WO2025247951A1 - Fyn kinase inhibitors, combinations and uses thereof - Google Patents

Abstract

The present invention relates to the field of medicine, in particular to products and uses thereof for activating or stimulating dendritic cell (DC), in particular for stimulating DC-mediated immunity, or for treating a disease or condition. More particularly, the invention relates to FYN kinase inhibitor(s), DC activated by a FYN kinase inhibitor, and DC defective for FYN, FGFR1, FGFR2 and/or HCK, as well as to uses thereof in the context of therapy. These products can further be used to prepare pharmaceutical compositions and combinations, as well as kits. The invention further relates to methods of prevention or treatment of a disease or condition involving anyone of the previously mentioned products.

Description

FYN KINASE INHIBITORS, COMBINATIONS AND USES THEREOF.

FIELD OF THE INVENTION

The present invention relates to the field of medicine, in particular to products and uses thereof for activating or stimulating dendritic cells (DCs), in particular for stimulating DC-mediated immunity, or for treating a disease or condition.

More particularly, the invention relates to FYN kinase inhibitors, DC activated by a FYN kinase inhibitor, and DC defective for FYN, FGFR1, FGFR2 and/or HCK such as Fv/z-silcnccd DC (in particular Fyn^v' DC), as well as to uses thereof in the context of therapy, preferably human therapy. These products can further be used to prepare compositions and combinations, in particular pharmaceutical compositions and combinations, as well as kits.

The invention further relates to methods of prevention or treatment of a disease or condition involving anyone of the previously mentioned products.

BACKGROUND OF THE INVENTION

In recent years targeted agents have shown clinical success in cancer patients. Among those, tyrosine kinase inhibitors (TKIs) are effective in the treatment of various neoplasms including hematological malignancies but also solid tumors such as lung cancers that overactivate the oncogenic kinase ALK (Shaw et al.). The advent of immunotherapies has generated a large panel of novel antineoplastic treatments. However, the most revolutionary evolvement in clinical oncology has been the introduction of immune checkpoint inhibiting monoclonal antibodies targeting CTLA-4 or the interaction between PD-1 and PD-L1, leading to an unprecedented control of multiple different cancer types (Andre, F et al.). Driven by this consideration, the inventors and others have developed screening methods to identify novel druggable immune checkpoints the inhibition of which enhances immunosurveillance. Various approaches focus on the identification of immunosuppressive signals expressed by cancer cells (such as PD-L1 or CD47), as well as on signaling routes that act on T cells to subvert their proliferation, infiltration of cancers, persistence within tumors and killing of malignant cells (Gu, S et al.,' Mair B et al.,' Freitas KA et al.,' Shifrut, E et al.,' Zhou, P et al.,' Belk, J. A. et al.,' Carnevale, J et al.).

However, very few studies investigated the targeting of relatively low-abundant dendritic cells (DCs) and the importance of DCs for the ignition of anticancer immune responses executed by T lymphocytes in the context of immunotherapy (Abratt, R et al.,' Liu, J et al.,' James, B.R. et al.,' Broz, M.L. et al.,' Spranger, S et al.,' de Mingo Pulido, A et al.,' Jaime-Sanchez, P et al.,' Li Dongming et al.), despite the need to develop additional rationale-based novel therapeutic strategies for patients.

The present invention now provides such new and advantageous therapeutic strategies.

SUMMARY OF THE INVENTION

Here, the inventors report the results of a large chemical drug screen which is based on immortalized DC precursors that have been differentiated into tumor reactive DCs (also herein identified as “cDCl” for “immature DCs of the type I conventional DC subtype”) and characterized both in vitro and in vivo. They advantageously herein demonstrate for the first time that the FYN protooncogene tyrosine kinase inhibitors as represented for example by TG100801, genetic ablation of FYN as well as its inhibition via monoclonal antibodies, enhances cancer immunosurveillance by inhibiting FYN kinase in dendritic cells (DC). In this setting, FYN operates as a druggable immune checkpoint that restrains the function of DCs. Similar results have been obtained by inhibiting Fgfrl, Fgfr2 or Hck (instead of Fyn) in dendritic cells (DC). In preclinical in vivo models, genetic and pharmacological inhibition of FYN has cDCl -dependent and T lymphocyte-mediated antineoplastic effects that synergize with PD-1 blockade immunotherapy.

Products and uses thereof for activating or stimulating dendritic cell (DC) in vivo, ex vivo or in vitro, in particular for stimulating DC-mediated immunity, or for treating a disease or condition in a subject, are advantageously herein described by inventors.

In a first aspect, inventors herein describe a FYN kinase inhibitor for use in enhancing, stimulating and/or increasing the immune response (immunity) in a subject in need thereof, e.g., in a subject suffering of a disease or condition, or in a subject at risk of developing a severe disease or condition, for example a cancer or an infection, by inducing and/or stimulating DC activation and/or maturation, in particular DC differentiation into activated DC, and/or by increasing antigen presentation by DC, thereby substantially improving treatment outcomes.

In a particular aspect, the FYN kinase inhibitor is described for use in prevention or treatment of cancer in a subject in need thereof. In another particular aspect, the FYN kinase inhibitor is described for use in prevention or treatment of an infection in a subject in need thereof. Also herein described is the in vitro or ex vivo use of a FYN kinase inhibitor to stimulate DC activation, increase antigen presentation by DC, and/or facilitate DC progenitor differentiation into an antigen reactive DC, i.e., into an immature DCs of the type I conventional DC subtype (“cDCl”).

In a particular aspect, the FYN kinase inhibitor is a small molecule selected from 4-chloro-3-[5- methyl-3-[4-(2-pyrrolidin-l-ylethoxy)anilino]-l,2,4-benzotriazin-7-yl]phenyl] benzoate

(TG100801), 4-chloro-3-[5-methyl-3-[4-(2-pyrrolidin-l-ylethoxy)anilino]-l,2,4-benzotriazin-7- yl]phenol (TG100572, CAS867334-05-2), pyrazolol[3,4-d] pyrimidine 1 (PPI, CAS No 172889- 26-8), pyrazolol[3,4-d] pyrimidine 2 (PP2, CAS No 172889-27-9), ARN25068 (CAS No 2649882-80-2), SU6656 (CAS No 330161-87-0), 1-NM-PP1 (CAS No 221244-14-0), 1- Naphthyl PPI (CAS No 221243-82-9), Saracatinib (CAS No 379231-04-6), and any functional derivative thereof such as for example the TG 100572 hydrochloride salt (CAS No 867331-64-4) of TG100572, inhibiting the functional expression of the Fyn gene of SEQ ID NO: 2, 3 or 4 or of the FYN protein of SEQ ID NO: 5, 6 or 7. In a particular aspect, the small molecule used as FYN kinase inhibitor is TG100801, PPI or PP2, or any functional derivative thereof.

In another particular aspect, the FYN kinase inhibitor is a FYN kinase antibody or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7, such as for example the Fyn (15) monoclonal antibody from Santa Cruz, or any functional equivalent thereof capable of targeting a sequence identical to, or comprising, SEQ ID NO: 65, such as for example the Anti-FYN (AA 1-206) Recombinant Antibody (CBXS-0025) from Creative Biolabs (https://www.antibody-creativebiolabs.com/anti-fyn-aa-l-206-antibody-cbxs- 0025-366076.htm), or the abl881 antibody from Abeam (https://www.abcam.com/en- us/products/primary-antibodies/fyn-antibody-fyn-01- abl 881 ?srsltid=AfmBOooj_3 lEhSN4QVNIOrbjUU9EhJjpYGvZqwu58U8Vpp5bSpRya7wk). Herein described FYN kinase antibodies are, as such, particular objects of the present invention.

Inventors also describe a composition comprising i) a FYN kinase inhibitor, a population of DC activated by a FYN kinase inhibitor, and/or a population of Fyn^v' DC, Fgfrl DC, Fgfr2'^/' DC and/or Hck' DC, and ii) a pharmaceutically acceptable support (also herein identified as an excipient, carrier or diluent), as well as said composition for use in the prevention or treatment of cancer or of an infection in a subject in need thereof.

Also herein described is a combination of i) a product selected from a FYN kinase inhibitor, a population of DC activated by a FYN kinase inhibitor, and a population of Fyn^v' DC, Fgfrl DC, Fgfr2'^/' DC and/or Hck' DC, and ii) T-cell immune check-point inhibitor(s), in particular anti- PD1 and/or anti-PDLl agent(s), as well as said combination for use as a medicament, in particular for use in the prevention or treatment of cancer or of an infection.

Thus, any one of the herein described small molecules, for example TG100801, PPI, PP2, or any functional derivative thereof, may be used in combination with T-cell immune check-point inhibitor(s), in particular anti-PDl and/or anti-PDLl agent(s), typically as a medicament, in particular in the prevention or treatment of cancer or of an infection.

Also herein described is the corresponding method of preventing or treating cancer or an infection in a subject in need thereof, wherein the method comprising a step of administering the subject with a FYN kinase inhibitor, a DC (or population of DC), a composition and/or a combination as described in the context of the present invention, thereby treating the subject.

Further herein described is a kit comprising any one or more of the herein described products, typically (a) one or several products selected from FYN kinase inhibitor(s), a DC activated by a FYN kinase inhibitor or a population of said DC, and a Fyn^v' DC, Fgfrl DC, Fgfr2'^/' DC or Hck, ^/_ DC, or a population of Fyn DC, Fgfrl DC, Fgfr2'^/' DC and/or Hck' DC, and (b) T-cell immune check-point inhibitor(s), in particular anti-PDl and/or anti-PDLl agent(s), preferably in different containers; or a composition comprising the combination of the product(s) (a) and the T-cell immune check-point inhibitor(s) (b), and a pharmaceutically acceptable carrier and material(s) for administering the product(s) (a) and/or the T-cell immune check-point inhibitor(s) (b) or for administering the composition. The kit may optionally comprise (c) additional distinct therapeutic agent(s), in particular an anti-cancer agent or an agent active against infection.

Typically, the kit also comprises instructions for using the inhibitor(s), cell(s), composition(s) or combination(s) according to the invention, in particular according to the disclosed methods.

Other advantages of the products and uses herein described are further indicated below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel therapeutic products, combinations of products, and pharmaceutical compositions comprising such products, as well as uses thereof in human medicine, preferably in oncology. These products are used for activating or stimulating dendritic cell (DC) in vivo, ex vivo or in vitro, in particular for stimulating DC-mediated immunity. In particular, inventors herein describe a FYN kinase inhibitor for use in enhancing, stimulating and/or increasing the immune response (immunity) in a subject in need thereof, e.g., in a subject suffering of a disease or condition, or in a subject at risk of developing a severe disease or condition, in particular a cancer or an infection, by inducing and/or stimulating DC activation and/or maturation, in particular DC differentiation into activated DC, and/or by increasing antigen presentation by DC, thereby substantially improving treatment outcomes.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject-matter disclosed herein belongs.

The following definitions may be useful to understand embodiments as presented herein.

The term “subject” or “patient” used interchangeably, refer to any single subject for which therapy is desired or that is participating in a clinical trial, epidemiological study or used as a control, including humans and mammalian veterinary patients.

The “subject” or “patient” is typically an animal, preferably a mammal. The subject can be a human or a non-human mammal such as a rodent, for example a mouse or a rat, a rabbit, a primate such as a monkey, a dog, a cat, a pork, a sheep, a bovid, an equine, for example a horse, or transgenic species thereof.

In a particular and preferred aspect, the mammal is a human being, whatever its age or sex. In some embodiments, the subject is an adult human subject. In some embodiments, the subject is a human child between the ages of birth and 18 years old. In a particular aspect, the subject is a child between the ages of birth and 15 years old having a pediatric cancer.

In a particular aspect, the subject is a subject having (suffering of) an infection.

In a preferred aspect, the subject is a subject having (suffering of) a cancer. Unless otherwise specified in the present disclosure, the cancer is characterized by malignant tumor and/or metastasis present for example in the brain, a bone, a lung, or the liver.

In a preferred embodiment, the subject is suffering of a lung cancer, preferably of a NSCLC.

In another preferred embodiment, the subject is suffering of a breast cancer or of a colorectal cancer.

In a particular aspect of the present invention, the subject is a subject undergoing a treatment of cancer. This patient is preferably a subject who does not respond (or in other terms who is resistant) to the treatment.

In a particular aspect, the patient is one who has been exposed, or who is still exposed, to a standard-of-care treatment with an anti-cancer agent and more preferably with an immunotherapeutic agent, for instance with an antibody targeting an immune checkpoint such as CTLA-4, PD-1, PD-L1 or TIGIT, an agonist of 0X40, CD40, STING or a Toll-like receptor, type-1 interferons or interleukins, said immunotherapeutic agent being optionally combined with a chemotherapeutic agent, a radiotherapy or an oncolytic virus.

The treatment may have occurred in a neoadjuvant setting (i.e. before surgery) or not (i.e. after surgery).

As used herein, “treating”, “treatment” or “treat” refers to therapeutic intervention in an attempt to alter the natural course of the subject being treated, and is typically performed for curative purpose. Desirable effects of treatment include, but are not limited to, preventing recurrence of disease, alleviation of symptoms, and diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In preferred embodiments, compositions and methods of the invention are used to delay development of a cancer or to slow the progression of a cancer, typically of tumor growth, or to delay development of an infection or to slow the progression of an infection.

Unless otherwise indicated, the terms “cancer”, “cancerous tumor”, “malignant tumor”, “tumor”, “neoplasia”, “cancer disease”, or “proliferative disease”, are herein used interchangeably. These terms refer to or describe the physiological condition in subjects that is typically characterized by unregulated cell growth. As used herein “cancer” refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth. As used herein “cancer” refers to solid tumors named for the type of cells that form them, as well as cancer of blood, bone marrow, or the lymphatic system. Examples of solid tumors include but are not limited to sarcomas, carcinomas and blastomas. Examples of cancers of the blood include but are not limited to leukemias, lymphomas and myeloma. The term “cancer” includes but is not limited to a primary cancer that originates at a specific site in the body. The term cancer also includes a cancer that has metastasized, i.e., that has spread from the place in which it started to other parts of the body, for example to the central nervous system (CNS) in particular to the brain, or to the bone, lung, or liver; a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of a different type from latter one.

In the context of the invention, a “tumor cell” is a cell obtained from a tumor or tissue of a subject suffering from a cancer, in particular from at least one of the herein identified cancers, preferably lung cancer, in particular non-small cell lung carcinoma (NSCLC), and exhibiting well-known hallmarks of cancer cells, e.g. sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. It is to be understood that the expression “tumor cells” used to identify cells obtained from a tumor of a subject, is also used, in the present description, to identify circulating tumor cells, cells obtained from a liquid tumor biopsy, cells obtained from a tumor bed, or cells obtained from a metastasis.

In some aspects, the cancer is refractory or resistant to treatment with a conventional therapeutic agent, for example with a tyrosine kinase inhibitor (TKI), or has progressed on, treatment with a conventional therapeutic agent, for example with a TKI.

In an aspect of the invention, the malignant tumor is selected from a sarcoma, a carcinoma, a blastoma, a lymphoma, a myeloma and a leukemia, for example from a lung cancer, a breast cancer and a colorectal cancer. In a preferred aspect, the cancer is a lung cancer, in particular a non- small cell lung cancer (NSCLC).

Unless otherwise indicated, the terms “infection” or “infectious disease” designate disorders, typically illness, caused by organisms such as for example bacteria, viruses, fungi or parasites. Signs and symptoms vary depending on the organism causing the infection, but often include fever and fatigue. Mild infections may respond to rest and home remedies, while some lifethreatening infections may need hospitalization. Hosts can fight infections using their immune systems. Mammalian hosts react to infections with an innate response, often involving inflammation, followed by an adaptive response. Specific medications used to treat infections include antibiotics, antivirals, antifungals, antiprotozoals and antihelminthics. Infectious diseases resulted in 9.2 million deaths in 2013 (about 17% of all deaths).

In an aspect of the invention, the infection is selected from an infection caused by a bacterium, virus, fungus, parasite or prion. The infection may be for example a respiratory infection (including the common cold, influenza, and pneumonia which can be caused by various viruses and bacteria), a gastrointestinal infection (for example by norovirus or rotavirus as well as any bacterial infection causing diarrhea and/or vomiting), acquired immunodeficiency syndrome (induced by human immunodeficiency virus), tuberculosis, malaria, Dengue fever, hepatitis (including hepatitis A, B and C), a sexually transmitted infection (such as for example chlamydia, gonorrhea or syphilis) or a vector-borne disease (for example Zika virus, Lyme disease, and West Nile virus) transmitted by vectors like mosquitoes and ticks.

A dendritic cell (also herein identified as a “DC”) is an antigen-presenting cell (also known as an accessory cell) of the mammalian immune system. A DC’s main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and adaptive immune systems.

FYN is a member of the Src family of tyrosine kinases typically associated with T-cell and neuronal signaling in development and normal cell physiology. Disruptions in these signaling pathways often have implications in the formation of a variety of cancers. As a proto-oncogene, FYN codes for proteins that help regulate cell growth. Changes in its DNA sequence transform it into an oncogene that leads to the formation of a different protein with implications for normal cell regulation.

The enzyme comprises 537 amino acids in a single polypeptide chain organized into 4 domains, with a molecular weight of 59 kilodaltons. The N-terminal membrane anchoring domain (SH4) possesses myristylated or palmitylated residues essential for proper enzyme trafficking and localization to the cytoplasmic membrane, immediately adjacent to this are the SH3 and SH2 domains, which play critical roles in the interaction of Fyn kinase with its protein targets, followed by a flexible linker connecting the C-terminal tyrosine kinase domain (SHI).

The SH2 domain of Fyn kinase binds phosphotyrosine-containing sequences and functions as a target recognition domain. The SH3 domain promiscuously complexes with polyproline peptides and plays a role in mediating protein-protein interactions between Fyn kinase and other polypeptides. Interaction between the SH3 and SH2 domains enhances the specificity of ligand binding and regulates the activity of the kinase (SH4) domain.

The well conserved SH4 domain contains two potential inhibitor binding sites, the first, sensitive to non-specific competitive inhibitors of ATP such as staurosporine, represents the actual ATP binding site of the kinase domain, whereas the second site, immediately adjacent to the ATP binding pocket, is oriented towards the SH2 domain. This second site may be occupied in such a way that ATP binding at the first site is not directly blocked. However, free access to the second site is blocked by the presence of ATP at the first site, and hence, inhibitors targeted to the second site display pseudo-competitive kinetics with respect to the availability of ATP. Some Fyn kinase inhibitors suffer from a lack of specificity and although many Fyn kinase inhibitors have been isolated, most significantly cross-react with other Src family enzymes. Some of the best known Fyn kinase inhibitors include: phenolic compounds such as rosmarinic acid, (-)-epigallocatechin gallate (EGCG) and myricetin; the pyrazolol[3,4-d] pyrimidines PPI and PP2, and various derivatives thereof; as well as other fused pyrimidine compounds such as benzyl 21-methoxy- 5,7,19-trioxa-2,13,24,26-tetraazapentacyclo[18.6.2.0³,¹¹.0⁴,⁸.0²³,²⁷]octacosa- l(26),3(l l),4(8),9,20,22,24,27-octaene-13-carboxylate (Janssen Pharmaceuticals, U.S. Patent No. 8,492,377) and CT5263 and CT5102 (CellTech). For example EGCG has moderate binding affinity to FYN kinase (see Saeki et al.), contrary to PPI or PP2 (cf. 2018 and Hanke et al., 1996). In humans, FYN enzyme is encoded by the FYN gene (p59-FYN, Slk, Syn, MGC45350, Gene ID 2534). The protein associates with the p85 subunit of phosphatidylinositol 3-kinase and interacts with the FYN-binding protein. Spliced transcript variants encoding distinct isoforms exist. Here, the term “FYN gene” or “FYN" refers in particular to any nucleotide sequence selected from SEQ ID NO: 2 (CCDS5094.1), 3 (CCDS 5095.1) or 4 (CCDS 5096.1), and the term “FYN enzyme” refers in particular to any amino acid sequence selected from SEQ ID NO: 5 (P06241-1), 6 (P06241-2) or 7 (P06241-3).

Fibroblast growth factor receptor 1 (FGFR1), also known as basic fibroblast growth factor receptor 1, fms-related tyrosine kinase-2 / Pfeiffer syndrome, and CD331, is a tyrosine kinase receptor whose ligands are specific members of the fibroblast growth factor family. Here, the term “FGFRI” refers in particular to any amino acid sequence selected from SEQ ID NO: 14 (Pl 1362- 1 - FGFRl-IIIc), 15 (11362-8), 16 (Pl 1362-17), 17 (Pl 1362-2), 18 (Pl 1362-9), 19 (Pl 1362-3), 20 (Pl 1362-10), 21 (Pl 1362-4), 22 (Pl 1362-11), 23 (Pl 1362-5), 24 (Pl 1362-12), 25 (Pl 1362- 6), 26 (Pl 1362-13), 27 (Pl 1362-7), 28 (Pl 1362-14), 29 (Pl 1362-15), 30 (Pl 1362-16), 31 (Pl 1362-18), 32 (Pl 1362-19 - FGFRl-IIIb), 33 (Pl 1362-20), or 34 (Pl 1362-21).

The FGFRI gene is located on human chromosome 8 at position pll.23 (i.e., 8pl l.23), has 24 exons, and codes for a precursor mRNA that is alternatively spliced at exons 8A or 8B thereby generating two mRNAs coding for two FGFR1 isoforms, FGFRl-IIIb (also termed FGFRlb) and FGFRl-IIIc (also termed FGFRlc), respectively. Although these two isoforms have different tissue distributions and FGF-binding affinities, FGFRl-IIIc appears responsible for most of functions of the FGFR1 gene while FGFRl-IIIb appears to have only a minor, somewhat redundant functional role. Somatic mutations and epigenetic changes in the expression of the FGFRI gene occur in and are thought to contribute to various types of lung, breast, hematological, and other types of cancers. Here, the term “FGFRI gene” or “FGFRI” refers in particular to any nucleotide sequence selected from SEQ ID NO: 8 (CCDS43730.1), 9 (CCDS43731.1), 10 (CCDS43732.1), 11 (CCDS55221.1), 12 (CCDS 55222.1- FGFRI Illb) or 13 (CCDS6107.2 - FGFRI IIIc).

Fibroblast growth factor receptor 2 (FGFR2) also known as CD332 (cluster of differentiation 332) is a protein that in humans is encoded by the FGFR2 gene residing on chromosome 10. Here, the term “FGFR2 gene” or “FGFR2” refers in particular to any nucleotide sequence selected from SEQ ID NO: 35 (CCDS31298.1 - FGFR2-IIIc), 36 (CCDS44485.1 - FGFR2-IIIb), 37 (CCDS44486.1), 38 (CCDS44487.1) or 39 (CCDS44488.1).

FGFR2 is a receptor having important roles in embryonic development and tissue repair, especially bone and blood vessels.

FGFR2 has two naturally occurring isoforms, FGFR2IIIb and FGFR2IIIc, created by splicing of the third immunoglobulin-like domain. FGFR2IIIb is predominantly found in ectoderm derived tissues and endothelial organ lining, i.e. skin and internal organs. FGFR2IIIc is found in mesenchyme, which includes craniofacial bone and for this reason the mutations of this gene and isoform are associated with craniosynostosis. FGFR2 has been shown to interact with Fibroblast growth factor 1 (FGF1). Mutations (changes) are associated with numerous medical conditions that include abnormal bone development (e.g. craniosynostosis syndromes) and cancer.

Here, the term “FGFR2” refers in particular to any amino acid sequence selected from SEQ ID NO: 40 (P21802-1 - - FGFR2-IIIc), 41 (P21802-2), 42 (P21802-3 - FGFR2-IIIb), 43 (P21802- 4), 44 (P21802-5), 45(P21802-6), 46 (P21802-8), 47 (P21802-14), 48 (P21802-15), 49 (P21802- 16), 50 (P21802-17), 51 (P21802-18), 52 (P21802-19), 53 (P21802-20), 54 (P21802-21), 55 (P21802-22) and 56 (P21802-23).

Tyrosine-protein kinase HCK is an enzyme that in humans is encoded by the HCK gene. Here, the term “HCK gene” or “HCK' refers in particular to any nucleotide sequence selected from SEQ ID NO: 57 (CCDS33460.1), 58 (CCDS54453.1), 59 (CCDS54455.1) or 60 (CCDS54456.1), and the term “HCK” refers in particular to any amino acid sequence selected from SEQ ID NO: 61 (P08631-1), 62 (P08631-2), 63 (P08631-3) or 64 (P08631-4).

HCK comprises five distinct domains which include two terminal domains and three SH domains. The N-terminal domain is important for lipid modifications and a C-terminal domain includes a regulatory tyrosine residue. Next, HCK comprises three highly conserved SH domains: SHI, SH2, and SH3. The catalytic SHI domain houses the kinase’s active site. The regulatory SH3 and SH2 domains are tightly bound together when HCK is in an inactive state. HCK is localized in the cytoplasm where it executes its functions as a kinase. HCK plays a key role during inflammation as it participates in actin-dependent processes like phagocytosis, membrane remodeling, and cell migration. It has also been shown that HCK participates in NLRP3 inflammasome formation and LPS-induced inflammatory response in mice. However, the mechanism of action is yet to be elucidated. HCK is part of a CXCL12/CXCR4 signaling axis that is partially responsible for the migration of leukemic cells in the bone marrow of patients with acute myeloid leukemia. HCK has also been implicated in driving cell survival in drug- tolerant cancer cells. Fyn⁷' DC or organisms, Fgfrl DC or organisms, Fgfr2⁷' DC or organisms and Hck⁷ DC or organisms designate genetically modified, more specifically knock-out (KO), dendritic cells or organisms, wherein the FYN, FGFR1, FGFR2 and HCK expression respectively is absent.

Tyrosine kinase inhibitors (TKI) are a group of pharmacologic agents that disrupt the signal transduction pathways of protein kinases by several modes of inhibition. Tyrosine kinases are enzymes responsible for the activation of many proteins by signal transduction cascades. The proteins are activated by adding a phosphate group to the protein (phosphorylation), a step that TKIs inhibit.

Recently, inventors developed a miniature immune system assay that allows for phenotypic drug screens aiming at the identification of immunostimulatory agents that positively impact dendritic cell (DC) antigen presentation. Here, they report a screen employing all available tyrosine kinase inhibitors that revealed the prodrug TG100801 as well as its active metabolite TG100572 as enhancers of DC antigen presentation. TG100801 was able to control tumor growth in immunocompetent mice but not in immunodeficient animals lacking mature T cells. Furthermore, they showed that TG100801 induced the activation of tumor antigen-reactive immature DCs of the type I conventional DC subtype (“cDCls”) but did not exhibit any direct effect on T cells. Depletion of cDCl reduced the therapeutic efficacy of TG100801 alone or in combination with PD-1 blockade. A genetic screen focusing on known tyrosine kinases inhibited by TG100801 revealed that the knockout of Fyn phenocopied the effect induced by TG100801. When injected into mice, Fyn⁷' DCs primed strong antigen- specific T cell activation analogue to TG100801- treated wildtype DCs. Treatment of Fyn DCs with TG100801 did not further increase their function. Through pharmacologic and genetic inhibition of FYN, inventors unveil a druggable DC-immune checkpoint that controls adaptive anticancer immunity.

In the context of the present invention, a FYN kinase inhibitor is a product inhibiting the expression, in particular the functional expression, of FYN, FGFR1, FGFR2 and/or HCK, preferably a product inhibiting the expression, in particular the functional expression, of at least FYN. In a preferred aspect, the FYN kinase inhibitor stimulates dendritic cells (DC) maturation and/or activation and/or increases antigen presentation by DC. This product may be a small molecule; a nucleic acid molecule; a peptide; an antibody, a derivative or any functional fragment thereof; an aptamer; or a ribozyme. In a particular preferred aspect, the FYN kinase inhibitor is a hydrophilic molecule, i.e., an inhibitor unable to cross cellular membranes, for example a molecule naturally comprising polar covalent bonds or wherein such bonds have been introduced.

The term “small molecule”, “drug” or “prodrug” designates a low molecular weight (preferably < 1000 daltons) organic or chemical compound or molecule that may regulate a biological process, in particular that bind specific biological macromolecule(s) and act as an effector, altering activity or function of the target. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.).

In a particular aspect, the FYN kinase inhibitor is a small molecule or compound inhibiting phosphorylation of the FYN kinase and/or FYN kinase activation.

Inventors herein identify in particular “TG100801” and its metabolite “TG100572”, as potent small molecule FYN (kinase) inhibitors. Functional derivatives of these compounds are within the scope of the claimed disclosure.

“TG100801” designates the “4-chloro-3-[5-methyl-3-[4-(2-pyrrolidin-l-ylethoxy)anilino]-l,2,4- benzotriazin-7-yl]phenyl] benzoate” (IUPAC name) product (CAS867331-82-6). Both names are herein used interchangeably. TG100801 is described further in Palanki et al.. A skilled person can refer to this document for the synthesis of TG100801.

“TG100572” designates the “4-chloro-3-[5-methyl-3-[4-(2-pyrrolidin-l-ylethoxy)anilino]-l,2,4- benzotriazin-7-yl]phenol” (IUPAC name) product (CAS867334-05-2). Both names are herein used interchangeably. TG100572 is described further in Doukas et al..

TG100801 and TG100572 may also exist as salts, solvates, esters and prodrugs thereof, such as for example the TG 100572 hydrochloride salt (Cat# HY-10185; CAS No 867331-64-4).

These compounds may contain asymmetric or chiral centers, and, therefore, exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the derivatives as well as mixtures thereof, including racemic mixtures, form part of the present invention. In addition, the present invention embraces all geometric and positional isomers. Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher’s acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. It is also possible that the derivatives may exist in different tautomeric forms, and all such forms are embraced within the scope of the invention.

All stereoisomers (for example, geometric isomers, optical isomers and the like) of the present compounds (including those of the salts, solvates, esters and prodrugs of the compounds as well as the salts, solvates and esters of the prodrugs), such as those which may exist due to asymmetric carbons on various substituent’s, including enantiomeric forms. Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The use of the terms “salt”, “solvate”, “ester”, “prodrug” and the like, is intended to equally apply to the salt, solvate, ester and prodrug of enantiomers, stereoisomers, rotamers, tautomers, positional isomers, racemates or prodrugs of the compounds herein described.

Polymorphic forms of the derivatives, including polymorphic forms of the salts, solvates, esters, prodrugs and stereoisomers of the derivatives, are intended to be included in the present invention. The term “pharmaceutically acceptable salts” or “pharmaceutically acceptable derivatives” is taken to mean an active ingredient, which comprises a compound of the invention or a derivative thereof, wherein the parent compound is modified by converting an existing acid or base moiety to its salt form, in particular if this salt form imparts improved pharmacokinetic properties on the active ingredient compared with the free form of the active ingredient or any other salt form of the active ingredient used earlier.

The term “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the non-toxic salts of the parent compound formed, e.g., from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002 ). In some embodiments, the compounds described herein include the N-oxide forms.

All references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.

The present invention also includes all salts of the compounds of the present invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

The compounds of the present disclosure can also be prepared as esters, for example, pharmaceutically acceptable esters. For example, a carboxylic acid function group in a compound can be converted to its corresponding ester, e.g., a methyl, ethyl or other ester. Also, an alcohol group in a compound can be converted to its corresponding ester, e.g., acetate, propionate or other ester.

The term “solvate” means a complex or aggregate formed by one or more molecules of a solute, i.e. a crystalline compound of the invention, and one or more molecules of a solvent. Such solvates typically have a substantially fixed molar ratio of solute and solvent. This term also includes clathrates, including clathrates with water. Representative solvents include, by way of example, water, methanol, ethanol, isopropanol, n-butanol, ethylene glycol, propylene glycol, ethyl acetate, tetrahydrofuran, 1,4-dioxane, acetonitrile and the like. If the solvent is water, the solvate formed is a hydrate, e.g. a mono- or dihydrate. If the solvent is alcohol, the solvate formed is an alcoholate, e.g., a methanolate or ethanolate. If the solvent is an ether, the solvate formed is an etherate, e.g., diethyl etherate.

The compounds of the present invention can be formulated and administered in a prodrug form. As used herein, the term “Prodrug” of the small molecules or compounds disclosed herein refers to species that have chemically- or metabolically-cleavable groups wherein, under physiological conditions in the living body (e.g., by oxidation, reduction, hydrolysis or the like, each of which is carried out enzymatically, or without enzyme involvement), the species become, provide, release, or are transformed into the biologically active compounds disclosed herein. In this manner, prodrugs can release in vivo the pharmaceutically active compounds disclosed herein in the form of a prodrug compound. Examples of prodrugs are compounds, wherein the amino group in a compound of the present invention is acylated, alkylated or phosphorylated, e.g., eicosanoylamino, alanylamino, pivaloyloxymethylamino or wherein the hydroxyl group is acylated, alkylated, phosphorylated or converted into the borate, e.g. acetyloxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaryloxy, alanyloxy or wherein the carboxyl group is esterified or amidated, or wherein a sulfhydryl group forms a disulfide bridge with a carrier molecule, e.g. a peptide, that delivers the drug selectively to a target and/or to the cytosol of a cell. These compounds can be produced from a compound of the present invention according to well-known methods. Other examples of prodrugs are compounds, wherein the carboxylate in a compound of the present invention is for example converted into an alkyl-, aryl-, choline-, amino-, acyloxymethylester, or linolenoyl-ester. Prodrugs are derivatives of the compounds disclosed herein.

Where tautomerism, e.g., keto-enol tautomerism, of compounds of the present invention or their prodrugs may occur, the individual forms, e.g., the keto or the enol form, are claimed separately and together as mixtures in any ratio. The same applies for stereoisomers, e.g., enantiomers, cis/trans isomers, conformers and the like.

If desired, isomers can be separated by methods well known in the art, e.g. by liquid chromatography. The same applies for enantiomers, e.g., by using chiral stationary phases. Additionally, enantiomers may be isolated by converting them into diastereomers, i.e., coupling with an enantiomerically pure auxiliary compound, subsequent separation of the resulting diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of a compound of the present invention may be obtained from stereoselective synthesis using optically pure starting materials.

In a particular aspect of the invention, the FYN kinase inhibitor small molecule is 4-chloro-3-[5- methyl-3-[4-(2-pyrrolidin-l-ylethoxy)anilino]-l,2,4-benzotriazin-7-yl]phenyl] benzoate

(TG100801), or 4-chloro-3-[5-methyl-3-[4-(2-pyrrolidin-l-ylethoxy)anilino]-l,2,4-benzotriazin- 7-yl]phenol (TG100572), or a functional derivative thereof capable of inhibiting the functional expression of the Fyn gene, in particular of any nucleotide sequence selected from of SEQ ID NO: 2, 3 or 4, or of the FYN protein, in particular of any amino acid sequence selected from of SEQ ID NO: 5, 6 or 7.

In another particular aspect, the FYN kinase inhibitor is a small molecule selected from pyrazolol[3,4-d] pyrimidine 1 (PPI, CAS No 172889-26-8), pyrazolol[3,4-d] pyrimidine 2 (PP2, CAS No 172889-27-9), ARN25068 (CAS No 2649882-80-2), SU6656 (CAS No 330161-87-0 could be considered as novel over D4221244-14-0), 1 -Naphthyl PPI (CAS No 221243-82-9), Saracatinib (CAS No 379231-04-6), and any functional derivative thereof inhibiting the functional expression of the Fyn gene of SEQ ID NO: 2, 3 or 4 or of the FYN protein of SEQ ID NO: 5, 6 or 7.

For example, the small molecule is PPI or any functional derivative thereof inhibiting the functional expression of the Fyn gene of SEQ ID NO: 2, 3 or 4 or of the FYN protein of SEQ ID NO: 5, 6 or 7, or PP2 or any functional derivative thereof inhibiting the functional expression of the Fyn gene of SEQ ID NO: 2, 3 or 4 or of the FYN protein of SEQ ID NO: 5, 6 or 7.

Anyone of the herein above described small molecules used as a FYN kinase inhibitor may be combined with any herein described T-cell immune check-point inhibitor(s), in particular a T-cell immune check-point inhibitor selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti- TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof, in particular an anti-PDl agent or an anti-PDLl agent.

For example, TG100801 may be combined with any herein described T-cell immune check-point inhibitor(s), in particular a T-cell immune check-point inhibitor selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti-TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof, in particular an anti-PDl agent or an anti- PDLl agent.

For example, PPI may be combined with any herein described T-cell immune check-point inhibitor(s), in particular a T-cell immune check-point inhibitor selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti-TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof, in particular an anti-PDl agent or an anti- PDLl agent.

For example, PP2 may be combined with any herein described T-cell immune check-point inhibitor(s), in particular a T-cell immune check-point inhibitor selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti-TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof, in particular an anti-PDl agent or an anti- PDLl agent.

In each case, the anti-PDl agent may be preferably an anti-PDl antibody selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab, or an anti- PDLl antibody selected from atezolizumab, avelumab and durvalumab, for example nivolumab. In yet another aspect, the FYN kinase inhibitor is a nucleic acid molecule (or “nucleotide sequence”).

The FYN kinase inhibitor can be a polynucleotide or an oligonucleotide. It is preferably an inhibitory nucleotide sequence, i.e., a nucleic acid that block transcription or translation, or a gene editing nucleic acid, or in other words a nucleic acid responsible for the genetic inhibition of FYN, FGFR1, FGFR2 or HCK, preferably for the genetic inhibition of FYN. These include short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acids, complementary DNA (cDNA) or guide RNA (gRNA usable in the context of a CRISPR/Cas system). In particular aspects of the invention, an inhibitory nucleotide sequence, such as a siRNA, targeting FGFR1, FGFR2 or HCK expression is used. In some preferred embodiments, an inhibitory nucleotide sequence targeting FYN expression is used. Interference with the function and expression of endogenous genes by double-stranded RNA such as siRNA has been shown in various organisms. See, e.g., A. Fire et al., “Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans” Nature 391 :806-811 (1998); J. R. Kennerdell& R. W. Carthew, “Use of dsDNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway,” CeJ 95:1017-1026 (1998); F. Wianni & M. Zernicka- Goetz, “Specific Interference with Gene Function by Double-Stranded RNA in Early Mouse Development,” Nat. Cell Biol. 2:70-75 (2000). siRNAs can include hairpin loops comprising self-complementary sequences or double stranded sequences. siRNAs typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches. Such double- stranded RNA can be synthesized by in vitro transcription of single- stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands. Double- stranded RNA targeting anyone of FYN, FGFR1, FGFR2 or HCK can also be synthesized from a cDNA vector construct in which a FYN, FGFR1, FGFR2 or HCK gene (e.g., human FYN gene) is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal. Double- stranded RNA targeting the gene of interest can be introduced into a cell by transfection of an appropriate construct.

Typically, RNA interference mediated by siRNA, miRNA, or shRNA is mediated at the level of translation; in other words, these interfering RNA molecules prevent translation of the corresponding mRNA molecules and lead to their degradation. It is also possible that RNA interference may also operate at the level of transcription, blocking transcription of the regions of the genome corresponding to these interfering RNA molecules. The structure and function of these interfering RNA molecules are well known in the art and are described, for example, in R. F. Gesteland et al., eds, “The RNA World” (3rd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2006), pp. 535-565, incorporated herein by this reference. For these approaches, cloning into vectors and transfection methods are also well known in the art and are described, for example, in J. Sambrook& D. R. Russell, “Molecular Cloning: A Laboratory Manual” (3rd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001), incorporated herein by this reference.

In addition to double stranded RNAs, other nucleic acid agents targeting anyone of FYN, FGFR1, FGFR2 or HCK can also be employed in the practice of the present invention, e.g., antisense nucleic acids. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific target mRNA molecule. In the cell, the single stranded antisense molecule hybridizes to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the translation of mRNA into protein, and, thus, with the expression of a gene that is transcribed into that mRNA. Antisense methods have been used to inhibit the expression of many genes in vitro. See, e.g., C J. Marcus- Sekur a, “Techniques for Using Antisense Oligodeoxyribonucleotides to Study Gene Expression,” Anal. Biochem. 172:289-295 (1988); J. E. Hambor et al., “Use of an Epstein-Ban Virus Episomal Replicon for Anti-Sense RNA-Mediated Gene Inhibition in a Human Cytotoxic T-Cell Clone,” Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014 (1988); H Arima et al., “Specific inhibition of Interleukin- 10 Production in Murine Macrophage-Like Cells by Phosphorothioate Antisense Oligonucleotides,” Antisense Nucl. Acid Drug Dev. 8:319-327 (1998); and W.-F. Hou et al., “Effect of Antisense Oligodeoxynucleotides Directed to Individual Calmodulin Gene Transcripts on the Proliferation and Differentiation of PC 12 Cells”, Antisense Nucl. Acid Drug Dev. 8:295-308 (1998), all incorporated herein by this reference. Antisense technology is described further in C. Lichtenstein & W. Nellen, eds., “Antisense Technology: A Practical Approach” (IRL Press, Oxford, 1997), incorporated herein by this reference. FYN, FGFR1, FGFR2 and HCK polynucleotide sequences from human and many other animals in particular mammals have been described in the art. Based on the known sequences, inhibitory nucleotides (e.g., siRNA, miRNA, or shRNA) targeting anyone of FYN, FGFR1 , FGFR2 or HCK can be readily synthesized using methods well known in the art.

Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integral number of base pairs between these numbers. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.) and Ambion, Inc. (Austin, Tex). In a particular aspect, the nucleic acid molecule is a recombinant DNA encoding a ribozyme. Ribozymes can also function as inhibitors of expression for use in the present invention. “Ribozymes” are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences: GUA, GUU and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes (see, e.g., Castanotto et al., 1994, Adv. in Pharmacology 25: 289-317 for a general review of the properties of different ribozymes).

In a particular and preferred aspect of the invention, the FYN kinase inhibitor is a nucleic acid molecule capable of inhibiting the functional expression of the Fyn gene, in particular the functional expression of SEQ ID NO: 2, 3 or 4, or of the FYN protein, in particular of SEQ ID NO: 5, 6 or 7. In other aspects, the FYN kinase inhibitor is a nucleic acid molecule capable of inhibiting the functional expression of the FGFR1 gene, in particular the functional expression of SEQ ID NO: 8, 9, 10, 11, 12 or 13, of the FGFR2 gene, in particular the functional expression of SEQ ID NO: 35, 36, 37, 38 or 39 or of the HCK gene, in particular the functional expression of SEQ ID NO: 57, 58, 59 or 60, or of the FGFR1 protein, in particular of SEQ ID NO: 14 to 34, of the FGFR2 protein, in particular of SEQ ID NO: 40 to 56, or of the HCK protein, in particular of SEQ ID NO: 61, 62, 63 or 64.

In another aspect, the FYN kinase inhibitor is a peptide or polypeptide molecule comprising amino acid residues.

As used herein the term “amino acid residue” refers to any natural/standard and non-natural/non- standard amino acid residue in (L) or (D) configuration, and includes alpha or alpha-disubstituted amino acids. It refers to isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, proline, serine, tyrosine. It also includes beta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha- aminoisobutyric acid (Aib), 4-amino-butyric acid, N- methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, cyclopentylalanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, cyclopentylglycine, cyclobutylglycine, cyclopropylglycine, norleucine (Nle), norvaline, 2-napthylalanine, pyridylalanine, 3 -benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3- fluorophenylalanine, 4-fluorophenylalanine, penicillamine, l,2,3,4-tetrahydro-isoquinoline-3- carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (hArg), N-acetyl lysine, 2-amino butyric acid, 2,4, -diaminobutyric acid (D- or L-), p- aminophenylalanine, N- methylvaline, selenocysteine, homocysteine, homoserine (HoSer), cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry/peptide chemistry.

Compounds used in the context of the present invention which include peptides may comprise replacement of at least one of the peptide bonds with an isosteric modification. Compounds of the present invention which include peptides may be peptidomimetics. A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent, but wherein one or more of the peptide bonds/linkages have been replaced, often by proteolytically more stable linkages. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many or all of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, potential for hydrogen bonding, etc. Typical peptide bond replacements include esters, polyamines and derivatives thereof as well as substituted alkanes and alkenes, such as aminomethyl and ketomethylene. For example, the peptide may have one or more peptide linkages replaced by linkages such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis or trans), -CH(OH)CH₂-, or -COCH2-, -N-NH-, -CH2NHNH-, or peptoid linkages in which the side chain is connected to the nitrogen atom instead of the carbon atom. Such peptidomimetics may have greater chemical stability, enhanced biological/pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.) and/or reduced antigenicity relative its peptide equivalent.

The present description further describes nucleic acid molecules which respectively encode the herein described peptides, polypeptides (including proteins) of the invention.

Such nucleic acid molecules are RNA or DNA that typically encode biologically active human FYN kinase inhibitor peptides or polypeptides, in particular human FYN kinase inhibitor peptides or polypeptides, or may be used to prepare recombinant forms thereof.

In another particular aspect, the FYN kinase inhibitor is an antibody or antigen-binding molecule, or a derivative or functional fragment thereof capable of binding to, and inhibiting the functional expression of, the FYN protein, in particular of SEQ ID NO: 5, 6 or 7, of the FGFR1 protein, in particular of SEQ ID NO: 14 to 34, of the FGFR2 protein, in particular of SEQ ID NO: 40 to 56, or of the HCK protein, in particular of SEQ ID NO: 61, 62, 63 or 64, preferably of the FYN protein of SEQ ID NO: 5.

The term “antibody” is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and antibody fragment so long as they exhibit the desired biological activity (e.g., inhibiting fibrosis). Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, V H regions (V H, V H-V H), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Antibodies according to the present invention can be of any class, such as IgG, IgA, IgDl, IgEl, IgMl or IgYl although IgG antibodies are typically preferred. Antibodies can be of any mammalian or avian origin, including human, murine (mouse or rat), donkey, sheep, goat, rabbit, camel, horse, or chicken. The antibodies can be modified by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or other modifications known in the art.

In general, techniques for preparing antibodies (including polyclonal antibodies, monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art, e.g., Harlow & Fane, Using Antibodies, A Eaboratory Manual, Cold Spring Harbor Eaboratory Press, Cold Spring Harbor, N.Y., 1998; Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); and Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, 1985. Additionally, antibodies according to the present invention can be fused to marker sequences, such as a peptide tag to facilitate purification; a suitable tag is a hexahistidine tag. The antibodies can also be conjugated to a diagnostic or therapeutic agent by methods known in the art. Techniques for preparing such conjugates are well known in the art. Other methods of preparing these monoclonal antibodies, as well as chimeric antibodies, humanized antibodies, and single-chain antibodies, are known in the art. In a particular aspect, the anti-FYN kinase antibody (also herein identified as anti-FYN or as FYN kinase antibody) specifically/selectively recognizes/binds to a human FYN protein, preferably to the peptide or protein comprising, or consisting in, an amino acid sequence of SEQ ID NO: 5, 6 or 7, or to an epitope thereof involved in the FYN kinase activity, in particular to an epitope sequence comprising, or consisting of, amino acids localized at positions 85 to 206 (TLFVALYDYEARTEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNY VAPVDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTFLIRESETTKGAYSLSIRD WDDMKGDHVKHYKIR, herein identified as SEQ ID NO: 65) of SEQ ID NO: 5.

This antibody preferably also neutralizes a biological activity of the targeted FYN protein, in particular of the targeted SEQ ID NO: 5, 6 or 7. Preferably, the monoclonal antibody decreases or inhibits FYN kinase activity in a subject as herein defined, typically a mammal, preferably a human being. Methods of making such antibodies are known in the art (See for example Cunningham et al., 2004 (PMID: 15269313)).

Examples of such advantageous FYN kinase antibodies preferably comprise:

1) a heavy chain comprising, or consisting of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the following sequence:

EVQLLESGGGLVQPGGSLRLSCAASTFSSYTTAWVRQMSWVRQAPGKGLEWVSTSTT YNDKVKGYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCQGTLVTVSSMV QLWGQGTLVTVSSASTKGPSVFPLAPSSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 66) and a light chain comprising, or consisting of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the following sequence: SVLTQPPSASGTPGQRVTISCSGSSVDSAVAWYQRKVSWYQQLPGTAPKLLIYSAPSRF SGSRHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCLSSGDVVLVLVLIFGGGTK LTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTE (SEQ ID NO: 67), or comprising amino acid sequences corresponding to the following six CDR sequences respectively consisting of: TFSSYTTAWVRQ (HCDR1 - SEQ ID NO: 68), STTYNDKVKG (HCDR2 - SEQ ID NO: 69), QGTLVTVSSMVQL (HCDR3 - SEQ ID NO: 70), corresponding to the CDRs of the heavy chain, and SVDSAVAWYQRK (LCDR1 - SEQ ID NO: 71), SAPSRFSGSR (LCDR2 - SEQ ID NO: 72) and LSSGDVVLVLVLI (LCDR3 - SEQ ID NO: 73) corresponding to the CDRs of the light chain, or (functionally equivalent) CDR sequences having at least 70%, 75%, 80%, 85% or 90% identity with them respectively, or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7; or 2) a heavy chain comprising, or consisting of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the following sequence:

EVQLLESGGGLVQPGGSLRLSCAASTFSSYGTAWVRQMSWVRQAPGKGLEWVSTST TYNDKVKGYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCQGTLVTVSSS VQLWGQGTLVTVSSASTKGPSVFPLAPSSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 74) and a light chain comprising, or consisting of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the following sequence:

SVLTQPPSASGTPGQRVTISCSGSSVDSAVAWYQRKVSWYQQLPGTAPKLLIYSAPRR FSGSRHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCLSSGVLVLQLVLIFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE TTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTE (SEQ ID NO: 75), or comprising amino acid sequences corresponding to the following six CDR sequences respectively consisting of: TFSSYGTAWVRQ (HCDR1 - SEQ ID NO: 76), STTYNDKVKG (HCDR2 - SEQ ID NO: 77), QGTLVTVSSSVQL (HCDR3 - SEQ ID NO: 78), corresponding to the CDRs of the heavy chain, and SVDSAVAWYQRK (LCDR1 - SEQ ID NO: 79), SAPRRFSGSR (LCDR2 - SEQ ID NO: 80) and LSSGVLVLQLVLI (LCDR3 - SEQ ID NO: 81), corresponding to the CDRs of the light chain, or (functionally equivalent) CDR sequences having at least 70%, 75%, 80%, 85% or 90% identity with them respectively, or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7; or

3) a heavy chain comprising, or consisting of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the following sequence:

EVQLLESGGGLVQPGGSLRLSCAASTFSSYGTAWVRQMSWVRQAPGKGLEWVSTST TYNDKVKGYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCQGTLVTVSSM VQLWGQGTLVTVSSASTKGPSVFPLAPSSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 82) and a light chain comprising, or consisting of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity with the following sequence:

SVLTQPPSASGTPGQRVTISCSGSSVDSAVAWYQRKVSWYQQLPGTAPKLLIYSAPSRF SGSRHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCLSSGVVVLVLVLVFGGGTK LTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTE (SEQ ID NO: 83), or comprising amino acid sequences corresponding to the following six CDR sequences respectively consisting of: TFSSYGTAWVRQ (HCDR1 - SEQ ID NO: 84), STTYNDKVKG (HCDR2 - SEQ ID NO: 85), QGTLVTVSSMVQL (HCDR3 - SEQ ID NO: 86), corresponding to the CDRs of the heavy chain, and SVDSAVAWYQRK (LCDR1 - SEQ ID NO: 87), SAPSRFSGSR (LCDR2 - SEQ ID NO: 88) and LSSGVVVLVLVLV (LCDR3 - SEQ ID NO: 89), corresponding to the CDRs of the light chain, or (functionally equivalent) CDR sequences having at least 70%, 75%, 80%, 85% or 90% identity with them respectively, or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7.

Anyone of the three (3) herein above described antibodies may be used as a FYN kinase inhibitor in the context of the invention (either as such or included in a composition or combination, in particular in a composition or combination for use, as claimed).

Anyone of the three (3) herein above described antibodies may be combined in the context of the present invention with any one of the herein described T-cell immune check-point inhibitor(s). This means for example that a FYN kinase antibody comprising i) a heavy chain comprising, or consisting of, the following sequence:

EVQLLESGGGLVQPGGSLRLSCAASTFSSYTTAWVRQMSWVRQAPGKGLEWVSTSTT YNDKVKGYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCQGTLVTVSSMV QLWGQGTLVTVSSASTKGPSVFPLAPSSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 66), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity thereto, and ii) a light chain comprising, or consisting of, the following sequence: SVLTQPPSASGTPGQRVTISCSGSSVDSAVAWYQRKVSWYQQLPGTAPKLLIYSAPSRF SGSRHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCLSSGDVVLVLVLIFGGGTK LTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTE (SEQ ID NO: 67), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity thereto, or comprising amino acid sequences corresponding to the following six CDR sequences respectively consisting of: TFSSYTTAWVRQ (HCDR1 - SEQ ID NO: 68), STTYNDKVKG (HCDR2 - SEQ ID NO: 69), QGTLVTVSSMVQL (HCDR3 - SEQ ID NO: 70), SVDSAVAWYQRK (LCDR1 - SEQ ID NO: 71), SAPSRFSGSR (LCDR2 - SEQ ID NO: 72) and LSSGDVVLVLVLI (LCDR3 - SEQ ID NO: 73), or (functionally equivalent) CDR sequences having at least 70%, 75%, 80%, 85% or 90% identity with them respectively, or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7, may be combined with any herein described T-cell immune check-point inhibitor(s), in particular a T-cell immune check-point inhibitor selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti-TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof, in particular an anti-PDl agent or an anti- PDL1 agent. The anti-PDl agent is preferably an anti-PDl antibody selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab, or an anti- PDL1 antibody selected from atezolizumab, avelumab and durvalumab, for example nivolumab.

This means also for example that a FYN kinase antibody a FYN kinase antibody comprising i) a heavy chain comprising, or consisting of, the following sequence:

EVQLLESGGGLVQPGGSLRLSCAASTFSSYGTAWVRQMSWVRQAPGKGLEWVSTST TYNDKVKGYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCQGTLVTVSSS VQLWGQGTLVTVSSASTKGPSVFPLAPSSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 74), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity thereto, and ii) a light chain comprising, or consisting of, the following sequence: SVLTQPPSASGTPGQRVTISCSGSSVDSAVAWYQRKVSWYQQLPGTAPKLLIYSAPRR FSGSRHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCLSSGVLVLQLVLIFGGGT KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE TTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTE (SEQ ID NO: 75), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity thereto, or comprising amino acid sequences corresponding to the following six CDR sequences respectively consisting of: TFSSYGTAWVRQ (HCDR1 - SEQ ID NO: 76), STTYNDKVKG (HCDR2 - SEQ ID NO: 77), QGTLVTVSSSVQL (HCDR3 - SEQ ID NO: 78), SVDSAVAWYQRK (LCDR1 - SEQ ID NO: 79), SAPRRFSGSR (LCDR2 - SEQ ID NO: 80) and LSSGVLVLQLVLI (LCDR3 - SEQ ID NO: 81), or (functionally equivalent) CDR sequences having at least 70%, 75%, 80%, 85% or 90% identity with them respectively, or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7, may be combined with any herein described T-cell immune check-point inhibitor(s), in particular a T-cell immune check-point inhibitor selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti-TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof, in particular an anti-PDl agent or an anti- PDL1 agent. The anti-PDl agent is preferably an anti-PDl antibody selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab, or an anti- PDL1 antibody selected from atezolizumab, avelumab and durvalumab, for example nivolumab. This means according to yet another example, that a FYN kinase antibody a FYN kinase antibody comprising i) a heavy chain comprising, or consisting of, the following sequence:

EVQLLESGGGLVQPGGSLRLSCAASTFSSYGTAWVRQMSWVRQAPGKGLEWVSTST TYNDKVKGYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCQGTLVTVSSM VQLWGQGTLVTVSSASTKGPSVFPLAPSSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK (SEQ ID NO: 82), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity thereto, and ii) a light chain comprising, or consisting of, the following sequence: SVLTQPPSASGTPGQRVTISCSGSSVDSAVAWYQRKVSWYQQLPGTAPKLLIYSAPSRF SGSRHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCLSSGVVVLVLVLVFGGGTK LTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTE (SEQ ID NO: 83), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identity thereto, or comprising the following six CDR sequences respectively consisting of: TFSSYGTAWVRQ (HCDR1 - SEQ ID NO: 84), STTYNDKVKG (HCDR2 - SEQ ID NO: 85), QGTLVTVSSMVQL (HCDR3 - SEQ ID NO: 86), SVDSAVAWYQRK (LCDR1 - SEQ ID NO: 87), SAPSRFSGSR (LCDR2 - SEQ ID NO: 88) and LSSGVVVLVLVLV (LCDR3 - SEQ ID NO: 89), or (functionally equivalent) CDR sequences having at least 70%, 75%, 80%, 85% or 90% identity with them respectively, or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7, may be combined with any herein described T-cell immune check-point inhibitor(s), in particular a T-cell immune checkpoint inhibitor selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti- TIGIT agent, anti-TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof, in particular an anti-PDl agent or an anti-PDLl agent. The anti-PDl agent is preferably an anti-PDl antibody selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab, or an anti-PDLl antibody selected from atezolizumab, avelumab and durvalumab, for example nivolumab.

For example, the FYN kinase antibody is the Fyn (15) monoclonal antibody from Santa Cruz or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7. Derivatives of the FYN kinase antibodies herein described includes variant antibodies remaining functional, i.e., capable of inhibiting (neutralizing or decreasing) a biological activity of the targeted FYN protein (enzyme) of SEQ ID NO: 5, 6 or 7, as explained herein above, typically in a mammal, preferably in a human being.

Variations in the (monovalent) antibodies herein described leading to such a variant antibody, can be made, for example, using any of the techniques and guidelines for conservative and nonconservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the monovalent antibody that results in a change in the amino acid sequence as compared with the native sequence antibody. Optionally the variation is by substitution of at least one amino acid with any other amino acid (including naturally occurring amino acids as well as amino acid analogs) in one or more of the domains of the antibody. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting (monovalent) antibody variants for activity exhibited by the “native” (or reference) monovalent antibody.

In an embodiment, the one or more mutations is in one or more of the CDRs disclosed herein.

In an embodiment, one or two residues in any one of the above-noted CDRs sequences are substituted. In a further embodiment, one residue in any one of the above-noted CDRs sequences is substituted.

“Identity” refers to sequence identity between two polypeptides. Identity can be determined by comparing each position in the aligned sequences. Methods of determining percent identity are known in the art, and several tools and programs are available to align amino acid sequences and determine a percentage of identity including EMBOSS Needle, ClustalW, SIM, DIALIGN, etc. As used herein, a given percentage of identity with respect to a specified subject sequence, or a specified portion thereof, may be defined as the percentage of amino acids in the candidate derivative sequence identical with the amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the Smith Waterman algorithm (Smith & Waterman, J. Mol. Biol. 147 147: 195-7 (1981)) using the BLOSUM substitution matrices (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992)) as similarity measures. A “% identity value” is determined by the number of matching identical amino acids divided by the sequence length for which the percent identity is being reported.

In one aspect, the antibody of the disclosure comprise C-terminal extensions or deletions of from 1 to 50, or more residues, for example 1 to 25, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids.

Additional C or N-terminal residues can be linkers that are used to conjugate the antibody of the disclosure to another moiety, or tags that facilitate the detection of the antibody. Such tags are well known in the art and include for example polyhistidine tags (His-tags), polyarginine tags, polyaspartate tags, polycysteine tags, polyphenylalanine tags, glutathione S-transferase (GST) tags, maltose binding protein (MBP) tags, calmodulin binding peptide (CBP) tags, Streptavidin/Biotin-based tags, HaloTag®, Profinity eXact® tags, epitope tags (such as FLAG, hemagglutinin (HA), HSV, S/Sl, c-myc, KT3, T7, V5, E2, and Glu-Glu epitope tags), reporter tags such as P-galactosidase (P-gal), alkaline phosphatase (AP), chloramphenicol acetyl transferase (CAT), and horseradish peroxidase (HRP) tags (see, e.g., Kimple et al., Curr Protoc Protein Sci. 2013; 73: Unit-9.9).

The FYN kinase inhibitor can also be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Compositions

Inventors herein describe a composition comprising i) a FYN kinase inhibitor, a population of DC activated by such a FYN kinase inhibitor, and/or a population of Fyn^v' DC, Fgfrl DC, Fgfr2 ⁷ DC and/or Hck' DC, and ii) a pharmaceutically acceptable support. A “composition” may contain one compound or a mixture of compounds. A “pharmaceutical composition” designates any composition useful or potentially useful in producing at least one physiological response in a subject to which such pharmaceutical composition is administered. The term refers concretely to a mixture of one or more of the active (therapeutic) agents described herein, or a pharmaceutically acceptable derivative thereof, for example a salt, polymorph, enantiomer, stereoisomer, solvate, tautomer, hydrate or prodrug of a compound of the invention as an active ingredient, and at least one pharmaceutically acceptable support, excipient, carrier or diluent. In some embodiments, the pharmaceutical composition comprises two or more pharmaceutically acceptable supports, excipients, carriers or diluents.

The products described herein are typically administered in admixture with one or more pharmaceutically acceptable supports, excipients, carriers or diluents in the form of a pharmaceutical composition.

Thus, “pharmaceutical composition” typically means one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.

The FYN kinase inhibitor is typically a FYN kinase inhibitor as herein above described.

Instead of, or in addition to, the FYN kinase inhibitor, and in addition to the pharmaceutically acceptable support (also herein identified as an excipient, carrier or diluent), the composition of the invention may comprise a dendritic cell (DC) activated by such a FYN kinase inhibitor.

Examples of such DC activated by a FYN kinase inhibitor as herein described (such as for example TG100801 and TG100572) are Fyn-silenced DC (in particular Fyn DC), FGFR1- silenced DC (in particular Fgfrl DC), FGFAG-silcnccd DC (in particular Fgfr2 ' DC) and HCK- silenced DC (in particular Hck' DC).

Fyn⁷ DC, Fgfrl⁷ DC, Fgfr2 ' DC and Hck⁷ DC are typically DC respectively knocked-out for the Fyn, Fgfrl, Fgfr2 or Hck gene.

A DC is considered as “activated” if it is reactive to antigen or more reactive to antigen than an immature DC which has not been exposed to a FYN inhibitor of the invention (for example to TG100801 or TG100572), i.e., capable of enhanced immune function (increased immunogenicity) when compared to said immature DC, for example capable of efficient antigen presentation to the immune system. The presentation is considered as efficient in particular if capable of enhancing, stimulating and/or increasing the immune response (immunity) in a subject, in particular in a subject suffering from a cancer (if the antigen is a tumor antigen) or an infection (if the antigen is from an infectious agent). Such an increased immunity will substantially improve treatment outcome.

In the context of the present invention, a DC is considered as “activated” if it expresses CD80 and CD86 (co- stimulatory) receptors and CD103, as well as MHC class II molecules, and if it does not express CD1 lb. Among viable leukocytes (“F4/80 MHC-II⁺CD1 lc⁺” cells), activated DC are identified as “F4/80 MHC-II⁺CDl lc⁺CD103⁺CDl lb- cells (or also simply as “CD103⁺CDl lb ” cells). The detection of the presence or expression of these markers may be easily performed by the person of ordinary skill in the art according to the teaching of Zhao et al..

DC efficient antigen presentation ability which is correlated to DC activation may be revealed also in vitro through IL-2 detection (as shown in the experimental section).

Tumor reactive DCs (also herein identified as “cDCl” for “immature DCs of the type I conventional DC subtype”) are particular activated DCs of the invention. These cells have been characterized both in vitro and in vivo in the context of the present invention (cf. experimental section). In their experiments, inventors have observed an increased DC-mediated cancer cell phagocytosis when cDCl are involved. These activated DC have been identified by inventors as capable of boosting tumor regression.

In a particular aspect of the invention, the composition comprises i) a population of DC defective for FYN. FGFR1, FGFR2 and/or HCK, in particular a population of Fyn DC, Fgfrl DC, Fgfr2 ^/_ DC and/or Hck' DC, and optionally a FYN kinase inhibitor as herein described, and ii) a pharmaceutically acceptable support.

The DCs according to the invention are eukaryotic cells, typically animal cells such as mammalian cells, e.g., human cells. The DCs of the invention are derived from the blood, bone marrow, lymph, or lymphoid organs (notably the thymus).

With reference to the subject to be treated, the DC of the invention may be autologous and/or allogeneic.

In a particular aspect, the DCs are isolated from a sample, e.g., a biological sample obtained from or derived from a subject, preferably from the subject to be treated, in particular a subject suffering from, or at risk of suffering from, a cancer or from an infection.

The terms “isolated”, “purified”, or “biologically pure” refer to a biological material (e.g., a cell) that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using antibodies-mediated enrichment from magnetic selection kits or cell sorting using flow cytometry. The DC, present in a subject or isolated from the subject, may be genetically modified (and is herein identified as an “engineered DC”), preferably modified to become defective for FYN, FGFR1, FGFR2 and/or HCK.

A DC defective for FYN, FGFR1, FGFR2 or HCK is a DC wherein said gene (or any functional nucleic acid sequence thereof such as those herein described) has been repressed, silenced or inactivated using an antisense technology as herein above described, such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in transient reduction of expression, or a gene editing technique which results in targeted gene inactivation or disruption, e.g., by induction of breaks and/or homologous recombination, and as a consequence in the repression, inhibition, or blockade of the FYN, FGFR1, FGFR2 or HCK activity.

As used herein, a “disruption” of a gene refers to a change in the sequence of the gene, at the DNA level. Examples include insertions, mutations, and deletions. The disruptions typically result in the repression and/or complete absence of expression of a normal or “wild type” product encoded by the gene. Exemplary of such gene disruptions are insertions, frameshift and missense mutations, deletions, knock-in, and knock-out of the gene or part of the gene, including deletions of the entire gene. Such disruptions can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, functional product, or any product, such as by insertion of a stop codon. Such disruptions may also occur by disruptions in the promoter or enhancer or other region affecting activation of transcription, so as to prevent transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.

Preferably, the inhibition of FYN, FGFR1, FGFR2 or HCK activity leads to the absence in the cell of substantial detectable activity of FYN, FGFR1, FGFR2 or HCK activity respectively.

Well-suited method to edit immune cells are notably described in Lucibello F, Menegatti S, Menger L. “Methods to edit T cells for cancer immunotherapy”. Methods Enzymol. 2020;631 :107-135.

In some embodiments, the gene disruption or repression is achieved using gene editing agents such as a DNA-targeting molecule, such as a DNA-binding protein or DNA-binding nucleic acid, or complex, compound, or composition, containing the same, which specifically binds to or hybridizes to the gene. In some embodiments, the DNA-targeting molecule comprises a DNA- binding domain, e.g., a zinc finger protein (ZFP) DNA-binding domain, a transcription activatorlike protein (TAL) or TAL effector (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA-binding domain, or a DNA-binding domain from a meganuclease. Zinc finger, TALE, and CRISPR system binding domains can be “engineered” to bind to a predetermined nucleotide sequence.

In some embodiments, the DNA-targeting molecule, complex, or combination contains a DNA- binding molecule and one or more additional domain(s), such as an effector domain to facilitate the repression or disruption of the gene. For example, in some embodiments, the gene disruption is carried out by fusion proteins that comprise DNA-binding proteins and a heterologous regulatory domain or functional fragment thereof. Typically, the additional domain is a nuclease domain. Thus, in some embodiments, gene disruption is facilitated by gene or genome editing, using engineered proteins, such as nucleases and nuclease-containing complexes or fusion proteins, composed of sequence-specific DNA-binding domains fused to, or complexed with, non-specific DNA-cleavage molecules such as nucleases. These targeted chimeric nucleases or nuclease-containing complexes carry out precise genetic modifications by inducing targeted double- stranded breaks or single-stranded breaks, stimulating the cellular DNA-repair mechanisms, including error-prone nonhomologous end joining (NHEJ) and homology-directed repair (HDR). In some embodiments the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), TALE nuclease (TALEN), an RNA-guided endonuclease (RGEN), such as a CRISPR-associated (Cas) protein, or a meganuclease. Such systems are well-known in the art (see, for example, U.S. Pat. No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Pat. Publ. 2014/0087426 and 2012/0178169; Boch et al. (2011 ) Nat. Biotech. 29: 135-136; Boch et al. (2009) Science 326: 1509-1512; Moscou and Bogdanove (2009) Science 326: 1501 ; Weber et al. (2011 ) PLoS One 6:el9722; Li et al. (2011 ) Nucl. Acids Res. 39:6315-6325; Zhang et al. (201 1 ) Nat. Biotech. 29: 149-153; Miller et al. (2011) Nat. Biotech. 29: 143-148; Lin et al. (2014) Nucl. Acids Res. 42:e47). Such genetic strategies can use constitutive expression systems or inducible expression systems according to well-known methods in the art.

Isolation of the DC includes one or more preparation and/or non-affinity based cell separation steps according to well-known techniques in the field. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.

In some embodiments, the cell preparation includes steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. Any of a variety of known freezing solutions and parameters in some aspects may be used. Typically, the cells are incubated prior to or in connection with genetic engineering for FYN, FGFR1, FGFR2 or HCK inhibition. The incubation steps can comprise culture, incubation, stimulation, activation, expansion and/or propagation. In some embodiments, inhibition of FYN, FGFR1, FGFR2 or HCK as per the invention can be performed using a FYN kinase inhibitor as previously described.

In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering. The incubation conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, and any other agents designed to stimulate the cells. In some embodiments, the stimulating conditions include temperature suitable for the growth of human DC, for example, at least about 25°C, generally at least about 30°C, and generally at or about 37°C. Optionally, the incubation may further comprise adding model antigen such as ovalbumin, cell lysate or lysate of an infectious particle such as bacteria, fungi or virus as source of antigens.

In some aspects, the methods include assessing expression of one or more markers on the surface of the engineered DC or DC being engineered. In one embodiment, the methods include assessing surface expression of one or more markers of DC’s activation, for example, by affinity-based detection methods such as by flow cytometry.

The pharmaceutical composition of the present invention may additionally comprise one or more other compounds as active ingredients, such as one or more additional therapeutic agent(s), preferably agent(s) known to be efficient against cancer or infection, or a prodrug compound or other known substance active against the disease or condition, preferably against cancer or infection.

Formulation and dosage

The pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention, typically in a therapeutically effective amount, preferably at least (a) a FYN kinase inhibitor, a population of DC activated by such a FYN kinase inhibitor, and/or a population of DC defective for FYN, FGFR1 , FGFR2 and/or HCK, in particular a population of Fyn DC, Fgfrl DC, Fgfr2 ^ DC and/or Hck' DC, and (b) pharmaceutically acceptable carrier(s), and optionally one or more additional therapeutic agents. The phrases “effective dosage or amount” and “dosage effective manner” refer to the amount of active compound, pharmaceutical agent or composition, sufficient to affect any one or more beneficial or desired outcomes sought by a researcher, veterinarian, medical doctor or other clinician, including biochemical, histological and/or behavioral symptoms, of a disease, its complications and intermediate pathological phenotypes presenting during development of the disease in a tissue, system, or subject.

For therapeutic use, a “therapeutically effective amount” refers to that amount of a drug, compound or pharmaceutical composition being administered which will relieve to some extent one or more of the symptoms of the disease, condition or disorder being treated, and will accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition.

In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, (4) relieving to some extent (or, preferably, eliminating) one or more signs or symptoms associated with the cancer, (5) decreasing the dose of other medications required to treat the disease, and/or (6) enhancing the effect of another medication, and/or (7) delaying the progression of the disease in a patient.

An effective dosage can be administered in one or more administrations.

The dosage regimen of the compounds, pharmaceutical compositions and combinations used in accordance with the disclosure vary depending on the particular compound(s) or salt thereof employed, and on a variety of factors including type, species, the age, weight, sex, and clinical condition, in particular renal and hepatic function, of the patient, the severity of the condition to be treated; the route of administration, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the disease or condition. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and also preferably causing complete regression of the disease, in particular of the cancer. Dosages of, for example, TG100801 or TG100572, as well as PPI, PP2, ARN25068, SU6656, 1- NM-PP1, 1-Naphthyl PPI, Saracatinib, and any functional derivative thereof inhibiting the functional expression of the Fyn gene of SEQ ID NO: 2, 3 or 4 or of the FYN protein of SEQ ID NO: 5, 6 or 7, used as the FYN kinase inhibitor can range from about 0.01 mg/kg of body weight per day to about 5000 mg/kg per day. In preferred aspects, dosages can range from about 1 mg/kg per day to about 1000 mg/kg per day or from about 0.1 mg/kg per day to about 100 mg/ weight per day, in particular in the range from 1 mg/kg per day to about 10 mg/kg per day. Thus, the actual amount per day for an adult mammal weighing 70 kg is usually between 70 and 700 mg, where this amount can be administered as a single dose per day or usually in a series of partdoses (such as, for example, two, three, four, five or six) per day, so that the total daily dose is the same.

In an aspect, the dose will be in the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, or continuous doses (which dose may be adjusted for the patient’s weight in kg, body surface area in m², and age in years). The dosage may be administered for example as a single dose (QD), or optionally may be subdivided into smaller doses, suitable for BID (twice daily), TID (three times daily) or QID (four times daily) administration.

The term “about” which is used to modify a numerically defined parameter means that the parameter may vary by as much as 10% above or below the stated numerical value for that parameter. For example, a dose of “about 5 mg” means 5 mg + 10%, i.e., the dose may vary between 4.5 mg and 5.5 mg.

An effective amount of a salt, solvate, polymorph, tautomer, enantiomer or stereoisomer of a compound of the invention can be determined as the fraction of the effective amount of the compound according to the invention per se.

In some instances, dosage levels at the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day.

An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. Improvement in survival and growth indicates regression. As used herein, the term “dosage effective manner” refers to amount of an active compound to produce the desired biological effect in a subject, tissue or cell.

In the context of oncology, the dosage regimen of any active ingredient herein described may be adjusted by the oncologist to provide the optimal therapeutic response to the patient. The compounds of the present invention may be administered in a wide variety of different dosage forms.

The (pharmaceutical) combinations or pharmaceutical compositions of the invention may be conveniently presented in unit dosage form suitable for single administration of precise amounts, and prepared by any of the methods well-known in the art of pharmacy.

Techniques for formulation and administration of the disclosed compounds of the disclosure can be found in Remington: the Science and Practice of Pharmacy, 19^th edition, Mack Publishing Co., Easton, PA (1995).

Therapeutic agents of the combination therapies of the present invention may be used in fastdissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986 by Liang and Chen (2001), the disclosure of which is incorporated herein by reference in its entirety. Solid formulations for oral administration for example may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Other suitable modified release formulations are described in U.S. Patent No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles may be found in Verma et al., Pharmaceutical Technology On-line, 25(2), 1-14 (2001).

In an embodiment, the compounds described herein, and the pharmaceutically acceptable salts thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable (inert) carrier or diluent in the form of one or more of, but not limited to, tablets, capsules (for example sustained release capsules and/or enteric coated capsules), lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, beverages, foods, other nutritional supplements, and the like.

Suitable pharmaceutically acceptable carriers (also herein identified as supports, excipients or diluents) may include one or more of solid fillers or diluents, sterile aqueous or organic solutions, various nontoxic organic solvents, etc.. Moreover, oral pharmaceutical compositions may be sweetened and/or flavored. As explained, the compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein. In general, the compounds of the invention may be present in such dosage forms at concentration levels ranging from about 0.1 percent to about 90 percent by weight. For oral administration, tablets may contain various excipients such as one or more of microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (such as corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulphate and talc may be employed. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; exemplary materials in this connection may also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and various combinations thereof. For enteral administration, examples of suitable preparations may also include implants including suppositories.

For parenteral administration (including intraperitoneal subcutaneous, intravenous, intradermal or intramuscular injection), solutions of compounds of the present invention in, for example, either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions may be buffered, if necessary or desirable, and the liquid diluent first rendered isotonic. These aqueous solutions may be suitable for intravenous injection purposes. The oily solutions may be suitable for intraarticular, intramuscular, and/or subcutaneous injection purposes. The preparation of such solutions under sterile conditions may be accomplished by standard pharmaceutical techniques known to those having ordinary skill in the art. For parenteral administration, examples of suitable preparations may include solutions, such as oily or aqueous or non-aqueous solutions, as well as suspensions, emulsions, and/or implants.

Compounds and combinations of the present invention may be formulated in sterile form in multiple or single dose formats. For example, the compounds of the present invention may be dispersed in a fluid carrier such as sterile saline and/or 5 percent saline dextrose solutions commonly used with injectables.

In another embodiment, the compounds of the present invention may be administered topically. Non-limiting examples of methods of topical administration include transdermal, buccal, or sublingual application. For topical applications, therapeutic compounds may be suitably admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, and/or a cream. Such topical carriers may include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, and/or mineral oils. Other possible topical carriers may include liquid petrolatum, isopropylpalmitate, polyethylene glycol, ethanol 95 percent, polyoxyethylenemonolaurate 5 percent in water, sodium lauryl sulphate 5 percent in water, and the like, and combinations thereof. In addition, materials such as surfactants, antioxidants, humectants, viscosity stabilizers, and the like, and combinations thereof, also may be added if desired.

When administered, the pharmaceutical compositions may be at or near body temperature. In some embodiments, the pharmaceutical compositions may be below body temperatures. In other embodiments, the pharmaceutical compositions may be above body temperatures.

Uses and therapeutic treatment

Inventors advantageously herein describe products and uses thereof for activating or stimulating dendritic cell (DC) in vivo, ex vivo or in vitro.

In a particular aspect, inventors herein describe the in vitro or ex vivo use of a FYN kinase inhibitor as described herein above to stimulate DC activation, increase antigen presentation by DC, and/or facilitate DC progenitor differentiation into an antigen reactive DC. The herein described FYN kinase inhibitors as well as DC populations activated by such FYN kinase inhibitors can be used as the active ingredient of a therapeutic vaccine composition (“vaccine”). Said activated DC populations exhibit an advantageously improved cancer-regressing efficacy in comparison to DCs that were not exposed to a FYN kinase inhibitor.

In a particular aspect, said FYN kinase inhibitor is an anti-FYN antibody as herein described, or a small molecule such as TG100801, TG100572, PPI, PP2, or any functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7.

In another particular and preferred aspect, inventors herein describe the in vivo use of a FYN kinase inhibitor as described herein above for stimulating DC-mediated immunity, or for treating a disease or condition in a subject.

A FYN kinase inhibitor as described herein above for use in enhancing, stimulating and/or increasing the immune response (immunity) in a subject suffering of a disease or condition, or in a subject at risk of developing a severe disease or condition, is in particular herein described. The FYN kinase inhibitor acts by inducing and/or stimulating dendritic cells (DC) maturation and/or activation, in particular DC differentiation into activated DC, and/or by increasing antigen presentation by DC.

A FYN kinase inhibitor stimulating DC activation and/or increasing antigen presentation by DC, for use in prevention or treatment of a disease in a subject in need thereof is in particular herein described. The disease is preferably a cancer or an infection. The herein above-described compositions are also described here for use as a medicament, in particular for use for the prevention or treatment of a disease, preferably of a cancer or of an infection, in a subject in need thereof.

In a particular aspect, the composition for use comprises i) a FYN kinase inhibitor, a population of DC activated by a FYN kinase inhibitor, and/or a population of DC defective for FYN, FGFR1, FGFR2 or HCK, for example of Fyn ' DC, Fgfrl DC, Fgfr2 DC and/or Hck DC, and ii) a pharmaceutically acceptable support.

In another particular aspect, the composition for use as a medicament comprises i) a FYN kinase inhibitor as herein above described which is not a small molecule (in particular when the use is for treating cancer), and ii) a pharmaceutically acceptable support.

In a preferred embodiment, the composition for use as a medicament comprises i) a population of DC activated by a FYN kinase inhibitor as herein above described, or a population of DC defective for FYN, FGFR1, FGFR2 or HCK, for example a population of Fyn DC, Fgfrl DC, Fgfr2' ' DC and/or Hck' DC, preferably a population of Fyn ¹ DC, and ii) a pharmaceutically acceptable support.

Also herein described is the corresponding method of preventing or treating a disease, preferably a cancer or an infection in a subject in need thereof, wherein the method comprising a step of administering the subject with a FYN kinase inhibitor, a defective DC (or population of defective DC), and/or a composition as described in the context of the present invention, thereby treating the subject.

Administration route

The compounds or pharmaceutically acceptable derivatives thereof, compositions and combinations of compounds of the invention may be administered enterally and/or parenterally. Parenteral administration involves in particular subcutaneous, intramuscular, intradermal, transdermal, intramammary, inhalational, pulmonary, intrapleural, nasal, intravenous, intraperitoneal and intrathecal routes. Enteral administration involves mainly oral, buccal (sublingual or perilinguale) and rectal routes. In some embodiments, the compound is administered intravenously, intra tumorally or orally. One skilled in the art will recognize the advantages of certain routes of administration. Combinations

Inventors herein advantageously describe a combination of i) a product selected from a FYN kinase inhibitor, a population of DC activated by a FYN kinase inhibitor, and a population of DC defective for FYN, FGFR1, FGFR2 and/or HCK, in particular a population of Fyn DC, Fgfrl DC, Fgfr2'^/' DC and/or Hck DC as herein above described, preferably a population of Fyn ⁷ DC, and ii) one or more T-cell immune check-point inhibitors (“IQ”).

As used herein, the terms “combination” or “combination therapy” refer to the administration of each therapeutic agent (of the combination therapy of the invention), either alone or in the form of a pharmaceutical composition or medicament, either sequentially, concurrently, or simultaneously.

As used herein, the term “sequential” or “sequentially” refers to the administration of each therapeutic agent of the combination therapy of the invention, either alone or in a medicament, one after the other, wherein each therapeutic agent can be administered in any order. Sequential administration may be particularly useful when the therapeutic agents in the combination therapy are in different dosage forms, for example, one agent is a tablet and another agent is a sterile liquid, and/or the agents are administered according to different dosing schedules, for example, one agent is administered daily, and the second agent is administered less frequently such as weekly.

As used herein, the term “concurrently” refers to the administration of each therapeutic agent in the combination therapy of the invention, either alone or in separate medicaments, the second therapeutic agent being administered immediately after the first therapeutic agent, and the therapeutic agents being administered in any order. In a particular embodiment the therapeutic agents are administered concurrently.

As used herein, the term “simultaneous” refers to the administration of each therapeutic agent of the combination therapy of the invention in the same medicament. As will be understood by those skilled in the art, the combination therapy may be usefully administered to a subject during different stages of their treatment.

In the context of the present description, a “combination” involves, in particular consists of, at least two products one of which is i) a product selected from a FYN kinase inhibitor, a population of DC activated by a FYN kinase inhibitor, and a population of DC defective for FYN, FGFR1, FGFR2 and/or HCK (also herein identified as “product i)”), and the second is ii) a T-cell immune check-point inhibitor (ICI) or a mixture of ICIs (also herein identified as “product ii)”). In the context of the present invention, the immune check-point inhibitor (ICI) is an inhibitor that blocks proteins called checkpoints that are preferably expressed by T cell.

The ICI may be selected for example from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti- TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Toll-like receptor, a type-1 interferon or an interleukin, and any mixture thereof.

In a preferred embodiment, the ICI is an anti-PD-1 or anti-PD-Ll agent.

Examples of anti-PD- 1 agents usable in the context of the present invention are pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, and nanobodies targeting PD-1. Examples of anti-PD-Ll agents usable in the context of the present invention are atezolizumab, avelumab, durvalumab, and nanobodies targeting PD-L1.

Examples of anti-CTLA-4 agents usable in the context of the present invention are ipilimumab, tremelimumab, and nanobodies targeting CTLA-4.

Examples of anti-TIGIT agents usable in the context of the present invention are vibostolimab, etigilimab, tiragolumab, domvanalimab, ociperlimab, and nanobodies targeting TIGIT.

Examples of anti-TIM-3 agents usable in the context of the present invention are MGB453, TSR- 022, BGBA425, RO7121661, ICAGN02390, RO724766, and nanobodies targeting TIM-3.

Examples of anti-LAG-3 agents usable in the context of the present invention are eftilagimod alpha, relatlimab, favezelimab, fianlimab, tebotelimab, and nanobodies targeting LAG-3.

Examples of anti- VISTA agents usable in the context of the present invention are JNJ-61610588, CA-170d, or nanobodies targeting VISTA.

Examples of agonists of 0X40 usable in the context of the present invention are rocatinlimab, BGB-A445, INCAGN01949 and ivuxolimab.

Examples of agonists of CD40 usable in the context of the present invention are Selicrelumab, APX005M, ChiLob7/4, ADC-1013, SEA-CD40 and CDX-1140.

Examples of agonists of STING usable in the context of the present invention are DMXAA, ASA404, ADU-S100/MIW815, MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BLSTING (BI 1387446), E7766, TAK-676, SNX281, SYNB1891 and GSK3745417.

Examples of agonists of Toll-like receptors usable in the context of the present invention are TQ- A3334, SHR2150, RO7119929, DSP-0509, BNT411, APR003, poly A:U, poly I:C, poly I:C plus polylysine (poly(ICLC)), CpG adjuvant, Bacillus Calmette-Guerin (BCG), monophosphoryl lipid A, imiquimod (R837), resiquimod (R848) and motolimod (VTX-2337).

Examples of type-1 interferons usable in the context of the present invention are recombinant type I IFNs such as recombinant IFN-a2. Examples of agonists of interleukins usable in the context of the present invention are bempegaldesleukin, a pegylated IL-2, siltuximab (anti-IL-6 antibody) and anakinra (IL-1R antagonist).

Examples of agonists of CD137 usable in the context of the present invention are urelumab (BMS- 663513), utomilumab (PE-05082566), PRS-343, Cinrebafusp alfa, RG7827, RO7122290, ADG106, INBRX-105/ES101, CTX-471, GenlO46/BNT311, MCLA-145, RG6076, RO7227166, MP0310, Genl042 /BNT312, AGEN2373, LVGN6051, ATOR-1017, STA551, ND-021/NM21-1480, GNC-038, Emfizatamab, DSP107, FS120, FS222, HGT-1030, ABL503 /TJ-L14B, IBI319, GNC-039, EU101, CB307, ABL111, TJ-CD4B, TJ-CLDN4B, TJ033721, GNC-035, PRS-344/S095012, BI 765179, QL301/QLF31907, ATG-101/YN-051/Ori-Bs-001, BT7480, PM1003, YH004, LBL-024, PM1032, HLX35/BNA035, HBM7008,

ABL105/YH32367, BGB-B167, ADG206 and PE0116.

If the immune check-point inhibitor is an anti-PDl agent, said anti-PD-1 agent is preferably an anti-PD-1 antibody selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab.

If the immune check-point inhibitor is an anti-PDL-1 agent, said anti-PDL-1 antibody is preferably selected from atezolizumab, avelumab and durvalumab.

The herein described combinations of a product i) and a product ii) are also described for use as a medicament, in particular for use for the prevention or treatment of a disease, preferably of a cancer or of an infection, in a subject in need thereof.

In a preferred aspect, the product i) and the T-cell immune check-point inhibitor(s) ii) are formulated for separate administration to a subject in need thereof.

Preferably, the product i) is administered to the subject in need thereof before the T-cell immune check-point inhibitor(s).

In a particular aspect, the product i) is a FYN kinase inhibitor and is formulated for systemic administration, preferably intravenous (I.V.) administration, or is a population of DC activated by a FYN kinase inhibitor, or a population of DC defective for FYN, FGFR1, FGFR2 and/or HCK, in particular a population of Fyn ^f DC, and is formulated for intra tumoral (I.T) administration.

Also herein described is the corresponding method of preventing or treating cancer or an infection in a subject in need thereof, wherein the method comprising a step of administering the subject with a combination as described in the context of the present invention, thereby treating the subject.

Kit

Further herein described is a kit comprising any one or more of the herein described products, typically (a) one or several products selected from FYN kinase inhibitor(s), a DC activated by a FYN kinase inhibitor or a population of said DC, and a population of DC defective for FYN, FGFR1, FGFR2 and/or Hck, such as a population of Fyn DC, Fgfrl DC, Fgfr2 ^ DC and/or Hck' DC, in particular a population of Fyn^v' DC, and (b) T-cell immune check-point inhibitor(s), in particular anti-PDl and/or anti-PDLl agent(s), preferably in different containers; or a composition comprising the combination of the product(s) (a) and the T-cell immune check-point inhibitor(s) (b), and a pharmaceutically acceptable carrier and material(s) for administering the product(s) (a) and/or the T-cell immune check-point inhibitor(s) (b) or for administering the composition or combination. The kit may optionally comprise (c) additional distinct therapeutic agent(s), in particular an anti-cancer agent or an agent active against infection.

Typically, the kit also comprises instructions for using the inhibitor(s), cell(s), composition(s) or combination(s) according to the invention, in particular according to the disclosed methods.

The kits described herein may be particularly suitable for administering different dosage forms, for example, oral and/or parenteral, for administering the separate active (in particular therapeutic) agents of the combination or composition at different dosage intervals, or for titrating the active (in particular therapeutic) agents of the combination or compositions against one another. To assist compliance, the kit typically includes directions (instructions) for administration and may be provided with a memory aid. The pharmaceutical composition or each element of the combination can be included in a container, pack, or dispenser. The kit may further comprise other materials that may be useful in administering the medicaments, such as diluents, filters, IV bags and lines, needles and syringes, and the like.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. All references cited in the present application are herein incorporated by reference. LEGEND TO THE FIGURES

Figure 1: Identification of TG100801 as an immunostimulatory tyrosine kinase inhibitor (TKI).

(A) Kinase targeting of the agents in the TKI chemical library. (B) Scheme of the in vitro antigen cross-presentation assay used for drug screening. Inducible-immortalized DC precursors (iniDCs) cultured in the presence of dexamethasone (DEX) and doxycycline (DOX) were differentiated into immature DCs (de-iniDC) by DEX and DOX withdrawal, and then treated with the TKIs from the chemical library for 16 h before being pulsed with OVA for 6 h. Cells were washed and co-cultured with the B3Z T cell hybridoma for additional 18 h. Then, supernatant was collected for the quantification of IL2 by ELISA. (C) The mean Log2 fold change (FC) of IL2 for each TKI treatment as compared with vehicle controls, as well as corresponding P values (obtained with t test) were used to generate the volcano plot. TKIs that led to significant changes of IL2 production for both parameters are depicted in red. (D) Kinase targeting of the screened TKIs ranked by antigen cross-presentation efficacy.

Figure 2: TG100801 induces dendritic cell (DC) activation.

De-iniDCs and Bone marrow dendritic cells (BMDCs) were treated with TG100801 at 1, 2 and 4 pM and then stimulated with the lysate of dying cancer cells before the assessment of the indicated surface markers by flowcytometry. The mean fluorescence intensity (MFI) of DC maturation markers (A-C, E-G) and the percentage of immature DCs of the type I conventional DC subtype (cDCl) (D, H, defined as CD103⁺CDl lb ) within all DCs are expressed as bar charts (mean + SD, n = 4). Statistical analysis was performed by means of two-way ANOVA.

Figure 3: TG100801 facilitates progenitor differentiation into cDCl.

IniDC precursors were differentiated into de-iniDCs by removal of dexamethasone (DEX) and doxycycline (DOX) (A), and bone marrow (BM) progenitors were cultured in the presence of GM-CSF for the differentiation of BMDCs (B). TG100801 at low dose was added during the DC differentiation process. Differentiated de-ini and BMDCs were pulsed with OVA and co-cultured with B3Z T cell hybridoma for the evaluation of antigen presentation efficacy measured by IL2 ELISA (C) or subjected to immunostaining to analyse the proportion of CD103⁺CDl lb cDCl (D) together with the indicated maturation markers by flow cytometry (E, F). Data are depicted as bar charts (mean + SD, n = 4) and statistical analysis was performed by means of two-way ANOVA. (G-I) De-iniDCs, BMDCs, or human monocyte-derived DCs (moDCs, from 3 healthy individuals) were treated with TG100801 at early stage of differentiation for the quantitative PCR analysis of DC subtype- specific transcription factor expression patterns. The mean fold change of mRNA expression level is shown in heatmaps (n = 4).

Figure 4: Systemic treatment of TG100801 changes immune infiltration in lung cancerbearing mice.

Orthotopic TCI NSCLC-bearing mice received two intraperitoneal (i.p.) injections of TG100801 or solvent controls at day 0 (when tumors became palpable) and at day 2. The blood, lung, tumordraining lymph node (tdLN), and spleen were harvested at day 7 and dissociated into single-cell suspensions for multiplex immunostaining and flow-cytometric analysis. The proportion of type 1 conventional DCs (A, cDCl, defined as F4/80“MHC-II⁺CDl lc⁺CD103⁺CDl lb“ among viable leukocytes) as well as the mean fluorescence intensity (MFI) of the indicated markers on all DCs (B-D, defined as F4/80 MHC-II⁺CD1 lc⁺ among viable leukocytes) were quantified and depicted as scattered box plots (n = 9 animals/group). The same samples were stained with a panel of antibodies to quantify the percentage of CD8⁺ T cells (E), regulatory T cells (F, Tregs, defined as CD4⁺CD25⁺Foxp3⁺), and their ratio (G), and are depicted as box plots. The CD8⁺ T-cell to Treg ratio was also analysed in the MCA205 fibrosarcoma-bearing mice following the same treatment protocol (H). Moreover, the ratio of PDl-positive cells within CD8⁺ T cells (I) and Tregs (J) was assessed. Statistical significance was calculated using one-way ANOVA test with Dunnett multiple comparisons, as compared with Sol.

Figure 5: Systemic treatment of TG100801 exerts cDCl -dependent tumor-regression against NSCLC and synergize with PD1 blocking antibody.

Luciferase-expressing TCI NSCLC cells (TCl_Luc) were intravenously (z.v.) injected into syngeneic mice to establish orthotopic lung cancers. When tumors became detectable by bioluminescence, mice were randomized and subjected to (i.v.) treatment of cytochrome c (Cyt c) to specifically deplete cDCl cells, and then to intraperitoneal (i.p.) treatment with solvent (Sol) or TG100801, as well as to subsequent treatments with anti-PD-1 blocking antibody (aPD-1) or the corresponding isotype control antibody (also) as illustrated in (A). Bioluminescence of orthotopic lung cancers was monitored by in vivo imaging. Representative images tracking the development of lung cancers in mice are shown in B and are quantified as total flux of photons over time expressed as growth curves in C, D (mean+SEM, n = 10 animals/groups). Overall survival was recorded and is depicted as Kaplan-Meier curves (E, F). Statistical significance was calculated by means of the type II ANOVA for tumor growth curves or log rank test for the survival curves. Figure 6: Target exploration for TG100801-mediated DC activation.

IniDCs stably expressing CRISPR-Cas9 (iniDC_Cas9) were transfected with guide RNAs (gRNAs) targeting the indicated kinases individually (A). Nontargeting gRNA (NonT) was used as control. Transfected precursor cells were differentiated into de-iniDC, which were then treated with TG100801 before being subjected to ovalbumin and subsequent co-culture with B3Z hybridoma T cells for the production of IL2 as the proxy for in vitro antigen cross-presentation capability. (B, C) Wild- type (Fyn^+/+) or Fyn knockout ( yn⁷) de-iniDCs were treated with different concentrations of TG100801 (1 or 2 pM) before being subjected to ovalbumin (OVA) or OVA peptide SIINFEKL (SL8 - SEQ ID NO: 1) and subsequent co-culture with B3Z hybridoma T cells for the quantification of IL2. (C) Fyn knockout (Fyn⁷ ) iniDCs that express Cas9 were further knocked out of Fgfrl, Fgfr2, or Hck (dKO) and differentiated into de-iniDCs, which were then treated with TG100801 before being subjected to the antigen presentation assay. IL2 production was quantified by ELISA and reported as scattered dot plot (mean + SEM, n = 5 replicates). Statistical significance was calculated by means of two-way ANOVA with FSD multiple comparisons test.

Figure 7: TG100801 stimulates DC mediated phagocytosis of cancer cells.

(A) TG100801 and TG100572 at the indicated concentrations were either added to de-iniDCs or BMDCs 16 h before incubation with OVA (protocol#!) and then washed out or they were added to the DC co-culture with B3Z hybridoma after OVA pulsing (protocol#2). Then, IL2 in cell culture supernatants was quantified by ELISA as an indication for DC antigen presentation. (B- D) IL2 quantity was normalized to the untreated conditions and is expressed as line chart (mean + SD, n = 4). (E) Following treatment with TG100801, DCs were cocultured with CellTracker- labelled cancer cells (viable or dying upon treatment with oxaliplatin). Then, phagocytosis was assessed by flow cytometry and is depicted as percentage of CellTr acker ⁺CDl lc⁺ events (mean + SD, n = 3). Statistical analysis was performed by means of two-way ANOVA.

Figure 8: No effect of TG100801 on T cell activation.

Mouse primary CD8⁺ T cells and B3Z hybridoma T cells were treated with vehicle or TG100801 at the indicated concentrations alone, or in combination with phorbol myristate acetate (PMA), for 16 h, followed by 5 h treatment with brefeldin A (BFA), and were then subjected to permeabilization and immunostaining for the analysis of intracellular cytokines using flow cytometry. The mean fluorescence intensity (MFI) is expressed as bar chart (mean + SD, n = 3). Figure 9: Systemic treatment with TG100801 induces T-cell-dependent regression of NSCLC.

(A) Luciferase-expressing murine non-small cell lung cancer (NSCLC) TCI cells (TCl_Luc) were i.v. injected into immunocompetent C57B1/6J or into immunodeficient nu/nu mice to establish orthotopic lung cancer. Once orthotopic lung tumors were detectable by bioluminescence, mice were randomized and subjected to intraperitoneal (z.p.) treatment with TG100801 or solvent (Sol) control, as illustrated in the scheme (A, D). Bioluminescence signal was monitored by in vivo imaging. Representative images are shown in B and E and the total photon flux was quantified over time as depicted in C, F (mean + SEM, n = 10 animals/group for C, and n = 6 animals/group for F). Statistical analysis was performed by means of type II ANOVA.

Figure 10: Adoptive transfer of Fyn KO DC enhances antitumor immune responses, improves tumor control, and synergizes with immune checkpoint inhibition.

(A) Experimental schematic showing CRISPR-Cas9-mediated knockout of Fyn in dendritic cells (DCs), adoptive transfer into tumor-bearing mice, and treatment with anti-PD-1 (aPD-1) immune checkpoint blockade. (B-C) Immature dendritic cells (iniDCs) stably expressing CRISPR-Cas9 (iniDC-Cas9) were transfected with guide RNAs targeting Fyn (sgFyn-#l, #2) or a non-targeting control (sgNonT). Transfected precursor cells were differentiated into either wild- type (Fyn / . sgNonT) or Fyn knockout (sgFyn-#l, #2) de-iniDCs. Differentiated DCs were pulsed with TCI tumor lysate and intravenously injected into syngeneic mice bearing orthotopic TCI lung tumors. DC transfer was interspersed with systemic administration of either aPD-1 or isotype control antibody (also), as indicated. Tumor burden was assessed by in vivo bioluminescence imaging and quantified as total photon flux (photons/sec). Tumor growth is shown as mean + SEM (n = 10 animals/group) (B). Overall survival is shown as Kaplan-Meier diagram (C). Statistical analysis was performed using Type II ANOVA for tumor growth and log-rank test for survival. Naive C57B1/6 mice were subcutaneously vaccinated with OVA-pulsed de-iniDCs bearing WT, knockout, or mutation of Fprl. The draining lymph node (dEN), spleen, and peripheral blood were collected and dissociated into single cell suspension for the stimulation with the MHC-L restricted OVA peptide SIINFEKL or T cell stimulation cocktails. After stimulation, the immune cells were subjected to H2Kb-OVA tetramer staining with T cell surface markers. The percentage of tetramer positive CD8+ T cells and central memory CD8 T cells (TCM), defined as CD8+CD44hiCD62Lhi, among all viable leukocytes were quantified and depicted as bar charts (mean + SEM, n = 4-6 animals/group). Statistical significance was calculated by means of two- way ANOVA with Dunnett's multiple comparisons test. Figure 11: Small-molecule Fyn inhibitors enhance antigen presentation in de-iniDCs at submicromolar concentrations in an MHC-I-dependent manner.

(A-I) Immature de-induced immortalized dendritic cells (de-iniDCs), either wild-type (WT), knockout for Fyn (Fyn-KO) or knockout for fi2-microglobulin (B2m-KO), were treated with a panel of Fyn inhibitors at the indicated concentrations. Cells were pulsed with ovalbumin and then co-cultured with B3Z hybridoma T cells. IL2 production was measured as a proxy for in vitro antigen cross-presentation. IL2 levels were quantified by ELISA and are presented as bar charts (mean + SD of quintuplicates) showing the efficacy of Fyn inhibitors to increase crosspresentation in WT, but not Fyn-KO or B2m-K0 de-iniDCs. Statistical significance was assessed using two-way ANOVA with Fisher’s Feast Significant Difference (ESD) multiple comparisons test.

Figure 12: Monoclonal Fyn antibody enters de-iniDCs and activates antigen presentation, mimicking the effects of TG100801.

Immature de-induced immortalized dendritic cells (de-iniDCs), either wild-type (WT) or knockout for Fyn (Fyn-KO) were treated with monoclonal anti-Fyn antibody (mAh) at 2 pg/mE for 12 h. (A,B) Antibody uptake was visualized by immunostaining employing AlexaFluor488 conjugated secondary antibody followed by epifluorescence microscopy. Representative images are shown in A and the fluorescence intensity of cytoplasmic signal was quantified in B. (C) The effect of anti-Fyn antibody on the cross-presentation efficacy of WT and Fyn-KO de-iniDCs was benchmarked against the chemical Fyn inhibitor TG100801 (TG). To this aim, de-iniDCs, either wild-type (WT) or knockout for Fyn (Fyn-KO) were treated with TG or the mAB at the indicated concentrations. (D) Cells were then pulsed with ovalbumin and co-cultured with B3Z hybridoma T cells. IL2 production was quantified by ELISA and is presented as bar charts (mean ± SD; quintuplicates) exhibiting equal efficacy of mAB and TG in WT DC and no further enhancement in a Fyn background.

Figure 13: Monoclonal Fyn Ab promotes activation and maturation of BMDCs, and promotes the frequency of cDCl and migratory cDCls.

(A-E) Bone marrow-derived DCs (BMDC) were treated with the Fyn inhibitor TG100801 (TG) or the monoclonal anti-Fyn antibody (mAB) at the indicated concentrations. Cells were then left stimulated with tumor lysate or were left untreated before collection and phenotyping via flow cytometry using the indicated markers. Treatment with both mAB and TG increased the % of CD103⁺CDl lb conventional type 1 dendritic cells (cDCl) (A) of the CCR7⁺XCR1⁺ migratory subtype (B). Moreover, mAB and TG treated DCs expressed CD40 (C), CD80 and CD86 (D) costimulatory and maturation markers as well as MHC-II (E), indicative of antigen presentation capability.

Figure 14: The Fyn mAh is favorable to T cell activation.

(A-F) Primary murine T cells isolated from mouse spleen were treated with Fyn inhibitor TG or the monoclonal anti-Fyn antibody (mAB) at the indicated concentrations. Cells were then stimulated with an anti-CD28 (aCD28) antibody for 1-3 days and T cell activation markers CD69 (A-C) and CD25 (D-F) were assessed by flow cytometry and showed that incubation with Fyn mAB does not decrease T cell activation by CD28.

Figure 15: Bulk RNA sequencing reveals gene clusters commonly upregulated in Fyn-KO, TG100801-treated, or anti-FYN antibody-treated DC, associated with immune navigation.

RNA was extracted from immature de-induced immortalized dendritic cells (de-iniDCs) either Fyn ^A (KO) or WT treated with the FYN inhibitor TG100801 (TG) or an anti-Fyn monoclonal antibody (mAb) for 24 h and subjected to bulk RNA sequencing. (A) Divisive hierarchical clustering of differentially expressed genes (DEGs) identifies gene clusters commonly upregulated in Fyn-KO and pharmacologically treated cells. (B,C) Gene ontology (GO) enrichment analysis of selected upregulated clusters reveals significant association with biological processes involved in leukocyte migration, chemotaxis and cell-cell adhesion. (D,E) Enrichment plots for KEGG pathways in gene clusters from panel A, shows pathways related to focal adhesion, cytokine-cytokine receptor interactions, and immune navigation.

Figure 16: K-means clustering of bulk RNA sequencing data reveals convergent transcriptional programs across Fyn-deficient and Fyn- inhibited dendritic cells.

(A) K-means clustering of differentially expressed genes in dendritic cells wild-type (WT), Fyn ^/_ (KO), and WT DCs treated with either the Fyn inhibitor TG100801 (TG) or an anti-Fyn monoclonal antibody (mAb). Six major gene clusters were identified based on expression similarity. (B-D) Gene Ontology (GO) enrichment analyses of four selected clusters confirmed overrepresentation of pathways involved in immune cell migration, T cell activation, leukocyte adhesion, and regulation of cell-cell and cell-substrate adhesion. EXAMPLES

Materials and methods

Cell Culture and related reagents

RPMI 1640 medium (Cat# 61870010), DMEM medium (Cat# 10566016), HEPES (CAT# 15630056), sodium pyruvate (Cat# 11360070), phosphate-buffered saline (PBS, Cat# 20012027), penicillin-streptomycin (Pen/Strep, 10,000 U/mL, Cat# 15140122), and TrypLE™ Express (Cat# 12604013) were purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS, Cat# F7524), P-mercaptoethanol (Cat# M3148), dexamethasone (Cat# D0700000), and doxycycline hyclate (Cat# D3000000) were purchased from Sigma (St. Louis, MO, USA). Recombinant murine GM-CSF (Cat# 315-03) was obtained from Peprotech (Cranbury, NJ, USA). Unless otherwise indicated, all plasticware was purchased from Corning. Life Sciences (Corning, NY, USA).

The parental iniDCs were kindly shared by Cornelia Richter and colleagues (Richter, C et al.), iniDCs stably expressing CRISPR Cas9 (iniDC_Cas9) were established by transduction with Edit-R lentiviral CAG-Blast-Cas9 nuclease particles (Cat# VCAS10129, Horizon Discovery, Waterbeach, UK) followed by cloning and validation as previously published (Zhao, L et al.). All gene-edited iniDC cell lines were generated by transfecting iniDC_Cas9 cells with specific crRNA + tracrRNA, followed by single cell sorting and immunoblotting for knockout validation. RPMI 1640 with 10% decomplemented FBS, 1 mM sodium pyruvate, 10 mM HEPES, and lx Pen/Strep was used as basic DC medium. P-mercaptoethanol (at a final concentration of 50 pM) and recombinant GM-CSF (at a final concentration of 10 ng/mL) was freshly added. IniDCs and derivative cell lines are immortalized under the induction of Dex/Dox (Dex at 100 nM + Dox at 2 pM). Dex/Dox removal (“deinduction”) led to a halt in proliferation and differentiation into immature DCs (“de-iniDCs”) that were used for experiments. The B3Z hybridoma T cells were kindly provided by Sebastian Amigorena and maintained with DC medium supplemented with P- mercaptoethanol (50 pM). TCI non-small cell lung cancer cells expressing luciferase (TCl_Luc) were cultured with DMEM medium containing 10% decomplemented FBS and lx Pen/Strep.

Chemicals and antibodies

All tyrosine kinase inhibitors were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Lipopolysaccharides (LPS, Cat# L2654) albumin from chicken egg white (OVA, Cat# A5503), and Cytochrome c from equine heart (Cat# C7752) were obtained from Sigma. In vivo neutralizing antibodies to PD-1 (Cat# BE0273), and corresponding isotype controls (Cat# BE0090) were purchased from BioXcell (Lebanon, NH, USA). Antibodies for ELISA, including alLip (Cat# 503502), biotin-conjugated alLip (Cat# 515801), aIL2 (Cat# 503702), biotin- conjugated aIL2 (Cat# 503804), IL6 (Cat# 504502), and biotin-conjugated aIL6 (Cat# 504602) came from Biolegend. Mouse monoclonal antibodies employed for high dimensional flow cytometry: aCD3 APC (Cat# 100236), aCDl lc APC (Cat# 117310), aCD45 Alexa Fluor 700 (Cat# 103116), aCD80 PE (Cat# 104708), aCD80 PercP-Cy5.5 (Cat# 104722), aCD86 APC- Fire750 (Cat# 105046 ), aCTLA-4 PE-Cy7 (Cat# 106314), aF4/80_BV785 (Cat# 123141), aMHC-II BV650 (Cat# 107641) were from Biolegend; aCD4 eFluor450 (Cat# 48-0042-82), aCD8a PercP-Cy5.5 (Cat# 45-0081-82), aCDl lb eFluor450 (Cat# 48-0112-82), aCDl lc APC- eFluor780 (Cat# 47-0114-82), aCD40 eFluor450 (Cat# 48-0402-82), aCD45 APC-eFluor780 (Cat# 103116), aCD69 E-Cy5 (Cat# 15-0691-82), aCD83 PE-Cy7 (Cat# 25-0839-42), aFOXP3 FITC (Cat# 11-5773-82), aPD-1 PE (Cat# 12-9985-82), aMHC-II PE (Cat# 12-5321-82), came from eBioscience/Life Technologies. Flowcytometry related monoclonal antibodies for human samples are detailed below. The Live/dead Yellow Fixable Dye (Cat# L34959) was from Life Technologies. FYN monoclonal antibody came from Santa Cruz (Cat# sc-434L, customized product, Sodium azide-free). The isotype control antibody corresponding to the FYN monoclonal antibody, mouse IgGl, K isotype antibody, came from Biolegend (Cat#: 400102). The following FYN inhibitors came from MedChemExpress: pyrazolol[3,4-d] pyrimidine 1 (PPI - Cat# HY- 13804; CAS No 172889-26-8), pyrazolol[3,4-d] pyrimidine 2 (PP2 - Cat# HY-13805; CAS No 172889-27-9), ARN25068 (Cat# HY-144290; CAS No 2649882-80-2), SU6656 (Cat# HY- B0789; CAS No 330161-87-0), 1-NM-PP1 (Cat# HY-13942; CAS No 221244-14-0), 1-Naphthyl PPI (Cat# HY-13941; CAS No 221243-82-9), TG 100801 (prodrug of TG 100572) (Cat# HY- 10186; CAS No 867331-82-6) or a salt thereof such as for example TG 100572 hydrochloride salt (Cat# HY-10185; CAS No 867331-64-4), and Saracatinib (Cat# HY- 10234; CAS No 379231- 04-6).

Tyrosine kinase inhibitor screen and in vitro antigen cross-presentation assay

For the screening, de-iniDCs (5xl0⁵ cells/mL) were seeded in 96-well u-bottom plates (200 pL/well) and treated with the agents of the Protein Tyrosine Kinase Compound Library (MedChemExpress) for 16 h before soluble OVA was added into the cell culture at a final concentration of 1 mg/mL and incubated for 4 h at 37 °C and 5% CO2. For other in vitro antigen presentation assays, BMDCs or de-iniDCs wild-type or their iniDC derivates carrying specific gene knockouts were treated with TKIs, as detailed in the figure legends and incubated with either soluble OVA or the OVA SIINFEKL peptide for 4 hours. The plates were then centrifuged at 500 g for 5 min, supernatant was removed and replaced with 200 pL/well of B3Z T cell hybridomas diluted to 5xl0⁵ cells/mL with DC medium, and co-incubated with DCs for 18 h at 37 °C before collecting the supernatant by spinning the plates at 500 g for 5 min and gently transferring 150 pL supernatant for the quantification of IL2 secretion by ELISA.

Transit gene knockout and generation of stable iniDC KO clones

All predesigned guidance RNAs (Edit-R synthetic crRNA), the non-targeting control#! (Cat# U- 007501-01-2), trans- activating CRISPR RNA (tracrRNA, Cat# U-002005-5000), as well as the DharmaFECT 1 transfection reagent (CAT# T-2001-04) were purchased from Horizon Discovery. CrRNA and tracrRNA were diluted as a 10 pM stock solution in Tris buffer (pH 7.4). Co-transfection of the crRNA:tracrRNA duplex was performed as previously published (Zhao, L et al. Le Naour, J et al.). IniDC_Cas9 cells were seeded in 6-well plates at IxlO⁶ / well in 2 mL DC medium without Dex/Dox. For each transfection, 25 nM crRNA and 25 nM tracrRNA were mixed in 100 pL RPMI 1640 medium and incubated 5 min at room temperature; 10 pL of DharmaFECT 1 transfection reagent was mixed in 100 pL RPMI 1640 medium and incubated 5 min at room temperature; then the two solutions were mixed and incubated for another 20 min before being added dropwise into iniDC_Cas9 cultures. Three days after transfection the cells were collected for FACS sorting to obtain clones carrying the KO of interest. For genetic screening purposes, the transfection reagent containing medium was replaced with fresh DC medium to let the cells recover overnight before the in vitro antigen cross-presentation assay.

IL2 ELISA

EEISA for IL2 was performed as previously published (Zhao, L et al.). In brief, capture antibody was diluted in 1 x ELISA coating buffer (diluted with water from 5x ELISA coating buffer obtained from Biolegend) at 1/500, applied 100 pL/well in 96-well high-binding assay plates (Coming), and incubated overnight at 4 °C; then the plates were washed 3 times with washing buffer (lx TBS with 0.1% Tween-20, 300 pL/well) and incubated with 150 pL/well blocking buffer (10% FBS + 1% BSA in PBS) for 1 h at room temperature to block unspecific binding sites. Then, plates were loaded with samples or serially-diluted standards and incubated for 2 h at room temperature. Afterwards the supernatant was discarded and plates were washed 4 times with washing buffer, then 100 pL/well of biotinylated detection antibody (1/500 diluted in blocking buffer) was added and incubated at room temperature for 1 h. The supernatant was discarded and plates were washed 4 times with washing buffer, then 100 pL/well of HRP- Avidin (Cat# 405103 from Biolegend, 1/1000 diluted in blocking buffer) was added and incubated at room temperature for 30 min. In the end, plates were washed 5 times and 100 pL/well of 1-Step™ Ultra TMB- ELISA substrate solution (Cat# 34028 from Life Technologies) was added for colorization. When the top standard wells turned dark blue (generally within 10 min), 50 pL/well of 0.5 M H2SO4 was used to stop the reaction. The absorbance at 450 nm was immediately measured using a BMG FLUOstar plate reader. Exact concentrations of the assayed cytokines were calculated by means of a standard curve and corresponding dilution factors of the sample.

Quantitative RT-PCR

RNA extraction from de-iniDCs, BMDCs, or human moDCs was performed with the GeneJET RNA Purification Kit (Life Technologies), following the manufacturers’ instructions. Reverse transcription from mRNA to cDNA was performed with the Maxima First Strand cDNA Synthesis Kit (Life Technologies), using approximately 2.5 pg total RNA as template. Real-time PCR reaction was performed on a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher) using the Power SYBR™ Green PCR Master Mix and corresponding settings. Gene-specific primers were designed by using the NCBI Primer-BLAST online application (https://www.ncbi.nlin.nih.gov/tools/primer"blast/) and synthesized by Eurofins Genomics. Primer sequences are listed in Table SI. qRT-PCR data was analyzed using the 2-^AACt method to obtain the fold change in gene expression that were normalized to expression levels of the housekeeping gene Gapdh.

Bulk RNA sequencing

Total RNA was extracted from approximately 1 x 10⁷ cultured de-iniDCs using the RNeasy Plus Mini Kit (Qiagen, Cat# 74134), following the manufacturer’s instructions. RNA quality assessment, library preparation, and sequencing were carried out by BMKGENE-Biomarker Technologies (BMK) GmbH (Munster, Germany). The resulting raw sequencing data files (fastq.gz) were processed using the RaNA-seq online platform (https://ranaseq.eu/) for quality control and generation of gene count tables. Differential gene expression and pathway analyses were performed using the iDEP web-based tool (http://bioinformatics.sdstate.edu/idep96/). Genes were included for analysis if they had a minimum expression threshold of 0.5 counts per million (CPM) in at least one library. The top 2000 most variable genes were used for division or k-means clustering and pathway enrichment analysis. Differential expression analysis was performed at the gene level using DESeq2. Genes were considered significantly differentially expressed if they showed a fold change of >2 and a false discovery rate (FDR) of <0.1. Pathway and network enrichment analyses were conducted using gene sets representing upregulated, downregulated, and all differentially expressed genes, with significance defined by a p-value <0.05. Animals and cancer models

All mice for experimentation were maintained at the Gustave Roussy Campus Cancer in a specific pathogen free (SPF), environmental controlled animal facility with 12 h light/dark cycles, receiving food and water ad libitum. All animal experiments were performed in compliance with the EU Directive 63/2010 and dedicated ethic protocols (Projects 2023_060) that was approved by the ethical committee of the Gustave Roussy Campus Cancer, CEEA IRCIV/IGR no. 26, registered at the French Ministry of Research). Female wild-type C57BL/6 mice (6~8 eight weeks old) and female athymic nude (ntt/ntt) mice were obtained from ENVIGO France (Gannat, France). For the TCI NSCEC model, wild type TCI Euc cells (5xl0⁵ in 100 pF PBS) were intravenously injected to mice. Tumor incidence and development were monitored by in vivo photonic imaging of tumor cell luciferase activity. When tumor incidence in the lung was detected at an exposure time of 4 min (6~7 days after cell injection), mice were randomized for treatment as described below. To perform bioluminescence imaging, mice were injected i.p. with 3 mg beetle luciferin potassium salt dissolved in DPBS (Promega, Madison, WI, USA), After 8 min (at peak bioluminescence signal) mice were anesthetized with vaporized isoflurane and photons were acquired on an IVIS Eumina III imaging system (Caliper Fife Sciences Inc., Hopkinton, MA, USA). In vivo imaging was conducted every 4-5 days with an exposure time starting with 4 min gradually decreased to 1 min when photon saturation occurred. Tumor bearing mice showing photon saturation at 1 min of exposure at small binning settings were euthanized.

For adoptive transfer, iniDCs stably expressing Cas9 were transfected with guide RNAs targeting Fyn (sgFyn-#l, #2) or a non-targeting control sgRNA (sgNonT). Following transfection, cells were cultured in the absence of Dex/Dox for three days to drive differentiation into de-iniDCs. To achieve tumor antigen loading for therapeutic vaccination, de-iniDCs were incubated for 2 hours with lysates prepared from an equal number of TCI -Euc cells. Cell lysates were generated by repeated freeze-thaw cycles followed by sonication. After antigen loading, cells were harvested, washed twice with cold PBS, and filtered through a 70 pm cell strainer to obtain a single-cell suspension. Processed de-iniDCs were resuspended in cold PBS and administered via intravenous injection (200 pF containing 2 x 10⁶ cells per mouse) into tumor-bearing mice.

For the detection of OVA-TCR expressing T cells, the H2Kb-OVA tetramer was assembled according to the Biolegend protocols: For 15 tests, 30 pF of H2Kb-OVA monomer (Cat# 280051) was mixed with 3.3 pF of streptavidin-PE (Cat# 405204), pipette to mix, and incubated on ice in the dark for 30 minutes. During the incubation, blocking solution was prepared by combining 80 pF of 1 mM D-Biotin (Cat# B4639, Sigma, diluted in PBS) and 6 pF of 10% (w/v) NaNs with 114 pF PBS. After the incubation, 2.4 pF of blocking solution was added to stop the reaction, which was further incubated at 2-8°C overnight. Before use, assembled tetramers were centrifuged at 2500xg for 5 minutes at 4°C and stored on ice in the dark. For tetramer staining, the OVA-SIINFEKL peptide- stimulated immune cells were washed with cold PBS and stained with a LIVE/DEAD fixable yellow dye for 25 minutes, followed by incubation with a 1/100 dilution of H2Kb-OVA tetramer in FACS buffer (200 pL/sample) at 4°C in the dark for 30 minutes. After washing, Fc receptors were blocked with a 1/200 Fc block in FACS buffer (50 pL/well) for 10 minutes. Cells were then spun down, the supernatant discarded, and stained with a mixture of antibodies targeting T cell surface markers (100 pL/well) for 30 minutes at 4°C in the dark. Following staining, cells were washed twice with 200 pL FACS buffer, fixed in 1% PFA for 20 minutes, washed twice, and acquired by FACS within 24 hours. Flow cytometric data acquisition was using a BD LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software.

Chemical and antibody treatment in vivo

Solvent (Sol) for chemicals is formulated as 10% Tween-80, 10% PEG400, and 4% DMSO in physiological saline. TG100801 was administrated i.p. at a dose of 5 mg/Kg, following the schedule specified in the figures and corresponding legends. In case of combination with checkpoint blockade, mice received i.p. injection of either 200 pg anti-PD-1 antibody, or 200 pg isotype antibody, at 8, 12 and 16 days after the first chemical treatments. For T cell depletion, mice received i.p. injections of 100 pg anti-CD8, or 200 pg of isotype antibody, one day before and the same day of pharmacological treatment or cell transfer, which were continued at a frequency of once a week for two weeks. For CD1 lb neutralization, mice received 100 pg anti- CD1 lb or equal amounts of isotype control antibody as scheduled for anti-CD8, but were treated every other day for the following 2 weeks. In some cases, mice were treated i.v. with 5 mg cytochrome C/mouse in PBS or PBS alone following the same schedule as anti-CDl lb.

Tissue dissociation and flowcytometry staining

Orthotopic TCI NSCLC cancers were established and tumor bearing mice were treated as described above. At day 3 after treatment, blood was collected from tumor bearing mice via cardiac puncture (under anesthesia with vaporized isoflurane) into 2 mL centrifuge tubes containing EDTA-K. Then, mice were euthanized for excising tumors and immune organs. The samples were collected in cold RPMI 1640 medium and kept on ice until dissociation. Blood was directly subject to erythrocyte elimination by using lx red blood cell lysing buffer (Biolegend); spleen and lymph nodes were mechanically dissociated and passed through 70 pm strainers (Coming) with the tip of a 1 mL syringe to generate single cell solutions; tumor-bearing lungs and excised s.c. tumors were digested in an enzymic buffer containing 1 mg/mL collagenase type IV (Life technologies) and DNase I (Sigma). The dissociated bulk cell suspension was resuspended in RPMI 1640, passed through 70 pm cell strainers and washed twice with cold PBS. Cells from spleen and lung were further treated with lx red blood cell lysis buffer to remove erythrocytes. Prior to surface staining of fluorescent antibodies, samples were incubated with LIVE/DEAD® Yellow Fixable dye to label damaged/dead cells, and incubated with antibodies against CD16/CD32 to block Fc receptors. For multiplex staining, cells were incubated with a panel of fluorescence-conjugated antibodies for 30 min surface staining in the dark. In the case of Foxp3 staining, the surface-labeled cells were permeabilized and fixed using a Foxp3/Transcription Factor Staining Buffer kit (Fife Technologies), and stained with the FOXP3 FITC antibody for another 30 min. Otherwise, surface-labeled cells were directly fixed with 4% PFA (Sigma). After 2 times wash, the cells were kept at 4 °C until flow cytometric analysis. Data were acquired on a BD ESRFortessa flow cytometer (BD Biosciences) and analyzed using the FlowJo software.

Statistical analysis

Statistical significance was calculated using the Graphpad Prism software (Version 9.0.2), by means of one-way or two-way ANOVA test (with FDR or Dunnett's multiple comparisons test), unpaired or paired Student t test, or Fisher’s exact test, as detailed in the corresponding figure legends. TumGrowth was used to analyze in vivo data (Enot, D.P. et al.) linear or log-transformed mixed-effects models for longitudinal comparison of tumor growth curves by type II ANOVA; cross-sectional analysis with Likelihood ratio test for comparing endpoint tumor size distribution; and Cox proportional hazards regression or logrank test for comparing survival curves. TumGrowth is freely available at Github/Kroemerlab. P values of 0.05 or less were considered to denote significance and were numerically annotated.

Results

A pharmacological drug screen identifies the tyrosine kinase inhibitor TG100801 as an enhancer of dendritic cell function

Recently, the inventors developed a dendritic cell genotype/phenotype screening platform that relies on the CRISPR/Cas9-mediated gene editing of conditionally immortalized immature dendritic cell (iniDCs) precursors, which are subsequently differentiated into immature DCs of the type I conventional DC (cDCl) subtype, which is particular relevant to cancer immunosurveillance (Zhao, L et al.). IniDC is a DC precursor cell line derived from the bone marrow of C57BL/6J mice engineered to express the SV40 large T cell antigen (SV40LgT) under the control of a modified tetracycline-regulated (Tet-on) system based on a reverse tetracycline transactivator (irtTA) fused to the ligand-binding domain (LBD) of a mutated glucocorticoid receptor (irtTA-GBD). The presence of doxycycline (DOX) and dexamethasone (DEX) induces the expression of SV40LgT, which simultaneously inactivates retinoblastoma (RB) and tumor protein 53 (TP53), hence rendering the cells immortal. The simultaneous removal of both DOX and DEX reverses the SV40LgT-mediated immortalization and allows for the differentiation of iniDCs into functional de-iniDCs (Richter, C et al.). Here, inventors treated de-iniDCs with the components contained in the Protein Tyrosine Kinase Compound Library (MedChemExpress) to assess the immunostimulatory potential of tyrosine kinase inhibitors (TKIs) targeting all druggable tyrosine kinase families (Fig. 1A). To this aim, TKI-treated de-iniDCs were pulsed with ovalbumin (OVA) protein and then co-cultured with B3Z T cell hybridoma expressing a T cell receptor (TCR) that recognizes the class I (Kb)-restricted peptide epitope of OVA (257-264, SIINFEKL). ELISA-quantifiable IL2 production was employed as a proxy for successful TCR engagement (Fig. IB). Enhanced antigen presentation efficacy measured as an increase in IL2 production by B3Z in response to the pre treatment of DCs with the agents from the Protein Tyrosine Kinase Compound Library was plotted against their p-value. Among the agents that induced high IL2 with great statistical significance inventors found 4-chloro-3-(5-methyl-3-{ [4- (2-pyrrolidin- 1 -ylethoxy)phenyl] amino } - 1 ,2,4-benzotriazin-7-yl)phenyl benzoate (TGI 00801 ) and 4-chloro-3-(5-methyl-3- { [4-(2-pyrrolidin- l-ylethoxy)phenyl] amino } - 1 ,2,4-benzotriazin-7- yl)phenol (TG100572) (Fig. 1C). TG100801 is a topically administered TKI that has reached the clinical testing phase in patients with macular degeneration, and TG100572 is the active metabolite that is produced by de-esterification of TG100801 when it penetrates the eye (Palanki, M.S. et al.). Topical TG100801 was shown to reduce retinal fluorescein leakage from the vasculature and to inhibit retinal edema measured by optical coherence tomography. Systemic delivery of the active metabolite TG100572 was reported to suppress laser-induced choroidal neovascularization in pre-clinical models. Furthermore, TG100572 was reported to induce cell death in proliferating endothelial cells in vitro, probably explaining the anti-neoangiogenic properties of the prodrug (Doukas, J et al.). Moreover, TG100801 and TG100572 have been shown to mediate broad spectrum inhibition of receptor tyrosine kinases at nanomolar level and of the Src kinase family proteins including FYN, LYN and LCK in the sub-nanomolar range (Doukas, J et al.).

TG100801 stimulates the differentiation of progenitor into cDCl

Next, the inventors validated the stimulatory effects of FYN inhibition on dendritic cells. To this aim, de-iniDCs, as well as primary BMDCs, were treated with TG100801 at different concentrations and then stimulated with tumor lysate. TG100801 induced the expression of activation markers such as CD80 and CD86 co-stimulatory receptors and that of MHC class II molecules in naive conditions, as well as to a certain extent in DCs that were stimulated with tumor lysate (Fig. 2A-C,E-G). Moreover, when added at low dose during differentiation of de- iniDCs, TG100801 stimulates the development of precursors into cells from the cDCl subpopulation, as indicated by an increase in the percentage of CD1O3⁺CD1 lb DCs (Fig. 2D, H). Using several distinct treatment protocols, the inventors observed that the immunostimulatory effects of TG100801 in co-culture assays with antigen-pulsed de-iniDC and B3Z T cells were only achieved when DCs were pre-exposed to the treatment and then washed before the addition of T cells. Immunostimulatory effects were entirely absent when TG100801 was added at a later timepoint to DC-B3Z co-cultures (Fig. 7A-D). Moreover, in additional co-culture assays, the phagocytosis of cancer cells by de-iniDC and BMDCs was increased by TG100801 in a dosedependent fashion (Fig. 7E). The treatment of B3Z T cells with high doses of TG100801 inhibited the production of both interferon gamma (IFNg) and IL2, suggesting an inhibitory effect on T cell engagement (Fig. 8). Altogether, these results indicate that TG100801 exerts immunostimulatory effects on DCs but immunosuppressive effects on T cells.

Inventors further observed that continuous treatment with low dose TG100801 during the differentiation of DCs from either iniDCs or primary bone marrow derived hematopoietic precursors increases their capacity to enhance antigen presentation and to stimulate cDCl differentiation together with the expression of CD80, CD86 and MHC class II (Fig. 3A-F). Furthermore, in de-iniDCs as well as primary BMDCs, the mRNA level of cDCl differentiationdriving transcription factors such as Batf3 Irf4 and Zfp366 exhibited a dose-dependent increase upon treatment with TGI 00801 at early stage of DC differentiation. Altogether, the aforementioned results showed that TG100801 stimulates DC activation and steers their differentiation into tumor reactive cDCl.

Systemic TG100801 treatment exerts cDCl-dependent tumor-regression and synergizes with immune checkpoint blockade

In the next step, the inventors treated TCI non-small lung cancer established orthotopically in immunocompetent C57B1/6J mice by two systemic injections of TG100801, a first time when tumors became detectable by in vivo imaging, and a second time two days later. One week after treatment onset blood, lung, tumor-draining lymph nodes (tdLNs), and spleens were collected for multiplex flowcytometry analysis, revealing that the percentage of cDCl cells was significantly increased in tdLN, blood and spleen, but decreased in the lung of TG100801 treated mice (Fig. 4A). DCs in tdLN, blood and spleen also showed an increase in the expression of CD80, CD86 and MHC class II, depending on tissue type (Fig. 4B-D). Moreover, TG100801 treated animals exhibited an increased abundance of CD8⁺ T cells and a decreased number of regulatory T cells (Treg) in many of the analysed tissues. This led to significant increase of the CD8⁺/Treg ratio in blood, lungs, tdLNs and spleens from TCI tumor-bearing animals (Fig. 4E-G). In mice with established subcutaneously MCA205 fibrosarcomas, injections of TG100801 caused an increase of the CD8⁺/Treg ratio in tdLNs and spleens, but not in non-tumor-draining lymph nodes (Fig. 4H). The percent of PD-1⁺ CD8⁺ cells was increased in the lungs of TCI tumor-bearing animals and TG100801 treatment led to further increase in this population in the lung and tdLN, whereas PD-1⁺ Tregs, despite being increased in tdLN, were reduced in the lung (Fig. 41, J). In conclusion, TG100801 alters the immune infiltrate by stimulating the activation and maturation of cDCl in tumor bearing mice. TG100801 also favourably impacts the CD8⁺/Treg ratio, while upregulating PD-1 on T lymphocytes.

Consistently, in TC-1 tumor-bearing mice systemic TG100801 reduced tumor growth, as monitored by in vivo imaging of intrathoracic luciferase-dependent chemoluminescence. TG100801 also increased overall survival of the animals. Such effects were entirely absent in immunodeficient nu/nu mice as well as in WT mice when either cytotoxic T cells (CTLs) were depleted with anti-CD8 monoclonal antibody (mAb) or the extravasation of myeloid cells was blocked by means of an anti-CDl lb mAb (Fig. 5A-C, Fig. 9A-D). Moreover, TG100801 sensitized TCI tumors to PD-1 blockade with a suitable (anti- PD-1) mAb. The combination of TG100801 and anti-PD-1 mAb reduced tumor growth below detection, de facto eradicating tumors in 80 % of the animals (Fig. 5A-C). The aforementioned results also suggest that Batf3- dependent cDCl cells are required for the anticancer effect of TG100801. To confirm this hypothesis, inventors depleted cDCl by means of the repeated intravenous injection of cytochrome c (Cyt c). Indeed, cDCl are the only cell type in the body that samples extracellular fluid, allowing it to reach their cytosol. For this reason, systemic Cyt c injection results in the specific depletion of cDCl cells, which activate the apoptosome and caspases, thus succumbing to apoptosis (Lin, M.L. et al.). However, Cyt c injection does not affect other DC subtypes or any other cell type (Zhao, L et al., 2023). As a result, Cyt c injections reduced the antitumor effects of TG100801 against orthotopic TCI NSCLC, thus reducing survival (Fig. 5D,F), underlining the importance of tumor reactive cDCl for TG100801-mediated immunogenicity. In sum, the aforementioned results support the contention that TG100108 stimulates cDCl-mediated immunity that can be further boosted by sequential PD-1 immune checkpoint inhibition. TG100801 targets the Fyn kinase immune checkpoint in DC to mediate anticancer immunity

Like many other kinase inhibitors, TG100801 has a rather broad target spectrum including several receptor tyrosine kinases and members of the Src kinase family. In order to identify the main pathway accounting for the immunostimulatory effect of TG100801-mediated inhibition, the inventors used CRSIPR/CAS9 mediated gene editing in iniDCs to generate transient knockouts of all kinases known to be affected by TG100801. The knockout of Fyn (and to a lesser extent that of Fgfrl Fgfr2, and Hck) mimicked the immunostimulatory effects of TG100801 in antigen presentation coculture assays, and TG100801 was not able to further improve the effect of Fyn depletion. This epistatic analysis indicates that genetic silencing of Fyn phenocopies the action of TG100801 (Fig. 6A). Stable knockout of Fyn yielding Fyn clones also caused an increase in OVA antigen presentation that could not be further enhanced by TG100801 (Fig. 6B). Next, inventors asked the question whether a combined inhibition of potentially functionally redundant kinases is necessary to trigger full-blown immunostimulation. To this aim, they compared sing Fyn knockout clones (genotype: Fyn ¹ ) with DCs that carry double knockouts affecting both Fyn and Fgfrl, Fgfr2 or Hck. The knockout of Fyn was sufficient to increase the immunogenicity of DCs similar to various doses (< 1 pM) of TG100801, and no added effect was observed in double knockouts (Fig. 6C).

Building on these findings, the inventors next assessed an array of chemically distinct FYN inhibitors, including pyrazolol[3,4-d] pyrimidines 1 (PPI) and 2 (PP2), ARN25068, SU6656, 1- NM-PP1, 1-Naphthyl PPI, TG 100572, TG 100801 and saracatinib. These compounds robustly enhanced antigen presentation in de-iniDCs in an MHC class Ldependent manner, reinforcing the conclusion that pharmacological FYN inhibition promotes antigen cross-presentation by DCs and highlighting the pathway-specific immunostimulatory potential of this approach (Fig. 11).

Consistent with these in vitro findings, the knockout of Fyn in dendritic cells (DCs) significantly enhanced antitumor immune responses when adoptively transferred into tumor-bearing mice. As compared to WT de-iniDCs, OVA pulsed Fyn^v and TG100801-treated WT de-iniDCs that were subcutaneously (s.c.) inoculated (Fig. 10) exhibited a similar increase in eliciting in vivo immune responses measured by quantifying the percentage of CD8 T cells carrying a H2b/SIINFEKL- specific T cell receptor (TCR), exhibiting a TCM phenotype, as well as producing IFNy in the draining lymph node (LN, Fig. 10). Using two independent sgRNAs targeting Fyn, adoptively transferred Fyn-deficient DCs improved tumor control in a syngeneic lung cancer model and exhibited a potent synergistic effect when combined with anti-PD-1 immune checkpoint blockade. Specifically, Fyn-deficient DCs sensitized TCI tumors to PD-1 blockade, leading to markedly improved therapeutic efficacy compared to either treatment alone. Notably, the combination of TG100801 and anti-PD-1 monoclonal antibody (mAh) resulted in near-complete tumor regression, achieving full tumor eradication in approximately 80% of treated animals. (Fig. 10).

Altogether, these results indicate that TG100801 mediates its anticancer effects primarily through the inhibition of Fyn in dendritic cells (DCs). Herein provided genetic and pharmacologic evidence converge to support the notion that Fyn functions as a negative regulator of antigen presentation and antitumor immunity in DCs. Its inhibition, either by TG100801 or by CRISPR/Cas9-mediated knockout, enhances the cross-presentation capacity of type I conventional dendritic cells (cDCls) and promotes their immunostimulatory phenotype. Furthermore, Fyn-deficient DCs confer potent therapeutic benefit upon adoptive transfer in tumor-bearing hosts, and synergize with PD-1 blockade to promote tumor regression and, in a substantial number of cases, immune-mediated tumor clearance.

A Fyn-targeting monoclonal antibody recapitulates and refines the immunostimulatory effect of small-molecule inhibition on DCs.

To confirm or further validate Fyn as a therapeutic target and to explore its translational potential, the inventors employed a monoclonal antibody (mAh) specifically targeting Fyn (concretely targeting amino acids localized at positions 85-206 (i.e., SEQ ID NO: 65) of human Fyn sequence of SEQ ID NO: 5, mapping to 6q21). The Fyn mAh was efficiently internalized by de-iniDCs within 12 hours of exposure and promoted antigen presentation to a degree comparable to TG100801 treatment (Fig. 12). Moreover, in primary bone marrow-derived dendritic cells (BMDCs), the Fyn mAh enhanced DC activation and maturation, leading to an increased frequency of conventional type 1 DCs (cDCls) and migratory cDCls (CCR7⁺XCR1⁺), phenocopying the immunostimulatory effects of TG100801 (Fig. 13). The Fyn mAh did not impair T cell activation markers such as CD69 and CD25, suggesting a selective uptake by DCs (Fig. 14). Fyn mAh may be advantageously used in combination with immunotherapy regimens. Transcriptomic profiling further revealed that both genetic deletion and pharmacologic inhibition of Fyn, via small molecule or mAh, induced overlapping gene expression programs in DCs, enriched in pathways related to leukocyte migration, chemotaxis, adhesion, and immune navigation (Fig. 15,16). These findings position Fyn as a previously unrecognized immune checkpoint in dendritic cells, the inhibition of which unleashes robust antitumor T cell responses by promoting the differentiation and activation of tumor-reactive cDCls. Together, these results establish Fyn as a critical regulator of dendritic cell function and an advantageous viable therapeutic target in cancer immunotherapy. By leveraging both genetic and pharmacologic approaches, the invention demonstrates that Fyn inhibition enhances antigen presentation, drives cDCl differentiation and migration, and sensitizes tumors to immune checkpoint blockade. The convergence of effects seen with small-molecule inhibitors as well as with anti-Fyn monoclonal antibody highlights the robustness and specificity of this pathway. In the context of the present invention, both small molecules and the antibodies used as FYN kinase inhibitors may be advantageously used in combination with an immunotherapy, anti-FYN antibodies being a preferred option in particular because they do not exert direct inhibitory effects on T cells.

Discussion

Most targeted agents in oncology have been developed to selectively induce cell death in malignant cells without causing adverse damage to healthy tissue. Nevertheless, this cancercentric view needs to be updated due to the fact that clinical effects of treatments that persist beyond therapy duration requires the stimulation of adaptive anticancer immunity (Petroni, G et al.). A number of cytotoxicants including anthracy clines, oxaliplatin and taxanes as well as targeted agents such as crizotinib and ceritinib induce immunogenic cell death (ICD) of malignant cells (Kroemer, G et al , Petrazzuolo, A et al.). ICD-emitted danger associated molecule patterns (DAMPs) are perceived by DCs that are driven to present tumor-associated antigens to T cells recruited into the tumor microenvironment. The inventors’ present work shows that FYN inhibition induces therapy-relevant stimulatory effects on DC-mediated tumor antigen presentation, thus sensitizing T cells for any possible subsequent ICI.

Here, the inventors used a miniature immune system assay to screen a chemical library of tyrosine kinase inhibitors and discovered in particular TG100801 and its active metabolite TG100572 as DC antigen presentation enhancers. Systemic treatment with TG100801 was able to reduce the growth of orthotopic lung cancers in mice, an effect that can be further boosted by subsequent PD-1 blockade. The antineoplastic effect of TG100801was dependent on the immune system as it was not observed in animals from which T cells were removed or in which the extravasation of myeloid cells was blocked. Mechanistically, TG100801 induced the activation of tumor reactive cDCls without any direct stimulatory effects on T cells. Consistently, the specific depletion of cDCl cells abolished the therapeutic action of TG100801 monotherapy as well as that of the combination of TG100801 with PD-1 blockade. Genetic inhibition of Fyn phenocopied the immunostimulatory effects of TG100801 on cDCl, and addition of the drug did not further increase antigen presentation by Fyn ¹ DCs, in support of the idea that TG100801 acts on Fyn to stimulate DC function.

The inventors’ observation of immunostimulatory effects of FYN inhibition in DCs contrast with prior reports indicating that FYN inhibition interferes with T cell development, activation and function (including the therapeutic performance of CAR T cells (Wu, L et al. Quin, Z et al. Groves, T et al.). Thus FYN has opposing effects on antigen presentation by DC and TCR engagement by T cells. However, in vivo, FYN inhibition by TG100801 could increase the T celldependent control of cancer. This might be explained by the rapid clearance of the active TG100801 metabolite, explaining why its topical ocular administration must be performed a minimum of three times per day to locally maintain effective drug levels, though does not reach detectable levels in the circulation (Doukas, J et al.). In the present study, TG100801 was employed for a short duration, and T cell function was further boosted by PD- 1 ICI one week later, when the drug TG100801 and its active metabolite should have been cleared and hence should not interfere any more with T cell function. The inventors were able to model this effect in vitro. Treatment of DC with TG100801, preferably followed by its washout before addition of T cells, led to enhanced activation of the DC-T dialogue, resulting in improved T cell function (Fig. 7). As shown here, initial FYN inhibition, followed by later PD- 1 blockade, mediated an advantageous effective tumor control.

Advantageous results obtained with TG100801 were reproduced with the following additional small molecules: PPI, PP2, ARN25068, SU6656, 1-NM-PP1, 1-Naphthyl PPI, and Saracatinib. In addition to small molecule inhibition, the inventors employed a monoclonal antibody (mAh) specifically targeting FYN and demonstrated that it also recapitulates the effects of TG100801 on dendritic cells. The FYN mAh efficiently entered de-iniDCs, enhanced antigen presentation, and promoted DC maturation. In bone marrow-derived DCs (BMDCs), the antibody increased the frequency of conventional type 1 dendritic cells (cDCl) and migratory cDCls (CCR7⁺XCR1⁺), closely mimicking the phenotype induced by TG100801.

The inventors conclude that hydrophilic FYN inhibitors (preferably those that are unable to cross cellular membranes), including FYN mAbs, are preferred inhibitors that can be administered in a way that only cDCl would be affected by the drugs, knowing that cDCl constitute the sole cell type that samples the extracellular fluid allowing to access external solutes to access their cytosol. Such hydrophilic FYN inhibitors can be selected from small molecules, peptides, peptidomimetics, nanobodies or antibodies. REFERENCES

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Claims

1. A FYN kinase inhibitor for use in stimulating the immune response in a subject suffering of a disease, by stimulating dendritic cells (DC) maturation and/or activation and/or by increasing antigen presentation by DC, wherein the disease is a cancer or an infection.

2. A FYN kinase inhibitor stimulating DC activation and/or increasing antigen presentation by DC, for use in prevention or treatment of a cancer or an infection in a subject in need thereof.

3. An in vitro or ex vivo use of a FYN kinase inhibitor to stimulate DC activation, increase antigen presentation by DC, and/or facilitate DC progenitor differentiation into an antigen reactive DC.

4. The FYN kinase inhibitor for use according to claim 1 or 2, or the in vitro or ex vivo use of claim 3, wherein the FYN kinase inhibitor stimulating dendritic cells (DC) maturation and/or activation and/or increasing antigen presentation by DC is selected from a nucleic acid molecule; a peptide; a small molecule; an antibody, a derivative or any functional fragment thereof inhibiting the functional expression of the Fyn gene of SEQ ID NO: 2, 3 or 4 or of the FYN protein of SEQ ID NO: 5, 6 or 7; and an aptamer; and is preferably a hydrophilic inhibitor.

5. The FYN kinase inhibitor for use, or in vitro or ex vivo use, according to claim 4, wherein the FYN kinase inhibitor small molecule is selected from 4-chloro-3-[5-methyl-3-[4-(2- pyrrolidin-l-ylethoxy)anilino]-l,2,4-benzotriazin-7-yl]phenyl] benzoate (TG100801), 4-chloro- 3-[5-methyl-3-[4-(2-pyrrolidin-l-ylethoxy)anilino]-l,2,4-benzotriazin-7-yl]phenol (TG100572), pyrazolol[3,4-d] pyrimidine 1 (PPI, CAS No 172889-26-8), pyrazolol[3,4-d] pyrimidine 2 (PP2, CAS No 172889-27-9), ARN25068 (CAS No 2649882-80-2), SU6656 (CAS No 330161-87-0), 1-NM-PP1 (CAS No 221244-14-0), 1-Naphthyl PPI (CAS No 221243-82-9), Saracatinib (CAS No 379231-04-6), and any functional derivative thereof inhibiting the functional expression of the Fyn gene of SEQ ID NO: 2, 3 or 4 or of the FYN protein of SEQ ID NO: 5, 6 or 7.

6. The FYN kinase inhibitor for use, or use, according to claim 4, wherein the FYN kinase inhibitor is a FYN kinase antibody or a functional derivative thereof inhibiting the functional expression of the FYN protein of SEQ ID NO: 5, 6 or 7.

7. A composition comprising i) a FYN kinase inhibitor as described in claim 5 or 6, a population of DC activated by a FYN kinase inhibitor as described in any one of claims 4 to 6, or a population of Fyn DC, and ii) a pharmaceutically acceptable support.

8. A composition according to claim 7 for use for the prevention or treatment of a cancer or of an infection, in a subject in need thereof.

9. A combination of i) a product selected from a FYN kinase inhibitor as described in claim 4 or 5, a population of DC activated by a FYN kinase inhibitor as described in any one of claims 4 to 6, and a population of Fyn ⁷ DC, and ii) T-cell immune check-point inhibitor(s).

10. The combination of claim 9, wherein the T-cell immune check-point inhibitor(s) is selected from an anti-PD-1 agent, anti-PD-Ll agent, anti-CTLA-4 agent, anti-TIGIT agent, anti-TIM-3 agent, anti-LAG-3 agent, anti- VISTA agent, an agonist of 0X40, CD40, CD137, STING or Tolllike receptor, a type-1 interferon or an interleukin, and any mixture thereof, and if the T-cell immune check-point inhibitor is an anti-PDl agent or an anti-PDLl agent, the anti-PDl agent is an anti-PDl antibody selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab and toripalimab, or an anti-PDLl antibody selected from atezolizumab, avelumab and durvalumab.

11. A combination according to claim 9 or 10 for use as a medicament.

12. The combination according to claim 11 for use for the prevention or treatment of a cancer or of an infection.

13. The combination according to claim 11 or 12, wherein the product i) and the T-cell immune check-point inhibitor(s) ii) are formulated for separate administration to a subject in need thereof.

14. The combination according to claim 13, wherein the product i) is administered to the subject in need thereof before the T-cell immune check-point inhibitor(s).

15. The combination for use according to anyone of claims 12 to 14, wherein the product i) is a FYN kinase inhibitor as described in any one of claims 4 to 6 and is formulated for systemic administration, preferably intravenous (I.V.) administration, or is a population of DC activated by a FYN kinase inhibitor as described in any one of claims 4 to 6, or a population of Fyn ⁷ DC, and is formulated for intra tumoral (LT) administration.

16. The FYN kinase inhibitor for use according to claim 1, 2, 4, 5 or 6, a composition for use according to claim 8, or a combination for use according to any one of claims 12 to 15, wherein the cancer is selected from a sarcoma, a carcinoma, a blastoma, a lymphoma, a myeloma and a leukemia, for example from a lung cancer, a breast cancer and a colorectal cancer, and is preferably a lung cancer, in particular a non-small cell lung cancer (NSCLC).

17. A kit comprising i) a compound selected from a FYN kinase inhibitor as described in any one of claims 4 to 6, a DC or population of DC activated by a FYN kinase inhibitor, a Fyn-¹ DC and a population of Fyn ⁷ DC, and ii) T-cell immune check-point inhibitor(s), in particular an anti- PD1 and/or anti-PDLl agent(s), in different containers.