WO2017212021A1 - Methods and pharmaceutical compositions for the treatment of cancer - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of cancer. Immunoreceptors play a crucial role in the regulation of immune homeostasis and inflammation. The inventors investigated how these receptors translate outside ligand interactions into opposite signals. The Src-family kinase Fyn was the crucial effector for inhibition of SHP-1, while Lyn/Lck were required for its activation downstream of Fc, B-cell and T-cell antigen receptors. The inventors demonstrated the distinct roles of antigen receptor-associated Src family kinases in regulating homeostatic and inflammatory conditions and thus offer means for modulating the immune system for promoting anti-cancer fighting. In particular, the present invention relates to a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Lyn/Lck inhibitor.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT

OF CANCER

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of cancer.

BACKGROUND OF THE INVENTION:

The ability of the immune system to detect and eliminate cancer was first proposed over

100 years ago. Since then, T cells reactive against tumor-associated antigens have been detected in the blood of patients with many different types of cancers, suggesting a role for the immune system in fighting cancer. Innate and adaptive immunity maintains effector cells such as lymphocytes and natural killer cells that distinguish normal cells from "modified" cells as in the case of tumor cells. To maintain homeostasis the immune system is controlled by a finely tuned network of regulatory mechanism (1). Among these, certains immunoreceptors have been shown to exert inhibitory and activating signal through ITAM motif (Yxx[L/I]x6-sYxx[L/I]) (2) depending on the valency of their respective ligand. Low valency interactions were shown to induce anergy and an inhibitory crosstalk with heterologous receptors thereby reducing the susceptibility to autoimmune and inflammatory diseases (3-10). By contrast, high valency ligand interactions promote an activating signal launching inflammatory and immune cascades to fight the inflammatory insult and restore homeostasis, but in case of ill-regulation or chronic stimulation can also result in autoimmune and inflammatory diseases (1, 10). The ITAM motif is found in the cytoplasmic domain of several transmembrane adapter molecules, such as the common γ subunit of FcR (FcRy), the Iga and Ig subunits of the BCR, and the γ, δ, ε and ζ subunits of the TCR-associated CD3 complex (1, 2), and in the cytoplasmic tail of other receptors, such as the FcyRIIA (11). Src-family kinases (SFKs) phosphorylate the ITAM motifs upon stimulus-induced receptor clustering leading to downstream effector recruitments and cell activation. However, how ITAM-bearing immunoreceptors translate ligand valency into opposite signals remains elusive. It also remains unclear why these receptors are associated with several SFKs, notably Lyn and Fyn (12-17).

SUMMARY OF THE INVENTION: The present invention relates to methods and pharmaceutical compositions for the treatment cancer. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Immunoreceptors play a crucial role in the regulation of immune homeostasis and inflammation. Depending on ligand valency they can promote either activating or inhibitory signals. The inventors investigated how these receptors translate outside ligand interactions into opposite signals. The Src-family kinase Fyn was the crucial effector for inhibition of SHP-1, while Lyn/Lck were required for its activation downstream of Fc, B-cell and T-cell antigen receptors. Mechanistically, a Fyn-PI3K-PKC axis turns off Lyn-mediated SHP-1 activation by shifting tyrosine into serine phosphorylation. These findings demonstrate the distinct roles of antigen receptor-associated Src family kinases in regulating homeostatic and inflammatory conditions and thus offer means for modulating the immune system for promoting anti-cancer fighting.

Accordingly, the first object of the present invention relates to a method of treating an autoimmune inflammatory disease in a subject in thereof comprising administering to the subject a therapeutically effective amount of a Lyn/Lck inhibitor. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In some embodiments, the subject suffers from a cancer selected from the group consisting of Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal- like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchiolo alveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythro leukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangio sarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant, Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medullo epithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplasia Disease, Myelodysplasia, Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, non-small cell lung cancer (NSCLC) which coexists with chronic obstructive pulmonary disease (COPD), Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath, meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastema, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial- stromal tumor, Synovial sarcoma, T-cell acute, lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T- cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Vemer Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

As used herein, the term "Lyn" has its general meaning in the art and refers to the LYN proto-oncogene, Src family tyrosine kinase encoded by the LYN gene (Gene ID: 4067) and is also known as JTK8; p53Lyn; p56Lyn. An exemplary human nucleic acid sequence for Lyn is accessibled in GenBank under the access number NM 001111097.2 (isoform B or NM 001111097.2 (isoform A). An exemplary human amino acid sequence for Lyn is accessible in GenBank under the accessible number NP 001104567.1 (isoform B), or NP 002341.1 (isoform A). Accordingly a "Lyn inhibitor" refers to any compound that is capable of inhibiting the activity or expression of Lyn. As used herein, the term "Lyn activity" includes any biological activity mediated by Lyn such as described in the EXAMPLE. In particular, the Lyn inhibitor of the present invention is particular suitable for abrogating kinase activity. Examples of Lyn inhibitors include but are not limited to polypeptides such as dominant- negative protein mutants, peptidomimetics, antibodies, ribozymes, antisense oligonucleotides, or other small molecules which specifically inhibit the activity or expression ofLyn.

As used herein the term "Lck" has its general meaning in the art and refers to the LCK proto-oncogene, Src family tyrosine kinase encoded by the LCK gene (Gene ID: 3932) and I s also known as LSK; YT16; IMD22; p561ck; pp581ck. An exemplary nucleic acid sequence for Lck is accessible in GenBank under the access number NM 001042771.2 or NM 005356.4. An exemplary amino acid sequence for Lck is accessible in GenBank under the access number NP 001036236.1 or NP 005347.3. Accordingly a "Lck inhibitor" refers to any compound that is capable of inhibiting the activity or expression of Lck. As used herein, the term "Lck activity" includes any biological activity mediated by Lck such as described in the EXAMPLE. In particular, the Lck inhibitor of the present invention is particular suitable for abrogating kinase activity. Examples of Lck inhibitors include but are not limited to polypeptides such as dominant- negative protein mutants, peptidomimetics, antibodies, ribozymes, antisense oligonucleotides, or other small molecules which specifically inhibit the activity or expression of Lck. In particular, the Lyn/Lck inhibitor is particularly suitable for promoting activation downstream of Fc, B-cell and T-cell antigen receptors and thus enhancing the immune system.

In some embodiments, the Lyn/Lck inhibitor is a small organic molecule.

In some embodiments, the inhibitor is a short hairpin RNA (shRNA), a small interfering

RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of Lyn/Lck. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

In some embodiments, the inhibitor is an intrabody having specificity for Lyn/Lck. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In another embodiment, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb. By a "therapeutically effective amount" of the inhibitor as above described is meant a sufficient amount to provide a therapeutic effect. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

According to the invention, the inhibitor is administered to the subject in the form of a pharmaceutical composition. Typically, the inhibitor may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The inhibitor can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In some embodiments, the Lyn/Lck inhibitor is administered in combination with an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. In particular, the immune checkpoint inhibitor of the present invention is administered for enhancing the proliferation, migration, persistence and/or cytoxic activity of CD8+ T cells in the subject and in particular the tumor- infiltrating of CD8+ T cells of the subject. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. As used herein the term "PD-1 inhibitor" as used herein refers to a compound, substance or composition that can inhibit the function of PD-1. For example, the inhibitor can inhibit the expression or activity of PD-1, modulate or block the PD-1 signaling pathway and/or block the binding of PD-1 to PD-L1 or PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.

A further object of the present invention relates to a method of screening a drug suitable for the treatment of cancer comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of Lyn/Lck. Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity or expression of Lyn/Lck. In some embodiments, the assay fist comprises determining the ability of the test compound to bind to Lyn/Lck. In some embodiments, a population of immune cells (mastocytes, monocytes, B cells, T cells...) is then contacted and activated so as to determine the ability of the test compound to inhibit the activation downstream of Fc, B-cell and T-cell antigen receptors. In some embodiments, the SHP-1 activation is determined and in particular the identification of serine into tyrosine phosphorylation. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. Assays for determining the test compound to dampen the immune signal response are well known in the art and are typically described in the EXAMPLE. It is to be understood that test compounds capable of inhibiting immune response, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intraantibodies), aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules. As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.

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.

EXAMPLE:

Material & Methods Study subjects.

Fourteen individuals (eight HI and six LN patients) were studied. The LN group was composed of 6 patients attending or referred to the Bichat's Hospital specialist nephrology unit between July 2014 and January 2016 meeting at least four ACR systemic lupus erythematosus criteria (32) presenting with active disease with nephritis proven by kidney biopsy (2 at class IV and 4 at class V) and in whom peripheral blood by venepuncture was obtained immediately prior to immunosuppressive therapy administration. All patients were female with age varying between 25 and 42. Ethical approval for this study was obtained from the Bichat Hospital Local Research. Ethics Committee and informed consent was obtained from all subjects enrolled.

Mice

C57BL/6 hFcyRIIA^Tg mice expressing the WT human FcyRIIA on CD 1 lb-positive cells were from Jackson Laboratory (JAX, Bar Harbor, ME, USA). Fyn ' hFcyRIIA¹⁸, Lyn^_/" hFcyRIIA^Tg were obtained by the intercross of hFcyRIIA^Tg mice with mice knockout for Fyn (JAX) or for Lyn (previously described in (15)). All mice carrying the hFcyRIIA transgene were used as heterozygous animals. Mice were bred and maintained at the mouse facilities of the Bichat Medical School campus. All experiments were performed in accordance with the French Council of Animal Care guidelines and national ethical guidelines of INSERM Animal Care Committee (Animal Use Protocol number 75-1596).

Nephrotoxic nephritis (NTN) mouse model.

NTN was induced by i.p. injection (200 μ1/20 g body weight) of rabbit anti-mouse glomerular basement membrane (GBM) in C57BL/6 hFcyRIIA^Tg, Lyn^_/", Fyn^_/", Fyn^_/" hFcyRIIA^Tg, Lyn ' hFcyRIIA^ mice (7 to 9 wk old). Briefly, mice were preimmunized i.p. with normal rabbit IgG (0.5 mg/20 g body weight) in CFA 5 days prior to i.p. administration of NTN serum. Blood samples were collected and animals were sacrificed at day 7 following NTN injection. Renal function parameters (urinary proteins and BUN), histological and immunohistological parameters were studied.

Collagen Antibody-Induced Arthritis (CAIA) model.

Arthritis was induced as described (9, 30) using the Arthrogen-CIA® Arthritogenic Monoclonal Antibody kit (Chondrex, Inc.). Mice were injected i.v. with anti-CII Ab cocktail (Day 0) followed by LPS (i.p.) 3 days later. Animals were injected i.p. with 10 mg/20 g body weight of 500 μg serum human IgA (purchased from Biomedicals)/20 g body weight or 100 μg AT-10 F(ab)'₂ or irrelevant mAb F(ab')₂ (clone 320) for 10 days at 2-day intervals. The first dose was administered 2 days prior to anti-CII Ab cocktail injection. Paw thickness was measured with a pocket thickness gauge. On day 10, animals were sacrificed and hind paws and knees were fixed in formalin or snap-frozen.

Cells and reagents.

Human blood samples (12 ml) were first submitted to red cell lysis and pellets of 10⁷ leukocytes were subjected RIPA buffer treatment (see below). BMM from 6- to 8-week-old mice were obtained after a 7-day culture with M-CSF (R&D systems). THP-1 and THP1- hFcyRIIA-R131⁺-CD14⁺ cell lines (kindly provided by Novimmune) (33) were maintained in RPMI-1640, 10% FCS and 50 μΜ β-mercaptoethanol or supplemented with 200 μg/ml Zeocin, 10 μg/ml blasticidin and 2 μg/ml puromycin (Invitrogen, France). Jurkat and Ramos human cell lines were maintained in RPMI-1640 supplemented with 10% FCS and antibiotics. FCS was removed from the culture medium immediately before stimulation as described (34). Mouse mAb anti-hFcyRII (clone AT- 10), anti-hCD3 (clone HIT-3a) or anti-hCD79a (clone ZL7-4) were purchased from Santa Cruz and used in their F(ab')₂ fragment forms. Mouse mAb anti-hFcaRI (clone A77) and irrelevant control mAb (320) were purified in-house and were used as F(ab')₂, as previously described (3, 9). For biochemical studies, rabbit anti-Syk, anti- Zap70, anti-SHP-1 , anti-Lyn, anti-Lck, anti-Fyn, anti-ER (all from Santa Cruz Biotechnology), anti-SHPl (phospho-Y536) (ECM Biosciences), and anti-SHPl (phospho- S591) (Abeam) were used. Anti-pERK, anti-pAKT, anti-pPKCa, anti-AKT and anti-PKCa were from Cell Signaling.

Cell stimulation.

For ITAMi signaling, 5xl0⁶ of monocytic cell lines (THP-1 -CD 14⁺-FcyRIIA⁺ or THP- 1-CD14⁺), Jurkat T cell and Ramos B cell lines (transfected with different siRNA) were incubated for 30 min with 10 μg/mL of anti-hFcyRII (clone AT- 10), anti-hCD3 (clone HIT-3a) or anti-hCD79a (clone ZL7-4) Abs F(ab')₂ fragments at 37°C, respectively. Cells were then incubated with or without LPS (10 ng/ml) and Flagellin (5 μg/ml) as described (35) or Pam3csk4 (1 μg/ml) for 1 hour for monocytic cell lines and 18 hours for Jurkat and Ramos. For ITAM signaling, cells were incubated with 10 μg/ml of anti-hFcyRII (clone AT- 10), anti-hCD3 (clone HIT-3a) or anti-hCD79a (clone ZL7-4) Abs F(ab')₂ at 4°C followed by an anti-κ light chain F(ab')₂ at 37° C for 18 h for cytokine measurement or 6 hours for intracellular IL-2 staining.

Analysis of intracellular cytokine staining.

After washing, Jurkat cells were incubated with or without anti-CD3 F(ab')₂ fragment or with preformed complexes of anti-CD3 F(ab')₂ plus anti-kappa F(ab')₂ fragments. Cells were then stimulated or not with flagellin (1 μg) for 6 hours. PMA (40 nM) and ionomycin (InM) were used as positive stimuli for 6 hours. Brefeldin A was added after 2 hours stimulation and maintained for 4 hours. The stimulation was stopped by adding 1 ml cold PBS. Intracellular cytokine staining was performed on fixed/permeabilized cells in residual permeabilization wash buffer (Biolegend, USA) using a conjugated antibody (anti-IL-2 PE or appropriate isotype control) for 20 min in the dark at room temperature as described (36). Data acquisition was performed using a BD Biosciences LSR Fortessa cytometer, and results were analyzed using Flow Jo analysis software (Tree Star).

Immunoprecipitation and immunoblotting.

Cells (5 x 10⁶ to 10⁷) were solubilized in RIPA lysis buffer containing 1% Nonidet P- 40/0.1% sodium dodecyl sulfate (SDS) as described⁸. For immunoprecipitation, cell lysates were incubated with 2 μg/ml of AT- 10 anti-FcyRIIA, A77 anti-FcaRI, HIT-3a anti-CD3 or ZL7-4 anti-CD79a mAbs and immunoprecipitated overnight at 4°C with Protein G-Sepharose (GE Healthcare). Samples were resolved by SDS polyacrylamide gel electrophoresis (10%), transferred to nitrocellulose membranes and immunob lotted with rabbit antibodies followed by goat anti-rabbit IgG (GE Healthcare) coupled to horseradish peroxidase. Membranes were developed by enhanced chemical luminescence treatment (Amersham Biosciences).

Enzyme-linked immunosorbent assay (ELISA).

IL-8 and IL-2 were measured in the supernatants of stimulated cells using ELISA kits (R&D Systems) according to the manufacturer's instructions.

Histological analysis.

For the kidney, paraffin-embedded sections 4 μιη in thickness were stained with PAS for morphological analysis. For immunohistochemistry, frozen kidney sections were incubated with biotinylated antibodies against rabbit IgG or mAb anti-mouse CD l ib, -mouse F4/80, - mouse CD3, and -mouse Ly6G (Becton Dickinson) for 1 hour at room temperature. When necessary, the primary antibody incubation was followed by incubation with anti-rabbit IgG or anti-goat IgG (Southern Biotech Associates). Slides were mounted with the Eukitt mounting medium (Electron Microscopy Sciences) and read with an upright microscope (DM2000; Leica) using the IM50 software (Leica). For colocalization experiments, frozen kidney sections were incubated overnight successively with each antibody (anti-phospho Syk^Y525"526 AF488 (Cell Signaling) and anti-phalloidin AF568 (Life Technologies) followed by incubation with streptavidin-Alexa Fluor 488 (Life Technologies) at room temperature. Tissue sections were mounted with Vectashield (Vector Laboratories). Slides were read with a laser-scanning confocal microscope (LSM 510; Carl Zeiss) at 630 magnification using the LSM Image Browser (Carl Zeiss). For formalin- fixed hind paws and mouse knees, the samples were decalcified and embedded in paraffin. Sagittal sections were stained with haematoxylin and eosin. Histological score of peri-articular exudates, synovitis and tissue loss were graded as 0 (normal), 3 (mild), 6 (moderate), or 9 (severe). The cumulative score of all features was used as histological score to represent overall disease severity. Sections were incubated with rabbit primary antibodies against mouse phospho (p)-Syk (Y525-526)(37), mouse p-SHPl (S591) (both from Abeam) or p-SHPl (Y536) (Assay Biotech). The presence of antibodies was detected with biotinylated anti-rabbit antibody (Southern Biotech) followed by peroxidase streptavidin and diaminobenzidine successive stains. Positive cells were quantified in hind paw sections with analysis software CaloPix (TRIBVN, Chatillon, France) after scanning with Aperio ScanScope CS (Leica Biosystems, Nanterre, France).

Gene expression and Real-time PCR.

RNA purification from ho mogen i zed_k i d ney s was performed by using RNAble (Eurobio). cDNA was obtained by reverse transcription using using Moloney murine leukaemia virus (Invitrogen). Samples were analyzed by real-time PCR with Taq Man® Gene Expression Master Mix (Applied Biosystem). Primers were purchased from Euro fins (Supplementary Table 1). Gene quantification was performed using a Chrom o4 Real-Time PCR Detection System (Bio-Rad Laboratories). Data were normalized to β-actin values.

siRNA transfections.

Experiments were performed using predesigned HP GenomeWide (Qiagen, Courtaboeuf, France) Single strand sense and antisense RNA nucleotides were annealed to generate an RNA duplex according to the manufacturer's instructions. Cells were incubated with 5 to 10 nM of each siRNA tested and 2 μΐ of Lipofectamine® RNAiMAX prepared according to the manufacturer's instructions (Invitrogen, Saint Aubin, France) for 48 or 72 hours at 37°C before use.

Primers and probe sequences for RT-qPCR.

Gene, accession number, sequence and location of primers and probes. F indicates a forward primer, R indicates a reverse primer, and P indicates a FAM-TAMRA probe.

Statistical analysis.

Statistical analyses were performed using GraphPad Prism software http://www.graphpad.com/scientific-software/prism/. All data were expressed as mean ± SEM. Statistical significance between two groups was examined by the Student's t-test or the Mann- Whitney test, while the one-way and two-way analysis of variance (ANOVA) with Bonferroni's, Holm-Sidak's or Newman-Keuls multiple comparisons test were used to evaluate multiple groups. -values of 0.05 were considered significant; values less than 0.05 are indicated in the figure legends.

Results and discussion:

To maintain homeostasis the immune system is controlled by a finely tuned network of regulatory mechanism (1). Among these, certains immunoreceptors have been shown to exert inhibitory and activating signal through ITAM motif (Yxx[L/I]x6-sYxx[L/I]) (2) depending on the valency of their respective ligand. Low valency interactions were shown to induce anergy and an inhibitory crosstalk with heterologous receptors thereby reducing the susceptibility to autoimmune and inflammatory diseases (3-10). By contrast, high valency ligand interactions promote an activating signal launching inflammatory and immune cascades to fight the inflammatory insult and restore homeostasis, but in case of ill-regulation or chronic stimulation can also result in autoimmune and inflammatory diseases (1, 10). The ITAM motif is found in the cytoplasmic domain of several transmembrane adapter molecules, such as the common γ subunit of FcR (FcRy), the Iga and Ig subunits of the BCR, and the γ, δ, ε and ζ subunits of the TCR-associated CD3 complex (1, 2), and in the cytoplasmic tail of other receptors, such as the FcyRIIA (11). Src-family kinases (SFKs) phosphorylate the ITAM motifs upon stimulus- induced receptor clustering leading to downstream effector recruitments and cell activation. However, how ITAM-bearing immunoreceptors translate ligand valency into opposite signals remains elusive. It also remains unclear why these receptors are associated with several SFKs, notably Lyn and Fyn (12-17). In order to address whether SFKs play a role in the induction of ITAMi or ITAM signaling, we downregulated Fyn or Lyn expression in representative human monocytic cell lines. These cells were either stimulated for ITAMi induction by divalent targeting, or for ITAM by multivalent crosslinking of FcyRIIA or FcaRI. ITAMi molecular signature was characterized by transient Syk recruitment followed by stable and prolonged SHP-1 recruitment. This signature required the presence of Lyn (but not Fyn), which were recruited to their respective immunoreceptors. In contrast, multivalent crosslinking of these receptors induced an ITAM molecular signature with a stable recruitment of Syk family kinases (but not SHP1), requiring the presence of recruited Fyn. Fyn silencing had no effect on ITAMi signaling but reversed the ITAM to an ITAMi signature, whereas Lyn silencing completely abolished ITAMi signaling without affecting ITAM. Functional consequences were then evaluated. Lyn but not Fyn is essential for the ITAMi inhibitory signals observed in FcR- mediated inhibition of LPS-mediated IL-8 production. By contrast, Fyn but not Lyn is essential for ITAM-mediated cell activation as measured by IL-8 production after multivalent engagement of FcyRIIA or FcaRI. To investigate whether other ITAM-bearing receptors could also deliver such opposite signals, divalent or multivalent targeting of TCR and BCR were performed using anti-CD3 or anti-CD79a F(ab')₂ fragments or complexed with anti-κ light chains in representative lymphocytic cell lines. Similarly to FcRs, while divalent targeting of TCR or BCR resulted in ITAMi signatures, multivalent crosslinking led to expected ITAM signature. As for FcR, Lck or Lyn was required for TCR- or BCR-mediated ITAMi signals, whereas Fyn was essential for ITAM configuration. Moreover, TCR-Fyn dissociation led to SHP-1 recruitment suggesting that Fyn could inhibit SHP-1 recruitment. We next evaluated the effect of such opposite signals on cytokine production. Whereas, TCR- and BCR-divalent targeting resulted in inhibition of IL-2 and IL-8 secretion induced by flagellin or Pam3csk4 which were dependent on Lck or Lyn, respectively, Fyn was required for these cytokine production induced upon TCR and BCR multivalent engagement. These results were confirmed by intracellular IL-2 detection on the Jurkat T cell line. Altogether, these data indicated that during ITAM signaling, Fyn would constrain the inhibitory signal. Strikingly, these results also extend to TCR and BCR that ITAM-bearing immunoreceptors undergo crosstalk with other receptors, thus dampening inflammatory responses through ITAMi signaling generated by low intensity signals (18). In addition, these results reveal that this effect is strictly regulated through the balance between non-redundant SFKs. These results mechanistically explain previous findings using weakly binding ligands or antibodies that trigger negative signals by TCR and BCR (19-23).

To address the mechanism by which Fyn deletion reverses ITAM to ITAMi, we explored a possible link between SHP-1 and Fyn under ITAM configuration. The inhibition observed under ITAM conditions in the absence of Fyn was reversed when SHP-1 was silenced and LPS-mediated IL-8 production was restored, suggesting that Fyn abrogated an inhibitory signal via the inactivation of SHP-1. Given that Y⁵³⁶ and S⁵⁹¹ residues are essential for SHP-1 activation/inhibition (24, 25), we analyzed whether Lyn and Fyn controlled SHP-1 function through differential phosphorylation of these residues by taking advantage of bone marrow- derived macrophages (BMM) from transgenic mice with the pro-autoimmune R131 isoform of the FcyRIIA (26). Whereas Lyn was responsible for Y536 phosphorylation of SHP-1 during FcyRIIA-ITAMi signaling, Fyn was responsible for S⁵⁹¹ phosphorylation of SHP-1 during FcyRIIA-ITAM signaling. Moreover, regarding the latter, the absence of Fyn resulted in Lyn- dependent Y⁵³⁶ phosphorylation of SHP-1, thus mimicking an ITAMi signal. To understand how Fyn, a tyrosine kinase, promotes SHP-1 phosphorylation on serine, we performed FcyRIIA-ITAM stimulation in the presence or absence of ER , PKC and PI3K inhibitors (14, 27). Whereas PI3K and PKC inhibitors completely blocked both SHP-1^S591 and PKC phosphorylation, the ERK inhibitor had no effect. In addition, PI3K and PKC inhibitors favoured SHP-1 ^Y536 phosphorylation under conditions of FcyRIIA-ITAM signalling, and this preference required the presence of Lyn. Together, these results indicate that during IT AM signaling, Fyn-mediated inactivation of SHP-1 through phosphorylation of the serine residue 591 by a PI3K-PKCa axis, thereby blocking its activation by Lyn.

To investigate the functional role of Lyn and Fyn in the regulation of IT AM signals in vivo, we first used a mild immune-complex nephritis model, the nephrotoxic nephritis (NTN), via administration of a rabbit serum anti-mouse glomerular basement membrane. This mild model was used because FcyRIIA^Tg and Lyn^_/~ mice have a well-established autoimmune phenotype (26, 28, 29). Consistent with the above-described role of Lyn in maintaining ITAMi- mediated immune homeostasis, NTN serum induced severe acute nephritis associated with high mortality one week after i.p. injections into Lyn^_/~FcyRIIA^Tg recipients only, whereas Lyn^_/", Fyn ^1' or Fyn '^~FcyRllA^Tg mice did not develop significant disease despite glomerular Ab deposits. Renal disease development in Lyn^_/" FcyRIIA^Tg mice was characterized by a marked increase in urinary protein and blood urea nitrogen concentration (BUN). Lyn^"/-FcyRIIA^Tg mice also exhibited severe renal injury involving extensive mesangial and capillary (subendothelial or even intracapillary) deposits associated with mild mesangial and endocapillary plus extracapillary proliferation. Glomerular lesions were characterized by an intense macrophage infiltrate and cytokine production. These effects involved activation of ITAM signaling, as demonstrated by in situ phosphorylation of the Y⁵²⁵ residue in Syk. The protective role of Lyn was confirmed in another autoimmune model, the collagen antibody-induced arthritis (CAIA) model. Here, targeting human FcyRIIA or FcaRI in Tg animals for ITAMi signaling (by AT- 10 F(ab')₂ or monomeric IgA (9, 30)) prevented disease development, and this protection required the presence of Lyn. In contrast, the aggravating role of Fyn was highlighted by the absence of lesions in both Fyn '^~FcyRllA^Tg and Fyn '^~FcaRl^Tg mice. Importantly, tissue analysis of phosphorylated kinases and phosphatases in joints of arthritic mice revealed that Lyn may protect the host against autoimmunity by constitutive phosphorylation of SHP-1 ^Y536 residue, whereas Fyn favours autoimmunity development by inactivation of SHP-1 through the phosphorylation of the SHP-1^S591 residue associated with induction of Syk^Y525~526 phosphorylation. Altogether, these results suggest that non-redundant SFKs determine the ITAMi/ITAM balance that governs the outcome of inflammatory and autoimmune responses.

To elucidate whether a naturally occurring inflammatory disease is influenced by Fyn, we first analyzed whether pro-autoimmune R131 FcyRIIA^Tg mice could develop overtime spontaneous nephritis as described for spontaneous autoimmune arthritis (26) and compared with animals deficient for Fyn. While 50-week-old FcyRIIA^Tg mice developed end-stage chronic kidney disease (with enhanced body weight, increased BUN levels, glomerular IgG deposits, sclerotic glomeruli, and fibrosis and kidney interstitial infiltration by CD l ib, F4/80 and CD3-positive cells), 50-week-old Fyn '^~FcyRllA^Tg mice were protected. This protective effect was associated with decreased levels of chemokines and mRNA expression coding for proinflammatory cytokines. Thus, the Fyn kinase is essential for R131 FcyRIIA-mediated autoimmune disease development. Finally, we analysed the steady-state activation of different IT AM signaling effectors in blood leukocytes from untreated lupus nephritis (LN) patients with severe disease clinically and morphologically classified as class IV (immune-active nephritis) and class V (less-active nephritis with membranous immune deposits only) compared with healthy individuals (HI). Consistent with the role of Lyn in homeostasis, Lyn was strongly associated with FcyRIIA in healthy individuals but weakly associated in patients, whereas Fyn and Syk were exclusively associated with FcyRIIA in LN patients. In contrast, SHP-1 and pSHP-l^Y536 were associated with FcyRIIA exclusively in cells from healthy individuals. Strikingly, pSHP- 1 ^S591 and pPKCa were exclusively observed in LN patient cell lysate samples, thus emphasizing an IT AM configuration. However, this inactive pSHP-l^S591 and pPKCa were not associated with FcyRIIA. Taken together, our observations demonstrate a balance of SFK fulfilling opposite signaling functions in FcR, TCR and BCRimmunoreceptor signaling. Lyn or Lck are crucial to maintain ITAMi-mediated homeostasis, whereas Fyn is essential for ITAM- mediated cell activation by inducing the PI3K-PKC signaling axis that inactivates SHP- 1 during inflammatory responses for most immunoreceptors.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Lyn/Lck inhibitor.

2. The method of claim 1 wherein the cancer is selected from the group consisting of Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angio myolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchiolo alveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythro leukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangio sarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant, Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplasia Disease, Myelodysplasia, Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, non-small cell lung cancer (NSCLC) which coexists with chronic obstructive pulmonary disease (COPD), Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath, meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastema, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute, lymphoblastic leukemia, T- cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Vemer Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

3. The method of claim 1 wherein the Lyn/Lck inhibitor is used for promoting the activation downstream of Fc, B-cell and T-cell antigen receptors.

4. The method of claim 1 wherein the Lyn/Lck inhibitor is a small organic molecule.

5. The method of claim 1 wherein the Lyn/Lck inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of Lyn Lck.

6. The method of claim 1 wherein the Lyn/Lck inhibitor is administered to the subject in combination with an immune checkpoint inhibitor.

7. The method of claim 1 wherein the Lyn/Lck inhibitor is administered to the subject in combination with a PD-1 inhibitor.

8. The method of claim 1 wherein the Lyn/Lck inhibitor is administered to the subject in combination with an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies anti-TIM- 3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.

9. A method of screening a drug suitable for the treatment of cancer comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of Lyn Lck.