cells targeting CD19, BCMA, CD22, CD20, CD30, GD2, HER2, EGFR, mesothelin, and PSMA, including those in late-stage development for solid tumors, and CAR-T cells expressing anti-CTLA-4 and anti-PD-1 antibodies.

[0249] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is a targeted agent selected from the group consisting of anti-angiogenic agents and a combination of two or more anti-angiogenic agents.

[0250] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is a targeted agent selected from CerRx, bevacizumab, conbercept, pegaptanib, ranibizumab, Endostar, axitinib, vandetanib, sorafenib, ponatinib, cabozantinib, nintedanib, rivoceranib, fruquintinib, ramucirumab, surufatinib, catequentinib, pyrotinib dimaleate, lenvatinib, sunitinib, regorafenib, aflibercept, M-23, Stainwei Biotech, hVEGF26-104 / RFASE, SIM-010603, HZB-1006, HB-0025, LYN-00101 , AMC-303, EVT-801 , HLX-06, JY-025, ramucirumab biosimilar, Sichuan Kelun Pharmaceutical, SYHA-1813, FAK / VEGFR3 inhibitors (cancer), CureFAKtor Pharmaceuticals, CX-1003, BMS- 817378, ST-1898, Beijing Scitech-MQ Pharmaceuticals, CYC-116, SOMCL-15-290, HLX-12, Chia Tai Tianqing Pharmaceutical, EOC-317, ICP-033, APL-102, ningetinib tosilate, altiratinib, vandetanib, FN-1501 , hydroxychloroquine + sorafenib (MDT, cancer), conbercept, PAN- 90806, fenretinide, MP-0250, dilpacimab, navicixizumab, IBI-302, BI-836880, HA121-28, ilorasertib, TT-00420, tesevatinib, MG-D-1609, NANT Hepatocellular Carcinoma Vaccine, KD- 035, VXM-01 , olinvacimab, gentuximab, BR-55, AK-109, brivanib alaninate, OTSGC-A24, telatinib, YSKB-1001 , pazopanib in combination with pembrolizumab, AL-2846, AL-8326, ENMD-2076, pamufetinib, emvododstat, muparfostat sodium, sevacizumab, GNR-011 , cediranib, famitinib L-malate, zanzalintinib, ibcasertib, lucitanib hydrochloride, sitravatinib, vorolanib, dovitinib, Ado-trastuzumab emtansine (Kadcyla), Brentuximab vedotin (Adcetris), Gemtuzumab ozogamicin (Mylotarg), Enfortumab vedotin (Padcev), Sacituzumab govitecan (Trodelvy), Trastuzumab deruxtecan (Enhertu), Belantamab mafodotin-blmf (Blenrep) (withdrawn from FDA approval in November 2022), Loncastuximab tesirine-lpyl (ZYNLONTA), Tisotumab vedotin-tftv (Tivdak), Mirvetuximab soravtansine (ELAHERE), and any combination of two or more of said targeted agents.

[0251] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is a chemotherapeutics agent that is a DNA modifying agent selected from a platinum-based compound, an antitumor antibiotic and a topoisomerase inhibitor.

[0252] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said DNA modifying agent is platinum-based and selected from lobaplatin, nedaplatin, miriplatin hydrate, lobaplatin, cisplatin, oxaliplatin, eptaplatin, heptaplatin, carboplatin, and dicycloplatin.

[0253] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said DNA modifying agent is an antitumor antibiotic selected from trabectidin, doxorubicin, mitomycin C, dactinomycin, daunorubicin, idarubicin, mitoxantrone, nemorubicin, bleomycin, and actinomycin D.

[0254] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is a DNA modifying agent selected from temozolomide, melphalan, chlormethine, pixantrone, bendamustine hydrochloride, bendamustine, polifeprosan 20 with carmustine, busulfex, fotemustine, carboplatin, treosulfan, prednimustine, melphalan flufenamide hydrochloride, thiotepa, melphalan, Delcath, apaziquone, dianhydrogalactitol, Kintara, glufosfamide, evofosfamide, Regulon, MGC-018, tinostamustine, mipicoledine, BP-C1 , pivekimab sunirine, OBI-3424, BTP-114, NC-4016, OPD-5 and LCB-71. In some embodiments, the present invention relates to the compound (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of patients with a neoplasm wherein said compound (I) as a first compound is for use in combination with at least a second compound according to the present invention, wherein said second compound is a topoisomerase inhibitor selected from class I inhibitor compounds etoposide and irinotecan, from class II inhibitors Topotecan and pixantrone, or from combinations thereof.ln some embodiments, the present invention relates to the compound (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of patients with a neoplasm wherein said compound (I) is for use in combination with at least a second compound according to the present invention, wherein said second compound is a cancer vaccine selected from GI4000 (NANT vaccine), Immutep (IMP- 321) NANT Colorectal Cancer Vaccine containing aldoxorubicin, NANT Hepatocellular Carcinoma Vaccine, NANT Squamous Cell Carcinoma Vaccine, NANT Triple Negative Breast Cancer Vaccine, NANT Melanoma Vaccine, NANT Merkel Cell Carcinoma Vaccine, NANT Head and Neck Squamous Cell Carcinoma Vaccine, NANT Pancreatic Cancer Vaccine and at least one combination thereof.

[0255] In another aspect, the present invention relates to compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) or said salt thereof is comprised at a concentration of between 1 and 10 mg / ml and administered via intravenous route either as a bolus injection or as a slow infusion in saline.

[0256] In one aspect, the present invention relates to a combination of a first compound which compound (I), or a pharmaceutically acceptable salt thereof, and a second compound selected from the group consisting of immuno-oncologic agents, immunotherapeutic agents, adoptive cell therapies, targeted agents, chemotherapeutic agents, cancer vaccines and a combination of at least two of said agents, therapies and / or vaccines, or in combination with radiotherapy, for use in the treatment of patients with a neoplasm as defined previously. In some embodiments, said second drug is a topoisomerase inhibitor selected from class I inhibitor compounds etoposide and irinotecan, from class II inhibitors topotecan and pixantrone, or from combinations thereof. In some embodiments, said second compound is comprised in an antibody-drug conjugate. In other words, an antibody-drug conjugate comprising said second compound. In some embodiments, said second compound is irinotecan comprised in sacituzumab thus forming the antibody-drug conjugate.

[0257] In a fourth aspect, the present invention relates to administration schemes, including a scheme where compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein compound (I) or said salt thereof is for use in a treatment that follows a weekly or biweekly administration of compound (I) or a pharmaceutically acceptable salt thereof for a period of 1 to 6 months.

[0258] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) or said salt thereof is for sequentially administration in a sequence of treatment starting with one or two priming intravenous injections of said compound (I) or of said salt thereof between 72h and 1h prior to the start of the administration of said second compound or said radiotherapy.

[0259] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) or said salt thereof is for sequentially administration in a sequence of treatment starting with administration of said compound (I) or of said salt by intravenous route as a priming dose between 1 and 10 mg / m2followed by a second dose of said compound (I) or of said salt thereof performed between 1h and 72h, particularly 24h or 48h, after said first priming dose at the start of the administration of said second compound or said radiotherapy, wherein further administrations of said compound (I) or of said salt thereof are equally spaced by 7 days during a period of 1 to 6 months.

[0260] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) or said salt thereof is for sequentially administration in a sequence of treatment starting with administration of said second compound or said radiotherapy, followed within betweem 1h and 192h thereafter, in particular 24h thereafter, or 168h thereafter, by intravenous injections of said compound (I) or of said salt thereof thereof, wherein further administrations of said compound (I) or of said salt thereof are equally spaced by 7 days during a period of 1 to 6 months.

[0261] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm acco according to the present invention, wherein said compound (I) or said salt thereof is for concomitant administration with administration of said second compound or said radiotherapy, wherein further administrations of said compound (I) or of said salt thereof start on either day 2 or day 9 and then take place equally spaced by 7 days during a period of 1 to 6 months.

[0262] In some embodiments, the present invention relates to the compound (I) or a water soluble organic or inorganic salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) or said water soluble organic or inorganic salt thereof is comprised in a pharmaceutical composition containg pharmaceuticaly acceptable excipients in admixture with said compound (I) or said water soluble organic or inorganic salt thereof at a concentration of between 1 and 10 mg / ml for administration via intravenous route either as bolus injection or as a slow infusion in saline.

[0263] In some embodiments, the present invention relates to the compound (I) or a water soluble organic or inorganic salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) is present as tri-sodium salt in water for injection at pH 6.5 to 7.8 at a concentration of between 1 and 10 mg / ml as a sterile solution.

[0264] In some embodiments, the present invention relates to the compound (I) or a water soluble organic or inorganic salt thereof for use in the treatment of patients with a neoplasm according to the present invention, further comprising a liposomal nanocarrier phospholipid.

[0265] In some embodiments, the present invention relates to the compound (I) or a water soluble organic or inorganic salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said liposomal nanocarrier phospholipid is cholesterol.

[0266] In some embodiments, the present invention relates to the compound (I) or a water soluble organic or inorganic salt thereof for use in the treatment of patients with a neoplasm according to the present invention, further comprising a positively charged peptide, in particular a poly-L- lysine comprising between 4 to 10 lysines, or a poly-L-arginine comprising between 4 to 10 arginines.

[0267] In some embodiments, the present invention relates to the compound (I) or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm according to the present invention, wherein said compound (I) or said salt thereof is comprised in a formulation using a miscellar or polymeric nanocarrier comprising an amphiphilic surfactant, in particlar selected from PEG, PLA, PCL, polypropylene oxide, poly-lysine, chitosan and any combination thereof, wherein said formulation is prepared as a parenteral or depot formulation, and wherein said compound (I) or said salt thereof is present at a concentration of between 1 and 10 mg / mL or of between 10 and 100 mg per g of nanocarrier.

[0268] The present invention also relates to a use of a compound (I) as defined herein for the manufacture of a drug, a medicament, or a pharmaceutical composition for treating a neoplasm.

[0269] Properties of compound (I)

[0270] In one or more embodiments, compound (I) is able to protect against fibrosis induced by neoplasm or administration of at least one anti-neoplastic agent. In one or more embodiments, compound (I) is able to overcome TLR4 overexpressing induced immunosuppression in cancer cells. Thanks to its capacity to reduce pro-immunosuppression factors, the compound of the invention may be used for the treatment of neoplasms characterized by undesirable excessive tumor immunosuppression.

[0271] In one or more embodiments, compound (I) is able to protect against neovascularization damages. Thanks to its capacity to reduce pro-angiogenic factors, the compound of the invention may be used for the treatment of cancers characterized by undesirable excessive angiogenesis.

[0272] In one or more embodiments, compound (I) is able to protect against fibrotic damages. Thanks to its capacity to reduce pro-fibrotic factors, the compound of the invention may be used for the treatment of cancers characterized by undesirable excessive fibrosis.

[0273] In one or more embodiments, compound (I) is able to overcome TLR4 overexpression induced chemoresistance in cancer cells.

[0274] In one or more embodiments, compound (I) is for use in a method for the treatment of inflammation induced by chemotherapy and / or radiotherapy (releasing TLR4 inducing DAMPs and PAMPs).

[0275] In one or more embodiments, compound (I) is configured to cause direct cell death of myd88 mutated tumors.

[0276] TLR4 overexpressing indications

[0277] In one or more embodiments, the neoplasm is associated with or resulting from chronic nonresolved inflammation mediated by TLR4 overexpression.

[0278] In one or more embodiments, the neoplasm is an head & neck cancer, a renal cancer, a soft tissue sarcoma, an uterus cancer, a renal cancer, a gastric cancer, a liver cancer, a mesothelioma, a pleural cancer, a sarcomatoid carcinoma, a peritoneal carcinomatosis, a pericardial cancer, an osteosarcoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), an ovarian cancer, a breast cancer, particularly a triple negative breast cancer, a pancreatic cancer, a myelofibrosis or a desmoid tumors.

[0279] In one or more embodiments, the fibrotic a neoplasm is a myelofibrosis, a desmoid tumor, a hepatocellular carcinoma, or a mesothelioma.

[0280] The invention is not limited by the type of neoplasm characterized by high expression of tolllike receptor 4 to be treated. Indeed, any neoplasm characterized by high expression of tolllike receptor 4 that is treatable (e.g., for which signs or symptoms of the disease are ameliorated upon treatment) via administration of compound (I) can be treated in an improved and more effective manner using compositions and methods of the invention.

[0281] Methods of treatment

[0282] The present invention further relates to a method for treating a neoplasm in a patient, the method comprising administering in the subject in need thereof an effective amount of compound (I) or a pharmaceutically acceptable salt thereof as defined herein.

[0283] In some embodiments, the invention provides methods for treating or delaying the progression of cancer in a patient comprising administering to the patient a therapeutically effective amount of compound (I).

[0284] In some embodiments, the invention provides methods for treating or delaying the progression of cancer in an individual comprising administering to the individual an effective amount of compound (I).

[0285] In some embodiments, the treatment results in a sustained response in the individual after cessation of the treatment. The methods described herein may find use in treating conditions where enhanced immune response is desired. Also provided herein are methods of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of compound (I).

[0286] The invention provides, in one embodiment, a method of reducing pro-fibrotic factors via inhibition of TLR4.

[0287] The invention provides, in one embodiment, a method of reducing pro-angiogenic factors via inhibition of TLR4.

[0288] The invention provides, in one embodiment, a method of reducing pro-metastatic factors via inhibition of TLR4.

[0289] The invention provides, in one embodiment, a method of reversing dendritic cell exhaustion via inhibition of TLR4.

[0290] The invention provides, in one embodiment, a method of reversing Natural Killer cell exhaustion via inhibition of TLR4.

[0291] In some embodiments, the invention provides a method of reversing CD8 T cell exhaustion (e.g. maintaining functionality of T cells exposed to excessive tumor related antigens) via inhibition of TLR4. In some embodiments the invention provides a method of shifting macrophage polarization from macrophage type M2 pro-tumoral to macrophage type M1 anti tumoral via inhibition of TLR4

[0292] Thus, the invention provides, in one embodiment, a method of reducing pro-tumor CD4 T cells via inhibition of TLR4.

[0293] TLR4 mediated resistance

[0294] In some embodiments, the individual has cancer that is resistant (e.g., has been demonstrated to be resistant) to one or more other forms of anti-cancer treatment (e.g., chemotherapy, immunotherapy, etc.). In some embodiments, resistance includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance includes progression of the cancer during treatment with chemotherapy. In some embodiments, resistance includes cancer that does not respond to traditional or conventional treatment with a chemotherapeutic agent. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.

[0295] In some embodiments, the present invention relates to a compound (I) as defined herein for use for treating a cancer in a subject resistant to chemotherapy.

[0296] In some embodiments, the present invention relates to a compound (I) as defined herein for use for treating a cancer in a subject resistant to target agent therapy.

[0297] In some embodiments, the present invention relates to a compound (I) as defined herein for use for treating a cancer in a subject resistant immunotherapy.

[0298] In some embodiments, the present invention relates to a compound (I) as defined herein for use for treating a cancer in a subject resistant to radiotherapy.

[0299] In some embodiments, the present invention relates to a compound (I) as defined herein for use for treating a cancer in a subject resistant to vaccine therapy.

[0300] In some embodiments, the present invention relates to a compound (I) as defined herein for use for treating a cancer in a subject resistant adoptive cell therapy.

[0301] Methods of combination treatment

[0302] Combination with immunotherapy In one or more embodiments, this other treatment is able to circumvent an immunosuppressive microenvironment of the neoplasm.

[0303] In one or more embodiments, the immuno-oncologic agents and / or the immunotherapeutic agents target is selected from CTLA-4 protein, PD-1 protein, PD-L1 protein, PD-L2 protein and / or at least a combination thereof. So, the agents, alone or as a combination, can target, preferably block, one or more proteins.

[0304] In one or more embodiments, the immuno-oncologic agents and / or the immunotherapeutic agents targeting TLR4 protein, PD-1 protein, PD-L1 protein, PD-L2 protein and / or at least a combination thereof, are monoclonal or bispecific antibodies.

[0305] In one or more embodiments, the antibodies against PD-1 are selected from cemiplimab, nivolumab, pembrolizumab, SHR1210, sintilimab, spartalizumab, tislelizumab, pidilizumab, BCD-100, toripalimab, PF-06801591 , AB122, AK105, AMG 404, BCD-100, Bl 754091 , F520, HLX10, HX008, JTX-4014, LZM009, MEDI0680, MGA012, Sym021 , TSR-042, PSB205, MGD019, MGD013, AK104, XmAb20717, RO7121661 , CX-188, serplulimab, zimberelimab, dostarlimab, nivolumab, prolgolimab, Biocad, geptanolimab, spartalizumab, QL-1604, toripalimab with albumin-bound paclitaxel, nofazinlimab, pimivalimab, IBI-318, AZD-2936, AZD-7789, EMB-02, RG-6139, HX-009, MVR-T3011, 609A, HLX-10 with cetuximab, RG-7769, HPV-E6-TCR-T cells, ezabenlimab, rosnilimab, LVGN-3616, TQB-2868, SYN-125, Sofusa anti-PD-1 , Yinkang-001 , AMG-256, GNR-051 , BAT-1308, ONO-4685, LBL-015, LNL-005, zeluvalimab, F-520, CC-90006 and at least one combination thereof.

[0306] In one or more embodiments, the antibodies against PD-L1 are selected from atezolizumab, durvalumab, avelumab, FS118, BCD-135, BGB-A333, CBT-502, CK-301 , CS1001 , FAZ053, HLX20, KN035, MDX-1105, MSB2311 , SHR-1316, TG-1501 , ZKAB001 , INBRX-105, MCLA- 145, KN046, M7824, LY3415244, sugemalimab, avelumab, socazolimab, adebrelimab, bintrafusp alfa, TQB-2450, KN-046, SHR-1701 , IBI-318, HB-0036, HLX-301 , PM-8002, PM- 8001 , PD-L1 t-haNK, ADG-104, LP-002, NANT Hepatocellular Carcinoma Vaccine, danburstotug, NM-01 , GR-1405, NANT Triple Negative Breast Cancer Vaccine, NANT Melanoma Vaccine, cosibelimab, MCLA-145, SAR-445710, PM-1022, SGN-PDL1V, dual anti- HER2 / anti-PD-L1 CAR-T cell therapy, GNC-035, PF-07257876, CYTO-102, Y-101 D, SKB- 337, TQB-2858, ATG-101 , IMM-2510, GS-19, LBL-024, QL-301, Q-1802, TST-005, PM-1003, IBI-323, RC-98, HB-0025, IBI-322, MT-6402, BCD-135, CDX-527, ABL-503, anti-PD-L1 CSR T-cell therapy (cancer), HLX-20, ND-021 , lodapolimab.

[0307] In one or more embodiments, the immuno-oncology or immunotherapeutic agent is selected from an anti-LAG3 and anti-CD223 agonist such as but not limited to ieramilimab, EMB-02, IBI-110, FS-118, IBI-323, encelimab, RG-6139, LBL-007, IMP-731 , relatlimab, Sym-022, HLX- 26, tebotelimab

[0308] In one or more embodiments, the immuno-oncology or immunotherapeutic agent is selected from a stimulator of STING (stimulator of interferon genes protein) or STING agonist such as but not limited to NBTXR-3, ADU-S100, IMSA-101 , exoSTING, ulevostinag, idronoxil, Noxopharm, TAK-500, ONO-7914, DN-015089, SNX-281 , BI-1387446, TAK-676, HG-381 , GSK-3745417, E-7766, SYN-STING, SB-11285, VB-85247, XMT-2068, XMT-2175

[0309] In one or more embodiments, the immuno-oncologic agents and / or the immunotherapeutic agents targeting PD-L1 protein are selected from small molecules inhibiting transcription PD- L1 genes, small molecules inhibiting translation of PD-L1 mRNA, and small molecules antagonist of PD-L1 protein.

[0310] In one or more embodiments, the immuno-oncology or immunotherapeutic agent is selected from an anti-LAG3 and anti-CD223 agonist such as but not limited to ieramilimab, EMB-02, IBI-110, FS-118, IBI-323, encelimab, RG-6139, LBL-007, IMP-731 , relatlimab, Sym-022, HLX- 26, tebotelimab.

[0311] In one or more embodiments, the immuno-oncology or immunotherapeutic agent is selected from a stimulator of STING (stimulator of interferon genes protein) or STING agonist such as but not limited to NBTXR-3, ADU-S100, IMSA-101 , exoSTING, ulevostinag, idronoxil, Noxopharm, TAK-500, ONO-7914, DN-015089, SNX-281 , BI-1387446, TAK-676, HG-381 , GSK-3745417, E-7766, SYN-STING, SB-11285, VB-85247, XMT-2068, XMT-2175.

[0312] In one or more embodiments, the immunotherapeutic agent is a CD3 T cell engaging bispecific antibody (BiTE).

[0313] In one embodiment, the BiTE is Tarlatamab (anti DLL3).

[0314] Combination with radiotherapy

[0315] The present invention provides methods for administering compositions and methods of the invention with (e.g., before, during, or after) radiation therapy. The invention is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to a subject. For example, the subject may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In some embodiments, the radiation is delivered to the subject using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife.

[0316] The source of radiation can be external or internal to the animal. External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animals. Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive. Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.

[0317] In one or more embodiments, the method for the treatment of neoplasm comprises an administration of ionizing radiations. In other words, the treatment of neoplasm involves administration of compound (I) and radiotherapy. The frequency of administration of compound (I) is one, two and respectively three weekly injections of compound (I) adapted to the radiotherapy frequency to prevent radiation induced fibrosis of the target organ. These combination schedules are based on human and animal pharmacokinetic data combined with decreased fibrosis data obtained in compound (I) treated dogs in a cardiac reperfusion model. Compound (I) is able to prevent induction of fibrosis in radiation exposed healthy tissue.

[0318] Combination with chemotherapeutics

[0319] In one or more embodiments, the chemotherapeutics agent is a DNA modifying agent (platinum based or antitumor antibiotics) or a topoisomerase inhibitor.

[0320] In one or more embodiments, the DNA modifying agent is platinum based and selected from lobaplatin, nedaplatin, miriplatin hydrate, lobaplatin, cisplatin, oxaliplatin, eptaplatin, heptaplatin, carboplatin, dicycloplatin.

[0321] In one or more embodiments, the DNA modifying agent is an antitumor antibiotics selected from trabectidin, doxorubicin, mitomycin C, dactinomycin, daunorubicin, idarubicin, mitoxantrone, nemorubicin, bleomycin, actinomycin D.

[0322] In one or more embodiments, the DNA modifying agent is selected from temozolomide, melphalan, chlormethine, pixantrone, bendamustine hydrochloride, bendamustine, polifeprosan 20 with carmustine, busulfex, fotemustine, carboplatin, treosulfan, prednimustine, melphalan flufenamide hydrochloride, thiotepa, melphalan, Delcath, apaziquone, dianhydrogalactitol, Kintara, glufosfamide, evofosfamide, Regulon, MGC-018, tinostamustine, mipicoledine, BP-C1 , pivekimab sunirine, OBI-3424, BTP-114, NC-4016, OPD-5, LCB-71. In one or more embodiments, the topoisomerase inhibitor is selected from class I inhibitor etoposide, irinotecan, or class II inhibitor topotecan, pixantrone.

[0323] Combination with targeted agents

[0324] In one or more embodiments, the targeted agent is a kinase inhibitor.

[0325] In one or more embodiments, the kinase inhibitor agent is an anti-angiogenic agent.

[0326] In one or more embodiments, the anti-angiogenic agent is selected from CerRx, bevacizumab, conbercept, pegaptanib, ranibizumab, endostar, axitinib, vandetanib, sorafenib, ponatinib, cabozantinib, nintedanib, rivoceranib, fruquintinib, ramucirumab, surufatinib, catequentinib, pyrotinib dimaleate, lenvatinib, sunitinib, regorafenib, aflibercept, M-23, Stainwei Biotech, hVEGF26-104 / RFASE, SIM -010603, HZB-1006, HB-0025, LYN-00101 , AMC-303, EVT-801 , HLX-06, JY-025, ramucirumab biosimilar, SYHA-1813, FAK / VEGFR3 inhibitors, CureFAKtor Pharmaceuticals, CX-1003, BMS-817378, ST-1898, CYC-116, SOMCL-15-290, HLX-12, Chia Tai Tianqing Pharmaceutical, EOC-317, ICP-033, APL-102, ningetinib tosilate, altiratinib, vandetanib, FN-1501 , hydroxychloroquine + sorafenib, conbercept, PAN-90806, fenretinide, MP-0250, dilpacimab, navicixizumab, IBI-302, BI-836880, HA121-28, ilorasertib, TT-00420, tesevatinib, MG-D-1609, NANT Hepatocellular Carcinoma Vaccine, KD-035, VXM-01 , olinvacimab, gentuximab, BR-55, AK-109, brivanib alaninate, OTSGC-A24, telatinib, YSKB- 1001 , pazopanib in combination with pembrolizumab , AL-2846, AL-8326, ENMD-2076, pamufetinib, emvododstat, muparfostat sodium, sevacizumab, GNR-011 , cediranib, famitinib L-malate, zanzalintinib, ibcasertib, lucitanib hydrochloride, sitravatinib, vorolanib, dovitinib and at least one combination thereof.

[0327] In one or more embodiments, the targeted agent is a proteasome inhibitor.

[0328] In one or more embodiments, the targeted agent is an epigenetic modulator.

[0329] In one or more embodiments, the targeted agent is a PARP inhibitor.

[0330] Combination with adoptive cell therapies

[0331] In one or more embodiments, the adoptive cells are selected from CAR-T cells, NK cells, Engineered T cells, Tumor-Infiltrating Lymphocytes and at least one combination thereof.

[0332] In one or more embodiments, the CAR-T cells are selected from the group consisting of tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, and other CAR-T therapies targeting CD19, BCMA, CD22, CD20, CD30, GD2, HER2, EGFR, mesothelin, and PSMA, including those in late-stage development for solid tumors. These CAR-T cells also include the ones expressing anti-CTLA- 4 and anti-PD-1 antibodies.

[0333] Combination with cancer vaccines

[0334] In one or more embodiments, the cancer vaccine comprises one or several antigen.

[0335] In one or more embodiments, the cancer vaccine is based on mRNA.

[0336] In one or more embodiments, the antigen is a pathogen-derived antigen or tumor derived antigen.

[0337] In one or more embodiments, the cancer vaccine is selected from GI4000, Immutep NANT Colorectal Cancer Vaccine containing aldoxorubicin, NANT Hepatocellular Carcinoma Vaccine, NANT Squamous Cell Carcinoma Vaccine, NANT Triple Negative Breast Cancer Vaccine, NANT Melanoma Vaccine, NANT Merkel Cell Carcinoma Vaccine, NANT Head and Neck Squamous Cell Carcinoma Vaccine, NANT Pancreatic Cancer Vaccine and at least one combination thereof.

[0338] Combination with hormonal therapy

[0339] In one or more embodiments, the anti-cancer agent targets hormone receptors or hormone synthesis pathways to inhibit the growth of hormone-dependent tumors (e.g., Tamoxifen for estrogen receptor-positive breast cancer, Abiraterone for prostate cancer).

[0340] Combination with gene therapy

[0341] In one or more embodiments, the anti-cancer agent is a gene therapy.

[0342] In one or more embodiments, the gene therapy is an oncolytic virus genetically engineered that selectively infect and kill cancer cells (e.g., Talimogene laherparepvec for melanoma).

[0343] In one or more embodiments, the gene therapy is CRISPR-Cas9 technology to correct genetic mutations or target cancer-specific genes.

[0344] Combination with nanoparticle

[0345] In one or more embodiments, the anticancer agents are delivered via nanoparticles to deliver drugs, genes, or other therapeutic agents directly to cancer cells, improving efficacy and reducing side effects (e.g., Liposomal doxorubicin). Treatment regimen and schedule

[0346] In one or more embodiments, compound (I) and the other therapy are sequentially administered. In other words, compound (I) is not administered at the same time as the anti- neoplastic agent. Thus, compound (I) is administered before or after the anticancer agent as shown in Table B.

[0347] In one or more embodiments, administration of compound (I) comprises a priming dose which is administered before the administration of the other therapy.

[0348] Schedules 1 to 4 in Table B describe a priming dose schedule followed by a concomitant second compound (I) IV injection with the other treatment. Further Compound (I) injections are equally spaced by 7 days. The minimum number of weekly spaced injections of compound (I) is 7 in the table examples. In the case of longer duration of other treatment, the number of compound (I) injections are adapted to cover the entire other treatment period.

[0349] Schedule 5: describes a priming dose of compound (I) administered 1h to 6h before the other treatment on day 1. Further compound (I) injections are equally spaced by 7 days. The minimum number of weekly spaced injections of compound (I) is 7 in the table examples. In case of longer duration other treatment, the number of compound (I) injections are adapted to cover the entire other treatment period.

[0350] In one or more embodiments, administration of compound (I) comprises a subsequent parental dose which is administered after administration of the other therapy.

[0351] Schedules 6 and 7 describe the start of other treatment 1 day, and respectively 7 days, before the first IV dose of compound (I). Subsequent compound (I) injections are equally spaced by 7 days. The minimum number of weekly spaced injections of compound (I) is 7 in the table examples. In the case of longer duration of other treatment, the number of compound (I) injections are adapted to cover the entire other treatment period.

[0352] Other schedules using a priming dose of compound (I) in Table B and other combinations schedules are possible depending on the other treatment. Table B: different sequential treatment schedules

[0353] IV: intravenous

[0354] Compound (I) priming dose and follow-up weekly doses are adapted to the antineoplastic treatment duration of the selected other treatment.

[0355] In one or more embodiments, compound (I) and the at least one anticancer agent are simultaneously administered. In other words, the compound (I) is administered at the same time as the anticancer agent.

[0356] Schedules 8 and 9 describe other treatment and compound (I) simultaneously started on day 1 day followed by a second dose of compound (I) IV either on day 2 or on day 8. Further compound (I) IV injections are equally spaced by 7 days. The minimum number of weekly spaced injections of compound (I) is 7 in Table B examples. In case of longer duration with the other treatment, the number of compound (I) injections are adapted to cover the entire other treatment period.

[0357] Administration and delivery

[0358] In another aspect, the invention provides a kit comprising i) a compound (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound (I) or a pharmaceutically acceptable salt thereof; ii) optionally a pharmaceutical solution to dissolve or dilute i), and iii) instructions to administer said compound (I) or said pharmaceutically acceptable salt thereof, or said pharmaceutical composition comprising the compound (I) or a pharmaceutically acceptable salt thereof.

[0359] Administration

[0360] The administration route can be topical, transdermal, oral, rectal, sublingual, intranasal, intrathecal, intratumoral or parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous and / or intradermal). Preferably, the administration route is oral or parenteral. More preferably, the administration route is intravenous when it concerns the treatment of neoplasm. The pharmaceutical composition is adapted for one or several of the above-mentioned routes. The pharmaceutical composition is preferably administered by injection or by intravenous infusion of suitable sterile solutions, or in the form of liquid or solid doses via the alimentary canal. More preferably, the pharmaceutical composition is administered by an injection route.

[0361] In one or more embodiments, the compound (I) is administered as a parenteral formulation in a single intravenous dose, preferably as an injection of 1.5 to 5.0 mg / m2once per week or less.

[0362] In one or more embodiments, compound (I) is administered as an oral formulation, preferably 5 to 50 mg / m2daily once or twice.

[0363] In one or more embodiments, compound (I) administered as an oral formulation at an amount of from 0.2 mg / kg body weight to 2 mg / kg body weight in concomitance and also on a sequential basis with its combination agent.

[0364] Formulation

[0365] In one or more embodiments, composition comprising compound (I), or a pharmaceutically acceptable salt thereof, for use in a method for the treatment of neoplasm, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated as solutions in pharmaceutically compatible solvents or as emulsions, suspensions or dispersions in suitable pharmaceutical solvents or vehicles, or as pills, tablets or capsules that contain solid vehicles in a way known in the art. Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. Formulations for rectal administration may be in the form of a suppository incorporating the active ingredient and carrier such as cocoa butter, or in the form of an enema. Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient. Nanomaterial formulations such as defined by the International Organization for Standardization (ISO) comprising nanoparticles as objects with dimensions of 1-100 nm and up to 1000 nm, because the physicochemical properties of the material at this scale differ from the bulk material. The nanocarriers are described in the review from Chariou P, ACS Nano. 2020 Mar 24; 14(3): 2678-2701 , Nanocarriers for the Delivery of Medical, Veterinary, and Agricultural Active Ingredients. Such nanocarriers can be in the form of liposomal nanocarriers 30 to 1000 nm in diameter. Biocompatible and biodegradable polymeric biomaterials such as polyacrylate (PAL), PEG, polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polyesters, chitosan polymers and polyurethanes can be used for the nanocarrier formulation of compound (I) and their pharmaceutically acceptable salts. Hydrogels as cross-linked hydrophilic polymers with a high-water retention capacity may be used as a reservoir of the active ingredient present at the core of the hydrogel for a controlled release of the active ingredient regulated by the physical properties of the hydrogel matrix (porosity, stiffness and swelling capacity). Micellar nanocarriers composed of amphiphilic surfactant molecules that spontaneously aggregate into spherical vesicles in an aqueous environment can be advantageously used with compound (I) and its salts, leading to small size polymeric micelles (20 to 80 nm) composed of PEG, PLA, PCL, polypropylene oxide, poly-l-lysine, or combinations of the above. Inorganic carriers such as mesoporous silica nanocarriers may be loaded with compound (I) and its pharmaceutically acceptable salts.

[0366] Every such formulation can also contain other pharmaceutically compatible and nontoxic auxiliary agents, such as, e.g. stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring substances. The formulations of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof. The pharmaceutical compositions are advantageously applied by injection or intravenous infusion of suitable sterile solutions or as oral dosage by the digestive tract. Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. (Guidelines for the administration of chemotherapy for malignant disease, v2.1.0 https: / / www.england.nhs. uk / mids-east / wp-content / uploads / sites / 7 / 2018 / 04 / guidelines- administration-chemotherapy-for-malignant-disease-v2-1-0.pdf)

[0367] EXAMPLES

[0368] The following examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0369] The following examples aim to demonstrate the efficacy of compound (I) in targeting TLR4- overexpressing tumors by determining its effect on multiple immunosuppressive, angiogenic, and fibrotic markers. Additionally, these examples illustrate the significance of combining compound (I) with relevant agents, particularly highlighting the importance of well-designed combination schedules.

[0370] The compound (I), was obtained as described in WO 95 / 014026 (biologically derived compound (I)) and WO 2008 / 059035 (synthetic compound (I)).

[0371] The biological derived compound (I) was obtained from Escherichia coli submitted to a mild alkaline pH at 37°C for several hours and the extract was acidified to cleave the dephosphorylated triacylated disaccharide. Compound (I) was extracted using a mixture of organic solvents and purified by multiple chromatography including ion-exchange and reverse phase preparative HPLC. The pure fractions were evaporated to dryness and compound (I) was solubilized in water for injection with the addition of dilute sodium hydroxide to lead to compound (I) sodium salt in water for injection.

[0372] Synthetic compound (I) was obtained in 17 steps as described in WO 2008 / 059035.

[0373] Preparation of compound (I) by chemical synthesis - Overview: the synthetic protocols are described in detail in PCT Application WO 2008 / 059035 A2 on pages 32 to 41. The purification protocol for the final product is described on page 52 — 53 of cited patent. Compound numbers used are those of the patent. Briefly, the allyl glycoside of D-glucosamine carrying a 3-O-benzyl group, a 4,6-O-benzylidene acetal and a N-trichloroethoxycarbonyl group (compound 3b, 4 steps from commercial D-glucosamine) is used as the precursor of both the glycosyl donor (compound 7b) and the glycosyl acceptor (compound 10b). Regioselective, reductive opening of the benzylidene acetal provides either the corresponding 6-O-benzyl derivative (compound 4b) or the 4-O-benzyl derivative (compound 8b). Compound 4b is then phosphorylated at the free 4-OH position, the allyl glycoside cleaved and the anomeric position activated as a trichloroacetimidate to provide glycosyl donor 7b. The amino group of compound 8b is then deprotected and acylated with (R)-3-benzyloxytetradecanoic acid to give the glycosyl acceptor carrying the free 6-OH group. The glycosidic bond formation is achieved by the reaction of 7b with 10b under Lewis acidic conditions, to provide the protected disaccharide 11a. The amino group of the non-reducing unit of 11a is then deprotected to amine 12d which is then acylated with (R)-3-dodecanoyloxytetradecanoic acid, to give the core structure of compound (I) as an allyl glycoside, compound 13b. The allyl glycoside is hydrolyzed, to give 14b, and the anomeric OH group phosphorylated using tetrabenzylpyrophosphate, to afford glycosyl phosphate 15b. The benzyl protecting groups are then cleaved by hydrogenolysis, to afford compound compound (I) in the fully protonated, acidic form. The final product is then purified by preparative reverse phase HPLC, and then submitted to a salt exchange on reverse phase support to form the sodium salt (pH ~7.4) and eluted using a water and isopropanol as described in the cited patent. After evaporation of the organo-aqueous solvent at low pressure (Rotavap), the final dried product is stored at 2 to 8°C.

[0374] Aliquots of the solid compound (I) are solubilized in DMSO or in water for injection at 1 mg / mL and respectively at 10 mg / mL. The compound (I) water solutions are further submitted to a sterile filtration using a 0.2 pm polysulfone filter and stored at 2 to 8°C.

[0375] Example 1 - Dose response antagonistic inhibition of TLR4 by compound (I)

[0376] Rationale

[0377] Current state of the art knowledge does consider compound (I) as TLR4 agonist. Below results are proving the contrary.

[0378] Material and method

[0379] HEK-293 cells stably expressing huTLR4 / MD-2 / CD14 (HEK-Blue™ hTLR4 cells) were obtained from InvivoGen as described in Pugin J, Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock, Blood, 2004, 104(13), 4071-4079, DOI 10.1182 / blood- 2003-04-1290.

[0380] TLR4-HEK293 cells expressing more than 90% surface TLR4 were used for the stimulation assays. On the day of the assay, TLR4-HEK293 cells were collected by trypsin treatment, resuspended in Dulbecco modified Eagle medium (DM EM; Gibco) containing 2% FCS and distributed into 96-well plates at the concentration of 60.000 cells per well.

[0381] Cell Stimulation Assays: HEK-Blue™ hTLR4 cells were seeded into a 96-well plate at the density of 6 x 104cells / well. Compound (I) was added to cells at the concentrations of 0, 0.013, 0.04, 0.12, 0.37, 1.1 , 3.3, 10 pg / mL diluted in culture media containing 5% FCS. E. coli K12 LCD25 LPS was used as a positive control for TLR4 stimulation. Markers of cell activation was alkaline phosphatase for HEK-Blue™ hTLR4 cells, with levels measured according to the protocol of the manufacturer. Cell stimulation experiments were performed in triplicates, and each experiment was repeated three times.

[0382] HEK-Blue™ hTLR4 were incubated with a fixed amount of E. coli K12 LCD25 LPS (10 ng / mL). Thirty minutes prior to LPS addition, compound (I) was added to cells at different concentrations (dose response ranging from 10 g / mL up to 1.37 ng / mL, diluted 1 :3 in culture media).

[0383] As control, compound (I) was added to cells without LPS. Alkaline phosphatase was measured in conditioned supernatants after 24h of incubation time using an alkaline substrate in a colorimetric assay. Compound (I) showed evidence of LPS antagonism in this assay. This is a representative experiment of three experiments with similar results.

[0384] Results

[0385] Table 1 : Compound (I) is a LPS antagonist in TLR4 / MD-2 / CD14(+) HEK293 Blue cells™. Calculated IC50 value of compound (I) is 26 x 10'9M (26 nM) and compound (I) is a TLR4 antagonist.

[0386] Example 2 - Compound (I) anti-proliferative effect against TLR4 overexpressing PDX models (CYPRE 3D)

[0387] Aim

[0388] The aim of this example is to demonstrate that compound (I) exhibits a stronger anti-tumor effect correlated with higher levels of TLR4 expression.

[0389] Model introduction

[0390] Multicellular tumor spheroids could lead to a cancer therapy breakthrough by modeling avascular tumor nodules in the laboratory. They could improve the predictive capacity of drug screening platforms, relieving cost and ethics concerns of human and animal trials. Primary cells and tumor explants address some concerns associated with cell lines, primarily introducing more tumor complexity. Primary cells are not pre-conditioned to the in vitro environment prior to drug screening, and the chances of mutations are greatly decreased. Tumor explants are even better at mimicking the tumor microenvironment as the cells contain some of the native extra-cellular matrix proteins as well as various support cells. In addition, using patient-derived cells and explants opens the door to personalized therapy, where drug and drug combinations can be pre-screened in vitro to determine efficacy prior to patient administration. While primary cells and tumor explants provide more physiologically relevant conditions compared to cell lines, they might not always be practical as they lack the reproducibility and verification potential of using cell lines.

[0391] Three-dimensional (3D) patient-derived xenograft (PDX) tumor models are increasingly being recognized as superior tools for studying tumor biology and developing cancer therapeutics compared to conventional 2D cell-derived xenograft (CDX) tumor models. PDX models maintain the histological and genetic heterogeneity of the original tumor, unlike CDX models, which typically represent a single clonal population of cells. PDX models also better recapitulate the tumor microenvironment, including stromal cells, extracellular matrix, and vasculature, and provide more accurate preclinical data for drug efficacy and toxicity testing. Furthermore, PDX models offer the possibility of personalized medicine by enabling the testing of patient-specific treatments. By using 3D PDX tumor models, researchers can more accurately model the complexity of human tumors and improve the translatability of preclinical studies to the clinic, ultimately leading to better treatment outcomes for cancer patients. Material and method

[0392] A hydrogel-grown spheroid 3D in vitro assay platform was used to evaluate compound (I) effect against a panel of patient-derived xenograft PDX tumor cell lines in an ex vivo tissue matrix hydrogel microenvironment with accompanying stromal (fibroblasts) and immune cell compartments.

[0393] The patterning technology engineered 3D tumor assays was assessed against PDX ImmunoOncology panel (PDX + HDF + PBMCs) in standard 96-well plates for compound (I) efficacy screening using a 7-dose format including the following compound (I) concentrations 0, 0.412, 1.235, 3.704, 11.111 , 33.333, and 100 pM.

[0394] PDX T umor cells were expanded in 2D cell culture and then grown in 3D hydrogels at a density of approximately 0.5 million cells per mL hydrogel along with human dermal fibroblasts coembedded at a density of 0.4 million cells per mL. T umor-Fibroblast co-cultures were grown to sufficient size and treated with pre-activated (CD3 / CD28) PBMCs along with compound (I). PBMCs were sourced from healthy donors at the University of Freiburg in Germany. Cell culture medium included RPMI 1640 supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin / Streptomycin, and 10 ng / mL lnterleukin-2 (IL2).

[0395] Spheroid growth determination

[0396] Images of spheroids in each well were captured on a standard brightfield microscope and analyzed on Imaged software. In Imaged, the area around each sphere was traced and quantified for each timepoint and later compared with its day 0 area to determine percent growth (%) growth. This practice normalized the data irrespective of neurosphere size variability. 3D growth measurements and compound (I) drug testing: Brightfield images of neurospheres were captured using an EVOS Microscope (Thermo Fisher Scientific) and individual spheres were tracked over time. Spheroid size was quantified on Imaged software by taking the sphere area.

[0397] Percent growth of each sphere was calculated by quantifying its current day size (i.e. , day n) and relating to its size at day 0 according to the following formula: [% growth = (Areadayn- Area ayo) / Area ayo]. In this way, each individual sphere was normalized to its initial day 0 size and average % growth could be determined for each timepoint and experimental condition. Neurospheres in VersaGel were exposed to varying concentrations of compound (I) (0, 5, 25, 125, and 625 pmol / L) in 0.5% DMSO and neurosphere media. Drug was administered on day 0 and day 3, followed by standard neurosphere culture up to 14 days.

[0398] Readout was performed using a high content imaging to quantify tumor growth as endpoint. Results

[0399] A series of cancer samples from patients selected based on TLR4 expression were assessed for their response to compound (I) at different concentrations. Read outs were Tumor Growth Inhibition (TGI in %).

[0400] Results following 3D tumor organoids exposure to a dose range of compound (I) are below (see additionally Figure 13):

[0401] • Tumor type: LIXFC2050 (Cholangiocarcinoma) with TLR4 expression level at 0.22 RPKM (Reads per kilo base per million mapped reads), TGI [%]: 4.4,

[0402] • Tumor type: OVXF899 (Ovarian cancer) with TLR4 expression level at 2.8 RPKM, TGI [%]: 4.8

[0403] • Tumor type: RXF1781 (Renal Cell Carcinoma) with TLR4 expression level at 6.16 RPKM, TGI [%]: 15

[0404] • Tumor type: MEXF622 (Melanoma) with TLR4 expression level at 7.55 RPKM, TGI [%]: 31.8

[0405] Compound (I) demonstrates divergent efficacy profiles contingent on tumor types with varying TLR4 expression levels. In instances of basal / low TLR4 expression, for example LIXFC2050 and OVXF899, compound (I) exhibits moderate potency with EC50 values of 41 pM and 10.1 pM respectively, alongside moderate tumor growth inhibition (TGI) ranging from 4.4% to 4.8%. Conversely, in tumors featuring high / overexpression of TLR4, for example RXF1781 and MEXF622), compound (I) displays enhanced efficacy with diminished EC50 values of 20.1 pM and 14.5 pM, and substantially heightened TGI percentages ranging from 15% to 31.8%.

[0406] Example 3 - Compound (I) in vivo efficacy against in humanized immunocompetent models of metastatic melanoma (A375 tumor overexpressing TLR4)

[0407] Material and method

[0408] An in vivo efficacy study has been performed by implanting A-375 human xenograft tumors into immunocompromised mice with human immune system.

[0409] Human CDX A-375 tumor cell line has been selected based on its rather elevated expression of toll like receptors especially TLR4 and its rather moderate (if not poor) response to immunotherapies such as anti-PD-L1 (mAb) and anti-PD-1 (mAb). A-375 is a cell line exhibiting epithelial morphology that was isolated from the skin of a 54-year-old, female patient with malignant melanoma. This cell line is used in immuno-oncology research and characterized as a highly invasive and spontaneously metastatic human malignant melanoma cell line. Female NXG mice aged 5-7 weeks were used for the study. These were purchased from Janvier Laboratoires (France) and had 7 days acclimatization period. Animals were identified by tail mark and housed in IVC cages (up to 5 per cage). All animals were allowed free access to a standard certified commercial diet and sanitized water during the study. The holding room was maintained under standard conditions: 18-24°C, 55-70% humidity and a 12h light / dark cycle. Female mice, 23-28 g, were implanted subcutaneously (s.c.) with A-375 tumor cells (1 x 107in Matrigel) on the flank. When tumors reached approximately 50 mm3animals were assigned to treatment groups and donor PBMCs injected.

[0410] Efficacy of compound (I) alone and in combination with checkpoint inhibitor anti-PD1 (pembrolizumab or atezolizumab?) on A-375 human tumor cell lines xenograft models on NXG mice has started when tumor had reached a size of 50 mm3size and after human PBMC engraftment. Treatment with compound (I) and combination of anti-PD-1 was started at day 7 following PBMC injection. A biweekly administration was performed via IV route for compound (I) and via IP route for mAbs. Injections of compound (I) and pembrolizumab were carried out separately starting with compound (I) followed by pembrolizumab (glass vial, white powder resolubilized with 0.9% sodium chloride for injection) with a 60-minute interval. The treatment was continued for 21 days. Efficacy of treatment was assessed by measuring tumor size twice per week over a period of 21 days.

[0411] This protocol used in this study has been approved by the Animal Welfare and Ethical Review Committee, and all procedures were carried out under the guidelines of the Animal (Scientific Procedures) Act 1986.

[0412] Treatment Scheme

[0413] The following treatment scheme is summarized in Table 2 hereunder. Vehicle is 0.9% NaCI and compound (I) formulation is 0.9% NaCI at pH 7.2. Treatments were done by the intravenous route, bolus, twice per week (BIW). Pembrolizumab was injected via the intraperitoneal route (IP).

[0414] Table 2: Experimental design, schedule and group constitutions

[0415] *mAb Pembrolizumab (anti-human PD-1 )

[0416] ** TU: Tumor cell injection 107cells in the flank (s.c. route)

[0417] Results

[0418] Of note, vehicle treated animals had similar growth of tumors confirming that this A375 model grows robustly in the presence of engrafted human PBMCs. In addition, seven days after injection of PBMCs (Day 0 - pre-treatment) blood was sampled via lateral tail vein and processed for FACS analysis of human CD45+ blood cells. All mice had engraftment levels within expected limits 7 days after injection. A sample of blood was taken during terminal sampling, see day 22, 29 days after PBMC injection, and processed for FACS analysis of human CD45+ blood cells.

[0419] Table 3: Comparative Tumor Growth Inhibition at Day 22

[0420] Furthermore, from results illustrated in figure 2, A375 tumor cells treated with anti-PD-1 Pembrolizumab shows an improvement over non-treated animal (vehicle group). And compound (I) treated animals group shows a better efficacy. And the combination therapy shows maximal efficacy, completely eradicating tumors. From results in Table 3, such combination of Pembrolizumab anti-PD-1 and compound (I) is exceptionally effective in reducing tumor volume as complete tumor eradication on day 22, indicating that compound (I) was able to overcome resistance to anti PD-1. Example 4 - Compound (I) in vivo efficacy against in humanized immunocompetent models of small cell lung cancer (SCLC NCI-H69 tumor overexpressing TLR4)

[0421] Goal

[0422] Assess the efficacy of compound (I) against humanized immunocompetent models bearing SCLC tumors.

[0423] Material and Method

[0424] Model

[0425] An in vivo efficacy study has been performed by implanting NCI-H69 from AxisBio human xenograft tumors into immunocompromised mice with human immune system.

[0426] NCI-H69 cell line has been established from the pleural fluid of a 55-year-old Caucasian male with small cell carcinoma of the lung prior to treatment. Inoculated into athymic mice, the cells form tumors with a typical histology of the original biopsy specimen. H69 is a typical small cell lung carcinoma (SCLC).

[0427] Protocol and study design

[0428] The purpose of this study was to assess the efficacy of compound (I) and checkpoint inhibitors in an NCI-H69 xenograft NXG-mouse model.

[0429] NXG mice aged 5-7 weeks were used for the study. These were purchased from Janvier (France) and had 7 days acclimatization period. Animals were identified by tail mark and housed in IVC cages (up to 5 per cage). All animals were allowed free access to a standard certified commercial diet and sanitized water during the study. The holding room was maintained under standard conditions: 18-24°C, 55-70% humidity and a 12h light / dark cycle.

[0430] All protocols used in this study have been approved by the Axis Bio Animal Welfare and Ethical Review Committee, and all procedures were carried out under the guidelines of the Animal (Scientific Procedures) Act 1986.

[0431] Female NXG mice were implanted with NCI-H69 tumor cells (1 x 107in Matrigel) on the flank. When tumors reached 50 mm3animals the animals were assigned to treatment groups and donor PBMCs injected.

[0432] Treatment scheme and schedule is described in Table 4: Treatment starts 7 days after PBMC injection. Experimental groups and routes (intravenous (IV), intraperitoneal (IP), twice per week (BIW) are listed below:

[0433] - Group 1 (G1): Vehicle: NaCI 0.9%, IV, BIW Group 2 (G2): compound (I) in saline, 2 mg / kg, IV BIW

[0434] Group 3 (G3): Pembrolizumab, 10 mg / kg IP; BIW

[0435] Results

[0436] First of all, vehicle treated animals had similar growth of tumors confirming that this NCI-H69 model grows robustly in the presence of engrafted human PBMCs. In addition, assessment of PBMC engraftment by FACS: Seven days after injection of PBMCs, i.e. Day 0 - pre-treatment) blood was sampled via lateral tail vein and processed for FACS analysis of human CD45+ blood cells. All mice had engraftment levels within expected limits 7 days after injection. Efficacy results are presented in Figure 3. Compound (I) induced by itself a robust anti-tumor effect, slightly better than for Pembrolizumab, as shown by the data in Table 4 and Figure 3.

[0437] Table 42: Treatment scheme (A) and comparative results (B) of Tumor Growth Inhibition (TGI) at Day 22

[0438] (A) Treatment Scheme

[0439] Tumor cells PBMC Sacrifice

[0440] Injection Injection terminal blood

[0441] T T

[0442] Treatments (+) D.y Do D3 D7 D10 D14 D17 D21 D22

[0443] 1) Vehicle, IV, BIW TU* Blood + + + + + + + Blood

[0444] 2) compound (I) IV BIW TU Blood + + + + + + + Blood

[0445] 3) Pembrolizumab** IP BIW TU Blood + + + + + + + Blood

[0446] * TU: Tumor cell injection 107cells in the flank (s.c. route) ** mAb Pembrolizumab (anti-human PD-1 )

[0447] (B) Comparative results

[0448] Compound (I) induced by itself a robust anti-tumor effect, slightly better than the anti-PD-1 mAb Pembrolizumab.

[0449] Example 5 - Differential in vivo efficacy of compound (I) depending on TLR4 expression level (HT29 Colon vs NCI-H69 SCLC)

[0450] Material and method

[0451] HT-29 is a human colon cancer cell line that was initially derived in 1964 from a 44-year-old Caucasian female. The cells form a tight monolayer and exhibit similarity to enterocytes from the small intestine. Ultrastructural features reported for HT-29 cells include microvilli, microfilaments, large vacuolated mitochondria with dark granules, smooth and rough endoplasmic reticulum with free ribosomes, lipid droplets, few primary and many secondary lysosomes. HT-29 is a human colorectal adenocarcinoma cell line with epithelial morphology. These cells are sensitive to the chemotherapeutic drugs 5-fluorouracil and oxaliplatin, which are standard treatment options for colorectal cancer.

[0452] NXG female mice were implanted with HT-29 cell line (1 x 107in Matrigel) on the flank and compared with NCI-H69 tumor cells (1 x 107in Matrigel) on the flank. When tumors reached 50 mm3, the animals were assigned to treatment groups, and donor PBMCs were injected.

[0453] Results

[0454] Table 3: Comparative Tumor Growth Inhibition (TGI) between low and high TLR4 expression level

[0455] The cell line NCI-H69 with the highest expression of TLR4 (4.99 RPKM), has the highest tumor growth inhibition, compared with HT29 (TLR4 3.92 RPKM)

[0456] Correlation coefficient between two sets of data measures the strength and direction of the linear relationship between them. The function takes two arrays of data as inputs and returns a value between -1 and 1 , where -1 indicates a perfect negative correlation, 0 indicates no correlation, and 1 indicates a perfect positive correlation r = (nZxy - ZxZy) I sqrt((nZxA2 - (Zx)A2)(nZyA2 - (Zy)A2)) where: r is the correlation coefficient n is the number of data points

[0457] Zxy is the sum of the product of each pair of corresponding values in the two data sets

[0458] Zx and Zy are the sums of the values in each data set

[0459] ZxA2 and ZyA2 are the sums of the squared values in each data set

[0460] Whilst r = 0.83 indicates a very high correlation between the expression of TLR4 and the T umor Growth Inhibition resulting from compound (I) treatment as stand alone Thus, targeting TLR4 overexpressing tumors with compound (I) is relevant from a therapeutic perspective because TLR4 is an important receptor for the activation of the innate immune system, which plays a critical role in the recognition and elimination of cancer cells.

[0461] Example 6A - Using compound (I) to enhance CD8+ T Cell Activation in CD8+ T Cell-

[0462] Deficient Tumors

[0463] Rationale

[0464] Active tumor-infiltrating CD8+ T cells are crucial for tumor cell killing. Tumor microenvironment regulates the anti-tumor immune responses through cytokines, kinases, and metabolic factors. Tumors promote environments with reduced number of activated CD8+ T cells. Therefore, any treatment that can activate and increase the number of CD8+ T cells is likely to exhibit antitumor activity.

[0465] Material and Method

[0466] Preparation of compound (I)

[0467] Described in Example 2

[0468] Experimental design

[0469] The experiments in examples hereunder were carried out using a Kibur Experimental Design as shown in figure 1. 18 combination treatments were tested with and without intravenous treatment with compound (I) corresponding to a total of 36 treatment conditions in two separate animal cohorts.

[0470] As shown in Table 6, the local treatments used in the KIBUR microdevice are listed: in the column “Compound A” show one negative control (vehicle), one compound (I) control and 8 different antineoplastic drugs and respectively in column “Compound B” in the microwells 2 to 10, the eight anticancer drugs were assessed alone, and in microwells 11 to 18, the same eight anti-cancer drugs are assessed as in combination with compound (I) at 30 pM. Table 4: Kibur distribution / com bi nation of treatment agents in each micro-well for both cohorts

[0471] Table 6 displays the distribution and agents and combination across 18 micro-wells. The microwells are numbered from 1 to 18 and each well contains one or two drugs. The first row of wells (wells 1-10) contains both single drugs, consisting of compound (I), Immune checkpoint inhibitors and chemotherapeutic agents. Immune checkpoint inhibitors (ICI) are anti-PD1 , anti- PDL1 , and anti-CTLA4. Chemotherapeutic agents are doxorubicin, cisplatin, etoposide, carboplatin, and topotecan.

[0472] The second row of micro-wells (wells 11-18) contains the same agents as in the first row, but each drug is combined with compound (I) at a concentration of 30 pM. Such a design helps to identify the most effective drug combination. Compound (I) and small molecule drugs were in a powder form. The antibody drugs were provided as a lyophilized powder.

[0473] Preparation of humanized mouse

[0474] Female NSG mice (NOD.Cg-Prkdcscid H2rgtm1Wjl / SzJ, supplied by Charles River Laboratories, 10-12 weeks of age) were humanized by intravenous injection with 1x107fresh

[0475] PBMC obtained from normal donors; PBMCs were obtained from 2 donors, each therapy setting comprised three mice for each donor. While the very limited experimental phase practically excludes humanization complications, several considerations are described here for completeness. PBMCs are derived from healthy, random, uncharacterized donors and are used freshly after isolation from leukoreduction system (LRS) chambers. While Charles River controls all technical aspects of PBMC handling after receipt, activity of these PBMCs may depend on the donor in general, its immune activation status at the time of blood withdrawal, as well as the donor / tumor model combination, and potential donor-to-donor variability in engraftment efficiency and / or the anti-tumor response cannot be excluded. Determination of engraftment efficiency (% human CD45+ cells in peripheral blood by flow cytometry) was conducted routinely as a quality control measure at the end of the experiment (Kibur device removal, day 4).

[0476] Development of graft-versus-host disease (GvHD) as a consequence of the engrafted human immune cell product is expected to be observed in the animals which may lead to individual animals reaching ethical endpoint criteria earlier than others. Development of GvHD is assessed by activity scoring. The time of onset of GvHD may vary depending on donor characteristics and injected cell number and cannot be reliably predicted for a particular study. Given Kibur experiments are limited to several days only, GvHD is not expected to become evident or relevant in these set of experiments.

[0477] Tumor and Device implantation

[0478] Mice were implanted with tumors on both flanks (double-sided) to ensure an appropriate number of tumors having a sufficient size at experiment start, i.e. day 0. At day 2, six Kibur devices were placed into systemic treated groups, and ten devices were placed into tumors of mice who did not receive prior systemic treatment. Therefore, an overall total of 16 Kibur devices were implanted.

[0479] In the context of drug treatment, priming dose refers to the use of a medication or intervention to prepare the body or target cells for a subsequent treatment. The priming dose effect in drug treatment can enhance the efficacy of the subsequent treatment, reduce side effects, or prevent resistance to the treatment (tumor sensitization). Such priming dose has been investigated as part of this study.

[0480] Therefore, 2 cohorts of animals have been implemented:

[0481] Cohort (A) of PMBC humanized NSG mice non-systemically pre-treated (non-primed)

[0482] Cohort (B) of PMBC humanized NSG mice systemically pre-treated (primed) with compound (I) via IV route. Compound (I) first dose was injected two days prior to Kibur microdevice implantation at 1 h following PMBC injection and the second compound (I) dose was injected IV just following Kibur microdevice implantation.

[0483] To summarize, the schedule hereunder has been followed:

[0484] Day 0: PBMC - from two donors Day 0: +1 h compound (I) IV 8 mg / kg (for compound (I) treated groups only)

[0485] Day 2: Implantation of Kibur device + compound (I) IV 8mg / kg (for compound (I) treated groups only)

[0486] Day 4: Explanation of tumors with Kibur device for analysis.

[0487] After a 48-hour incubation period, tumors were explanted.

[0488] Drug Formulation and Microdevice Loading

[0489] The small molecule test articles were combined with polyethylene glycol (PEG) to form a drug- polymer mixture, which could then be loaded into drug delivery reservoirs. All products were sprayed with 70% ethanol to maintain sterility and reduce endotoxin contamination.

[0490] The preparation of small molecule drugs proceeded per the following protocol:

[0491] 1. Weigh out the necessary amount of drug on a microscale and put it in a sterile vial.

[0492] 2. Calculate the amount of PEG needed to achieve a 20% drug 180% PEG (w / w) formulation.

[0493] 3. Add just enough solvent to dissolve the drug / PEG mixture. Vortex until the solute dissolves completely.

[0494] 4. Evaporate the solvent on a rotary evaporator at 30°C.

[0495] The antibody drugs were loaded “as-is” without any formulation. Microdevices were then loaded with the 18 drug or drug-polymer mixtures. The loading schema of the drugs in the microdevice microwells is provided in Figure 1.

[0496] Results

[0497] Table 5: Effect of compound (I) and various synergistic combination on CD8 activation

[0498] As shown in Table 7, compound (I) is able to significantly activate CD8 as compared to the PEG alone corresponding to the negative untreated control, as a stand-alone and in combination with topotecan, etoposide, or carboplatin. Table 6: Effect of compound (I) priming on compound (I) and most synergistic combinations on CD8 activation

[0499] In the context of a priming dose administration of compound (I), results in Table 8 indicate that combinations with etoposide, topotecan, carboplatin and aPD1 (4.55) have shown significant CD8 increase compared to results in Table 7 without the priming dose of compound (I). Surprisingly, a priming dose administration of compound (I) prior to its own use as stand-alone leads to significant CD8 increase (5.96). Similarly, a priming dose administration of compound (I) prior to etoposide use as a stand-alone leads to the highest CD8 increase (11.76). Lastly, a priming dose administration of compound (I) prior to use of aCTLA4 as a stand-alone leads to a significant CD8 increase (6.48) which was not observed in the context of no priming dose administration of compound (I) (0.97). Compound (I) could serve as a treatment in the context of tumors with low level of activated CD8+ T cells.

[0500] Example 6B - Using compound (I) to alleviate CD8+ T Cell Exhaustion in High- Exhaustion Tumor

[0501] Rationale

[0502] High expression of the immunosuppressive marker PD-1 indicates exhausted CD8 T cells that are incapable of recognizing and destroying tumor cells. Therefore, any treatment that can reduce this checkpoint is likely to exhibit antitumor activity.

[0503] Material and method

[0504] The method used is the same as the method used in Example 6A. Results

[0505] Results are in Table 9 and Table 10 hereunder. The high expression of PD-1 immunosuppressive marker PD-1 induced by the tumor was reversed by compound (I) to a level equivalent to aPD1 and aPD-L1.

[0506] Table 7: Compound (I) and Immune checkpoint inhibitors impact on PD-1 expression (without compound (I) priming dose)

[0507] Table 10: Compound (I) priming dose administration and impact on PD-1 expression

[0508] Compound (I) has shown surprisingly levels of PD-1 reversion equivalent to the standard of care a-PD-1 and a PD-L1. Compound (I) could serve as a treatment in the context of tumors with high exhausted CD8 T cells.

[0509] Example 7 - Using compound (I) to enhance Dendritic Cell Activation in DC-Deficient

[0510] Tumors

[0511] Rationale

[0512] Dendritic cells (DCs) are antigen-presenting cells that play a crucial role in the initiation and regulation of immune responses. DCs are essential for the activation of T cells, which are important for the elimination of cancer cells. In the context of cancer treatment, DCs have been shown to be relevant in terms of anti-tumor efficacy (Kaneno R, Shurin GV, Tourkova IL, Shurin MR. Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations. J Transl Med. 2009 Jul 10;7:58). Therefore, a drug able to activate dendritic cells is relevant in terms of anti-tumor efficacy, as it can enhance the immune response against cancer cells and improve the overall therapeutic efficacy of cancer treatment. The activation of DCs can lead to the release of alarmin protein from dying tumor cells, which can indirectly activate other immune cells. Immune cells can be powerful regulators of tumor growth and disease progression. The potential role of professional antigen-presenting cells in cancer such as dendritic cells and macrophages may present exogenous antigens through major histocompatibility complex class I molecules to CD8+ T cells, a process referred to as cross presentation. The activation of DCs can be measured by the expression of CD11+. As CD11c+ cells are in direct cell-cell contact with CD8+ T cells within the primary tumor and are associated with an active anti-tumor immune microenvironment and favorable prognosis, CD11c has been investigated in the Kibur model. Material and method

[0513] The method used is the same as the method used in Example 6A.

[0514] Results

[0515] As illustrated in Table 11, CD11c is upregulated not only by compound (I) itself as stand-alone (0.31) but also in combination with etoposide (0.41), topotecan (0.77), aPD1 (0.84), and aPD- L1 (0.91).

[0516] Table 11 : Effect of compound (I) and most synergistic combinations on CD11c related Dendritic Cells activation Table 12: Effect of compound (I) priming on compound (I) and most synergistic combinations on CD11c related Dendritic Cells activation

[0517] In the context of priming with compound (I), Table 12, combinations with aPD-L1 (31.22) and with aCTLA-4 (9.33) have shown the highest CD11c increase, i.e highest activation of dendritic cells. Surprisingly, a priming with compound (I) prior to its own use as stand-alone leads to significant CD11c decrease (5.96). On the other hand, a priming with compound (I) prior to a combination with topotecan (8.15), with carboplatin (7.77) and doxorubicin (6.87) leads to significant CD11c increase.

[0518] Combination treatment or by pre-treatment (priming) compared to vehicle or compound (I) alone. Compound (I) can effectively reactivate the immune system, therefore reducing the immunosuppression induced by the tumor (low level of Dendritic cells activation). This property is especially surprising in the context of some combinations and in the context of priming. In other words, compound (I) could serve as a good immune activating agent, effectively complementing therapies that exhibit limitations in addressing dendritic cell mediated immunosuppression or inducing it.

[0519] Example 8 Using compound (I) to alleviate macrophage M2 polarization in high M2 tumors (marker F4 / 80 in vivo)

[0520] Rationale

[0521] Macrophages are immune cells that play a crucial role in the body's response to infection and inflammation. They can be classified into two broad categories based on their function: pro- inflammatory macrophages and anti-inflammatory macrophages. Pro-inflammatory macrophages, also known as M1 macrophages, are involved in the early stages of the immune response. On the other hand, anti-inflammatory macrophages, also known as M2 macrophages, are involved in the later stages of the immune response, particularly in the resolution of inflammation and tissue repair. Both pro-inflammatory and anti-inflammatory macrophages are important for maintaining the balance between inflammation and tissue repair. Pro- and anti-inflammatory macrophages play important roles in various diseases. An imbalance in their function can lead to the development or progression of diseases. In cancer, pro-inflammatory macrophages can help to initiate an immune response against the tumor cells, but chronic inflammation can also promote tumor growth. Imbalances can lead to the development or progression of diseases, and targeting macrophage function may be a potential therapeutic approach for certain diseases.

[0522] F4 / 80 is a marker associated with M2 macrophages, which are known to promote tumor growth and suppress immune responses. Reducing the expression of F4 / 80 indicates a shift from a pro-tumorigenic M2 phenotype to an anti-tumorigenic M1 phenotype, suggesting likely antitumor efficacy.

[0523] Protocol

[0524] Same as Example 6A

[0525] Results

[0526] Table 13: Effect of compound (I) on M2 marker F4 / 80

[0527] As indicated in Table 13, compound (I) is able to significantly reduce M2 marker compared to non-treated (no priming). Surprisingly, a priming treatment with compound (I) allows a significant reduction of this marker, even on the PEG non-treated condition.

[0528] Example 9 - Using compound (I) to manage the ratio of macrophage M1 and M2 (ex vivo)

[0529] Rationale

[0530] Same as previous Example 8

[0531] Material and method

[0532] Compound (I) was supplied as a powder (batch CM2020_0366 purity 98%). To demonstrate the ability of the compound (I) compound to reprogram M1 or M2 macrophages, the experiments were performed on primary human macrophages as described in Figure 4. Macrophages were generated from primary human peripheral blood monocytes, purified by anti-CD14 magnetic sorting and stimulated for 5 days with CSF-1 (100 ng / mL). The immature macrophages (MO) thus generated were then polarized for 2 or 4 days into pro-inflammatory macrophages (M1) by adding LPS (100 ng / mL) + IFNg (20 ng / mL) or into anti-inflammatory macrophages (M2) by adding IL-4 / IL-13 (20 ng / mL). The efficiency of M1 and M2 polarization was quantified by flow cytometry by regarding the expression level of pro- (CD80 and CD86) and anti-inflammatory (CD206, CD163, CD200R, CD209) membrane markers. The results are expressed as mean of fluorescence index (MFI). The effect of compound (I) on macrophage polarization was assessed on the polarization induction and on the reprogramming of already polarized macrophages (4 donors). To define the quantity of compound (I) to be added to M0, M1 and M2 macrophages, an evaluation of the compound toxicity was performed. For this, different concentrations of the molecule of interest (6 different concentrations from 0.1 to 30 pM) were added at day 7 and the evaluation of cell death was carried out by flow cytometry using DAPI labelling two days later (d9). These experiments were performed on two different donors. Statistical analysis was performed using a one-way ANOVA test and significance was considered when P values were lower than 0.05. The results are expressed as the mean ± SEM. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 , ns (not significant) according to a oneway ANOVA.

[0533] Toxicity assay of compound (I) on M0, M1 and M2 human primary macrophages

[0534] Method

[0535] The toxicity of compound (I) compound was evaluated on already polarized M0, M1 and M2 macrophages. For this purpose, different concentrations of compound (I) between 0.1 and 30 pM (6 concentrations) were added at d7 and the evaluation of cell death was carried out by flow cytometry using DAPI labelling two days later (d9). These experiments were performed on two different donors.

[0536] Results

[0537] Compound (I) showed no toxicity on M0, M1 and M2 macrophages up to 10 pM, Figure 5. This concentration of compound (I) [10 pM] was used to evaluate the ability of the molecule to inhibit the induction of M1 or M2 macrophage polarization.

[0538] Evaluation of the ability of compound (I) to inhibit the induction of M1 or M2 macrophage polarization

[0539] Method

[0540] Compound (I), whose concentration was defined previously, was added 16 hours before the induction of polarization. The pro- and anti-inflammatory polarization of macrophages was analyzed at d7 using flow cytometry by measuring the expression of pro-inflammatory (CD80, CD86) and anti-inflammatory (CD206, CD163, CD200R, CD209) membrane markers.

[0541] Results

[0542] The results of the experiment performed on several donors are presented in Figures 5 and 6. The treatment with compound (I) strongly decreased the expression of pro-inflammatory markers (CD80, CD86) in M1 macrophage polarization, Figure 5. Interestingly, compound (I) drastically decreased the CD163 expression level in MO and M2 conditions. Moreover, compound (I) partially dampened the expression of CD209, CD206 and CD200R during M2- macrophage polarization, Figure 6. The compound (I) compound thus appears to be able to modulate M1 and M2 macrophage polarization.

[0543] Compound (I) can surprisingly modulate macrophage polarization in a way to prevent immunosuppression by tumor M2 related macrophages.

[0544] The results clearly demonstrated that compound (I) is non-toxic on the different subpopulations of human macrophages tested (MO, M1 and M2), and this up to 10 pM. Compound (I) cannot reprogram MO, M1 or M2 macrophages. In contrast, compound (I) can dampen the induction of M1 or M2 macrophage polarization. Indeed, when compound (I) was added as a priming dose, compound (I) showed a surprising priming effect. Compound (I) treatment drastically decreases the CD163 expression level in MO and M2 macrophages. Therefore, targeting CD163-positive macrophages, using an administration of a priming dose of compound (I) is a legitimate approach for cancer therapy.

[0545] Example 10 - Pharmacodynamic effects of compound (I) on immunological markers in humanized immunocompetent SCLC NCI-H69 model overexpressing TLR4

[0546] Material and method

[0547] Material and method are described in Example 4

[0548] Treatment

[0549] A biweekly (BIW) administration was performed via IV route for the vehicle (0.9% NaCI) and for compound (I) dissolved in 0.9% NaCI. Treatment groups, doses, routes, regimens are indicated in Table 14.

[0550] Treatment Scheme

[0551] Table 14: Design, schedule and groups

[0552] ** TU = Tumor cell injection 107cells in the flank (s.c. route) (+) = Treatments

[0553] # 12 mice per group : 7 mice for pharmacodynamic measurement and 5 for terminal sampling.

[0554] Treatment was continued for 21 days.

[0555] Terminal study sampling (Day 10 PD and endpoint efficacy animals)

[0556] Whole blood was collected via cardiac puncture and processed to serum.

[0557] Tumor tissue was resected and divided into 2 portions: (a) one portion (approximately 50 mg) was snap frozen and retained at -80°C prior to analysis; (b) The remaining tumor tissue was processed to a single cell suspension by manual disruption and digestion protocols, before being analyzed using two FACS panels, detailed below.

[0558] Panel 1 : Viability, hCD45, hCD3, hCD4, hCD8, hCD25, hCD127, PD-1, CTLA4, TLR4

[0559] The following cell types were identified by this panel:

[0560] - Human T-cells (viable, hCD45+, hCD3+)

[0561] - Human helper T-cells (viable, hCD45+, hCD3+, hCD4+)

[0562] - Human cytotoxic T-cells (viable, hCD45+, hCD3+, hCD8+)

[0563] - Activated Human T-regs (viable, hCD45+, hCD3+, hCD25+, hCD127+)

[0564] - Checkpoint modulators / exhaustion: CTLA-4, PD-1 expression on all of the above cell phenotypes

[0565] - TLR4 level in tumor cells: (viable, hCD45-, TLR4+)

[0566] Panel 2: Viability, hCD45, hCD3, hCD14, hCD16, hCD80, hCD163, hCD11c, hCD83, hCD56, hCD206

[0567] The following cell types were identified by this panel: - Human monocytes (viable, hCD45+, hCD3-, hCD14+, hCD16 - activation state were identified via high or low CD16 expression)

[0568] - Human M1 macrophages: (viable, hCD45+, hCD3-, hCD14+, hCD80+)

[0569] - Human M2 macrophages: (viable, hCD45+, hCD3-, hCD14+, hCD163+, hCD206+)

[0570] - Human dendritic cells: (viable, hCD45+, hCD3-, hCD11c activation identified by hCD83+) Human NK cells: hCD3-, hCD16+, hCD56+ or - (high or diminished expression was assessed).

[0571] Stained samples were analyzed using a Thermo Attune NXT apparatus, with side scatter and forward scatter initially used for gating. Full analysis was carried out using Flowjo software.

[0572] For terminal study sampling, whole blood was collected via cardiac puncture and the following samples taken:

[0573] - Whole blood was processed and stained for FACS analysis of mCD45 and hCD45 cells.

[0574] - Serum was processed from the remainder and stored at Axis Bio for possible future analysis.

[0575] - Tumor tissue was resected and divided into 2 sections: one was snap frozen and retained and the other was formalin fixed

[0576] Pharmacodynamic Samples (PD)

[0577] On day 18 of treatment, the decision was made to sample all groups for FACS, due to a combination of tumor volume and early onset signs of host versus graft disease.

[0578] On day 18, the remaining animals were culled and tumor tissue excised and digested to a single cell suspension. Cells isolated from each animal were divided in two equal portions for Panel 1 and Panel 2 biomarkers quantification and stained using the following antibodies:

[0579] Panel 1 : Viability, hCD45, hCD3, hCD4, hCD8, hCD25, hCD127, PD-1 , CTLA4, TLR4

[0580] Panel 2: Viability, hCD45, hCD3, hCD14, hCD16, hCD80, hCD163, hCD11c, hCD83, hCD56, hCD206

[0581] In vivo pharmacodynamic immunological markers sampling after sacrifice at Day 18

[0582] Key oncology immunology markers that have been assessed and shown significant changes include the following (Table 15):

[0583] NK cells (viable, hCD45+, hCD3-, hCD16+, hCD56+) were significantly increased in tumor tissue from animals treated with compound (I)

[0584] M1 macrophages (viable, hCD45+, hCD3-, hCD14+, hCD80+) were significantly increased in tumor tissue from animals treated after compound (I) treatment CD4+ cells (viable, hCD45+, hCD3+, hCD4+) were decreased in tumor tissue from animals treated after compound (I) treatment.

[0585] CD8+ cells (viable, hCD45+, hCD3+, hCD8+) were increased in tumor tissue from animals treated after compound (I) treatment.

[0586] DCs dendritic cells (viable, hCD45+, hCD3-, hCD11 c activation identified by hCD83+) were increased in tumor tissue from animals treated after compound (I) treatment.

[0587] Key oncology immunology markers ratios have also been determined (Table 16)

[0588] M1 / M2 ratio increase dramatically after compound (I) treatment. In the context of anticancer treatment, the M1 / M2 ratio is a measure of the balance between M1 and M2 macrophages in the tumor microenvironment. A lower M1 / M2 ratio, characteristic of TLR4 overexpressing tumors, often indicates poor prognosis in cancer patients, while a higher M1 / M2 ratio is associated with a better prognosis.

[0589] CD4 / CD8 ratio decrease dramatically after compound (I) treatment. In the context of anticancer treatment, this ratio is a measure of the balance between CD4 immunosuppressive pro tumoral and CD8 anti tumoral T cells in the tumor microenvironment. A higher CD4 / CD8 ratio, characteristic of TLR4 overexpressing tumors, often indicates poor prognosis in cancer patients, while a lower ratio is associated with a better prognosis.

[0590] Table 15: Key clinical oncology markers in tumor tissues, values for compound (I) are determined as relative change to vehicle index (set as 1) - NK+ (activated anti tumoral natural killer cells), CD4+ (activated pro-tumoral immunosuppressive CD4 T cells), M1+ (type 1 activated anti tumoral macrophages), CD8+ (activated anti tumoral CD8 T cells), DCs+ (activated anti tumoral dendritic cells)

[0591] Table 16: Key clinical oncology markers ratios CD4 / CD8 and M1 / M2

[0592] Overall NGX mice bearing NCI-H69 tumors and populated with human PBMCs upon intravenous treatments with compound (I) aloneresulted in key intratumoral changes. - A significant decrease in tumor volume

[0593] - A significant increase in CD3+ and CD8+ intratumoral cells

[0594] - A major change in immune cell sub-population infiltration into tumor tissue of which the majority appeared to have an anti-tumor phenotype.

[0595] - A major change in tumor tissue infiltrating macrophages phenotypes which are re-polarized (a) towards an anti-tumoral phenotype M1 (increase) and (b) simultaneously show a significant decrease of the pro tumoral phenotype M2.

[0596] Such results indicate a very good ability of compound (I) to reduce the tumor induced immunosuppression.

[0597] Example 11 - Using compound (I) to enhance tumor sensitization for adoptive cell therapies

[0598] Rationale compound (I) immunological effect against the tumor allows for a combination with adoptive cell therapies based on T cells, NK cells and Dendritic cells

[0599] Material and methods

[0600] Same material and method as in Example 10

[0601] Results

[0602] Table 17: Key immunological markers activated by compound (I) (extracted data from Example 10)

[0603] (*) relative change compared to vehicle group

[0604] (**) relative to total population of activated dendritic cells (DCs)

[0605] (***) relative to total population of activated NK cell population

[0606] Compound (I), which demonstrates the ability to activate various immune cell subsets including CD8+ T cells, dendritic cells (DCs), NK cells, and T cells, holds significant relevance for combination therapy with the cell-based immunotherapies like CAR-T cell therapy, CAR-NK cell therapy, dendritic cell therapy, and T-cell receptor (TCR) therapy (Table 17).

[0607] Table 18: CD8+ immunological marker from Kibur experimentation - without compound (I) priming in Example 1 , results as relative change(*) compared to vehicle group

[0608] Table 19: CD11+ and CD3+ immunological marker from Kibur experimentation - without compound (I) priming in Example 1 , results as relative change(*) compared to vehicle group

[0609] First of all, compound (I) induces a substantial increase in CD8+ T cells (approximately 3.2 - fold, Table 17). This is a crucial factor for combination therapies with CAR-T cell and TCR therapies. CAR-T cell therapy relies on the activation and proliferation of CD8+ cytotoxic T cells engineered to target cancer-specific antigens. By using compound (I) to boost CD8+ T- cell activity, it may enhance the efficacy of CAR-T cell therapies.

[0610] Secondly, compound (I) also activates dendritic cells (Table 19). DCs play a pivotal role in presenting tumor antigens to T cells, initiating an immune response against cancer. Combining compound (I) with dendritic cell therapy can further enhance the antigen-presenting capacity of DCs, potentially improving the response to dendritic cell-based treatments.

[0611] Thirdly, compound (I) activates NK cells, which are natural killer cells known for their ability to target and destroy cancer cells. This property makes compound (I) relevant for combination with CAR-NK cell therapies. By using compound (I) in conjunction with CAR-NK cells, it might amplify the cytotoxic potential of these immune cells against cancer.

[0612] Lastly, the combination of compound (I) with T-cell and NK cell therapies may result in synergistic effects. Compound (I) primes the immune system, increasing the pool of cytotoxic immune cells, while cell therapies engineer these cells to be highly specific for cancer targets. This combined approach could lead to more robust and targeted antitumor responses.

[0613] Compound (I) might help reduce immunosuppression, particularly in the tumor microenvironment. By enhancing the activity of immune cells like CD8+ T cells (Table 18) and NK cells, it can counteract immunosuppressive factors often present in solid tumors, creating a more favorable environment for the effectiveness of cell-based therapies. Example 12 - Using compound (I) to alleviate inflammation, fibrosis, angiogenesis and metastasis markers in tumor (multicellular human in vitro stromal & vascular models)

[0614] Material and method compound (I) was assessed in a Non-small Cell Lung Cancer (NSCLC) in vitro panel of 2 human primary cell-based systems. Eurofins Discovery Biomaps NSCLC Systems & Translational Biomarker Readouts for Immuno-Oncology was used for the immune-onco profiling of compound (I) in the following two systems https: / / www.eurofinsdiscovery.com / solution / biomap-oncology-models:

[0615] (A) StroNSCLC, Host Stromal-Tumor Microenvironment, Oncology, Oncology NSCLC, using H1299 NSCLC cell line + Peripheral blood mononuclear cells + Primary human fibroblasts: read out CD106 / VCAM-1 , CD87 / uPAR, Collagen III, CXCL10 / IP-10, EGFR, HGF, PAI-I, PBMC, Cytotoxicity, sGranzyme B, sIFN-y, slL-10, slL-13, SIL-17A, slL-2, slL-4, slL-6, sMDC, sPGE2, SRB, sTNFa, sVEGF, tPA, uPA

[0616] (B) VascNSCLC, Host Vascular-Tumor Microenvironment, Oncology, Oncology NSCLC, using H1299 NSCLC cell line + Peripheral blood mononuclear cells + Primary human endothelial cells: read out CCL2 / MCP-1 , CD106 / VCAM-1 , CD40, CD69, CD87 / uPAR, CXCL10 / IP-10, PAI-I, PBMC Cytotoxicity, sGranzyme B, sIFN-y, slL-10, slL-13, SIL-17A, slL-2, slL-4, slL-6, sMDC, SRB, sTNFa). These systems are designed to model complex human tissue and disease biology to assess the impact an agent has in an immune-suppressed tumor microenvironment model.

[0617] The NSCLC panel is an in vitro phenotypic profiling technology that screens test agents in human primary cell-based systems modeling complex tissue and disease states. These systems are constructed with several human primary cell types from healthy donors, and stimulated with cytokines or growth factors to capture the relevant signaling networks that occur in human tissue or pathological conditions. The Non-small Cell Lung Cancer (NSCLC) panel was developed to model the tumor-mediated immunosuppression that occurs in the tumor-microenvironment (TME) of cancer patients. To recapitulate the complex signaling networks that manifest in the multi-component TME, this in vitro system is a co-culture of human primary immune cells with primary tissue cells in the presence of a specific cancer cell line. These co-cultures are stimulated with sub-mitogenic levels of T cell receptor (TCR) ligands to prime but not maximally activate T cells and model immune cells recruited to the intra-tumoral microenvironment. The NSCLC panel models the in vitro interactions between the immune-stromal (fibroblasts) and immune-vascular (endothelial cells) environments in the context of non-small lung cancer (NCI-H1299 NSCLC cell line). The interactions between tumor cells, stimulated immune cells (peripheral blood mononuclear cells [PBMC]), and the host stromal network (human neonatal dermal fibroblasts [HDFn]) are captured in the StroNSCLC system. In parallel, the VascNSCLC system captures the interactions between tumor cells, activated immune cells and the vascular tissue (human umbilical vein endothelial cells [HLIVEC]). The biomarkers selected for the NSCLC panel inform on activities related to inflammation, immune-function, tissue remodeling and metastasis in the context of a host tumor-microenvironment.

[0618] StroNSCLC in vitro system

[0619] Human cell types: Peripheral blood mononuclear cells, H1299 NSCLC cell line, Dermal fibroblasts

[0620] Stimulation: TCR ligands (sub-mitogenic levels)

[0621] Disease / Tissue Relevance: Host stromal-immune-tumor microenvironment biology relevant for immuno-oncology (l-O) and oncology indications

[0622] - Biomarker Readouts: CD106 / VCAM-1, CD87 / uPAR, Collagen III, CXCLIO / IP-IO, EGFR, HGF, PAI-1 , PBMC Cytotoxicity, sGranzyme B, sIFN- gamma, slL-10, sIL- 13, SIL-17A, slL-2, slL-4, slL-6, sMDC, sPGE2, SRB, sTNF- alpha, sVEGF, tPA, uPA

[0623] System description: The StroNSCLC system is a co-culture of human primary fibroblasts, immune and HI 299 cells. This stromal tumor microenvironment (TME) model is stimulated via the T cell receptor for 48-hrs to recapitulate the activation of tumor infiltrating immune cells in the context of an immunosuppressive cancer cell line and normal host stromal tissue. Biomarker readouts capture impacts on translationally relevant immune, matrix remodeling and inflammation related intratumoral responses. Compound (I) was profiled at 4 concentrations in triplicate to detect immuno-oncology (1-0) relevant immune restorative effects versus oncology-relevant inhibitory activities.

[0624] VascNSCLC in vitro system

[0625] Human cell types: H1299 NSCLC cell line, Venular endothelial cells, Peripheral blood mononuclear cells

[0626] Stimulation: TCR ligands (sub-mitogenic levels)

[0627] Disease / Tissue Relevance: Host vascular- immune-tumor microenvironment biology relevant for immuno- oncology (l-O) and oncology indications - CCL2 / MCP-I, CD106 / VCAM-1 , CD40, CD69, CD87 / uPAR, CXCL10 / IP10, PBMC Cytotoxicity, sGranzyme B, IFN-gamma, slL-10, slL-13, SIL-17A, slL-2, slL-4, slL-6, sMDC, SRB, sTNF-alpha.

[0628] The VascNSCLC system is a co-culture of human primary endothelial, immune and HI 299 cells. This vascular tumor microenvironment (TME) model is stimulated via the T cell receptor for 48-hrs to recapitulate the activation of tumor infiltrating immune cells in the context of an immunosuppressive cancer cell line and normal host vascular tissue. Biomarker readouts capture impacts on translationally relevant immune, angiogenesis and inflammation related intratumoral responses. Compound (I) was profiled at 4 concentrations in triplicate to detect immuno-oncology (1-0) relevant immune restorative effects versus oncology-relevant inhibitory activities.

[0629] Output

[0630] Quantitative measurements of biomarker activities across this panel, along with comparative analysis of the biological activities of known bioactive agents in the NSCLC Reference Database to predict the safety, efficacy and function of these test agents. A signature profile that is reflective of the changes in protein biomarker readouts within an individual system was generated for compound (I) at 4 different concentrations and the biomarker readouts selected for therapeutic and biological relevance as predictive for disease outcomes effects, was validated using different agents with known clinical efficacy. Each readout is measured quantitatively by immune-based methods that detect protein (e.g., ELISA) or functional assays that measure viability. NSCLC readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules and enzymes. In total, the 42 biomarker readouts in the NSCLC panel capture the biological changes that occur within the physiological context of each particular BioMAP system.

[0631] The NSCLC panel allows a focused in vitro evaluation of compound (I) in a model system that includes the hierarchical signaling networks that exist between interacting pathways and cells in a disease relevant setting. To represent the changes that are occurring in these complex systems, biomarker readouts with the ability to integrate and distinguish changes in different regulatory networks have been selected. Specific NSCLC panel activities have been correlated to in vivo biology based on their mechanism of action and target selectivity and provide disease indication guidance and combination feasibility for a diverse set of target classes.

[0632] A profile plot, which is an overlay of the signature of four concentrations of compound (I) profiled in triplicate. Biomarkers that have reached statistical significance (p < 0.01) and a defined NSCLC panel criteria are annotated on the plot and categorized below the plot into oncology-specific biological classifications (immune-related activities, inflammation-related activities, matrix remodeling activities, angiogenesis-related activities and tumor-related activities).

[0633] Results

[0634] The Profile plot of compound (I) represented on Figure 7 shows that: compound (I) is active with 16 annotated readouts; compound (I) impacts inflammation and angiogenesis related activities decreasing VCAM- 1 , IP-10, sMDC and modulating sTNFa); compound (I) impacts fibrosis related matrix remodeling activities decreasing EGFR, Collagen III, and decreasing one angiogenesis-related activity uPAR compound (I) impacts immunosuppression related activities decreasing slL-4, sGranB, sIL- 2; increased slL-13, slL-6 and modulating slL-10.

[0635] In the Biomaps NSCLC Systems & Translational Biomarker Readouts for Immuno-Oncology assay, compound (I) is compared to Pembrolizumab which is selected as benchmark from the NSCLC list of drugs. Pembrolizumab (MK-3475, Keytruda) is an lgG4-k humanized monoclonal antibody used in cancer immunotherapy to block the programmed death receptor- 1 (PD-1) from binding with its ligands PD-L1 and PD-L2 that mediate immune tolerance leading to boosted immune responses. Pembrolizumab is approved for the treatment of patients with melanoma, NSCLC, HNSCC, cHL, urothelial carcinoma, and MSI-H colorectal and gastric cancers. Compound (I) was assessed at a concentration of 18 pM and the selected reference benchmark Pembrolizumab at 50 pg / ml.

[0636] There are two common activities in the VascNSCLC system (increased slL-6 and decreased sMDC).

[0637] There are 18 differentiating biomarkers (not shown) within the following systems: StroNSCLC (VCAM-1, uPAR, Collagen III, IP-10, EGFR, slL-10, sMDC, sVEGF) and VascNSCLC (IP-10, sGranB, slL-10, slL-2, slL-4, CD69, sIFNy, Pcyto, sTNFa, SIL-17A).

[0638] Differentiating biomarkers are defined when one profile has a readout outside of the significance envelope with an effect size above 20% (|log10 ratio) > 0.1), and the readout for the other profile is either inside the envelope or in the opposite direction.

[0639] In summary, compound (I) is active and noncytotoxic at the concentrations tested in the NSCLC panel modeling human tumor TME biology. Compound (I) had 16 activities detected across both systems of this panel with seven biomarkers annotated in the StroNSCLC and nine in the VascNSCLC. Compound (I) impacted multiple immune-related biomarkers, including slL-6 that was increased in both systems and slL-13 which was increased in VascNSCLC at the lowest concentration only. Compound (I) also decreased slL-4, sGranB, slL-2, and slL-10. Inflammation related activities of compound (I) included decreased VCAM- 1 , IP-10, sMDC and modulated sTNFa. In the StroNSCLC system, compound (I) decreased two matrix remodeling biomarkers, EGFR and Collagen III, and one angiogenesis-related activity, uPAR.

[0640] Overlay of compound (I) at its top tested concentration of 18 pM and the selected reference benchmark Pembrolizumab at 50 pg / mL revealed two common activities in the VascNSCLC system: increased in slL-6 and decreased in sMDC. There were 18 differentiating biomarkers identified between these two profiles. In these systems, Pembrolizumab has the capacity to increase cytokine levels such as I FNy, slL-2 and slL-10, indicative of restored immune function.

[0641] Compound (I) surprisingly again changed the immunosuppression profile: reduction of pro Th2 immunosuppressive markers slL4 and slL10 of these panels and reduction of modulating markers on a complementary basis to anti-PD-1. This suggests that compound (I) modulates immune biology via a mode of action distinct from PD-1 blockade.

[0642] Example 13 - Compound (I) mitigation of pro-angiogenic and immunosuppressive factors in TLR4-overexpressing tumor

[0643] As a reminder, this s a treatment of Small Cell Lung Cancer tumor model NCI-H69 (overexpressing TLR4) according to different conditions by compound (I).

[0644] Material and method

[0645] The following protocol was used for cultivating and harvesting cells: NCI-H69 cells were cultivated in RPMI-1640 Medium with fetal bovine serum to a final concentration of 10%. NCI- H69 cells were grown after 4 passages in multi-well plates and cells were incubated with either vehicle (negative control) or compound (I) (10 pM = 11.35 pg / mL) for 20 min and respectively for 12h. A total of 5 million cells were used for each condition in triplicate and were transferred into a 1.5 mL Eppendorf tubes. Following centrifugation at 4-8°C, supernatants were harvested quickly and as completely as possible and immediately snap-frozen and stored at -80°C prior to analysis. Pellets were washed once with ice-cold 1x PBS (Phosphate-buffered saline, 0-4°C) and after PBS removal, the dry pellets were immediately snap-frozen and stored at -80°C until extraction and analysis for proteomics and phosphoproteomics: Proteins of 12 human NCI-H69 cell samples were extracted with scioExtract buffer (Sciomics) using the extraction SOPs. After quality control of the samples, the bulk protein concentration was determined by BCA assay.

[0646] Overview of samples analyzed for protein concentrations: NCI-H69 Untreated, 12 hours, Cell pellet 1 (control. 12h) = 3.38 mg protein / mL; NCI-H69 Untreated, 12 hours, Cell pellet 2 control.

[0647] 12h 3.41 mg protein / mL; NCI-H69 Untreated, 12 hours, cell pellet 3 control. 12h 3.72 mg protein / mL; NCI-H69 + compound (I), 12 hours, Cell pellet 1 compound (l).12h 3.47 mg protein / mL; NCI-H69 + compound (I), 12 hours, Cell pellet 2 compound (l).12h 3.42 mg protein / mL; NCI-H69 + compound (I), 12 hours, Cell pellet 3 compound (l).12h 3.56 mg protein / mL; NCI-H69 Untreated, 20 min, Cell pellet 1 control. 20 min 3.71 mg protein / mL; NCI- H69 Untreated, 20 min, Cell pellet 2 control. 20 min 3.62 mg protein / mL; NCI-H69 Untreated, 20 min, cell pellet 3 control. 20 min 3.64 mg protein / mL; NCI-H69 + compound (I), 20 min, Cell pellet 1 compound (I). 20 min 3.64 mg protein / mL; NCI-H69 + compound (I), 20 min, cell pellet 2 compound (I). 20 min 3.35 mg protein / mL; NCI-H69 + compound (I), 20 min, Cell pellet 3 compound (I). 20 min 3.19 mg protein / mL.

[0648] Sample labelling: The samples were labelled at an adjusted protein concentration for two hours with scioDye 2 (Sciomics). After two hours, the reaction was stopped. Excess dye was removed and the buffer exchanged to PBS. All labelled protein samples were stored at -20° C until use.

[0649] Sample incubation: The 12 samples were analyzed on 12 scioDiscover antibody microarrays (Sciomics GmbH, Heidelberg, Germany) targeting 1 ,438 different proteins with 1 ,929 antibodies. Each antibody is represented on the array in four replicates. The arrays were blocked with scioBlock (Sciomics GmbH, Heidelberg, Germany) on a Hybstation 4800 (Tecan, Austria) and afterwards the samples as well as scioPhosphomix 1 were incubated. Via scio- Phosphomix 1 , information on protein specific phosphorylation levels at serine, threonine and tyrosine residues can be obtained. After incubation, the slides were thoroughly washed with 1x PBSTT, rinsed with 0.1x PBS as well as with water and subsequently dried with nitrogen. Data acquisition and analysis: Slide scanning was conducted using a Powerscanner (Tecan, Austria) with constant instrument laser power and PMT settings. Spot segmentation was performed with GenePix Pro 6.0 (Molecular Devices, Union City, CA, USA). Acquired raw data were analyzed using the linear models for microarray data (LIMMA) package of R- Bioconductor after uploading the median signal intensities. For normalization, a Cyclic Loess normalization was applied. For analysis of the samples a one-factorial linear model was fitted via least squares regression with LIMMA, resulting in a two-sided t-test or F-test based on moderated statistics. All presented p values were adjusted for multiple testing by controlling the false discovery rate according to Benjamini and Hochberg. Proteins were defined as differential for |logFC| > 0.5 and an adjusted p value < 0.05.

[0650] Differences in protein abundance or phosphorylation level between different samples or sample groups are presented as log-fold changes (logFC) calculated for the basis 2. In a study comparing samples versus control a logFC = 1 means that the sample group had on average a 21= 2 fold higher signal than the control group. logFC = -1 stands for 2"1= 1 / 2 of the signal in the sample as compared to the control group.

[0651] Compound (I) effect against pro-angiogenic factors

[0652] Table 20 provides a comprehensive view of the dynamic changes in pro-angiogenic markers under various conditions and time points, offering insights into their regulation and potential implications for angiogenesis-related processes.

[0653] Table 20: Key pro-angiogenic markers assessed at different time points (20 min, 12h, longitudinal), at different location (supernatant or whole sample) and different read-out level (proteome, phosphoproteome).

[0654] Table 20 provides an overview of key pro-angiogenic markers assessed at various time points (20 minutes, 12 hours, and longitudinally) and in different sample locations (supernatant or whole sample). The markers are evaluated at different read-out levels, including the proteome and phosphoproteome. The "YES" designation indicates a significant down-regulation with a Log2FC value of less than 0.5 and a significant p-value. Conversely, "NS" signifies no significant effect.

[0655] The markers evaluated in this table include:

[0656] - ANGP2 (Angiopoietin-2): ANGP2 is significantly reduced in terms of proteomic expression on a longitudinal level. ANGP2 is a key regulator of angiogenesis, involved in the formation of new blood vessels. It can destabilize blood vessels, leading to the growth of new capillaries, and is associated with various diseases, including cancer. OLR1 (Oxidized Low-Density Lipoprotein Receptor 1): compound (I) treatment leads to significant OLR1 down-regulation in the supernatant sample at 20 minutes, whole sample at 20 minutes, whole sample at 12 hours, and longitudinally. For info, OLR1 plays a role in the uptake of oxidized low-density lipoproteins (LDL) and is associated with various cellular processes. In angiogenesis, it can impact the recruitment of endothelial cells.

[0657] VEGFa (Vascular Endothelial Growth Factor A): compound (I) treatment leads to significant down-regulation (YES) in the supernatant sample at 20 minutes, whole sample at 20 minutes, whole sample at 12 hours, and longitudinally. VEGFa is a well-known and potent pro-angiogenic factor, stimulating the growth and permeability of blood vessels. It plays a crucial role in angiogenesis and vascularization.

[0658] VEGFc (Vascular Endothelial Growth Factor C): compound (I) treatment leads to significant down-regulation (YES) in the whole sample at 20 minutes and longitudinally, while no significant effect (NS) is observed in the other conditions. VEGFc is another member of the VEGF family and is known to promote lymphangiogenesis, the formation of lymphatic vessels.

[0659] VEGFd (Vascular Endothelial Growth Factor D): compound (I) treatment leads to significant down-regulation (YES) in the whole sample at 20 minutes, but no significant effect (NS) in other conditions. VEGFd is closely related to VEGFc and plays a role in lymphangiogenesis. It is involved in the growth and maintenance of lymphatic vessels.

[0660] FGF2 (Fibroblast Growth Factor 2): compound (I) treatment leads to significant downregulation (YES) in the supernatant at 20 minutes and the whole sample at 12 hours, while no significant effect (NS) is observed in other conditions. FGF2 is a growth factor that can stimulate angiogenesis. It promotes the proliferation and migration of endothelial cells, contributing to blood vessel formation.

[0661] VEGFR1 (Vascular Endothelial Growth Factor Receptor 1): compound (I) treatment leads to significant down-regulation (YES) in the whole sample at 20 minutes, whole sample at 12 hours, and longitudinally, while no significant effect (NS) is observed in the other conditions. VEGFR1 is a receptor for VEGF-A and plays a role in angiogenesis by mediating the effects of VEGFA. factors

[0662] The list of tumor immunosuppressive factors with significant downregulation (log FC< 0 and p- value < 0.01) per compound (I) treatment from the 12h proteome sample and from the longitudinal assessment:

[0663] Chemokines CCL5, CCL8, CXCL5, CXL10

[0664] Immune checkpoint PD1 Th2 immunosuppressive cytokines IL13, IL4 and TSLP

[0665] Example 14 - Inhibiting vascular angiogenesis with compound (I)

[0666] Rationale

[0667] As already described in the definition section, vascular angiogenesis refers to the formation of new blood vessels from pre-existing ones. Tumors exploit vascular angiogenesis to sustain their rapid growth and create pathways for cancer cells to enter the bloodstream, leading to the spread of cancer to distant organs.

[0668] Material and method

[0669] To assess the ability of compound (I) to inhibit vascular angiogenesis, we tested its ability to inhibit the proliferation of Telomerase-immortalized microvascular endothelial cells (TIME) ATCC® PCS-100-030TM. Proliferation viability assays were performed by XTT assays or cell counting with a Coulter Counter. Statistics Student’s t-test was used for comparison of continuous variables. Significance was defined as P < 0.05.

[0670] Results:

[0671] Table 21 : Dose response effect of compound (I) on the growth of TIME cells

[0672] *average two separate experiments in triplicate

[0673] Surprisingly, even if compound (I) is not known as being anti-angiogenic, at 30 pM (Table 21), it shows a significant reduction of the TIME growth (down to 78.22%).

[0674] Example 15 - Inhibiting lymphangiogenesis with compound (I)

[0675] Rationale

[0676] Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing ones, significantly promotes metastasis by providing a route for tumor cells to disseminate from the primary site to distant organs. Tumor-secreted factors such as VEGF-C and VEGF-D stimulate lymphatic endothelial cells, enhancing lymphatic vessel growth and increasing permeability, facilitating tumor cell entry into the lymphatic system. Once within the lymphatics, cancer cells are transported to regional lymph nodes and eventually into the systemic circulation, leading to the establishment of secondary tumors. Additionally, the lymphatic microenvironment promotes immune evasion and supports a pro-metastatic niche, further aiding metastatic progression.

[0677] Material and method

[0678] To assess the ability of compound (I) to inhibit lymphangiogenesis, we tested its ability to inhibit the proliferation of Lymphatic endothelial cells (LEC) from PromoCell. Proliferation viability assays were performed by XTT assays or cell counting with a Coulter Counter. Statistics Student’s t-test was used for comparison of continuous variables. Significance was defined as P < 0.05.

[0679] Table 22: Dose response effect of compound (I) on the growth of LEC cells *average two separate experiments in triplicate

[0680] Surprisingly, even if compound (I) is not known as being anti-lymphangiogenic, between 1 pM and 30 pM as indicated in Table 22, compound (I) shows a significant reduction of the LEC growth, down to 80.45%.

[0681] Example 16 - Overcoming Axitinib-resistant & immunosuppressive angiogenesis with compound (I)

[0682] Material and method

[0683] In order to evaluate the potential of compound (l)for synergistic interaction with the anti- angiogenic agent axitinib (Selleckchem, No.S1005, CAS: 319460-85-0), experiments were conducted to determine whether compound (I) could reverse the resistance observed in the 0-786 tumor cell line, a well-known model of kidney cancer, which is typically unresponsive to axitinib treatment. Thus, 50000 0-786 renal cancer cells were seeded in 24-well plates and incubated for 24 or 48 hours in the presence of 30 pM compound (I) and two concentrations of axitinib. Cells were counted with a Coulter counter and t-test was used for significance of growth inhibition or cell death in compound (I) samples versus control. Proliferation viability assays were performed by XTT assays or cell counting with a Coulter Counter. Statistics Student’s t-test was used for comparison of continuous variables. Significance was defined as P < 0.05.

[0684] Results

[0685] Table 23: Dose response effect of Axitinib combined with compound (I) against Axitinib resistant 0-786 cells *average two separate experiments in triplicate

[0686] Surprisingly, even if compound (I) is able to strongly restore sensitivity to axitinib, with a growth reduced down to almost 50% at 5 pM axitinib dose (Table 23). Compound (I) allows to overcome resistance to anti angiogenic treatment axitinib.

[0687] All in all, compound (I) has surprisingly shown anti-angiogenic properties, at the cellular and molecular level, as stand-alone and in combination. A combination with anti-angiogenic treatments against cancer is of great potential for commercial success.

[0688] Example 17 - Overcoming Sunitinib-resistant & immunosuppressive angiogenesis with compound (I)

[0689] Rationale

[0690] Tyrosine kinase inhibitors (TKIs) represent a vital class of targeted therapy in cancer treatment. One illustrative example of a TKI is sunitinib, a multi-targeted inhibitor known for its effectiveness in treating angiogenic cancers, such as renal cell carcinoma and gastrointestinal stromal tumors.

[0691] Material and method

[0692] In order to evaluate the potential compound (I) for synergistic interaction with the anti- angiogenic agent sunitinib, we conducted experiments to determine whether compound (I) could reverse the resistance observed in the 0-786 tumor cell line, a well-known model of kidney cancer, which is typically unresponsive to sunitinib treatment. Cell culture and treatment: 50000 0-786 renal cancer cells were seeded in 24-well plates and incubated for 24 or 48 hours in the presence of 30 pM compound (I) and two concentrations of sunitinib. Cells were counted with a Coulter counter and T-test was used for significance of growth inhibition or cell death in compound (I) samples versus control. Proliferation viability assays were performed by XTT assays or cell counting with a Coulter Counter. Statistics Student’s t-test was used for comparison of continuous variables. Significance was defined as P < 0.05.

[0693] Results

[0694] Table 24: Dose response effect of Sunitinib combined with compound (I) combined with on the growth of 0-786 cells resistant to Sunitinib

[0695] ‘average two separate experiments in triplicate

[0696] Surprisingly, even if compound (I) is able to strongly restore sensitivity to sunitinib, with a growth reduced down to almost 50% at 5 pM sunitinib dose (Table 24). Compound (I) allows to overcome resistance to TKI treatment sunitinib.

[0697] Example 18 - Inhibiting fibrosis in various in vitro multicellular and tissue-specific human models with compound (I)

[0698] Material and method

[0699] BioMAP Fibrosis panels MyoF 89-0003-0011 , REMYyoF 89-0003-0040, SAEMyoF 89-0003- 0042 from Eurofins Discovery, https: / / www.eurofinsdiscovery.com / catalog / biomap-fibrosis- panel / 89-0016-0904 are in vitro phenotypic profiling technology assays that screens test agents in human primary cell-based systems modeling complex tissue and disease states. The Fibrosis panel was developed to model the fibrotic and inflammatory processes that drive fibrosis in tissue-specific contexts. Fibrosis panel is constructed with one or more human primary cell types from healthy donors, and stimulated with cytokines or growth factors to capture the relevant signaling networks that occur in human tissue or pathological conditions. The Fibrosis panel allows a focused in vitro evaluation of the impact of an agent in a model system that includes the hierarchical signaling networks that exist between interacting pathways and cells in a disease relevant setting. To represent the changes that are occurring in these complex systems, biomarker readouts with the ability to integrate and distinguish changes in different regulatory networks have been selected. Specific Fibrosis panel activities have been correlated to in vivo biology, can distinguish compounds based on mechanism of action and target selectivity and provide disease indication guidance and combination feasibility for a diverse set of target classes.

[0700] The Fibrosis panel includes 3 systems in Table 25, which are stimulated to model TGFp and TNFa driven myofibroblast differentiation during chronic inflammation and wound healing in different tissue settings. SAEMyoF system corresponds to interstitial lung diseases, including pulmonary fibrotic diseases such as idiopathic pulmonary fibrosis (IPF), which is modeled by co-culturing small airway epithelial cells and adult fibroblasts.

[0701] REMyoF system corresponds to fibrotic disease of the kidney associated with end-stage renal failure captured in the consisting of a co-culture of renal proximal tubule epithelial cells and adult fibroblasts.

[0702] MyoF system corresponds to the impact on fibroblasts alone which can be interrogated using the monoculture.

[0703] Table 25: Description of 3 fibrosis models SAEMyoF, MyoF and REMyoF Biomarker readouts capture impacts on translationally relevant matrix remodeling, tissue repair, wound healing and inflammation related responses in the diseased kidney. Quantitative measurements of biomarker activities across this panel, along with comparative analysis of the biological activities of known bioactive agents in the Reference Database are used to predict the safety, efficacy and function of these test agents.

[0704] Readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules and enzymes. Readouts were measured quantitatively by immune-based methods that detect protein (ELISA) as well as functional assays that measure viability. In total, the Fibrosis panel contains 54 biomarker readouts that capture the hallmark biological changes that occur within the physiological context of each panel.

[0705] A signature profile that is reflective of the changes in protein biomarker readouts was generated for compound (I) on each three individual systems. Biomarker readouts selected for therapeutic and biological relevance are predictive for disease outcomes or specific drug effects, have been validated using agents with known clinical efficacy.

[0706] Differentiating biomarkers are defined when one profile has a readout outside of the significance envelope with an effect size > 20% (|log10 ratio) > 0.1), and the readout for the other profile is either inside the envelope or in the opposite direction.

[0707] Results

[0708] Fibrosis panel results are shown in Figure 11 , which is an overlay of the signatures of four concentrations of compound (I) profiled in triplicate. Biomarkers that have reached statistical significance (p < 0.01) and a defined criteria are annotated on the plot and categorized below the plot into fibrosis-specific biological classifications (myofibroblast activation-related activities, fibrosis-related matrix activities, tissue remodeling / wound healing activities, and inflammation-related activities). First of all, compound (I) is not cytotoxic at the concentrations tested in this study (0.65 to 18 pM). In addition, compound (I) is active with 20 annotated readouts and mediated changes in key biomarker activities are listed by biological and disease classifications: compound (I) impacts inflammation-related activities (increased VCAM-1 , IP-10, l-TAC, MCP-1, IL-8), compound (I) impacts myofibroblast activation-related activities (decreased N-Cadherin; modulated aSMA), fibrosis-related matrix activities (decreased Collagen I, Collagen III) compound (I) increased TIMP-1 , MMP-1 , Collagen IV, Decorin), and tissue remodeling / wound healing activities (increased EGFR, tPA, Keratin 8 / 18) compound (I) decreased N-Cad, collagene I and collagene-ll I. The Profile plot on Figure 11 is followed by an overlay of one concentration of a test agent with one concentration of Pirfenidone, a benchmark selected from the Fibrosis panel benchmark list. Pirfenidone is an antifibrotic compound approved for the treatment of idiopathic pulmonary fibrosis (I PF) and is currently under investigation for use in the treatment of unclassifiable interstitial lung disease (uILD). In this overlay comparison, common or differentiating biomarker activities are annotated and listed by system, along with a description of the benchmark compound. The comparison of compound (I) at 18 pM with the selected reference benchmark antifibrotic pirfenidone which was assessed at 1700 pM (to observe these changes pirfenidone concentration was tested at a very high concentration, almost 100-fold higher than compound (I) concentration). compound (I) and pirfenidone have two common activities, one in SAEMyoF with a decrease in Collagen III and one in MyoF with a decrease in Collagen I. compound (I) and pirfenidone have 14 differentiating activities within the following systems: o SAEMyoF (IP-10, EGFR, MMP-1) o MyoF (Collagen IV, IL-8, Decorin, MMP-1 , TIMP-1 , PAI-1) o REMyoF (MCP-1, EGFR, Keratin 8 / 18, MMP-1 , tPA).

[0709] In summary, compound (I) was active and noncytotoxic at the concentrations tested in this study. There were 20 biomarker activities detected across the three systems that model different aspects of fibrosis-related biology. Some biomarkers, such as EGFR and MMP-1 , were impacted by compound (I) in all systems in which they are assessed. In contrast, other fibrosis-related biomarkers were impacted by compound (I) in a context-dependent manner in select systems only. Specifically, compound (I) decreased aSMA in the pulmonary fibrosis- related SAEMyoF system while the same biomarker was increased in the renal fibrosis-related REMyoF system. Alpha smooth muscle actin (aSMA) is a hallmark biomarker of activated myofibroblasts, considered a key cell type mediating pathogenic fibrosis in multiple organs. Additionally, compound (I) decreased levels of Collagen-Ill and N-cadherin in the SAEMyoF system, but had no effect on these biomarkers in the REMyoF system.

[0710] Together these data suggest that compound (I) surprisingly had a great anti-fibrotic impact across multiple systems.

[0711] Example 19 - Inhibiting fibrosis and ischemia in vivo with compound (I)

[0712] Material and method

[0713] The effect of compound (I) was assessed on myocardial infarct size (IS) model in open-chest barbital anesthetized dogs. This cardiac model was selected as it resembles an exaggerated “cliche” to the inflammatory events occurring after or during lung cancer treatments - e.g. radiotherapy or small molecules anti-cancer treatments - leading to the burst production of inflammatory cytokines following cardiac reperfusion.

[0714] In this experiment five groups of 6 dogs were treated with either vehicle or 0.2, or 1 , or 5 or 50 pg / kg of compound (I) 24 hours prior to coronary artery occlusion and reperfusion:

[0715] Control Group (n=6): These animals were treated with vehicle 24 hours prior to the infarct size experiment.

[0716] Group compound (I) 0.2 pg / kg (n=6): These animals were treated with compound (I) 0.2 pg / kg i.v. 24 hours prior to the infarct size experiment.

[0717] Group compound (I) 1 pg / kg (n=6): These animals were treated with 1 pg / kg of compound (I), 24 hours prior to the infarct size experiment.

[0718] Group compound (I) 5 pg / kg (n=6): These animals were treated with 5 pg / kg of compound (I), 24 hours prior to the infarct size experiment.

[0719] Group compound (I) 50 pg / kg (n=6): These animals were treated with 50 pg / kg of compound (I), 24 hours prior to the infarct size experiment.

[0720] Dogs weighing approximately 20 kg were anesthetized with a combination of 20 mg / kg of pentobarbital and 200 mg / kg of barbital and supplemented as needed throughout the experiment. All dogs were instrumented for the measurement of hemodynamics and were subjected to 60 minutes of left anterior descending (LAD) coronary artery occlusion and 3 hours of reperfusion. Subsequently, the dogs were surgically prepared by placing an IV line in the femoral vein and artery and a Millar catheter in the left ventricle (LV) for the measurement of LV pressures and LV dP / dt. The chest was then opened at the 4th or 5th intercostal space and the heart suspended in a pericardial cradle. The left anterior descending (LAD) coronary artery was isolated just distal to its first diagonal branch for subsequent occlusion and reperfusion and a catheter was placed into the left atrium for the injection of radioactive microspheres to measure coronary collateral blood flow during LAD occlusion and blood flow at 3 hours of reperfusion. At the termination of the experiment, Patent blue dye was injected into the left atrial catheter with the LAD reoccluded and saline into the occluded area to delineate the nonischemic area (blue-colored) and the area at risk for infarction (normal colored). The heart was then fibrillated and removed from the dog for the subsequent measurement of IS by using triphenyl tetrazolium chloride histochemical stain, which dyes non- infarcted tissue bright red and leaves the infarcted tissue a grey color. After storage overnight in formaldehyde, the tissue was washed and IS determined gravimetrically and expressed as a % of the area at risk (IS / AAR) or left ventricle (IS / LV). Tissue pieces from the non-ischemic and ischemic area were also placed in tubes and counted in a gamma counter to determine regional myocardial blood flow during occlusion and at 3 hours of reperfusion.

[0721] Infarct size is proportional to the fibrosis area. Radioactive microspheres were used to measure regional myocardial blood flow at 30 minutes of occlusion and at 3 hours of reperfusion and triphenyl tetrazolium chloride staining was used to determine myocardial infarct size histochemically.

[0722] Results

[0723] The hemodynamic results (heart rate, mean blood pressure, rate pressure product, left ventricular dP / dt) reveal that there were no significant differences between the control and the four compound (l)-treated groups at any time point throughout the experiment (Table 26).

[0724] Table 26: Hemodynamic values. All values are the mean ± SEM (n=6). p>0.05 by one way ANOVA at each time point when comparing Compound (l)-treated dogs to the control group. Abbreviations: Trans = Transmural, Occ = Occlusion; Rep = Reperfusion Similarly, there were no differences in blood gas values, pH, pCC>2 and pC>2 throughout the study (Table 27).

[0725] Table 27: Blood gas values. All values are the mean ± SEM (n=6). Abbreviations: Occ = Occlusion; Rep = Reperfusion. p>0.05 by a one way ANOVA at each time point when comparing Compound (I)- treated dogs with the control group.

[0726] The transmural blood flow values are shown in Table 28. There were no significant differences in collateral blood flow during occlusion or LAD blood flow at 3 hours of reperfusion between the 5 groups.

[0727] Table 28: Transmural blood flow values (mL / min / g). All values are the mean ± SEM (n=6)

[0728] Abbreviations: Trans = Transmural, Occ = Occlusion; Rep = Reperfusion. p>0.05 by a one way ANOVA at each time point when Compound (I) were compared to the control group.

[0729] The area at risk for infarction (AAR / LV) was also not different between groups (Figure 12(A)).

[0730] Thus, there were no differences in the 2 major determinants of the intensity of ischemia during occlusion, TCCBF or AAR / LV, which suggests that any effect of compound (I) on infarct size is occurring independent of these factors and is a true cardioprotective effect.

[0731] The infarct size data are summarized in Figures 12(B), 12(C), and 12(D) below, and in Table 29. The ischemic size (IS) in actual grams was only significant at the 5 ug / kg dose (Figure 12(B)).

[0732] When the results were normalized (Figure 12(C)), the 3 highest doses of compound (I) (1 , 5, and 50 p.g / kg) produced an approximately 50% reduction in IS expressed and normalized as a % of the AAR or LV weight. Compound (l)-treated dogs at 0.2, 1 , 5, and 50 p.g / kg had a 25.7%, 53.2%, 51.6%, and 50.2% reduction in infarct size (IS / AAR) as compared to the control group respectively. These reductions did reach statistical significance (unpaired t test) for doses of 1 , 5, and 50 p.g / kg.

[0733] Table 29: Infarct size data. All values are the mean ± SEM (n=6).

[0734] * indicates p<0.05 (unpaired Student’s t-test)

[0735] Abbreviations : LV= Left ventricular, AAR = Area at risk, IS = Infarct size, p values were calculated by a one way ANOVA at each time point when comparing compound (I)- treated dogs with the control group

[0736] Figure 12(A) illustrates the area at risk as percent (%) of the left ventricular weight in the control and compound (l)-treated dogs. All values are the mean ± SEM of six dogs / group. There were no significant differences observed between groups.

[0737] Figure 12(B) shows the effect of four different doses (0.2, 1, 5 and 50 pg / kg, i.v.) of compound (I) and saline vehicle given 24 hours prior to the acute experiment on myocardial infarct size in grams (g). Only the 5 pg / kg dose produced a significant reduction in infarct size. *P< 0.05 versus the control group. All values are the mean ± SEM of six dogs / group.

[0738] Figure 12(C) illustrates the effect of four different doses (0.2, 1 , 5 and 50 pg / kg, iv) of compound (I) and saline vehicle given 24 hours prior to the acute experiment on myocardial infarct size (IS) expressed as a percent (%) of the area at risk (AAR). The three higher doses of compound (I) produced nearly equivalent reductions in IS / AAR. *P<0.05 versus the control group. All values are the mean ± SEM of six dogs / group.

[0739] Figure 12(D) shows the effect of four different doses (0.2, 1 , 5 and 50 pg / kg, iv) of compound (I) and saline vehicle given 24 hours prior to the acute experiment on myocardial infarct size (IS) expressed as a percent (%) of the left ventricular weight (LV). The three higher doses of compound (I) produced nearly equivalent reductions in IS / LV. *P<0.05 versus the control group. All values are the mean ± SEM of six dogs / group.

[0740] Figure 12(E) shows the transmural coronary collateral blood flow (TCCBF, mL / min / g) at 30 minutes of occlusion in the control and the compound (l)-treated groups. There were no significant differences between groups. All values are the mean ± SEM of six dogs / group.

[0741] A single parenteral administration of compound (I) prior to a planned ischemic / reperfusion event reduces the infarct size in animals. Compound (I) astonishingly appears to be protective in the low microgram range (i.e. 0.2 to 5 pg / kg). Cardioprotective effects are linked to the production of cytokines which then induces overexpression of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). This is known to induce the synthesis of cyclic GMP (cGMP) and then the activation of cGMP-kinase which is subsequently phosphorylated and induces the opening of KATP channels. Due to potassium outflow, myocardial cells become hyperpolarized. This results in a shortening of action potential of the cells and decreased ATP consumption. Then, calcium influx is reduced. Therefore, in case of cellular damage muscular cells are protected from death.

[0742] Example 20 - Therapeutic regimen with and without loading / priming dose of compound (I)

[0743] Rationale

[0744] In the clinical context, a loading or priming dose is an initial higher dose of a drug, compound (I), that may be given at the beginning of a course of treatment before dropping down to a lower maintenance dose. Loading doses are a means to quickly achieve therapeutic drug concentrations or prompt an immediate clinical response. Loading doses are larger than maintenance doses and are usually administered as a single bolus, although some drugs may require multiple loading doses administered over several hours to days. Material and method

[0745] The proposed therapeutic regimen is a sequence of treatment starting with one or two loading injections of compound (I) (as stand-alone) between 48h and 24h prior to the first combination treatment with chemotherapy, targeted therapy or immunotherapy with monoclonal antibodies such as but not limited to anti CTLA4, anti-PD1 or anti-PD-L1.

[0746] A maintenance treatment with compound (I) will follow or combine with chemotherapy, targeted therapy or immunotherapy with a weekly or biweekly maintenance dose of compound (I) for a period of 4 to 8 weeks.

[0747] The compound (I) loading dose is comprised between 1 and 10 mg / m2via IV route either as bolus injection or a slow infusion in saline.

[0748] The maintenance dose is done as a weekly or biweekly IV injection between 1 and 10 mg / m2via IV route either as bolus injection or a slow infusion in saline. Optionally if the patient responds well to the treatment, the dose may be increased.

[0749] Results Table 30 exemplifies the extent to which a priming (equivalent to loading dose) is beneficial for all combinations and even for the use of compound (I) as stand-alone.

[0750] Table 30: Impact of priming vs non-priming measured as Ratio Example 21 - Priming tumor with compound (I) to enhance subsequent treatment efficacy

[0751] Rationale

[0752] Compound (I) agent may be used to sensitize the tumor cells to the effects of chemotherapy or immunotherapy by administering a priming dose. A priming dose is a single IV injection of compound (I) administered prior to the start of anticancer treatment. Such treatment priming can be an effective strategy to enhance the efficacy of subsequent treatments and improve patient outcomes.

[0753] Material and method

[0754] Refer to Example 6A and 20

[0755] Priming effect on apoptosis

[0756] Table 31 : Pro-apoptotic effect of compound (I) based on CC3 marker with and without priming

[0757] Table 31 shows that administration of a priming dose of compound (I) leads to a significant increase in the percentage of cells positive for CC3 compared to the no priming condition (14.82% vs 2.75%). This suggests that compound (I) has a priming effect on the activation of the apoptotic pathway, making the cells more susceptible to undergo apoptosis. This effect is specific to compound (I) since the negative control (PEG) did not show any significant change in the percentage of CC3 positive cells with or without priming. Priming dose administration of compound (I) has a significant impact on the pro-apoptotic properties of the treatment agents, with a substantial increase in the percentage of CC3 (a marker of apoptosis) observed in most cases. Priming effect on immunosuppressive PD-1 (in combination with anti-PD-1)

[0758] As seen in the Table 32, treatment with compound (I) as stand-alone leads systematically pretreatment with compound (I) increasing tumor PD-1 expression could sensitize the tumor to further treatment with anti-PD-1. By increasing the expression of PD-1 on the surface of tumor cells, the tumor becomes more visible to the immune system, making it easier for the immune cells to recognize and attack the tumor. This leads to an enhanced response to subsequent anti-PD-1 (aPD1) therapy, potentially improving patient outcomes.

[0759] Table 32: PD1 expression based on different treatment conditions and combinations

[0760] The Table 32 provides data on the PD1 expression effects of compound (I), aPD-1 as standalone and the compound (I) + anti-PD-1 in combination.

[0761] First of all, these data show that priming with compound (I) does not affect PD1 expression for the PEG negative control. Surprisingly, in the context of no priming, compound (I) is able to strongly reduce PD1 expression down to 18.49% and much less in the context of priming (29.51 %). Similarly, in the context of no priming, aPD1 is able to strongly reduce PD1 expression down to 17.87% and much less in the context of priming (25.54%). On the other hand, The level of PD1 expression reduction with compound (I) as stand-alone is surprisingly the same level as for a PD1 without priming. More surprisingly, combination between compound (I) and aPD1 without priming with compound (I) resulted in a higher level of PD1 (66.7%). In contrast, priming with compound (I) followed by a combination between compound (I) and aPD-1 have shown the strongest PD-1 reduction.

[0762] The data suggest that a priming dose administration of compound (I) allows to get the best aPD1 combination effect against PD-1 expression. Example 22 - Synergistic in vivo efficacy combination of compound (I) with anti-PD-L1 against TLR4 overexpressing tumor

[0763] Material and method

[0764] Material and methods are described in Example 3

[0765] Results

[0766] Table 33: comparative Tumor Growth Inhibition (TGI) between different in vivo tumors

[0767] Table 33 shows TGI for compound (I) alone and in combination with anti-PD-L1 on NCI-H69 with TLR4 overexpression. The analysis of coefficient of correlation, as described in Example 5, indicates that there is very high correlation between TLR4 level of overexpression and the Tumor Growth Inhibition for both respective compound (I) as a standalone (83% of correlation) and compound (I) in combination with anti-PD-L1 (95% of correlation).

[0768] Thus, targeting TLR4 overexpressing tumors with compound (I) is relevant in the context of combination with anti PD-L1.

[0769] Example 23- Synergistic combination between compound (I) and anti-PD-L1 on a number of pharmacodynamic factors

[0770] Material and method

[0771] Kibur experimental protocol is described in Example 6A.

[0772] Results

[0773] Table 34: Compound (I) and combination with anti-PD-L1 effect on CD11c DC marker and Ki67 Tumor proliferation marker (priming condition) from the Kibur experimentation in Example 1 As shown in Table 34, between compound (I) and anti-PD-L1 is synergistic as well on the reduction of tumor proliferation Ki67 and the Dendritic cells activation.

[0774] Example 24 - Sensitizing TLR4-overexpressing tumors to Anti-PD-L1 therapy with compound (I)

[0775] Material and method is described in Example 13

[0776] Results

[0777] As seen in the Table 35 below, treatment with compound (I) leads to a significant increase of PD-L1 expression on the surface of tumor cells.

[0778] Table 35: Expression of PD-L1 (PD1 L1) from NCI-H69 cell line treated with compound (I). Overexpression of PD-L1 from longitudinal phosphoproteome sample (after 12 hours)

[0779] Such a tumor upregulation of PD-L1 induced by compound (I) is a valuable approach to sensitize tumors to anti-PD-L1 treatment. When compound (I) upregulates PD-L1 on tumor cells, it increases the expression of the PD-L1 protein on the surface of the tumor cells. Upregulation of PD-L1 on tumor cells via a priming effect of compound (I) makes it easier for anti- PD-L1 therapy to target the tumor cells, as the increased expression of PD-L1 makes them more visible to the immune system.

[0780] Example 25 - Sensitizing TLR4-overexpressing tumors to Anti-PD-L1 therapy with compound (I)

[0781] Material and method:

[0782] Method is described in Example 13

[0783] Results

[0784] Table 36: Expression of PD-L2 (PD1 L2) from NCI-H69 cell line treated with compound (I). Overexpression of PD-L2 from longitudinal phosphoproteome sample (after 12 hours)

[0785] Tumor upregulation induced by compound (I) of PD-L2 is a valuable approach to sensitize tumors to anti-PD-L2 treatment. When compound (I) upregulates PD-L2 on tumor cells, it increases the expression of the PD-L2 protein on the surface of the tumor cells. This can make it easier for anti-PD-L2 therapy to target the tumor cells, as the increased expression of PD-L2 makes them more visible to the immune system.

[0786] Example 26 - in vivo synergistic effects of cisplatin and compound (I) on TLR4+ overexpressing tumor cell with and without priming

[0787] Material and Method

[0788] Kibur Method and protocol are described in Example 6A.

[0789] Results

[0790] Table 37: CC3, Ki67 and PD1 markers - without compound (I) priming

[0791] Results as elative change compared to vehicle group

[0792] As shown in the Table 37, in the context of no priming with compound (I), the combination "Cisplatin + compound (I)" has the highest impact on the CC3 apoptotic marker, indicating a significant effect on the specific biological process measured by CC3. On the other hand, the combination "Cisplatin + compound (I)" results in the lowest Ki67 value, suggesting the lowest level of cell proliferation among the conditions. Lastly, combination between Cisplatin and compound (I) is similar to compound (I) stand-alone.

[0793] Table 38:8 CC3, Ki67 and PD1 markers - with compound (I) priming

[0794] Results as relative change compared to vehicle group Table 38 presents data on the effects of various treatment agents on three markers in the context of compound (I) priming: CC3, Ki67, and PD-1. "Cisplatin + compound (I)" demonstrates the most substantial impact on the CC3 marker. For cell proliferation (Ki67), "Cisplatin + compound (I)" results in the lowest value, indicating the least proliferation, while "Cisplatin" ranks second. In terms of PD-1 expression, "Cisplatin" exhibits the highest value, possibly influencing the immune response. "Cisplatin + compound (I)" has the lowest PD-1 value. These findings underscore the differential effects of each treatment condition on these specific markers, providing valuable insights into their biological impacts.

[0795] In the context of cancer overexpressing TLR4, combination between compound (I) and cisplatin are surprisingly synergistic on multiple immunological markers, with and without compound (I) priming.

[0796] Example 27 - Sensitizing TLR4-mediated cisplatin resistance with compound (I)

[0797] Rationale

[0798] Recent studies have shown that TLR4 overexpression modulates survival and chemoresistance in multiple cancer cell lines. The effect of compound (I) on cisplatin efficacy was tested against Small Cell Lung Cancer tumor resistant to cisplatin NCI-H69 CPR.

[0799] Material and method

[0800] A range of concentrations was tested on (0, 1, 3, 10, 30 pM) at different time points, (24, 48, 78 hours). We considered inhibition lower than or equal to 10% to be insignificant.

[0801] Results

[0802] The effect of compound (I) on H69 or H69 CPR (Cisplatin resistant cells) is dose dependent with an IC25 of 30 pM after 24 hours or 48 hours incubation, although better efficacy is observed after 24 hours incubation for H69 CPR cells (Table 39).

[0803] Table 39: Viability of NCI-H69 CPR in combination with cisplatin

[0804] Therefore, treatment with compound (I) surprising blocks TLR4 and restores sensitivity to cisplatin in models resistant to cisplatin. Example 28 - in vivo synergistic effects of carboplatin and compound (I) on TLR4+ overexpressing tumor cell with and without priming

[0805] Material and method

[0806] Protocol is described in Example 6A

[0807] Results

[0808] Table 40: CD8, F4 / 80 and FoxP3 immunological marker without compound (I) priming

[0809] (*) relative change compared to vehicle group

[0810] Table 41 : CD11c, F4 / 80 and FoxP3 immunological marker without compound (I) priming

[0811] (*) relative change compared to vehicle group

[0812] First of all, compound (I) can be successfully combined (with and without priming) to carboplatin, showing synergy in terms of immune reaction with CD8 and CD11c increase (Table 40), immunosuppression reversion with F4 / 80, FoxP3 and CD206 reduction (Table 41) and in terms of tumor proliferation based on reduction .

[0813] Considering synergistic combination with cisplatin and carboplatin, one can extrapolate to platinum-based chemotherapeutics such as oxaliplatin with and without priming against TLR4 overexpressing tumors.

[0814] Example 29 - in vivo synergistic effects of doxorubicin and compound (I) on TLR4+ overexpressing tumor

[0815] Material and method

[0816] Protocol is described in Example 6A. Results

[0817] Table 42: CC3, CD11c, CD3 markers (with compound (I) priming)

[0818] Even if a combination between compound (I) and doxorubicin has not shown synergy at the in vitro level, it has shown and surprising and high level of synergy in terms of pro-apoptotic effect against the tumor (CC3 marker synergistically increased to 143.9), in terms of immune response activation (CD11c DC marker synergistically increased to 5.02) and in terms of Lymphocyte T cells population (Almost 10 times CD3 marker increase). These results are not to be anticipated in vivo by the skilled person (Table 42).

[0819] In light of these examples, it can be reasonably postulated that compound (I) exhibits an unexpected capacity for synergistic interaction when co-administered with anti-tumor antibiotics.

[0820] Example 30 - in vivo synergistic effects of topoisomerase inhibitor class I and compound (I) on TLR4+ overexpressing tumor with and without priming

[0821] Rationale

[0822] Topoisomerase inhibitors play a vital role in cancer therapy by disrupting the DNA replication process, ultimately halting cancer cell growth and division, and thus contributing to the success of cancer treatments.

[0823] Material and method

[0824] Protocol is described in Example 6A.

[0825] Results

[0826] Table 43: CD11c, CD3, CD8, FoxP3 and PD-1 immunological markers - without compound (I) priming

[0827] (*) relative change compared to vehicle group

[0828] Table 44: CD11c, FoxP3, Ki67 and PD-1 markers - with compound (I) priming

[0829] (*) relative change compared to vehicle group

[0830] Results

[0831] First of all, compound (I) can be successfully combined (with and without priming) to topotecan, showing synergy in terms of immune re-action with CD11c, CD3 and CD8 increase (Tables 43 and 44). On the other hand the deleterious effect of topotecan induced increase of immunosuppressive markers FoxP3 and PD-1 is significantly reduced thanks to a combination with compound (I), therefore improving topotecan therapeutic window.

[0832] Example 31 - in vivo synergistic effects of topoisomerase inhibitor class II and compound (I) on TLR4+ overexpressing tumor with and without priming Material and method

[0833] Protocol is described in Example 6A.

[0834] Results

[0835] Table 45: CD11c, CD3, CD8, FoxP3 and PD-1 immunological markers - without compound (I) priming Results as relative change compared to vehicle group

[0836] Table 46: CD11c, FoxP3, Ki67 and PD-1 markers - with compound (I) priming

[0837] Results as relative change compared to vehicle group

[0838] First of all, compound (I) can be successfully combined (with and without priming) to etoposide, showing synergy in terms of immune re-action with CD11c, CD8 increase (Table 45), reduction of immunosuppressive markers FoxP3 and tumor proliferation marker Ki67 (Tables 45 and 46). PD-1 is significantly reduced thanks to a combination with compound (I).

[0839] In light of the illustrations provided in Examples 30 and 31 , it can be reasonably postulated that compound (I) exhibits an unexpected capacity for synergistic interaction when coadministered with topoisomerase inhibitors belonging to both class I and class II.

[0840] Example 32 - Listing of TLR4 overexpressing tumors

[0841] Source = Eurofins Discovery

[0842] Expression above 1 considered as overexpression, very significant above 3

[0843] CELL LINE (TUMOR-TISSUE TYPE): TLR4=expression level

[0844] AGS (Stomach): TLR4=6.793; T24 (Bladder): TLR4=6.747; HS766T (Pancreas): TLR4=6.278; SKMEL3 (Melanoma): TLR4=6.275; SNB19 (Glioma (astrocytoma)): TLR4=6.223; HS821T (Sarcoma (Ewing's)): TLR4=6.024; J82 (Bladder): TLR4=6.016; MDAMB231 (Breast): TLR4=6.015; HL60 (Leukemia (acute promyelocytic)): TLR4=5.952; RPMI8226 (Myeloma (B- cell, plasmacytoma)): TLR4=5.926; CALU6 (Lung (NSCLC)): TLR4=5.789; HS863T (Sarcoma (Ewing's)): TLR4=5.716; HT1080 (Sarcoma (fibrosarcoma)): TLR4=5.618; DU4475 (Breast): TLR4=5.546; DBTRG05MG (Glioma (glioblastoma multiforme)): TLR4=5.446; HS695T (Melanoma): TLR4=5.286; WM115 (Melanoma): TLR4=5.241 ; HS822T (Sarcoma (Ewing's)): TLR4=5.234; LS123 (Colon): TLR4=5.212; HS729 (Sarcoma (rhabdomyosarcoma)): TLR4=5.175; TE125T (Sarcoma (rhabdomyosarcoma)): TLR4=5.125; SJSA1 (Osteosarcoma): TLR4=5.12; THP1 (Leukemia (acute monocytic)): TLR4=5.118; HPAC (Pancreas): TLR4=5.101 ; HS698T (Colon): TLR4=5.058; DKMG (Glioma): TLR4=5.044; SW480 (Colon): TLR4=5.042; SW684 (Sarcoma (fibrosarcoma)): TLR4=5.038; HS934T (Melanoma): TLR4=5.017; HS683 (Glioma): TLR4=5.016; NCIH69 (Lung (SCLC)): TLR4=4.992; TUR (Lymphoma (histiocytic)): TLR4=4.926; EB2 (Lymphoma (Burkitt's)): TLR4=4.877; HS852T (Melanoma): TLR4=4.806; NUDUL1 (Lymphoma (undifferentiated, nonBurkitt's type)): TLR4=4.781 ; SW1417 (Colon): TLR4=4.746; SKMEL28 (Melanoma): TLR4=4.736; SNU423 (Liver): TLR4=4.731 ; H4 (Glioma (neuroglioma)): TLR4=4.724; A375 (Melanoma): TLR4=4.698; T98G (Glioma (glioblastoma multiforme)): TLR4=4.673; HS604T (Lymphoma (Hodgkin's)): TLR4=4.67; U138MG (Glioma (glioblastoma)): TLR4=4.666; SW1353 (Osteosarcoma (chondrosarcoma)): TLR4=4.66; HS746T (Stomach): TLR4=4.655; MV411 (Leukemia (biphenotypic B myelomonocytic)): TLR4=4.648; TF1 (Leukemia (erythroleukemia)): TLR4=4.647; PSN1 (Pancreas): TLR4=4.645; ASPC1 (Pancreas): TLR4=4.612; U2OS (Osteosarcoma): TLR4=4.606; HS819T (Osteosarcoma (chondrosarcoma)): TLR4=4.598; SW620 (Colon): TLR4=4.587; REC1 (Lymphoma (Mantle cell, B-cell non-Hodgkin))): TLR4=4.57; SW982 (Sarcoma (synovial)): TLR4=4.561 ; CAKI2 (Kidney): TLR4=4.559; HS706T (Sarcoma (giant cell)): TLR4=4.554; YAPC (Pancreas): TLR4=4.532; HS739T (Breast): TLR4=4.529; COLO201 (Colon): TLR4=4.525; SUDHL4 (Lymphoma (B-cell non-Hodgkin)): TLR4=4.525; TCCSUP (Bladder): TLR4=4.52; HT (Lymphoma (B-cell diffuse mixed)): TLR4=4.519; HS281T (Breast): TLR4=4.518; BXPC3 (Pancreas): TLR4=4.508; SU8686 (Pancreas): TLR4=4.503; JEKO1 (Lymphoma (Mantle cell)): TLR4=4.46; NCIH747 (Colon (cecum)): TLR4=4.453; HS840T (Head & neck (pharyngeal papilloma)): TLR4=4.442; SH4 (Melanoma): TLR4=4.434; M059J (Glioma (glioblastoma)): TLR4=4.432; PC3 (Prostate): TLR4=4.425; HS578T (Breast): TLR4=4.419; HS294T (Melanoma): TLR4=4.41 ; SW403 (Colon): TLR4=4.405; SW116 (Colon): TLR4=4.4; DAUDI (Lymphoma (Burkitt's)): TLR4=4.4; HT1376 (Bladder): TLR4=4.382; SW948 (Colon): TLR4=4.355; HS229T (Lung (NSCLC)): TLR4=4.348; HELA (Cervix): TLR4=4.334; A2058 (Melanoma): TLR4=4.317; HS688AT (Melanoma): TLR4=4.312; G361 (Melanoma): TLR4=4.306; BHT101 (Thyroid): TLR4=4.299; EB3 (Lymphoma (Burkitt's)): TLR4=4.296; HS445 (Lymphoma (Hodgkin's)): TLR4=4.285; HS742T (Breast (scirrhous adenocarcinoma)): TLR4=4.284; HS839T (Melanoma): TLR4=4.282; SKMES1 (Lung (NSCLC, squamous cell carcinoma)): TLR4=4.28; CAL54 (Kidney): TLR4=4.272; SW48 (Colon): TLR4=4.266; SW1463 (Colon (rectum)): TLR4=4.264; A172 (Glioma (glioblastoma)): TLR4=4.263; HS936TC1 (Melanoma): TLR4=4.249; NAMALWA (Lymphoma (Burkitt's)): TLR4=4.248; HT1197 (Bladder): TLR4=4.229; SNU1 (Stomach): TLR4=4.223; NTERA2CLD1 (Testis): TLR4=4.219; NCIH508 (Colon (cecum)): TLR4=4.217; SUDHL10 (Lymphoma (large cell)): TLR4=4.212; CAL27 (Head & neck (squamous cell carcinoma, tongue)): TLR4=4.204; CAMA1 (Breast): TLR4=4.201 ; NCIH520 (Lung (NSCLC, squamous cell carcinoma)): TLR4=4.18; MDAMB175VII (Breast): TLR4=4.175; JAR (Placenta (choriocarcinoma)): TLR4=4.168; SJRH30 (Sarcoma (rhabdomyosarcoma)): TLR4=4.16; BT474 (Breast): TLR4=4.159; ALI565 (Breast): TLR4=4.151 ; SW954 (Vulva (squamous cell carcinoma)): TLR4=4.146; SW1271 (Lung (SCLC)): TLR4=4.144; OE19 (Head & neck (esophageal carcinoma)): TLR4=4.141 ; NALM6 (Leukemia (B-cell precursor, ALL)): TLR4=4.13; SW156 (Kidney (hypernephroma)): TLR4=4.119; CCRFCEM (Leukemia (acute lymphoblastic)): TLR4=4.118; HUTU80 (Duodenum): TLR4=4.113; LS513 (Colon (cecum)): TLR4=4.104; A498 (Kidney): TLR4=4.103; NCIH446 (Lung (SCLC)): TLR4=4.094; M DAM B453 (Breast): TLR4=4.094; A101 D (Melanoma): TLR4=4.083; CHP212 (Neuroblastoma): TLR4=4.081 ; U118MG (Glioma (glioblastoma)): TLR4=4.074; DMS273 (Lung (SCLC)): TLR4=4.07; HS895T (Melanoma): TLR4=4.066; SKPNDW (Sarcoma (neuroectodermal tumor, retroperitoneal)): TLR4=4.065; A253 (Head & neck (epidermoid carcinoma, salivary gland)): TLR4=4.061 ; LS1034 (Colon (cecum)): TLR4=4.061 ; EM2 (Leukemia (chronic myelogenous)): TLR4=4.05; HOS (Osteosarcoma): TLR4=4.048; SKNDZ (Neuroblastoma): TLR4=4.044; MJ (Lymphoma (cutaneous T-cell)): TLR4=4.043; HUPT4 (Pancreas): TLR4=4.037; HPAFII (Pancreas): TLR4=4.03; A431 (Head & neck (epidermoid carcinoma)): TLR4=4.03; T173 (Osteosarcoma): TLR4=4.029; LS411 N (Colon (cecum)): TLR4=4.025; OVCAR3 (Ovary): TLR4=4.013; VCAP (Prostate): TLR4=3.998; CORL105 (Lung (NSCLC)): TLR4=3.998; NCIH510A (Lung (SCLC)): TLR4=3.997; SCC4 (Head & neck (squamous cell carcinoma, tongue)): TLR4=3.991 ; JRT3T35 (Leukemia (acute T-cell)): TLR4=3.99; SKOV3 (Ovary): TLR4=3.989; SKBR3 (Breast): TLR4=3.975; MG63 (Osteosarcoma): TLR4=3.974; SW579 (Thyroid (squamous cell carcinoma)): TLR4=3.971 ; HLF (Liver): TLR4=3.968; SNU5 (Stomach): TLR4=3.967; 7860 (Kidney): TLR4=3.967; SW626 (Ovary): TLR4=3.964; JEG3 (Placenta (choriocarcinoma)): TLR4=3.959; SW1088 (Glioma (astrocytoma)): TLR4=3.958; CMLT1 (Leukemia (T-cell, CML)): TLR4=3.954; MALME3M (Melanoma): TLR4=3.95; NCIH1688 (Lung (SCLC)): TLR4=3.947; SR (Lymphoma (large cell immunoblastic)): TLR4=3.944; DU145 (Prostate): TLR4=3.94; SKMEL1 (Melanoma): TLR4=3.938; KU812 (Leukemia (chronic myelogenous)): TLR4=3.935; SIHA (Cervix (squamous cell carcinoma)): TLR4=3.931 ; SKNEP1 (Kidney): TLR4=3.927; SKNAS (Neuroblastoma): TLR4=3.926; TE441T (Sarcoma (rhabdomyosarcoma)): TLR4=3.923; HT29 (Colon): TLR4=3.923; COLO205 (Colon): TLR4=3.92; RS411 (Leukemia (acute lymphoblastic)): TLR4=3.911 ; WM2664 (Melanoma): TLR4=3.908; D0HH2 (Lymphoma (B-cell)): TLR4=3.903; 22RV1 (Prostate): TLR4=3.902; CROAP2 (Lymphoma): TLR4=3.897; REH (Leukemia (acute lymphocytic, non-B, non-T)): TLR4=3.884; SW872 (Sarcoma (liposarcoma)): TLR4=3.88; SNUC2B (Colon (cecum)): TLR4=3.878; C33A (Cervix): TLR4=3.875; PFSK1 (Glioma): TLR4=3.874; JURKAT (Leukemia (acute lymphoblastic)): TLR4=3.874; T47D (Breast): TLR4=3.87; SUDHL16 (Lymphoma (large cell, B-cell)): TLR4=3.87; SUDHL8 (Lymphoma (large cell)): TLR4=3.869; HS343T (Breast): TLR4=3.866; RL (Lymphoma (non-Hodgkin's)): TLR4=3.865; SUPT1 (Lymphoma (T-cell lymphoblastic)): TLR4=3.862; COLO320HSR (Colon): TLR4=3.859; SW962 (Vulva): TLR4=3.858; UMUC3 (Bladder): TLR4=3.858; C32 (Melanoma): TLR4=3.857; WIDR (Colon): TLR4=3.852; BEWO (Placenta (choriocarcinoma)): TLR4=3.852; JIYOYE (Lymphoma (Burkitt's)): TLR4=3.849; D283MED (Medulloblastoma): TLR4=3.847; H9 (Lymphoma): TLR4=3.84; SAOS2 (Osteosarcoma): TLR4=3.839; C32TG (Melanoma): TLR4=3.836; G292CLONEA141 B1 (Osteosarcoma): TLR4=3.831 ; HCT15 (Colon): TLR4=3.831 ; RPMI7951 (Melanoma): TLR4=3.826; HUCCT1 (Liver (cholangiocarcinoma, bile duct)): TLR4=3.824; IM9 (Myeloma (multiple)): TLR4=3.823; RL952 (Uterus / endometrium): TLR4=3.821 ; SW13 (Adrenal gland): TLR4=3.821 ; MHHPREB1 (Lymphoma (B-cell lymphoblastic non-Hodgkin)): TLR4=3.819; RAJI (Lymphoma (Burkitt's)): TLR4=3.818; BCP1 (Lymphoma (B-cell)): TLR4=3.818; SHP77 (Lung (SCLC)): TLR4=3.818; VAESBJ (Sarcoma (epitheloid)): TLR4=3.817; HLE (Liver): TLR4=3.816; KLE (Uterus / endometrium): TLR4=3.816; MEG01 (Leukemia (chronic myelogenous)): TLR4=3.813; SW1783 (Glioma (astrocytoma)): TLR4=3.812; U87MG (Glioma (glioblastoma)): TLR4=3.811 ; COLO829 (Melanoma): TLR4=3.806; L428 (Lymphoma (Hodgkin's)): TLR4=3.802; NCIH82 (Lung (SCLC)): TLR4=3.8; SW837 (Colon (rectum)): TLR4=3.799; NCIH596 (Lung (NSCLC)): TLR4=3.797; TT (Thyroid): TLR4=3.795; CAKI1 (Kidney): TLR4=3.792; K562 (Leukemia (chronic myelogenous)): TLR4=3.789; D341MED (Medulloblastoma): TLR4=3.788; SCC25 (Head & neck (squamous cell carcinoma, tongue)): TLR4=3.788; MOLT16 (Leukemia (acute lymphoblastic)): TLR4=3.785; WI38 (Lung (normal fibroblasts)): TLR4=3.784; SUDHL6 (Lymphoma (large cell, diffuse mixed histiocytic & lymphocytic, follicular B-cell)): TLR4=3.783; EFM19 (Breast): TLR4=3.781 ; SKUT1 (Sarcoma (leiomyosarcoma, uterus))): TLR4=3.78; CAPAN2 (Pancreas): TLR4=3.777; BC1 (Lymphoma (B-cell)): TLR4=3.772; ZR751 (Breast): TLR4=3.77; RDES (Sarcoma (Ewing's)): TLR4=3.768; MC116 (Lymphoma (undifferentiated)): TLR4=3.757; SCC9 (Head & neck (squamous cell carcinoma, tongue)): TLR4=3.755; G401 (Kidney): TLR4=3.748; 5637 (Bladder): TLR4=3.746; A704 (Kidney): TLR4=3.746; HS675T (Colon): TLR4=3.742; SKNFI (Neuroblastoma): TLR4=3.741 ; BE2C (Neuroblastoma): TLR4=3.738; LNCAP (Prostate): TLR4=3.737; CALU1 (Lung (NSCLC, epidermoid carcinoma)): TLR4=3.734; MOLT3 (Leukemia (acute lymphoblastic)): TLR4=3.733; SW900 (Lung (SCLC, squamous cell carcinoma)): TLR4=3.732; ARH77 (Myeloma (B-cell leukemia / plasma cell leukemia)): TLR4=3.731 ; A427 (Lung (NSCLC)): TLR4=3.73; OE21 (Head & neck (squamous cell carcinoma, esophagus)): TLR4=3.73; FADU (Head & neck (squamous cell carcinoma, pharynx)): TLR4=3.726; MDAMB436 (Breast): TLR4=3.722; C4I (Cervix): TLR4=3.72; PANC1 (Pancreas): TLR4=3.717; CAOV3 (Ovary): TLR4=3.716; CCFSTTG1 (Glioma (astrocytoma)): TLR4=3.714; CFPAC1 (Pancreas): TLR4=3.714; BFTC905 (Bladder): TLR4=3.713; U266B1 (Myeloma (B-cell)): TLR4=3.711 ; DAOY (Medulloblastoma): TLR4=3.71 ; DB (Lymphoma (B- cell)): TLR4=3.708; NCIH441 (Lung (NSCLC)): TLR4=3.708; SKLMS1 (Sarcoma (leiomyosarcoma)): TLR4=3.708; LS174T (Colon): TLR4=3.706; CGTHW1 (Thyroid): TLR4=3.699; CROAP5 (Lymphoma (B-cell)): TLR4=3.695; ST486 (Lymphoma (Burkitt's)): TLR4=3.694; DMS114 (Lung (SCLC)): TLR4=3.683; KG1 (Leukemia (acute myelogenous)): TLR4=3.679; KHOS240S (Osteosarcoma): TLR4=3.674; HT3 (cervix (retinoblastoma)): TLR4=3.673; MEWO (Melanoma): TLR4=3.666; HEL9217 (Leukemia (erythroleukemia)): TLR4=3.663; HS611T (Lymphoma (Hodgkin's)): TLR4=3.662; MDAMB415 (Breast): TLR4=3.661 ; RKOAS451 (Colon): TLR4=3.655; DMS53 (Lung (SCLC)): TLR4=3.649; CA46 (Lymphoma (Burkitt's)): TLR4=3.645; RD (Sarcoma (rhabdomyosarcoma)): TLR4=3.641 ; 769P (Kidney): TLR4=3.639; MOLT4 (Leukemia (acute lymphoblastic)): TLR4=3.639; MIAPACA2 (Pancreas): TLR4=3.639; HEC1A (Uterus / endometrium): TLR4=3.638; KATOIII (Stomach): TLR4=3.635; NCIH661 (Lung (NSCLC)): TLR4=3.631 ; BV173 (Leukemia (B-cell precursor, CML)): TLR4=3.631 ; HCT116 (Colon): TLR4=3.624; ES2 (Ovary (clear cell carcinoma)): TLR4=3.623; CHAGOK1 (Lung (NSCLC)): TLR4=3.619; BT549 (Breast): TLR4=3.617; NCIH295R (Adrenal gland): TLR4=3.616; A204 (Sarcoma

[0845] (rhabdomyosarcoma)): TLR4=3.616; CAL62 (Thyroid): TLR4=3.614; ME180 (Cervix (epidermoid carcinoma)): TLR4=3.612; HS888SK (Osteosarcoma): TLR4=3.609; SCABER (Bladder (squamous cell carcinoma)): TLR4=3.607; MCF7 (Breast): TLR4=3.606; CASKI (Cervix (epidermoid carcinoma)): TLR4=3.604; BPH1 (Prostate (benign prostatic hyperplasia)): TLR4=3.603; PA1 (Ovary): TLR4=3.596; MS751 (Cervix (epidermoid carcinoma)): TLR4=3.593; RKOE6 (Colon): TLR4=3.589; DOTC24510 (Cervix): TLR4=3.588; SNU16 (Stomach): TLR4=3.581 ; RKO (Colon): TLR4=3.574; NCIH292 (Lung (NSCLC)): TLR4=3.573; A549 (Lung (NSCLC)): TLR4=3.559; G402 (Kidney): TLR4=3.556; A673 (Sarcoma (Ewing's)): TLR4=3.554; AN3CA (Uterus / endometrium): TLR4=3.554; C4II (Cervix): TLR4=3.552; ACHN (Kidney): TLR4=3.548; A7 (Melanoma): TLR4=3.547; RAMOSRA1 (Lymphoma (Burkitt's)): TLR4=3.535; BT483 (Breast): TLR4=3.527; A388 (Head & neck (epidermoid carcinoma)): TLR4=3.524; NCIH460 (Lung (NSCLC)): TLR4=3.518; MDAMB468 (Breast): TLR4=3.514; 647V (Bladder): TLR4=3.512; CHL1 (Melanoma): TLR4=3.492; 639V (Bladder (ureter)): TLR4=3.491 ; DETROIT562 (Head & neck (pharyngeal carcinoma)): TLR4=3.484; IMR32 (Neuroblastoma): TLR4=3.48; CEMC1 (Leukemia (acute lymphoblastic)): TLR4=3.459; HEPG2 (Liver): TLR4=3.451 ; MCIXC (Neuroblastoma): TLR4=3.445; CAPAN1 (Pancreas): TLR4=3.44; MT3 (Colon): TLR4=3.428; BM1604 (Prostate): TLR4=3.427; CORL23 (Lung (NSCLC, large cell carcinoma)): TLR4=3.422; HMCB (Melanoma): TLR4=3.417; Y79 (Eye (retinoblastoma)): TLR4=3.417; MESSA (Sarcoma (uterine)): TLR4=3.413; BT20 (Breast): TLR4=3.405; SLIDHL5 (Lymphoma (large cell, diffuse mixed histiocytic)): TLR4=3.394; SKO007 (Myeloma (B-cell)): TLR4=3.393; HUT102 (Lymphoma (cutaneous T-cell)): TLR4=3.39; HCT8 (Colon): TLR4=3.381 ; GA10 (Lymphoma (Burkitt's)): TLR4=3.372; KPL1 (Breast): TLR4=3.361 ; RPMI6666 (Lymphoma (Hodgkin's)): TLR4=3.351 ; T84 (Colon): TLR4=3.35; DLD1 (Colon): TLR4=3.338; OE33 (Head & neck (esophageal carcinoma)): TLR4=3.333; COLO320DM (Colon): TLR4=3.313;

[0846] Example 33 - In vivo efficacy study vs Humanized NCI-N87 gastric cancer

[0847] Introduction

[0848] Overexpression of HER2 and TLR4 are commonly found in gastric cancer and is associated with a poor prognosis. One of the treatment is the antibody Trastuzumab (Roche) It works by specifically targeting this protein on the surface of cancer cells. By binding to HER2, trastuzumab blocks the activation of this protein, thereby preventing the growth and proliferation of cancer cells.

[0849] One approach to alternative or complementary gastric cancer treatment could involve using TLR4 antagonists to counteract the effects of TLR4 overexpression. And as shown previously, Compound (I) is a TLR4 antagonist and one of its potential mechanism is the inhibition of the TLR4 promoting effect in cancer which involves also HMGB1 protein:

[0850] Material and Method

[0851] Solutions

[0852] A stock solution at a concentration of 10 mg / mL Compound (I) was prepared by dissolving 9,9 mg of Compound (I) in 990 pL of 0.0176M NaOH. pH of the stock solution was then measured and adjusted to 7, 0-7, 5.

[0853] A dosing solution at 0.4 mg / mL of Compound (I) was prepared by adding 78,4 pL of the stock solution to 1881,6 pL sterile saline (0,9% NaCI). Intavenous dosing was carried out at 5 mL / kg to give a dose of 2 mg / kg.

[0854] Trastuzumab was bought from the supplier and injected in animals as is through the intraperitoneal route

[0855] Animals A total of 63 female NXG mice aged 5-8 weeks were used for the study. These were purchased from Janvier Laboratories and were allowed 7 days acclimatisation before entering the study. Animals were housed in IVC cages (up to 5 per cage) with individual mice identified by tail mark. All animals were allowed free access to a standard certified commercial diet and sanitised water during the study. The holding room was maintained under standard conditions: 20-24°C, 45-65% humidity and a 12h light / dark cycle.

[0856] All protocols used in this study have been approved by the Axis Bio Animal Welfare and Ethical Review Committee, and all procedures were carried out under the guidelines of the Animal (Scientific Procedures) Act 1986.

[0857] Tumor cell lines

[0858] NCI-N87 Cell Line Derived Xenograft is a human gastric adenocarcinoma cell line that was originally derived from a patient with poorly differentiated adenocarcinoma of the stomach Researchers have used NCI-N87 cells to study various aspects of gastric cancer biology. NCI- N87 cells are overexpressing Toll-like receptor 4 (TLR4) which is important in promoting the immune response in various cancers and TLR4 is highly expressed in a stage-dependent manner in gastric cancer

[0859] This cell line has also been used to test the efficacy of monoclonal antibodies, such as trastuzumab, that target HER2 and have been approved for the treatment of HER2-positive gastric cancer. The NCI-N87 CDX mouse model can be applied to studies involving apoptosis induction, circulating biomarkers, inhibition of migration and invasion, microtubule-inhibition (e.g. Nab-paclitaxel) and anti-tumor combination therapies (trastuzumab + capecitabine + oxaliplatin).

[0860] NCI-N87 cells (5 x1061 :1 with Matrigel) were implanted on the rear dorsum of female NXG mice (n=63). When tumours reached approximately 150 mm3animals were assigned to three treatment groups (Table 47). PBMCs from 2 donors were isolated, processed and dosed IV to the animals immediately prior to therapeutic dosing. Table 47

[0861] FACS

[0862] At the end of the treatment period (day 26), animals were culled and tumor tissue from those treated with vehicle or DHN198 were resected and digested to a single cell suspension using collagenase and DNase. The single cell suspension was divided into 2 equal aliquots and each aliquot stained using the following antibody panels for FACS analysis:

[0863] - Antibody panel 1 - Lymphocytes

[0864] Stained cells were run on an Attune NXT flow cytometer (thermos)apparatus and measures analysed using Becton, Dickinson Flowjo v10 software. Initial gating was carried out using forward and side scatter.

[0865] Results

[0866] Tumor Volume

[0867] Statistical analyses of tumor volumes on female NXG mice bearing NCI-N87 tumors were performed on data from the final day of study (day 26). Figure 14 shows the average tumor volume of NCI-N87 tumor bearing female NXG mice treated with vehicle control and each treatment group.

[0868] Tumor progression volumes were assessed every 2ndto 4thday up to sacrifice of mice, the results are summarized in table 48. Table 48

[0869] Tumors on animals treated with vehicle grew steadily during the study (Figure 14). Treatment with trastuzumab at 10mg / kg on day 1 and 16 resulted in NCI-87 tumors that were statistically smaller than vehicle controls (p=0.032, ANOVA; Table 49). Treatment with Compound (I) at 2 mg / kg BIW significantly controlled growth of NCI-N87 tumors (p=0.0048, ANOVA; Table 49).

[0870] There was no statistical difference in NCI-N87 tumor volume between animals treated with trastuzumab or Compound (I). Table 49 - Mean starting and Day 26 volume of NCI-N87 tumors implanted in female NXG mice. Values shown are mean ±SD; n=7 for all for all groups. Statistical analyses carried out using One-way ANOVA with Dunnett’s multiple comparisons test.

[0871] The percentage T / C (test versus control) values in table Table 49 were calculated as described below:

[0872] Mean tumour volume on day 26-Mean starting volume % T / C Value = - - -5- x100

[0873] Mean vehicle tumour volume on day 26-Mean vehicle starting volume FACS

[0874] Results from FACS analysis are summarized in figures 15 and 16 for panel 1 and in figures 17 and 18 for panel 2.

[0875] From results on figures 15 and 16, treatment with Compound (I) resulted in a highly significant increase in hCD45+ cell infiltration in NCI-N87 tumors (0.27 versus 10.01 % of viable cells; p=0.019, 2-tailed T-test). A significant increase in the proportion of lymphocytes (hCD3+ cells) within NCI-N87 tumours (25.64 versus 42.64; p=0.02, 2 tailed T-test) (associated significant decrease in CD3- cells was also observed) can be determined. A significant decrease in T-reg cell infiltration in NCI-N87 tumours (13.69 versus 2.74 % of CD4+ cells; p=0.01 , 2-tailed T-test) and a significant decrease in viable NCI-N87 tumour cells (97.87 versus 88.46% of total viable cells; p=0.02, 2-tailed T-test) can be observed

[0876] From results on figures 17 and 18, treatment with Compound (I) resulted in a significant increase in the proportion of M1 macrophages (1.90 versus 3.98 % of macrophages cells; p=0.047, 2-tailed T-test) and a highly significant increase in the proportion of N1 neutrophils (16.00 versus 29.74 % of macrophages cells; p=0.004, 2-tailed T-test).

Claims

1. CLAIMS1. Compound (I)or a pharmaceutically acceptable salt thereof for use in the treatment of patients with a neoplasm characterized by high expression of toll-like receptor 4 in the neoplasm, wherein high expression means an RPKM of 1 or higher, particularly 2 or higher, 3 or higher, 4 or higher, more particularly 4.5 or higher.

2. Compound (I) or a salt thereof for use in the treatment of patients with a neoplasm according to claim 1 , wherein said neoplasm is further characterized by at least one of the following blood and / or tumor pathological features: patients exhibiting pretreatment an abnormally reduced level of activated CD8-positive cytotoxic T lymphocytes (CD8+ cytotox) in the blood, wherein an abnormally reduced level means less than 800, in particular from 300 to 800, more particularly from 50 to 300, CD8-positive cytotoxic T lymphocytes per pL blood, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of exhausted CD8- positive cytotoxic T lymphocytes in the blood, wherein an abnormally increased level means more than 50, in particular from 50 to 200, more particularly from 200 to 600, exhausted CD8-positive cytotoxic T lymphocytes per pL blood, wherein said exhaustedCD8-positive cytotoxic T lymphocytes are characterized by the expression of the immunological markers PD-1 , Tim-3, Lag 3 and / or RGMb, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of CD4-positive T regulatory lymphocytes in the blood, wherein an abnormally increased level means more than 50, in particular from 50 to 200, more particularly from 200 to 500, CD4- positive T regulatory lymphocytes per pL blood, wherein said CD4-positive T regulatory lymphocytes are characterized by the expression of the immunological marker FoxP3, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced concentration of activated CD8-positive cytotoxic T lymphocytes compared to activated CD4-positive T regulatory lymphocytes in the blood, wherein an abnormally reduced concentration means that the ratio of CD8-positive cytotoxic T lymphocytes to CD4-positive T regulatory lymphocytes is 1 or lower, in particular between 0.75 and 1 , more particularly lower than 0.75, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced level of activated dendritic cells in the blood, wherein an abnormally reduced level means less than 100, in particular from 50 to 100, more particularly from 10 to 50, CD11c-positive cells per pL blood, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced level of activated natural killer cells, hereafter NKs, in the blood, wherein an abnormally reduced level means less than 500, in particular from 200 to 500, more particularly from 50 to 200, CD56-positive cells per pL blood, in particular as determined by flow cytometry; patients exhibiting pretreatment an abnormally reduced level of polarized macrophages type 1 , hereafter M1 , in the blood, wherein an abnormally reduced level means less than 300, in particular from 150 to 300, more particularly from 50 to 150, M1 cells per pL blood, wherein said M1 cells are characterized by the expression of the immunological markers CD80 and / or CD163, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of polarized macrophages type 2, hereafter M2, in the blood, wherein an abnormally increased level means more than 100, in particular from 100 to 200, more particularly from 200 to 500, M2 cells per pL blood, wherein said M2 cells are characterized by the expression of the immunological markers CD206, CD163, CD200, CD200R and / or CD209, in particular as determined by flow cytometry or immunohistochemistry;patients exhibiting pretreatment an abnormally reduced concentration of polarized macrophages type 1 compared to polarized macrophages type 2 in the blood, wherein an abnormally reduced concentration means that the ratio of M1 / M2 is 1 or lower, in particular between 0.75 and 1 , more particularly below 0.75, in particular as determined by flow cytometry or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of immunosuppressive cells in the tumor, wherein an abnormally increased level means that the concentration of the immunological marker PD-L1 in the tumor is 2 ng / pL or higher, in particular between 2 and 5 ng / pL, more particularly between 5 and 20 ng / mL, and / or of the immunological marker PD-L2 in the tumor is 2 ng / pL or higher, in particular between 2 and 5 ng / pL, more particularly between 5 and 15 ng / mL, in particular wherein the concentration of PD-L1 and / or PD-L2 is determined by ELISA or immunohistochemistry; patients exhibiting pretreatment an abnormally increased level of TME angiogenesis in the tumor, wherein an abnormally increased level means that the concentration of the growth factors VEGFa and / or VEGFc is 50 pg / pL or higher, in particular between 50 and 100 pg / pL, more particularly between 100 and 200 pg / pL, in particular as determined by ELISA; patients exhibiting pretreatment an abnormally increased level of TME fibrosis, wherein an abnormally increased level means that at least one of the following conditions is met: (i) in the case where said neoplasm is lung cancer, the Ashcroft score is 2 or higher, in particular between 2 and 5, more particularly between 5 and 8, in particular as determined by fibrotic histopathological assessment; (ii) the fibrotic stain score is 5% or higher, in particular between 5% and 15%, more particularly more than 15%, in particular as determined by fibrotic histopathological assessment using staining with Sirius Red, Fast Green, or via the Masson trichrome procedure; (iii) the concentration of the growth factor TGFbeta in the tumor is 5 ng / mL or higher, in particular between 5 and 10 ng / mL, more particularly between 10 and 20 ng / mL, in particular as determined by ELISA; (iv) the concentration of the growth factor CTGF in the tumor is 5 ng / mL or higher, in particular between 5 and 10 ng / mL, more particularly between 10 and 50 ng / mL, in particular as determined by ELISA, and (v) the concentration of the FGF growth factors in the tumor is 20 pg / mL or higher, in particular between 20 and 50 pg / mL, more particularly between 50 and 100 pg / mL, in particular as determined by ELISA; and patients exhibiting pretreatment an abnormally increased level of at least one of the following metastatic markers in the blood or tumor, respectively: (i) more than 1circulating tumor cell (CTC) per mL of blood, in particular between 1 and 50 cells / mL, more particularly between 50 and 200 cells / mL, in particular as determined by liquid biopsy or flow cytometry; (iii) a concentration of the epithelial-mesenchymal transition (EMT) marker MMP9 of 50 ng / mL or more in the tumor, in particular between 50 and 100 ng / mL, more particularly between 100 and 300 ng / mL, in particular as determined by ELISA.

3. Compound (I) or a salt thereof for use in the treatment of patients with a neoplasm according to claim 1 , wherein said neoplasm is caused by and / or associated with chronic non-resolved inflammation, chronic non-resolved infection, and / or fibrosis.

4. Compound (I) or a salt thereof for use in the treatment of patients with a neoplasm according to claim 1 , wherein said neoplasm is selected from head & neck cancer, oesophageal cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, uterus cancer, ovarian cancer mesothelioma, non-small cell lung cancer, small cell lung cancer, triple negative breast cancer, soft tissue sarcoma, osteosarcoma, fibrosarcoma, myelofibrosis, and desmoid tumors.

5. Compound (I) or a salt thereof for use in the treatment of patients with a neoplasm according to claim 1 , wherein said treatment is a second-line treatment after discontinuation of a first-line treatment of said neoplasm with a different anti-neoplasm drug.

6. A combination of (i) compound (I) or a salt thereof, and (ii) a second compound selected from the group consisting of immuno-oncologic agents, immunotherapeutic agents, adoptive cell therapies, targeted agents, chemotherapeutic agents, cancer vaccines and a combination of at least two of said agents, therapies and / or vaccines, or in combination with radiotherapy, for use in the treatment of patients with a neoplasm as defined in any one of claims 1 to 4.

7. The combination for use according to claim 6, wherein said second compound is selected from an immuno-oncologic agent and an immunotherapeutic agent, in particular wherein said second compound targets CTLA-4, PD-1 , PD-L1 , PD-L2 and / or a combination thereof.

8. The combination for use according to claim 7, wherein said second compound is a monospecific or bispecific antibody-based drug compound.

9. The combination for use according to claim 7, wherein said second compound is selected from molecules inhibiting transcription PD-L1 genes, small molecules inhibiting translation of PD-L1 mRNA, and small molecules being antagonists of PD-L1 protein.

10. The combination for use according to claim 7, wherein said second compound is a molecule selected from peptides, fusion proteins and nucleic acid strands in their naked form or linked to a toxic payload, cytotoxic agent or radioisotope.

11. The combination for use according to claim 6, wherein said immuno-oncologic agents and / or the immunotherapeutic agents are adoptive cells.

12. The combination for use according to claim 11 , wherein said adoptive cells are selected from the group consisting of CAR-T cells, NK cells, engineered T cells, tumor-infiltrating lymphocytes and a combination of at least two of said cells.

13. The combination for use according to claim 6, wherein said second compound is a targeted agent selected from the group consisting of anti-angiogenic agents, antibody-drug conjugates, targeted radioligands, and a combination of two or more angiogenic agents.

14. The combination for use according to claim 6, wherein said second compound is a chemotherapeutic agent that is a DNA modifying agent selected from a platinum-based compound, an antitumor antibiotic and a topoisomerase inhibitor.

15. The combination for use according to claim 14, wherein said DNA modifying agent is platinum-based and selected from lobaplatin, nedaplatin, miriplatin hydrate, lobaplatin, cisplatin, oxaliplatin, eptaplatin, heptaplatin, carboplatin, and dicycloplatin.

16. The combination for use according to claim 14, wherein said DNA modifying agent is an antitumor antibiotic selected from trabectidin, doxorubicin, mitomycin C, dactinomycin, daunorubicin, idarubicin, mitoxantrone, nemorubicin, bleomycin, and actinomycin D.

17. The combination for use according to claim 14, wherein said second drug is a topoisomerase inhibitor selected from class I inhibitor compounds etoposide and irinotecan, from class II inhibitors topotecan and pixantrone, or from combinations thereof.

18. The combination for use according to claim 17, wherein said second compound is comprised in an antibody-drug conjugate, in particular using a topoisomerase inhibitor as said second compound, in particular wherein said second compound is irinotecan comprised in sacituzumab.

19. The compound (I) or a salt thereof for use according to any one of claims 1 to 5, or the combination for use according to any one of claims 6 to 18, wherein said compound (I) or said salt thereof is comprised at a concentration of between 1 and 10 mg / mL and administered via intravenous route either as bolus injection or as a slow infusion in saline.

20. The compound (I) or a salt thereof for use according to any one of claims 1 to 5, or the combination for use according to any one of claims 6 to18, wherein compound (I) or said salt thereof is for use in a treatment that comprising a weekly or biweekly administration of compound (I) or a salt thereof for a period of 1 to 6 months.

21. The combination for use according to any one of claims 6 to 18, wherein said compound (I) or said salt thereof is for sequential administration in a sequence of treatments starting with one or two intravenous priming dose injections of said compound (I) or of said saltthereof between 72h and 1h prior to the start of the administration of said second compound or said radiotherapy.

22. The combination for use according to any one of claims 6 to 18, wherein said compound (I) or said salt thereof is for sequential administration in a sequence of treatments starting with administration of said compound (I) or of said salt thereof by intravenous route as a priming dose between 1 and 10 mg / m2followed by a second dose of said compound (I) or of said salt thereof performed between 1h and 72h, particularly 24h or 48h, after said first priming dose at the start of the administration of said second compound or start of said radiotherapy, wherein further administrations of said compound (I) or of said salt thereof are equally spaced by 7 days during a period of 1 to 6 months.

23. The combination for use according to any one of claims 6 to 18, wherein said compound (I) or said salt thereof is for sequential administration in a sequence of treatments starting with administration of said second drug or said radiotherapy, followed within 1h and 192h thereafter, in particular 24h thereafter, or 168h thereafter, by intravenous injections of said compound (I) or of said salt thereof, wherein further administrations of said compound (I) or of said salt thereof are equally spaced by 7 days during a period of 1 to 6 months.

24. The combination for use according to any one of claims 6 to 18, wherein said compound (I) or said salt thereof is for concomitant administration with administration of said second drug or said radiotherapy, wherein further administrations of said compound (I) or of said salt thereof start on either day 2 or day 9 and then take place equally spaced by 7 days during a period of 1 to 6 months.

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