WO2021088846A1

WO2021088846A1 - Fgfr4/pd-1 combination treatments

Info

Publication number: WO2021088846A1
Application number: PCT/CN2020/126383
Authority: WO
Inventors: Klaus Hoeflich; Oleg Schmidt-Kittler; Megan HATLEN; Zhaoxiang REN
Original assignee: Cstone Pharmaceuticals (Suzhou) Co., Ltd.; Blueprint Medicines Corporation
Priority date: 2019-11-04
Filing date: 2020-11-04
Publication date: 2021-05-14
Also published as: CN114728058A

Abstract

A combination therapeutic regimens for treatment of a condition of a subject that would benefit from modulating the immune function. Provided herein are methods of using a combination therapy that comprises a PD-1 axis binding antagonist that blocks the interaction between PD-1 and PD-L1 and fisogatinib to treat a condition of a subject that would benefit from modulating the immune function. Also provided herein are a combination, a composition, a medicament, agent or kit of a PD-1 axis binding antagonist that blocks the interaction between PD-1 and PD-L1 and fisogatinib for use in the treatment of a condition of a subject that would benefit from modulating the immune function, in particular for use in the treatment of cancer.

Description

FGFR4/PD-1 COMBINATION TREATMENTS

FIELD OF INVENTION

The present invention relates to combination therapies useful for the treatment of a condition of a subject that would benefit from modulating the immune function. In particular, the invention relates to a combination therapy which comprises a PD-1 axis binding antagonist and fisogatinib, which is a FGFR4 inhibitor.

BACKGROUND OF THE INVENTION

Immunotherapies are one approach to treat hyperproliferative disorders. A major hurdle that scientists and clinicians have encountered in the development of various types of cancer immunotherapies has been to break tolerance to self-antigen (cancer) in order to mount a robust anti-tumor response leading to tumor regression. Unlike traditional development of small and large molecule agents that target the tumor, cancer immunotherapies target cells of the immune system that have the potential to generate a memory pool of effector cells to induce more durable effects and minimize recurrences.

The programmed death 1 (PD-1) receptor and PD-1 ligands 1 and 2 (PD-L1 and PD-L2, respectively) play integral roles in immune regulation. Expressed on activated T cells, PD-1 is activated by PD-L1 (also known as B7-H 1) and PD-L2 expressed by stromal cells, tumor cells, or both, initiating T-cell death and localized immune suppression, potentially providing an immune-tolerant environment for tumor development and growth. Conversely, inhibition of this interaction can enhance local T-cell responses and mediate antitumor activity in nonclinical animal models.

A growing body of evidence from preclinical and clinical results suggests that targeted immune checkpoints are becoming the most promising approach to treating cancer patients. The programmed cell death molecule 1 is one of the immunological checkpoint proteins, which plays a major role in limiting T cell activity, which provides a major immune resistance mechanism by which tumor cells can escape immune surveillance. The interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells negatively regulates the immune response and attenuates anti-tumor immunity. The expression of PD-L1 on tumors is associated with decreased survival in esophageal, pancreatic and other types of cancer, highlighting this pathway as a new promising target for tumor immunotherapy.

Current therapies for advanced hepatocellular carcinoma (HCC) are marginally effective. Although immunotherapy has the potential to elicit non-toxic, systemic, long-lived anti-tumor activity, there are in fact several unique properties of HCC that limit the efficacy of immune-based therapies. Specifically, the most challenging barrier to immune-based therapy in HCC is the unique immunobiology of the liver. The liver in both healthy and diseased states has a plethora of regulatory mechanisms that sustain the immunosuppressive milieu of the liver and serve as an obstacle to effective HCC immunotherapy (Oncoimmunology 1: 1, 48-55; January/February 2012) . Recently, it was unexpectedly found that fisogatinib described in WO2015/061572 has immune modulating capabilities. In particular, fisogatinib was found to downregulate the immune gene, CCL20, and to induce T-cell infiltration, which makes it highly capable of counteracting the immunosuppressive mechanisms found in HCC and other cancers. Thus, fisogatinib is an attractive agent to be used in combination with a PD-1 axis binding antagonist.

Though there have been many recent advances in the treatment of cancer, there remains a need for more effective and/or enhanced treatment of an individual suffering the effects of cancer. The combinations and methods herein that relate to combining therapeutic approaches for enhancing anti-tumor immunity address this need.

BRIEF SUMMARY OF THE INVENTION

This invention relates to combination therapeutic regimens for treatment of a condition of a subject that would benefit from modulating the immune function.

Provided herein are methods of using a combination therapy that comprises a PD-1 axis binding antagonist that blocks the interaction between PD-1 and PD-L1 and fisogatinib to treat a condition of a subject that would benefit from modulating the immune function.

In some embodiments, the methods comprise administering to the subject a combination therapy which comprises a pharmaceutically effective amount of an antibody that blocks the interaction between PD-1 and PD-L1 and a pharmaceutically effective amount of fisogatinib. In some embodiments, the condition is immune-related diseases or conditions, tumor, cancer or a chronic viral infection.

Also provided herein are combinations of a PD-1 axis binding antagonist that blocks the interaction between PD-1 and PD-L1 and fisogatinib for use in the treatment of a condition of a subject that would benefit from modulating the immune function, in particular for use in the treatment of cancer. In some embodiments, the invention provides a composition, medicament or agent comprising a PD-1 axis binding antagonist for use in combination with fisogatinib for treating a cancer. In some embodiments, the invention provides a composition, medicament or agent comprising fisogatinib for use in combination with a PD-1 axis binding antagonist for treating a cancer.

Also, provided herein are uses of a PD-1 axis binding antagonist (such as an antibody that blocks the interaction between PD-1 and PD-L1) and fisogatinib for the manufacture of a medicament for the treatment of a condition of a subject that would benefit from modulating the immune function, in particular for use in the treatment of cancer. In some embodiments, the medicaments comprise a kit, and the kit also comprises a package insert comprising instructions for using the PD-1 axis binding antagonist in combination with fisogatinib to treat a cancer in a subject.

Other embodiments provide use of a PD-1 axis binding antagonist in the manufacture of medicament for treating a cancer in a subject when administered in combination with fisogatinib and use of fisogatinib in the manufacture of a medicament for treating a cancer in a subject when administered in combination with a PD-1 axis binding antagonist.

Also provided are kits comprising a first container, a second container and a package insert, wherein the first container comprises at least one dose of a medicament comprising a PD-1 axis binding antagonist, the second container comprises at least one dose of a medicament comprising fisogatinib, and the package insert comprises instructions for treating a subject using the medicaments.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the PD-1 axis binding antagonist can be selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist; preferably, it is an antibody or antigen-binding fragment thereof; preferably, the antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof; more preferably, the antibody is an anti-PD-L1 antibody; most preferably, the anti-PD-L1 antibody is a monoclonal antibody or an antigen binding fragment thereof, which specifically binds to PD-L1 and blocks the binding of PD-L1 to PD-1.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain CDR sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39, and 41. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33 and 35. All the sequences including the heavy chain CDR sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39, and 41, as well as the light chain CDR sequences selected from the group consisting of SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33 and 35 are also disclosed in the CN106432501A, which is incorporated herein by reference as part of the disclosure of the present application.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of:

a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5;

b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15 and/or SEQ ID NO: 17;

c) a heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; and

d) a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39 and/or SEQ ID NO: 41.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the antibody or antigen-binding fragment thereof further comprises a light chain variable region selected from the group consisting of:

a) a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 11;

b) a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23; and

c) a light chain variable region, SEQ ID NO: 31, SEQ ID NO: 33 and/or SEQ ID NO: 35.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49; and fisogatinib.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the subject is a human; preferably, the subject has received at least one round of a prior therapy; more preferably, the subject has up-regulated PD-L1 expression.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the prior therapy is surgical resection, transplantation, local regional therapy (including but not limited to cryotherapy, radioembolization, radiotherapy, external beam radiation, transarterial chemoembolization (TACE) , transarterial embolization (TAE) , microwave hyperthermia, laser ablation, radiofrequency ablation (RFA) , percutaneous intrahepatic chemotherapy administration, and percutaneous ethanol injection (PEI) ) , systemic therapy (including but not limited to chemotherapy (e.g. cytotoxic agent) , target therapy (agent works by targeting the cancer’s specific genes, proteins, or the tissue environment that contributes to cancer growth and/or survival) , immunotherapy (such as : checkpoint inhibitors (immune stimulating agent, immune blocking agent) , adoptive cell transfer, monoclonal antibodies, treatment vaccines, cytokines, bacillus calmette-guérin) , hormone therapy (agent slows or stops the growth of cancer that uses hormones to grow) , and any of combinations of chemotherapy , target therapy , immuno-oncology therapy, hormone therapy or local regional therapy) , and/or best supportive care.

In some embodiments of the above methods, uses, combinations, compositions, medicaments or kits, the condition is immune-related diseases or conditions, tumor, cancer or a chronic viral infection. In some embodiments, the condition is cancer, specifically, the cancer is a solid tumor or a blood cancer. In some embodiments, the cancer is re-current or metastatic, is locally advanced and/or metastatic or relapsed or refractory.

In some embodiments of the above methods, combinations, compositions, medicaments, uses or kits, the PD-1 axis binding antagonist is formulated as a liquid medicament, especially as injection, for example, at a dose of about 150 mg, about 500 mg, or about 1500 mg; and fisogatinib is formulated, especially as a tablet, for example, at a dose of about 300 mg, about 400 mg, or about 600 mg. In some embodiments, the PD-1 axis binding antagonist and fisogatinib are administered sequentially in either order or simultaneously.

In some embodiments of the above methods, combinations, compositions, medicaments, uses or kits, the PD-1 axis binding antagonist is administered once every threeweeks during the treatment cycle, at a dose of about 1200 mg.

In some embodiments of the above methods, combinations, compositions, medicaments, uses or kits, fisogatinib is administered once every day during the treatment cycle, at a dose of about 300 mg, about 400 mg, or about 600 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a series of two graphs. Figure 1A shows the production of CCL20 by various human hepatocellular carcinoma cell lines when treated with increasing concentrations of fisogatinib and Figure 1B shows the correlation of cellular FGF19 expression to CCL20 production or output for these cell lines.

Figure 2 is a graph showing that treatment with fisogatinib (BLU-554) decreases the chemoattractivity of JHH7 and HEP3B supernatant for regulatory T-cells (Treg) .

Figure 3A is a series of two graphs that show the percent of tumor volume change over time in an immune deficient JHH7 xenograft model with and without treatment with fisogatinib (BLU-554) .

Figure 3B is a series of two graphs that show the percent of tumor volume change over time in immune competent JHH7 xenograft model with and without treatment with fisogatinib (BLU-554) . The combined figures show that while fisogatinib can induce tumor stasis in an immune deficient setting, its antitumoral effect is stronger in the presence of an intact immune system, resulting in tumor shrinkage.

Figure 4 shows that fisogatinib treatment induces T-cell infiltration of JHH7 tumors.

Figure 5 is a volcano plot showing that several immune genes are modulated in cells after treatment in vitro with fisogatinib (BLU-554) .

Figure 6 is a graph showing that fisogatinib and/or anti-PD-L1 antibody Ab-1 (mono or combo) exhibited significantly enhanced PBMCs-mediated tumor killing efficacy in rhIFN-γ pre-treated Hep-3B. *p<0.05; **p<0.01; ***p<0.001.

DETAILED DISCLOSURE OF THE INVENTION

The following disclosure and exemplary embodiments are presented to enable one of ordinary skill in the art to make and use the subject invention. Various modifications to the embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the subject invention is not intended to be limited to the embodiments shown, but are to be accorded the widest scope consistent with the principles and features described herein.

I. Definitions

So that the invention may be more readily understood, some technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

"About" when used to modify a numerically defined parameter (e.g., the dose of a PD-1 axis binding antagonist or fisogatinib, or the length of treatment time with a combination therapy described herein) means that the parameter may vary by as much as 10%below or above the stated numerical value for that parameter. For example, a dose of about 5 mg/kg may vary between 4.5 mg/kg and 5.5 mg/kg.

As used herein, including the appended claims, the singular forms of words such as "a, " "an, " and "the, " include their corresponding plural references unless the context clearly dictates otherwise.

"Consists essentially of, " and variations such as "consist essentially of" or "consisting essentially of, " as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition. As a non-limiting example, a PD-1 axis binding antagonist that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, which do not materially affect the properties of the binding compound.

"Patient" or "subject" refers 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 any organism, preferably an animal, more preferably humans and mammalian veterinary patients such as rat, mouse, rabbit, cattle, horses, dogs, and cats.

"Administration" as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.

"Treatment" or "therapy" of a condition includes preventing or mitigating a condition, reducing the rate at which a condition arises or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, healing a condition, or a combination of the above.

For cancer, "treatment" or "therapy" can refer to inhibiting or slowing the growth, reproduction, or metastasis of a tumor or malignant cell, or some combination of the above. For a tumor, "treatment" or "therapy" includes clearance of all or part of a tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of tumor progression, or some combination of the above.

"Treat" or "treating" a cancer as used herein means to administer a combination therapy of a PD-L1 antagonist and another therapeutic agent to a subject having a cancer, or diagnosed with a cancer, to achieve at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth. Positive therapeutic effects in cancer can be measured in a number of ways (See, W.A. Weber, J. Nucl. Med. 50: 1 S-10S (2009) ) .

In some embodiments, the treatment achieved by a combination of the invention is any of partial response (PR) , complete response (CR) , overall response (OR) , progression free survival (PFS) , disease free survival (DFS) and overall survival (OS) . PFS, also referred to as "Time to Tumor Progression" indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients have experienced a CR or PR, as well as the amount of time patients have experienced stable disease (SD) . DFS refers to the length of time during and after treatment that the patient remains free of disease. OS refers to a prolongation in life expectancy as compared to naive or untreated subjects or patients. In some embodiments, response to a combination of the invention is any of PR, CR, PFS, DFS, OR, or OS that is assessed using Response Evaluation Criteria in Solid Tumors (RECIST) 1. 1 response criteria.

The treatment regimen for a combination of the invention that is effective to treat a cancer patient may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject. While an embodiment of any of the aspects of the invention may not be effective in achieving a positive therapeutic effect in every subject, it should do so in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the chi2-test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test) , Jonckheere-Terpstra-test and the Wilcoxon-test.

As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of tumor, remission of a immune-related disease (e.g., PD-L1 associated disease, cancer) , decreasing symptoms resulting from a immune-related disease (e.g., cancer) , increasing the quality of life of those suffering from a immune-related disease (e.g., cancer) , decreasing the dose of other medications required to treat a immune-related disease (e.g., cancer) , delaying the progression of a immune-related disease (e.g., cancer) , curing an immune-related disease (e.g., cancer) , and/or prolong survival of patients having a immune-related disease (e.g., cancer) .

"Ameliorating" means a lessening or improvement of one or more symptoms as compared to not administering the present combination, compositions, medicaments or kits. "Ameliorating" also includes shortening or reduction in duration of a symptom.

As used herein, an "effective dosage" or "effective amount" of drug, compound, or pharmaceutical composition is an amount that is sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing incidence or amelioration of one or more symptoms of various immune-related diseases or conditions (such as for example advanced cancer) , decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the a immune-related disease of patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to 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. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

For example, for the use of a drug, compound, combination, or pharmaceutical composition disclosed in the present invention, a therapeutically effective amount refers to a dose or concentration, at which the drug, compound, combination, or pharmaceutical composition can eliminate all or part of the tumor, inhibit or slow tumor growth, inhibit mediating the growth or reproduction of cells in a cancerous state, inhibit tumor cell metastasis, alleviate any symptoms or markers associated with a tumor or cancer condition, prevent or delay the progression of a tumor or cancer condition, or some combination of the above.

As used herein, "in combination with" refers to administration of one treatment modality in addition to another treatment modality. As such, "in combination with" refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.

"Treatment regimen" , "dosing protocol" and dosing regimen are used interchangeably to refer to the dose and timing of administration of each therapeutic agent in a combination of the invention.

"Dysfunction" in the context of immune dysfunction, refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes the common elements of both exhaustion and/or anergy in which antigen recognition may occur, but the ensuing immune response is ineffective to control infection or tumor growth.

"Dysfunctional" , as used herein, also includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into down-stream T-cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.

"Anergy" refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T-cell receptor (e.g. increase in intracellular Ca ²⁺ in the absence of ras-activation) . T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of costimulation. The unresponsive state can often be overridden by the presence of Interleukin-2. Anergic T-cells do not undergo clonal expansion and/or acquire effector functions.

"Exhaustion" refers to T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells.

Exhaustion prevents optimal control of infection and tumors. Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) as well as cell intrinsic negative regulatory (costimulatory) pathways (PD-1, B7-H3, B7-H4, etc. ) .

"Enhancing T-cell function" means to induce, cause or stimulate a T-cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T-cells. Examples of enhancing T-cell function include: increased secretion of gamma-interferon from CD8+ T-cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) relative to such levels before the intervention. In one embodiment, the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art.

A "T cell dysfunctional disorder" is a disorder or condition of T-cells characterized by decreased responsiveness to antigenic stimulation. In a particular embodiment, a T-cell dysfunctional disorder is a disorder that is specifically associated with inappropriate increased signaling through PD-1. In another embodiment, a T-cell dysfunctional disorder is one in which T-cells are anergic or have decreased ability to secrete cytokines, proliferate, or execute cytolytic activity. In a specific aspect, the decreased responsiveness results in ineffective control of a pathogen or tumor expressing an immunogen. Examples of T cell dysfunctional disorders characterized by T-cell dysfunction include unresolved acute infection, chronic infection and tumor immunity.

"Tumor immunity" refers to the process in which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated, and the tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.

"Immunogenicity" refers to the ability of a particular substance to provoke an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the clearance of the tumor cells by the immune response. Examples of enhancing tumor immunogenicity include treatment with a PD-1 axis binding antagonist and fisogatinib.

"Sustained response" refers to the sustained effect on reducing tumor growth after cessation of a treatment. For example, the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration at least the same as the treatment duration, at least 1.5X, 2.0X, 2.5X, or 3.0X length of the treatment duration.

A "disorder" is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.

"Cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

"Immune-related disease" in the present invention refers to any disease which is linked or due to a dysfunction, in particular a dysregulation, such as an over-activation of the immune system. The immune-related disease according the invention is preferably selected from the group consisting of an inflammatory disease, in particular a chronic inflammatory disease, an inflammatory skin disease, an auto-immune disease and an allergic disease.

"Disease associated with or associated with PD-L1" in the present invention means any condition which is caused, aggravated or otherwise related due to an increase or decrease in expression or activity of PD-L1 (e.g., human PD-L1) .

"Tumor" as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms) .

"Tumor burden" also referred to as "tumor load" , refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of tumor (s) , throughout the body, including lymph nodes and bone narrow. Tumor burden can be determined by a variety of methods known in the art, such as, e.g. by measuring the dimensions of tumor (s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., ultrasound, bone scan, computed tomography (CT) or magnetic resonance imaging (MRI) scans.

The term "tumor size" refers to the total size of the tumor which can be measured as the length and width of a tumor. Tumor size may be determined by a variety of methods known in the art, such as, e.g. by measuring the dimensions of tumor (s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., bone scan, ultrasound, CT or MRI scans.

"Cancer" , "cancerous" , or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In some embodiments, the cancer is characterized by amplified FGFR-4. In some embodiments, the cancer is characterized by overexpression of FGFR-4. In some embodiments, the cancer is characterized by amplified FGF-19. In some embodiments, the cancer is characterized by overexpression of FGF-19. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer) , lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, including hepatocellular carcinoma (HCC) , bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, nodular melanomas, multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL) ; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia) ; chronic lymphocytic leukemia (CLL) ; acute lymphoblastic leukemia (ALL) ; hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD) , as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors) , Meigs' syndrome, brain, as well as head and neck cancer, and associated metastases. In some embodiments, cancers that are amenable to treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin’s lymphoma (NHL) , renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi’s sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC) , and HCC. Yet, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers. In some embodiments, the cancer is HCC. In some embodiments, the HCC is characterized by amplified FGFR-4. In some embodiments, that HCC is characterized by overexpression of FGFR-4. In some embodiments, the cancer is characterized by amplified FGF-19. In some embodiments, the cancer is characterized by overexpression of FGF-19.

"RECIST 1. 1 Response Criteria" as used herein means the definitions set forth in Eisenhauer et al., E. A. et al., Eur. J Cancer 45: 228-247 (2009) for target lesions or non-target lesions, as appropriate based on the context in which response is being measured.

"Sustained response" means a sustained therapeutic effect after cessation of treatment with a therapeutic agent, or a combination therapy described herein. In some embodiments, the sustained response has a duration that is at least the same as the treatment duration, or at least 1 .5, 2.0, 2.5 or 3 times longer than the treatment duration.

"Sample, " as used herein, refers to a composition that is obtained or derived from a subject and/or individual of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof.

By "tissue sample" or "cell sample" is meant a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease tissue/organ. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

A "reference sample" , "reference cell" , "reference tissue" , "control sample" , "control cell" , or "control tissue" , as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual. For example, healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to a tumor) . In another embodiment, a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual. In even another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the subject or individual.

For the purposes herein a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis, provided that it is understood that the same section of tissue sample may be analyzed at both morphological and molecular levels or analyzed with respect to both polypeptides and polynucleotides.

By "correlate" or "correlating" is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.

The word "label" when used herein refers to a detectable compound or composition. The label is typically conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or an antibody, and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which results in a detectable product.

An "effective response" of a patient or a patient's "responsiveness" to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and progression free survival) ; resulting in an objective response (including a complete response or a partial response) ; or improving signs or symptoms of cancer.

A patient who "does not have an effective response" to treatment refers to a patient who does not have any one of extending survival (including overall survival and progression free survival) ; resulting in an objective response (including a complete response or a partial response) ; or improving signs or symptoms of cancer.

An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F (ab') 2, Fv) , single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies) , and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or subclass thereof) , and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes) , e.g., IgG1, lgG2, lgG3, lgG4, lgA1 and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "antigen binding fragment" or "antigen binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., PD-L1) . Antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include Fab; Fab'; F (ab') 2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., Nature 341: 544-546, 1989) , and an isolated complementarity determining region (CDR) .

Examples of antigen-binding fragments include, but are not limited to, such as diabody, Fab, Fab', F (ab') 2, Fv fragment, disulfide-stabilized Fv fragment (dsFv) , (dsFv) ₂, bispecific dsFv (dsFv-dsFv') , disulfide stabilized bifunctional antibody (dsdiabody) , single chain antibody molecule (scFv) , scFv dimer (bivalent bifunctional antibody) , bivalent single chain antibody (BsFv) , multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies and bivalent domain antibodies. The antigen binding fragment can bind to the same antigen as the parent antibody. In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody, ligated to a framework region from one or more different human antibodies.

A "Fab" fragment of an antibody refers to that portion of the antibody molecule consisting of a light chain (including the variable region and the constant region) bound to the variable region and the constant region of a heavy chain by disulfide bonds.

A "Fab'" fragment refers to a Fab fragment that contains a portion of the hinge region.

"F (ab') ₂" refers to a dimer of Fab.

The "Fc" of an antibody refers to the portion of the antibody consisting of the combination of the second and third constant regions of the heavy chain bound by disulfide bonds. The Fc portion of an antibody is responsible for a variety of different effector functions such as ADCC and CDC but does not function in antigen binding.

The "Fv" segment of an antibody refers to the smallest antibody fragment that contains the complete antigen binding site. The Fv fragment consists of the variable region of a light chain and the variable region of a heavy chain.

"Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to one another directly or via a peptide chain (Huston JS et al, Proc Natl Acad Sci USA, 85: 5879 (1988) ) .

"Single-chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

"Camelized single domain antibody" , "heavy-chain-only antibody" or "HCAb" refers to an antibody containing two V _H domains and no light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally derived from camelidae (camels, dromedaries and llamas) . Although the light chain is deleted, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al. Nature. Jun 3; 363 (6428) : 446-8 (1993) ; Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, " Immunogenetics. Apr; 54 (1) : 39-47 (2002) ; Nguyen VK. et al., Immunology. May; 109 (1) : 93-101 (2003) ) . The variable region of a heavy chain antibody (VHH domain) is the smallest known antigen-binding unit generated by adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .

A "nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant regions, CH2 and CH3.

A "diabody" includes a smaL1 antibody fragment with two antigen-binding sites, wherein the fragment contains a V _H domain and a V _L domain (V _H-V _L or V _H-V _L) linked together on the same polypeptide chain (See, Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15; 90 (14) : 6444-8 (1993) ; EP 404097; WO 93/11161) . The linker between the two domains is so short that the two domains on the same chain cannot be paired with each other, the two domains are forced to pair with the complementary domains of another chain, and thereby forming two antibody-binding sites. These two antibody-binding sites may target the same or different antigens (or epitopes) .

A "domain antibody" refers to an antibody fragment consists only one heavy chain variable region or one light chain variable region. In certain cases, two or more V _H domains are covalently joined by a polypeptide linker and form a bivalent domain antibody. The two V _H domains of a bivalent domain antibody may target the same or different antigens.

In some embodiments, " (dsFv) ₂" comprises three peptide chains: two V _H moieties are linked by a single polypeptide linker and bound to two V _L moieties by disulfide bonds.

In some embodiments, a "bispecific ds diabody" comprises V _L1-V _H2 (linked by a polypeptide linker) and V _H1-V _L2 (also linked by a polypeptide linker) , bound to one another via disulfide bonds between V _H1 and V _L1.

"Bispecific dsFv" or "dsFv-dsFv" comprises three polypeptide chains: a V _H1-V _H2 moiety, wherein the heavy chains are linked by a polypeptide linker (e.g. a long flexible linker) and bound to V _L1 and V _L2 moieties, respectively, via disulfide bonds. Each heavy chain and light chain bound by disulfide bonds has a different antigen specificity.

In some embodiments, the "scFv dimer" is a bivalent diabody or bivalent scFv (BsFv) comprising the dimerization of two V _H-V _L moieties, the V _H of one moiety coordinate with the V _L of another moiety and form two binding sites which can target the same antigens (or epitope) or to different antigens (or epitopes) . In other embodiments, the "scFv dimer" is a bispecific diabody comprising V _L1-V _H2 (linked by a polypeptide linker) associated with V _H1-V _L2 (also linked by a polypeptide linker) , wherein V _H1 and V _L1 coordinate, V _H2 and V _L2 coordinate, and each coordinated pair has a different antigen specificity.

An antibody, an antibody conjugate, or a polypeptide that "preferentially binds" or "specifically binds" (used interchangeably herein) to a target (e.g., PD-L1 protein) is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a PD-L1 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other PD-L1 epitopes or non-PD-L1 epitopes. It is also understood that by reading this definition, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

In some embodiments, the antibodies or antigen-binding fragments thereof of the present application specifically bind to human and/or monkey PD-L1 and has a binding affinity (K _D) of ≤ 10 ^-6M (e.g. ≤5×10 ^-7M, ≤2×10 ^-7M, ≤10 ^-7M, ≤5×10 ^-8M, ≤2×10 ^-8M, ≤10 ^-8M, ≤5×10 ^-9M, ≤ 2×10 ^- ⁹M, ≤10 ^-9M, ≤ 10 ^-10M, about 10 ^-10M, 10 ^-10M to 10 ^-9M, 10 ^-10M to 10 ^-8.5M or10 ^-10M to 10 ^-8M) . K _D in the present application refers to the ratio of the dissociation rate to the binding rate (koff/kon) , may be determined by surface plasmon resonance methods, for example, using an instrument such as Biacore.

The ability to "block binding" or "compete for the same epitope" in the present application refers to the ability of an antibody or an antigen-binding fragment thereof to inhibit the binding interaction between two molecules (e.g. human PD-L1 and anti-PD-L1 antibody) to any detectable degree. In some embodiments, an antibody or the antigen-binding fragment thereof that blocks binding between two molecules may inhibit the binding interaction between the two molecules to at least 50%. In some embodiments, such inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

The "epitope" as used herein refers to the specific group of atoms or amino acids on an antigen molecule to which an antibody binds. Two antibodies may bind to the same epitope on an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment thereof provided herein blocks the binding of an exemplary antibody, such as 1.4.1, 1.14.4, 1.20.15, and 1.46.11, to human PD-L1, then the antibody or antigen-binding fragment thereof may be considered to bind the same epitope as those exemplary antibodies.

A specific amino acid residue in an epitope is mutated by, for example, alanine scanning mutagenesis, and a mutation that reduces or prevents protein binding is identified. "Alanine scanning mutagenesis" is a method that may be used to identify some residues or regions of a protein affecting the interaction of an epitope with other compounds or proteins to which it binds. Residues or a group of target residues in the protein are substituted by neutral or negatively charged amino acids (most preferably alanine or polyalanine or conservative amino acids) . Any mutation in the amino acid residue that reduces the binding of the protein or a codon mutation that encodes it exceeds a threshold or a mutation that minimizes the binding of the protein compared to other mutations is likely present in the epitope to which the protein binds. In some embodiments of the present application, an epitope, important for a PD-L1 antibody, comprises at least one of the following amino acid residues: E58, E60, D61, K62, N63 and R113.

A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD) ) ; and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273: 927-948) . As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.

A "CDR" of a variable domain are amino acid residues within the variable region that are identified in accordance with the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, and/or conformational definitions or any method of CDR determination well known in the art. Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, e.g., Chothia et al., Nature 342: 877-883, 1989. Other approaches to CDR identification include the "AbM definition, " which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now

) , or the "contact definition" of CDRs based on observed antigen contacts, set forth in MacCallum et al., J. Mol. Biol., 262: 732-745, 1 996. In another approach, referred to herein as the "conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283: 1 156-1 166, 2008. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.

"Isolated antibody" and "isolated antibody fragment" refers to the purification status and in such context means the named molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term "isolated" is not intended to refer to a complete absence of such material or to an absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with experimental or therapeutic use of the binding compound as described herein.

"Monoclonal antibody" or "mAb" or "Mab" , as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567) . The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991 ) Nature 352: 624-628 and Marks et al. (1991 ) J. Mol. Biol. 222: 581 -597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 1 16: 731.

"Chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

"Human antibody" refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, "mouse antibody" or "rat antibody" refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

"Humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. The prefix "hum" , "hu" or "h" is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although some amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

"Conservatively modified variants" or "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc. ) , such that the changes can frequently be made without altering the biological activity or other desired property of the protein, such as antigen affinity and/or specificity. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed. ) ) .

"Homology" refers to sequence similarity between two polypeptide sequences when they are optimally aligned. When a position in both of the two compared sequences is occupied by the same amino acid monomer subunit, e.g., if a position in a light chain CDR of two different Abs is occupied by alanine, then the two Abs are homologous at that position. The percent of homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared x 100. For example, if 8 of 10 of the positions in two sequences are matched or homologous when the sequences are optimally aligned then the two sequences are 80%homologous. Generally, the comparison is made when two sequences are aligned to give maximum percent homology. For example, the comparison can be performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.

As used herein, "PD-L1" refers to a programmed cell death ligand 1 (PD-L1, see for example Freeman et al. (2000) J. Exp. Med. 192: 1027) . The amino acid sequence of a representative human PD-L1 is NCBI Accession No.: NP_054862.1, and the nucleic acid sequence of a representative human PD-L1 is NCBI Accession No.: NM_014143.3. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found in many tumors or cancer cells. PD-L1 binds to the receptor PD-1 or B7-1 expressed on activated T cells, B cells and bone marrow cells. Binding of PD-L1 to its receptor triggers signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Thus, PD-L1 plays a major role in suppressing the immune system in some events, such as pregnancy, autoimmune diseases, tissue transplantation, and is thought to allow tumors or cancer cells to evade immune checkpoints and evade immune responses.

A mature PD-L1 lacks the presecretory leader sequence, also referred to as leader peptide The terms "PD-L1" and "mature PD-L1" are used interchangeably herein, and shall be understood to mean the same molecule unless otherwise indicated or readily apparent from the context.

"PD-1 axis binding antagonist " refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis -with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing) . As used herein, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.

"PD-1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition) . In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. "PD-L1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1 , B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B7-1. In one embodiment, a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L1 so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition) .

In any of the treatment method, medicaments and uses of the present invention in which a human subject is being treated, the PD-L1 antagonist blocks binding of human PD-L1 to human PD-1. PD-L1 antagonists useful in the any of the treatment methods, medicaments, and uses of the present invention include a monoclonal antibody (mAb) which specifically binds to PD-L1, and preferably specifically binds to human PD-L1. The mAb may be a human antibody, a humanized antibody or a chimeric antibody, and may include a human constant region. In some embodiments the human constant region is selected from the group consisting of lgG1, lgG2, lgG3 and lgG4 constant regions, and in preferred embodiments, the human constant region is an lgG1 or lgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F (ab') 2, scFv and Fv fragments.

In some embodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, an anti-PD-L1 antibody is the antibody claimed in PCT Application No. PCT/CN2016/093560.

As used herein, "anti-PD-L1 antibody" refers to an antibody that is capable of specifically binding to PD-L1 (e.g., human or monkey PD-L1) with sufficient affinity for diagnostic and/or therapeutic use. As used herein, an anti-human PD-L1 mAb or a diagnostic anti-hPD-L1 mAb refers to a monoclonal antibody that specifically binds to mature human PD-L1.

"PD-L2 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1.

"PD-L1" expression as used herein means any detectable level of expression of PD-L1 protein on the cell surface or of PD-L1 mRNA within a cell or tissue. PD-L1 protein expression may be detected with a diagnostic PD-L1 antibody in an IHC assay of a tumor tissue section or by flow cytometry. Alternatively, PD-L1 protein expression by tumor cells may be detected by PET imaging, using a binding agent (e.g., antibody fragment, affibody and the like) that specifically binds to PD-L1. Techniques for detecting and measuring PD-L1 mRNA expression include RT-PCR and real-time quantitative RT-PCR. One approach employs a simple binary end-point of positive or negative for PD-L1 expression, with a positive result defined in terms of the percentage of tumor cells that exhibit histologic evidence of cell-surface membrane staining. A tumor tissue section is counted as positive for PD-L1 expression is at least 1 %, and preferably 5%of total tumor cells. In another approach, PD-L1 expression in the tumor tissue section is quantified in the tumor cells as well as in infiltrating immune cells, which predominantly comprise lymphocytes. The percentage of tumor cells and infiltrating immune cells that exhibit membrane staining are separately quantified as < 5%, 5 to 9%, and then in 10%increments up to 100%. For tumor cells, PD-L1 expression is counted as negative if the score is < 5%score and positive if the score is > 5%. PD-L1 expression in the immune infiltrate is reported as a semi-quantitative measurement called the adjusted inflammation score (AIS) , which is determined by multiplying the percent of membrane staining cells by the intensity of the infiltrate, which is graded as none (0) , mild (score of 1, rare lymphocytes) , moderate (score of 2, focal infiltration of tumor by lymphohistiocytic aggregates) , or severe (score of 3, diffuse infiltration) . A tumor tissue section is counted as positive for PD-L1 expression by immune infiltrates if the AIS is > 5.

The level of PD-L1 mRNA expression may be compared to the mRNA expression levels of one or more reference genes that are frequently used in quantitative RT-PCR, such as ubiquitin C.

In some embodiments, a level of PD-L1 expression (protein and/or mRNA) by malignant cells and/or by infiltrating immune cells within a tumor is determined to be "overexpressed" or "elevated" based on comparison with the level of PD-L1 expression (protein and/or mRNA) by an appropriate control. For example, a control PD-L1 protein or mRNA expression level may be the level quantified in nonmalignant cells of the same type or in a section from a matched normal tissue.

"FGFR-4" or "FGFR-4 protein" refers to any form of the FGFR-4 protein, including wild type and all variant forms (including, without limitation, mutant forms and splice variants) . The FGFR-4 protein is a product of the FGFR-4 gene, and the FGFR-4 protein therefore includes any protein encoded by any form of the FGFR-4 gene, including all aberrations, e.g., point mutations, indels, translocation fusions, and focal amplifications.

"Inhibitor" refers to a compound that inhibits an enzyme such that a reduction in activity of the enzyme can be observed, e.g., in a biochemical assay. In some embodiments, an inhibitor has an IC 50 of less than about 1 μΜ, less than about 500 nM, less than about 250 nM, less than about 100 nM, less than about 50 nM, or less than about 10 nM. An inhibitor of FGFR-4 refers to a compound that inhibits FGFR-4.

"Overexpressed, " as used herein, means there is production of a gene product in a sample that is substantially higher than that observed in a population of control samples (e.g. normal tissue) .

"Selective" refers to a compound that inhibits the activity of a target protein, e.g., FGFR-4, more potently than it inhibits activity of other proteins. In this instance, the isoforms FGFR-1, FGFR-2, FGFR-3, and FGFR-4 are all considered distinct proteins. In some embodiments, a compound can inhibit the activity of the target protein, e.g., FGFR-4, at least 1.5, at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 500, or at least 1000 or more times potently than it inhibits the activity of a non-target protein.

"Pharmaceutically acceptable" indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

II. METHODS, USES AND MEDICAMENTS

This invention relates to a method for treatment of a condition of a subject that would benefit from modulating the immune function.

Provided herein is a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and fisogatinib. Fisogatinib is a selective FGFR4 inhibitor, which unexpectedly was found to have an effect on the expression of genes whose corresponding protein may have immune modulating capabilities. More specifically, fisogatinib was shown to decrease the expression of certain genes (CCL20) which can impact immune cells, potentially weakening the repellant layer surrounding the tumor. The fact that fisogatinib weakens the repellant layer may allow T-cells to readily infiltrate the tumor, making their activiation by a PD-1 axis binding antagonist more advantageous and thus, providing a superior combination. Also provided herein is a method of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and fisogatinib.

Also, provided herein are uses of a PD-1 axis binding antagonist (such as an antibody that blocks the interaction between PD-1 and PD-L1) and fisogatinib for the manufacture of a medicament for the treatment of a condition of a subject that would benefit from modulating the immune function, in particular for use in the treatment of cancer. In some embodiments, the medicaments comprise a kit, and the kit also comprises a package insert comprising instructions for using the PD-1 axis binding antagonist in combination with the fisogatinib to treat a cancer in a subject.

PD-1 Axis Binding Antagonists

For example, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PDL1 binding antagonist and a PDL2 binding antagonist. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

In some embodiments of the above methods, combinations, compositions, medicaments, uses or kits, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

In some embodiments of the above methods, combinations, compositions, medicaments, uses or kits, the PD-1 binding antagonist is an anti-PD-L1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody) or an antigen binding fragment thereof. In some embodiments of the above methods, combinations, compositions, medicaments, uses or kits, exemplary fully human monoclonal antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11 are provided, the CDR sequences of which are shown in Table 1. And the heavy chain or the light chain variable region sequences are also listed below.

Table 1

1.4.1-VH (30511) : (SEQ ID NO: 43 is an amino acid, SEQ ID NO: 44 is a nucleic acid) heavy chain CDR1-3: SEQ ID NOs: 1, 3, 5 are amino acid sequences and SEQ ID NOs: 2, 4, 6 are nucleic acid sequences.

Section V: IGHV4-39*01

Section D: IGHD1-26*01

Section J: IGHJ4*02

1.4.1-VL (30027) : (SEQ ID NO: 45 is an amino acid and SEQ ID NO: 46 is a nucleic acid) light chain CDR1-3: SEQ ID NOs: 7, 9, 11 are amino acid sequences and SEQ ID NO: 8, 10, 12 are nucleic acid sequences:

Section V: IGLV3-1*01

Section J: IGLJ2*01

1.14.4-VH (29812) : (SEQ ID NO: 47 is an amino acid, SEQ ID NO: 48 is a nucleic acid) heavy chain CDR1-3: SEQ ID NOs: 13, 15, 17 are amino acid sequences and SEQ ID NOs: 14, 16, 18 are nucleic acid sequences:

Section V: IGHV3-23*01

Section D: IGHD5-5*01

Section J: IGHJ4*02

1.14.4-VL and 1.46.11-VL (29841) : (SEQ ID NO: 49 is an amino acid, SEQ ID NO: 50 is a nucleic acid) Light chain CDR1-3: SEQ ID NOs: 19, 21, 23 are amino acid sequences and SEQ ID NO: 20, 22, 24 are nucleic acid sequences:

Section V: IGLV3-21*02

Section J: IGLJ2*01

1.20.15-VH (30712) : (SEQ ID NO: 51 is an amino acid, SEQ ID NO: 52 is a nucleic acid) light chain CDR1-3: SEQ ID NOs: 25, 27, 29 are amino acid sequences and SEQ ID NOs: 26, 28, 30 are nucleic acid sequences:

Section V: IGHV4-39*01

Section D: Not determined

Section J: IGHJ4*02

1.20.15-VL (29907) : (SEQ ID NO: 53 is an amino acid, SEQ ID NO: 54 is a nucleic acid) Light chain CDR1-3: SEQ ID NOs: 31, 33, 35 are amino acid sequences and SEQ ID NO: 32, 34, 36 are nucleic acid sequences:

Section V: IGLV3-1*01

Section J: IGLJ2*01

1.46.11-VH (30626) : (SEQ ID NO: 55 is an amino acid, SEQ ID NO: 56 is a nucleic acid) light chain CDR1-3: SEQ ID NOs: 37, 39, 41 are amino acid sequences and SEQ ID NOs: 38, 40, 42 are nucleic acid sequences:

Section V: IGHV3-23*01

Section D: IGHD5-5*01

Section J: IGHJ4*02

1.46.11-VL (29841) : (SEQ ID NO: 49 is an amino acid, SEQ ID NO: 50 is a nucleic acid) light chain CDR1-3: SEQ ID NOs: 19, 21, 23 are amino acid sequences and SEQ ID NOs: 20, 22, 24 are nucleic acid sequences:

Section V: IGLV3-21*02

Section J: IGLJ2*01

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a heavy chain CDR sequence selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39 and 41.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33, and 35.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5; a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 17; a heavy chain variable region comprising SEQ ID NO : 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; and a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39 and/or SEQ ID NO: 41.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a light chain variable region selected from the group consisting of a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 11; a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23; and a light chain variable region comprising SEQ ID NO: 31 SEQ ID NO: 33 and/or SEQ ID NO: 35.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise:

a) A heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 11;

b) A heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO 15 and/or SEQ ID NO: 17; and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23;

c) A heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; and a light chain variable region comprising SEQ ID NO: 31, SEQ ID NO: 33 and/or SEQ ID NO: 35;

d) A heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39 and/or SEQ ID NO: 41; and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23.

A person skilled in the art will understand that the CDR sequences provided in Table 1 can be modified to contain one or more substitutions of amino acids, thereby resulting in enhanced biological activity such as enhanced binding affinity to human PD-L1. For example, a library of antibody variants (e.g., Fab or FcFv variants) can be produced and expressed with phage display technique, and screened for antibodies that have affinity to human PD-L1. In another example, the binding of the antibody to human PD-L1 can be simulated using computer software and the amino acid residues forming the binding interface on the antibody can be identified. Substitution of these residues can be avoided to prevent the decreasing in binding affinity, or these residues can be targeted for substitution to form a stronger binding. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, at least one (or all) of the substitutions in the CDR sequences are conservative substitutions.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments comprise one or more CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity of the sequences listed in Table 1, ) and in the meantime retaining similar or even higher binding affinity to human PD-L1 than its parental antibody, the said parental antibody has substantially the same sequence, but the corresponding CDR sequences have 100%sequence identity to the sequences listed in Table 1.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-1 antibodies and antigen-binding fragments thereof are fully human. The fully human antibodies do not have the issues in the human body such as immunogenicity or reduced binding affinity as often observed with the humanized antibodies.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the fully human anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, and SEQ ID NO: 55, and a homologous sequence with at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity; and/or a light chain variable region, wherein the light chain variable region is selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 49, and SEQ ID NO : 53, and a homologous sequence with at least 80%(e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity. These fully human antibodies retain the binding affinity to human PD-L1, preferably at a level similar to the exemplary antibodies: 1.4.1, 1.14.4, 1.20.15 and 1.46.11.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the fully human anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a) a heavy chain variable region comprising SEQ ID NO: 43; and a light chain variable region comprising the SEQ ID NO: 45; b) a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49; c) a heavy chain variable region comprising SEQ ID NO: 51; and a light chain variable region comprising SEQ ID NO: 53; or d) a heavy chain variable region comprising SEQ ID NO: 55; and a light chain variable region comprising SEQ ID NO: 49.

The present application also includes antibodies and antigen-binding fragments thereof that compete for the same epitope as the anti-PD-L1 antibody and antigen-binding fragments thereof of the present application. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibody blocks the binding of 1.4.1, 1.14.4, 1.20.15 and 1.46.11 to human or monkey PD-L1 at an IC ₅₀ value (i.e. the half inhibitory concentration) less than 10 ^-6M, less than 10 ^-7M, less than 10 ^-7.5M, less than 10 ^-8M, less than 10 ^- ^8.5M or less than 10 ^-9M or less than 10 ^-10M. The IC ₅₀ values are determined by competitive assays such as ELISA assays, radioligand competition binding assays, and FACS analysis.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof of the present application are capable of specifically binding with human PD-L1 with a binding affinity (Kd) of ≤10 ^-6M (e.g. ≤5x10 ^-7M, ≤2x10 ^-7M, ≤10 ^-7M, ≤5x10 ^-8M, ≤2x10 ^-8M, ≤10 ^- ⁸M, ≤5x10 ^-9M, ≤2x10 ^-9M, ≤10 ^-9M, 10 ^-10M, about10 ^-10M, 10 ^-10M to10 ^-8.5M or 10 ^-10M to 10 ^- ⁸M) . The binding affinity is measured by plasmon resonance binding assay. The binding affinity can be expressed by K _D value, which is calculated as the ratio of the dissociation rate to the binding rate (koff/kon) when the binding of the antigen to the antigen-binding molecule reaches equilibrium. The antigen-binding affinity (e.g. KD) can be appropriately determined by a suitable method known in the art, including using an instrument such as Biacore plasmon resonance binding assay (see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006) .

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof of the present application bind with human PD-L1 at an EC ₅₀ (i.e., the half binding concentration) of 0.1 nM-100 nM (e.g., 0.1 nM-50 nM, 0.1 nM-30 nM, 0.1 nM-20 nM, or 0.1 nM-10 nM or 0.1 nM-1nM) . Binding of the antibody to human PD-L1 can be determined by methods known in the art such as sandwich methods such as ELISA, Western blot, FACS or other binding assays. In an illustrative example, the antibody to be tested (i.e., the primary antibody) is allow to bind to the immobilized human PD-L1 or a cell expressing human PD-L1, the unbound antibody is then washed away, a labeled secondary antibody is introduced, which is capable of binding with the primary antibody, thus the bound primary antibody can be detected. The detection can be performed on a microplate reader when immobilized PD-L1 is used, or can be performed by using FACS analysis when cells expressing human PD-L1 are used. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof of the present application bind to human PD-L1 at an EC ₅₀ (i.e., 50%effective concentration) of 1 nM to 10 nM or 1 nM to 5 nM (as determined by FACS analysis) .

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof of the present application inhibit the binding of human PD-L1 to its receptor at an IC ₅₀ of 0.2 nM to 100 nM (e.g. 0.2 nM to 50 nM, 0.2 nM to 30 nM, 0.2 nM to 20 nM, 0.2 nM to 10 nM, or 1 nM to 10 nM) , which is measured by competitive assays.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof of the present application inhibit the binding of human PD-L1 to its receptor, and thereby provide biological activities include, for example inducing production of cytokines from the activated T cells (such as CD ⁴⁺ T cells and CD ⁸⁺ T cells) , inducing the proliferation of the activated T cells (such as CD ⁴⁺ T cells and CD ⁸⁺ T cells) and reversing the inhibitory function of regulatory Treg. Exemplary cytokines include IL-2 and IFNγ. The term "IL-2" refers to interleukin 2, which is a cytokine signaling molecule in the immune system that regulates the activities of white blood cells (e.g. leukocytes) . The term "interferon gamma (IFNγ) " is a cytokine produced by natural killer (NK) cells, NK T cells, CD ⁴⁺and CD ⁸⁺T cells, which is an critical activator of macrophages and an inducer of major histocompatibility complex (MHC) molecule expression. The production of cytokines can be determined by methods known in the art, such as ELISA. These methods can also be used to detect T cell proliferation, including the [ ³H] thymidine incorporation assay.

The anti-PD-L1 antibodies and antigen-binding fragments thereof are specific for human PD-L1. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof do not bind with PD-L2 (e.g. human PD-L2) . For example, the binding affinity to PD-L2 is even lower than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %or 1%of the binding affinity to human PD-L1.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof bind to monkey PD-L1 at an EC ₅₀ (determined by ELISA) of no more than 100 nM, for example, no more than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof bind to monkey PD-L1 at an EC50 of about 1 nM -10 nM.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof do not bind to murine PD-L1, but bind to monkey PD-L1 with binding affinity similar to that of human PD-L1. For example, binding of the exemplary antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11 to murine PD-L1 is not detectable by conventional binding assays such as ELISA or FACS analysis, while in accordance with the detection of ELISA or FACS, these antibodies bind to monkey PD-L1with similar affinity or EC50 value to that of human PD-L1.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof have reduced or eliminated effector function. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof have a constant region of IgG4 isotype, which has reduced or eliminated effector function. Effector functions such as ADCC and CDC can result in cytotoxicity to cells expressing PD-L1. Many cells, including normal cells, are capable of expressing PD-L1. In order to avoid potential undesired toxicity to these normal cells, some embodiments of the antibodies and antigen-binding fragments thereof of the invention have reduced or even eliminated effector functions. A number of assays are known to evaluate ADCC or CDC activity, such as Fc receptor binding assay, complement C1q binding assay, and cell lysis method, which can be readily selected by those skilled in the art. Without wishing to be bound by theory, but it is believed that antibodies with reduced or eliminated effector functions such as ADCC and CDC cause no or minimize cytotoxicity to cells expressing PD-L1, such as those normal cells, thus undesirable side effects are avoided. At the same time, the tumor cells expressing PD-L1 bind to the anti-PD-L1 antibody and thus cannot escape from the immune checkpoint and thus can be recognized and eliminated by the immune system.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof of the present application have reduced side effects. For example, the anti-PD-L1 antibodies and antigen-binding fragments thereof may have fully human IgG sequence and thus the immunogenicity thereof is lower than humanized antibodies. For another example, the anti-PD-L1 antibodies and antigen-binding fragments thereof can be in IgG4 format to eliminate ADCC and CDC.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof of the present application have the advantage that they can be used in combination with immunogenic substances, such as tumor cells, purified tumor antigens, and cells transfected with encoding immune stimulating cytokines, tumor vaccines. Furthermore, the anti-PD-L1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemotherapy and radiation therapy, target-based small molecule therapy, and other emerging immune checkpoint modulator therapies. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments thereof can be used as the base molecule for antibody-drug conjugates, bispecific or multivalent antibodies.

The anti-PD-L1 antibody and antigen-binding fragment thereof described in the present application may be a monoclonal antibody, a polyclonal antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody or an anti-idiotypic antibody. Recombinant antibodies are antibodies that are produced in vitro using recombinant methods rather than in animals. A bispecific antibody or a bivalent antibody is an artificial antibody having a fragment of two different monoclonal antibodies, which are capable of binding two different antigens. "Bivalent" antibodies and antigen-binding fragments thereof include two antigen-binding sites. The two antigen binding sites may bind to the same antigen or bind to a different antigen respectively, in which case the antibody or antigen binding fragment is "bispecific. "

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-1 antibodies and antigen-binding fragments thereof of the present application are fully human antibodies. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the fully human antibodies are produced using recombinant methods. For example, a transgenic animal such as a mouse can be prepared to carry a transgene or transchromosome of a human immunoglobulin gene and thus capable of producing fully human antibodies after immunization with an appropriate antigen, such as human PD-1. The fully human antibody can be isolated from such transgenic animal, or alternatively, can be prepared by hybridoma technology, the spleen cells of the transgenic animal are fused with an immortalized cell line to produce hybridoma cells that secretes the fully human antibody. Exemplary transgenic animals include, but are not limited to, Omni rat, whose expression of the endogenous rat immunoglobulin gene is inactivated and simultaneously engineered to contain functional recombinant human immunoglobulin locus; Omni mouse, whose expression of the endogenous mouse immunoglobulin gene is inactivated and simultaneously engineered to contain recombinant human immunoglobulin locus having a J-locus deletion and a C-kappa mutation.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein are camelized single chain domain antibodies, diabody, scFv, scFv dimers, BsFv, dsFv, (dsFv) 2, dsFv-dsFv', Fv fragment, Fab, Fab', F (ab') 2, ds difunctional antibody (dsdiabody) , Nanobody, domain antibody or bivalent domain antibody.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein further comprise an immunoglobulin constant region. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises a CH1, CH1-CH2 or CH1-CH3 region. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the immunoglobulin constant region can further comprise one or more modifications to achieve the desired properties. For example, the constant region can be modified to reduce or eliminate one or more effector functions to enhance FcRn receptor binding or to introduce one or more cysteine residues.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the anti-PD-L1 antibodies and antigen-binding fragments thereof further comprise a conjugate. It is contemplated that the antibodies or antigen-binding fragments thereof of the present invention may be linked to a variety of conjugates (see, for example, "Conjugate Vaccines" , Contributions to Microbiology and Immunology, JMCruse and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugates may be linked to the antibodies or antigen-binding substances by covalent attachment, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or addition, etc. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the epitope binding portion that can be used to bind one or more conjugates. For example, such a site may comprise one or more reactive amino acid residues, such as cysteine residues and histidine residues, to facilitate covalent attachment to the conjugate. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the antibody may be linked to the conjugate indirectly or via another conjugate. For example, the antibodies or antigen-binding fragments thereof may bind to biotin and then indirectly bind to a second conjugate that is linked to avidin. The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety or a cytotoxic moiety. Examples of detectable labels may include fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphate enzyme, luciferase, glucoamylase, lysozyme, glucose oxidase or β-D-galactosidase) , radioisotopes (e.g. ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³⁵S, ³H, ¹¹¹In, ¹¹²In, ¹⁴C, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹⁷⁷Lu, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, and ³²P, other lanthanides, luminescent labels) , chromophore, digoxigenin, biotin/avidin, DNA molecule or gold for detection.

In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the conjugate can be a pharmacokinetic modifying moiety such as PEG which helps to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In some embodiments of the above methods, combinations, uses, compositions, medicaments, agents or kits, the conjugate may be a purification moiety such as a magnetic bead. A "cytotoxic moiety" can be any agent that is detrimental to cells or that can damage or kill the cells. Examples of cytotoxic moiety include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogues, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g. mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU) , cyclophosphamide , busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin) , anthracycline antibiotics (e.g. daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (e.g. dactinomycin (formerly known as actinomycin) , bleomycin, mithramycin, and anthramycin (AMC) ) and anti-mitotic agents (e.g. vincristine and vinblastine) .

The antibody prepared from the cells herein may be purified by the purification method such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out, and affinity chromatography, wherein the affinity chromatography is the preferred purification technique. The species of the antibody and the Fc domain of any immunoglobulin present in the antibody determine whether protein A is suitable as an affinity ligand. Protein A can be used in the purification of the antibodies based on the human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) . Protein G is suitable for all murine isotypes and human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) . Agarose is the most commonly used attachment matrix for affinity ligand, but other matrices are available as well. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene may achieve faster flow rates and shorter processing times compared with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX. TM resin (J. T. Baker, Phillipsburg, N. J. ) . Other protein purification techniques can also be determined based on the antibodies obtained. Depending on the desired antibodies, other protein purification techniques, such as fractionation on an ion-exchange column, ethanol precipitation, reverse-phase HPLC, silica gel chromatography, heparin agarose gel chromatography based on anion-or cation-exchange resin (e.g. polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available.

After any preliminary purification step (s) , the mixture comprising the antibody of interest and contaminants may be subjected by low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at low salt concentration (e.g. from about 0 to 0.25 M salt concentration) .

Exemplary anti-PD-1 antibodies include but are not limited to nivolumab, cemiplimab, pembrolizumab, camrelizumab (SHR1210) , sintilimab (IBI308) , tislelizumab (BGB-A317) , toripalimab (JS 001) , and AMP 224 (GlaxoSmithKline) . Exemplary anti-PD-L1 antibodies include but are not limited to avelumab, durvalumab, atezolizumab, the antibody claimed in PCT Application No. PCT/CN2016/093560, KN035 (3D Medicines) , and CK-301 (Checkpoint Therapeutics) .

In some embodiments of the above described method, use, combination, composition, agent, or kit, the fisogatinib modulates the CCL20 gene after administration alone and before administration of the PD-1 axis binding antagonist, and/or during concurrent administration with the PD-1 axis binding antagonist. In some embodiments, fisogatinib decreases the amount of CCL20 gene expression.

In other embodiments of the above described method, use, combination, composition, agent, or kit, the fisogatinib promotes influx of T-cells into a tissue that is the subject of treatment, after administration alone and before administration of the PD-1 axis binding antagonist, and/or during concurrent administration with the PD-1 axis binding antagonist.

In other embodiments of the above described method, use, combination, composition, agent, or kit, the fisogatinib decreases the ability of regulatory T cells (TREGs) to suppress the immune system.

FGFR4 inhibitor

In some embodiments of the above methods, combinations, compositions, medicaments, uses or kits, the FGFR4 inhibitor is fisogatinib, which has the following structure:

or a pharmaceutically accceptable salt thereof.

Methods of Treatment or USE

Therapeutic Agent

In one aspect of the invention, the invention provides a method for treating a condition of a subject that would benefit from modulating the immune function comprising administering to the subject a pharmaceutically effective amount of a PD-1 axis binding antagonist and a pharmaceutically effective amount of fisogatinib .

Any of the PD-1 axis binding antagonists described herein may be used in the methods of the present disclosure.

In some embodiments, the PD-1 axis binding antagonist is A PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain CDR sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39, and 41.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33 and 35.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of:

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of:

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 11;

b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 17; and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23;

c) a heavy chain variable region comprising the SEQ ID NO: 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; and a light chain variable region comprising SEQ ID NO: 31, SEQ ID NO: 33 and/or SEQ ID NO: 35; or

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of: SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51 and SEQ ID NO: 55, and homologous sequences with at least 80%, (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 53, and homologous sequences with at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.

a) a heavy chain variable region comprising SEQ ID NO: 43; and a light chain variable region comprising SEQ ID NO: 45;

b) a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49;

c) a heavy chain variable region comprising SEQ ID NO: 51; A chain variable region comprising SEQ ID NO: 53; or

d) a heavy chain variable region comprising SEQ ID NO: 55; and a light chain variable region comprising SEQ ID NO: 49.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49.

In some embodiments, the antibody or antigen-binding fragment thereof is a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv) 2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49.

Indications and Subjects

The methods described herein may find use in treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer. Also provided herein are methods of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and fisogatinib. Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and fisogatinib.

In further aspects, provided herein are methods of treating infection (e.g., with a bacteria or virus or other pathogen) . In some embodiments, the infection is with virus and/or bacteria. In some embodiments, the infection is with a pathogen. In some embodiments, the infection is an acute infection. In some embodiments, the infection is a chronic infection.

In some embodiments, the individual has been treated with fisogatinib before the combination treatment with a PD-1 axis binding antagonist and fisogatinib.

In some embodiments, the individual has cancer that is resistant (has been demonstrated to be resistant) to one or more PD-1 axis antagonists. In some embodiments, resistance to PD-1 axis antagonist includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance to PD-1 axis antagonist includes progression of the cancer during treatment with the PD-1 axis antagonist. In some embodiments, resistance to PD-1 axis antagonist includes cancer that does not response to treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.

In another aspect, the individual has cancer that expresses (has been shown to express e.g., in a diagnostic test) PD-L1 biomarker. In some embodiments, the patient's cancer expresses low PD-L1 biomarker. In some embodiments, the patient's cancer expresses high PD-L1 biomarker. In some embodiments of any of the methods, assays and/or kits, the PD-L1 biomarker is absent from the sample when it comprises 0%of the sample.

In some embodiments of any of the methods, assays and/or kits, the PD-L1 biomarker is present in the sample when it comprises more than 0%of the sample. In some embodiments, the PD-Ll biomarker is present in at least 1%of the sample. In some embodiments, the PD-L1 biomarker is present in at least 5%of the sample. In some embodiments, the PD-L1 biomarker is present in at least 10%of the sample.

In some embodiments of any of the methods, assays and/or kits, the PD-L1 biomarker is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH, and combinations thereof.

In some embodiments of any of the methods, assays and/or kits, the PD-L1 biomarker is detected in the sample by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC) . In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody. In some embodiments, the PD-L1 biomarker is detected as a weak staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected as a moderate staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected as a strong staining intensity by IHC. In some embodiments, the PD-L1 biomarker is detected on tumor cells, tumor infiltrating immune cells, stromal cells and any combinations thereof. In some embodiments, the staining is membrane staining, cytoplasmic staining or combinations thereof.

In some embodiments of any of the methods, assays and/or kits, the absence of the PD-L1 biomarker is detected as absent or no staining in the sample. In some embodiments of any of the methods, assays and/or kits, the presence of the PD-L1 biomarker is detected as any staining in the sample.

The efficacy of any of the methods described herein (e.g., combination treatments including administering an effective amount of a combination of a PD-1 axis binding antagonist and fisogatinib) may be tested in various models known in the art, such as clinical or pre -clinical models. Suitable pre-clinical models are exemplified herein and further may include without limitation ID8 ovarian cancer, GEM models, B16 melanoma, RENCA renal cell cancer, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma models of cancer.

For any of these exemplary models, after developing tumors, mice are randomly recruited into treatment groups receiving combination anti-PDL1 and fisogatinib treatment or control treatment. Tumor size (e.g., tumor volume) is measured during the course of treatment, and overall survival rate is also monitored.

In another aspect, provided herein are methods for modulating immune function in a subject having cancer comprising administering an effective amount of a combination of a PD-1 axis binding antagonist and fisogatinib.

In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a sample of the patient's cancer as examined using a diagnostic test) has elevated levels of T cell infiltration. As used herein, T cell infiltration of a cancer may refer to the presence of T cells, such as tumor-infiltrating lymphocytes (TILs) , within or otherwise associated with the cancer tissue. It is known in the art that T cell infiltration may be associated with improved clinical outcome in certain cancers (see, e.g., Zhang et al, N. Engl. J. Med. 348 (3) : 203-213 (2003) ) .

In some embodiments of the methods of the present disclosure, the individual has a T cell dysfunctional disorder. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cell anergy or decreased ability to secrete cytokines, proliferate or execute cytolytic activity. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cell exhaustion. In some embodiments of the methods of the present disclosure, the T cells are CD4+ and CD8+ T cells. Without being bound by theory, fisogatinib treatment may increase T cell (e.g., CD4+ T cell, CD8+ T cell, memory T cell) priming, activation and/or proliferation relative to prior to the administration of the combination. In some embodiments, the T cells are CD4+ and/or CD8+ T cells.

In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a sample of the patient's cancer is examined using a diagnostic test) has low levels of T cell infiltration. In some embodiments, the cancer (in some embodiments, a sample of the patient's cancer is examined using a diagnostic test) has no detectable T cell infiltrate. In some embodiments, the cancer is a non-immunogenic cancer (e.g., non-immunogenic colorectal cancer and/or ovarian cancer) . Without being bound by theory, fisogatinibtreatment may increase T cell (e.g., CD4+ T cell, CD8+ T cell, memory T cell) priming, activation and/or proliferation relative to prior to the administration of the combination.

Any cancers described herein may be used in the methods.

In some embodiments, the subject or individual is a human.

Dose Levels and Dosage Regimens

An effective amount of the PD-1 axis binding antagonist and fisogatinib may be administered for prevention or treatment of disease. The appropriate dosage of the PD-1 axis binding antagonist and/or fisogatinib may be determined based on the type of disease to be treated, the type of the PD-1 axis binding antagonist and fisogatinib, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician. In some embodiments, combination treatment with fisogatinib and PD-1 axis binding antagonists (e.g., anti-PD-1 or anti-PDL1 antibody) are synergistic, whereby an efficacious dose of fisogatinib in the combination is reduced relative to efficacious dose of the fisogatinib as a single agent.

As a general proposition, the therapeutically effective amount of the antibody or antigen-binding fragment thereof provided herein administered to human will be in the range of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg) of patient body weight whether by one or more administrations. In some embodiments, the antibody or antigen-binding fragment thereof used is 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg About 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg or about 100 mg/kg administered daily. In some embodiments, the antibody or antigen-binding fragment thereof is administered at about 50 mg/kg or less, 10 mg/kg or less, 5 mg/kg or Less, 1 mg/kg or less, 0.5 mg/kg or less or 0.1 mg/kg or less.

In some embodiments, the administration dosage may change over the course of treatment. For example, in some embodiments the initial administration dosage may be higher than subsequent administration dosages. In some embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.

A particular dose can be administered at multiple intervals, such as once a day, twice daily or more, twice or more per month, once a week, once every two weeks, once every three weeks, once a month, or every two months or more. In certain embodiments, the dosage administered can vary with the course of the treatment. For example, in certain embodiments, the initial dose administered can be higher than the subsequent dose. In certain embodiments, the administered dose is adjusted during the course of treatment depending on the response of the subject to be administered. The dosage regimen can be adjusted to achieve an optimal response (e.g., a therapeutic response) . For example, a single dose may be administered or multiple divided doses administered over a period of time.

In general, a suitable daily dose of fisogatinib will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day. For example, the dose could be between 10 and 2000 mg per day. Alternatively, the dose can be between 100 and 1000 mg per day, or between 200 and 600 mg per day. If desired, the effective daily dose of the active compound may be administered as one, two, three, four, or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

In some embodiments, the combination therapy of the invention comprises administration of a PD-1 axis binding antagonist and fisogatinib. The PD-1 axis binding antagonist and fisogatinib may be administered in any suitable manner known in the art. For example, The PD-1 axis binding antagonist and fisogatinib may be administered sequentially (at different times) or concurrently (at the same time) . In some embodiments, the PD-1 axis binding antagonist is in a separate composition as fisogatinib. In some embodiments, the PD-1 axis binding antagonist is in the same composition as fisogatinib.

The PD-1 axis binding antagonist and fisogatinib may be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, fisogatinib is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

In some embodiments, each therapeutic agent in a combination therapy of the invention may be administered simultaneously (i.e., in the same medicament) , concurrently (i.e., in separate medicaments administered one right after the other in any order) or sequentially in any order. Sequential administration is particularly useful when the therapeutic agents in the combination therapy are in different dosage forms (one agent is a tablet or capsule and another agent is a sterile liquid) and/or are administered on different dosing schedules, e.g., a chemotherapeutic that is administered at least daily and a biotherapeutic that is administered less frequently, such as once weekly, once every two weeks, or once every three weeks.

In some embodiments, fisogatinib is administered before administration of the PD-L1 antagonist, while in other embodiments, fisogatinib is administered after administration of the PD-L1 antagonist.

In some embodiments, at least one of the therapeutic agents in the combination therapy is administered using the same dosage regimen (dose, frequency and duration of treatment) that is typically employed when the agent is used as monotherapy for treating the same cancer. In other embodiments, the patient receives a lower total amount of at least one of the therapeutic agents in the combination therapy than when the agent is used as monotherapy, e.g., smaller doses, less frequent doses, and/or shorter treatment duration. Each small molecule therapeutic agent in a combination therapy of the invention can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration.

The therapeutic agents in a combination therapy of the invention may be administered by continuous infusion, or by doses at intervals of, e.g., daily, every other day, three times per week, or one time each week, two weeks, three weeks, monthly, and bimonthly, etc. A total weekly dose is generally at least 0.05 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 10 mg/kg, 1 00 mg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg body weight or more.

Each therapeutic agent in a combination therapy of the invention may be administered either alone or in a medicament (also referred to herein as a pharmaceutical composition) which comprises the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients and diluents, according to standard pharmaceutical practice.

In some embodiments, the methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy) , chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc. ) . In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation.

A combination therapy of the invention may be used prior to or following surgery to remove a tumor and may be used prior to, during or after radiation therapy. In some embodiments, a combination therapy of the invention is administered to a patient who has not been previously treated with a biotherapeutic or chemotherapeutic agent. In other embodiments, the combination therapy is administered to a patient who failed to achieve a sustained response after prior therapy with a biotherapeutic or chemotherapeutic agent, i.e., is treatment-experienced.

A combination therapy of the invention is typically used to treat a tumor that is large enough to be found by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan. In some embodiments, a combination therapy of the invention is used to treat an advanced stage tumor having dimensions of at least about 200 mm ³, 300 mm ³, 400 mm ³, 500 mm ³, 750 mm ³, or up to 1000 mm ³.

In some embodiments, a combination therapy of the invention is administered to a human patient who has a cancer that tests positive for PD-L1 expression. In some embodiments, PD-L1 expression can be detected using a diagnostic anti-human PD-L1 antibody, or antigen binding fragment thereof, in an IHC assay on an FFPE or frozen tissue section of a tumor sample removed from the patient. Typically, the patient's physician would order a diagnostic test to determine PD-L1 expression in a tumor tissue sample removed from the patient prior to initiation of treatment with the PD-L1 antagonist and VEGFR inhibitor, but it is envisioned that the physician could order the first or subsequent diagnostic tests at any time after initiation of treatment, such as for example after completion of a treatment cycle. Selecting a dosage regimen (also referred to herein as an administration regimen) for a combination therapy of the invention depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the subject being treated. Preferably, a dosage regimen maximizes the amount of each therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each biotherapeutic and chemotherapeutic agent in the combination depends in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available.

Determination of the appropriate dosage regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, the patient's clinical history (e.g., previous therapy) , the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

Pharmaceutical Compositions, Medicaments and Kits

The present invention also provides a composition which comprises a PD-1 axis binding antagonist and fisogatinib as described above and a pharmaceutically acceptable excipient or carrier.

The pharmaceutically acceptable excipient or carrier for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial materials, and the like. Osmotic materials, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, adjuvants or non-toxic auxiliary substances, other components known in the art or various combinations of the above.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, odorants, thickeners, colorants, emulsifiers or stabilizers such as sugar and cyclodextrin. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, sorbitan, butyl Anisole, butylated hydroxytoluene and/or propyl gallate.

Further, the pharmaceutically acceptable excipient or carrier can include, for example, an aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, for example, a liquid solvent, a suspension, an emulsifier or a solid form suitable for the production of a liquid solvent, suspension or emulsifier.

The present invention also provides a medicament which comprises a PD-1 axis binding antagonist and fisogatinib as described above and a pharmaceutically acceptable excipient or carrier.

In some embodiments, a medicament comprising an anti-PD-L1 antibody as the PD-1 axis binding antagonist may be provided as a liquid formulation or prepared by reconstituting a lyophilized powder with sterile water for injection prior to use.

The PD-1 axis binding antagonist and fisogatinib medicaments described herein may be provided as an article of manufacture or a kit which comprises a first container and a second container and a package insert. The first container contains at least one dose of a medicament comprising an PD-1 axis binding antagonist, the second container contains at least one dose of a medicament comprising fisogatinib, and the package insert, or label, which comprises instructions for treating a patient for cancer using the medicaments. In some embodiments, the article of manufacture or kit further comprises package insert comprising instructions for suing the PD-1 axis binding antagonist in conjunction with fisogatinib to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. The first and second containers may be comprised of the same or different shape (e.g., vials, syringes and bottles) and/or material (e.g., plastic or glass) .

The article of manufacture or 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. In some embodiments of article of manufacture or the kit, the PD-1 axis binding antagonist is an anti-PD-L1 antibody and the instructions state that the medicaments are intended for use in treating a patient having a cancer that tests positive for PD-L1 expression by an IHC assay.

In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent) . Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.

Any of the PD-1 axis binding antagonists described herein may be used in the pharmaceutical compositions, medicaments and kits of the present disclosure. For example, in some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody or antigen-binding fragment thereof which comprises: a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49.

These and other aspects of the invention, including the exemplary specific embodiments listed below, will be apparent from the teachings contained herein.

The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

III. EXAMPLES

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1

Correlation of CCL20 with FGF19 expression and inhibition by fisogatinib

CCL20 ELISA Assay

Hepatocellular carcinoma cell lines assigned as positive for FGF19 expression (FGF19 expression is greater than 0.1 FPKM by Broad CCLE RNA-Seq) or assigned as negative for FGF19 expression (FGF19 expression is less than 0.1 FPKM by Broad CCLE RNA-Seq) were plated in the appropriate media and treated with varying concentrations of fisogatinib for 72 hours at 37℃. After fisogatinib incubation, supernatant from each sample was collected and mixed 1: 1 with Assay Diluent RD1-57, in a new plate as recommended (R&D Systems, Inc. Catalog Number DM3A00) . This plate was then incubated for 2 hours at room temperature. Next, 4 washes were performed using the recommended buffer and human MIP-3α conjugate was added to each sample. Samples incubated for 2 hours at room temperature before washes were repeated; samples were then incubated with Substrate Solution for 30 minutes at room temperature, protected from light. Next, Stop Solution was added to each sample and within 30 minutes of this addition, plates were read using a microplate reader set to 450 nm to determine the optical density of each sample. Cell lines were obtained from ATCC, the Japanese Collection of Research Bioresources (JCRB) , or the RIKEN BioResource Research Center. Results are shown in Figure 1.

Example 2

Effects of Fisogatinib on Regulatory T-cell Migration

Hepatocellular carcinoma cell lines assigned as positive for FGF19 expression (FGF19 expression is greater than 0.1 FPKM by Broad CCLE RNA-Seq) were plated in the appropriate media and treated with vehicle or 1 μM fisogatinib for 72 hours at 37℃. After fisogatinib incubation, supernatant from each sample was collected and transferred to a new well of a 24-well plate. A

6.5 mm

with a 5.0 μm pore polycarbonate membrane insert (Sigma-Aldrich, Catalog Number CLS3421-48EA) was placed in each well, resting atop the plated supernatant. Next, thawed peripheral blood CD4+/CD25+ regulatory T-cells (AllCells, LLC, Catalog Number PB009-4F) were placed in each transwell insert, in media devoid of cytokines and chemokines, and allowed to incubate for 18 hours at 37℃. After 18 hours, the transwell insert was carefully removed. The remaining cells, supernatant, and media were collected and spun at 1, 500 rpm in a tabletop microcentrifuge for 5 minutes to pellet migrated cells from each sample. The pelleted cells were resuspended in PBS, plated, and incubated at room temperature for 30 minutes.

solution (Promega, Catalog Number G7570) was then added 1: 1 to each well of cells and the mixture was shaken for 2 minutes at 350 rpm. Next, the plate was allowed to incubate at room temperature for 10 minutes, after which point, luminescence for each sample was recorded using a microplate reader. Recombinant human CCL20/MIP-3 alpha protein (R&D Systems, Inc. Catalog Number 360-MP-025) was used as a positive control for regulatory T-cell migration. Individual data is plotted with the bar representing the sample mean; the standard error of the mean (SEM) is depicted. Cell lines were obtained from ATCC, the Japanese Collection of Research Bioresources (JCRB) , or the RIKEN BioResource Research Center. Results are shown in Figure 2.

Example 3

Fisogatinib assessment in immune deficient xenograft of JHH7 mice

To determine if immune system plays a role in the anti-tumor function of fisogatinib, 100 female Nu/Nu mice were inoculated with 5 million cells of the JHH7 hepatocellular carcinoma cell line (JCRB) per mouse. Cells were prepared in a 0.1 mL suspension with 0.1 mL of BD Matrigel. 2 animals were designated as sentinels; 2 animals were designated as extras. Inoculated animals were monitored daily by general clinical observation throughout the study period. Body weight was recorded twice weekly before randomization and recorded every day during the dosing period and the dose was adjusted per body weight. Relative change of body weight (RCBW) of each mouse was calculated according to the following formula: RCBW (%) = (BWi –BW0) /BW0×100; BWi is body weight on a specific day; BW0 is body weight on the first day of administration. Average and standard deviation of RCBW of each group was also calculated. Tumor area (length × width) was measured two times per week using digimatic calipers throughout the study period and tumor volume was calculated based on the following formula: tumor volume = (length × width2) /2. The data was graphically represented as the percent of initial tumor volume (%) . 50 animals were selected for the efficacy study with fisogatinib once their tumors reached the appropriate size (100-300 mm ³) . The 50 animals were randomized (blocked randomization/using Excel software) and divided into the following groups for compound administration: vehicle; fisogatinib at 30 mg/kg BID, PO; fisogatinib at 100 mg/kg BID, PO; fisogatinib at 200 mg/kg BID, PO. All animals were monitored daily for clinical observation (animal mortality, appearance, spontaneous activity, body posture, and food and water intake. Any lesions and adverse reactions were recorded) . Any animals showing signs of debilitation, marked body weight loss (>20%) , cachexia or large tumors that would inhibit an animal’s ability to eat and drink or mobility were euthanized immediately. Any animals with severely ulcerated, infected or severely hemorrhagic tumors, or tumors whose estimated weight exceeded 20%of the body weight were euthanized. The study was terminated after 21 days of treatment. Results of the immune deficient JHH7 mice are shown in Figure 3A.

Fisogatinib assessment in immune competent xenograft of JHH7 mice

Female NSG ^TM‐SGM3 mice were myeloablated according to procedures recommended by The Jackson Laboratory. Following myeloablation, the mice were reconstituted via tail-vein injection with human CD34+ cord blood cells from one of three donors. Once the human CD45+ percentage of the peripheral blood of the animals reached > 25%, approximately 105-120 days (3.5-4 months) post-reconstitution, 30 mice were inoculated with 5 million cells of the JHH7 hepatocellular carcinoma cell line (JCRB) per mouse in a 1: 1 mixture with BD Matrigel. Inoculated animals were monitored daily by general clinical observation throughout the study period. Body weight was recorded twice weekly before randomization and recorded every day during the dosing period and the dose was adjusted per body weight. Relative change of body weight (RCBW) of each mouse was calculated according to the following formula: RCBW (%) = (BWi –BW0) /BW0×100; BWi is body weight on a specific day; BW0 is body weight on the first day of administration. Average and standard deviation of RCBW of each group was also calculated. Tumor area (length × width) was measured two times per week using digimatic calipers throughout the study period and tumor volume was calculated based on the following formula: tumor volume= (length × width2) /2. The data was graphically represented as the percent of initial tumor volume (%) . 18 animals were selected for the efficacy study with fisogatinib once their tumors reached the appropriate size (54-120 mm3) . The 18 animals were randomized (blocked randomization/using Excel software) and divided into the following groups for compound administration: vehicle; fisogatinib at 200 mg/kg BID, PO. All animals were monitored daily for clinical observation (animal mortality, appearance, spontaneous activity, body posture, and food and water intake. Any lesions and adverse reactions were recorded) . Any animals showing signs of debilitation, marked body weight loss (>20%) , cachexia or large tumors that would inhibit an animal’s ability to eat and drink or mobility were euthanized immediately. Any animals with severely ulcerated, infected or severely hemorrhagic tumors, or tumors whose estimated weight exceeded 20%of the body weight were euthanized. The study was terminated after 41 days of treatment. Results of the immune competent JHH7 mice are shown in Figure 3B.

As shown in Figure 3, the immune competent animal, compared to that in the immune-deficient tumor model, a same dose of fisogatinib shows higher anti-tumor activity within the immune competent JHH7 mouse tumor model transplanted with CD34+ hematopoietic stem cell, in which the human immune system including T-lymphocyte is reconstructed in the immune-deficient mouse. The transplanted tumor is JHH-7 human HCC cell with amplified and highly expressed FGF19, which is relevant to the clinical indication. These results indicate that immune system plays a role in the anti-tumor function of fisogatinib (Figure 5) . As PD-L1 antibody can specifically block the signal pathway of PD-1/PD-L1, inhibit the immune negative regulation signal, restore the activity of T-lymphocyte and enhance the immune response, thus, based on these results combining fisogatinib with anti-PD-L1 antibody is expected to provide a superior effect over monotherapy.

Example 4

Fisogatinib enhances the infiltration of T-lymphocyte in the tumor

The effects of fisogatinib on the infiltration of T-lymphocyte in the tumor samples from Example 3 were examined using CD3 immunohistochemistry. Formalin-fixed paraffin-embedded tumor samples were processed according to procedures recommended by HistoTox Labs, Inc. These samples were then sectioned at approximately 5 microns, positioned on glass slides, and stained by immunohistochemistry (IHC) for human T-cell co-receptor CD3. IHC-stained glass slides were then scanned using an Aperio AT2 whole slide scanner. Whole slide images were annotated to delineate regions of interest (ROI) . Exclusions were applied to remove non-tumor tissue, necrosis, and artifacts (folds and tears) . Using the previously described ROIs, an algorithm was applied on the IHC-stained samples using Visiopharm (VIS) image analysis software. A count-based thresholding method was applied to measure CD3 positive cells within each ROI. CD3 nuclear density was calculated using the following formula: CD3 Nuclear Density = (#CD3-positive cells /Tumor Area) *1000000. The results show that fisogatinib increases the infiltration of T-lymphocyte in the tumor with apparently increased CD8+ and CD4+ T-lymphocyte (Figure 6) in the JHH-7 immune reconstructed (with FGF19 amplification and highly expression) xenograft tumor model, which indicates that inhibiting the FGF19/FGFR4 pathway may regulate the anti-tumor immunity. Thus, fisogatinib can potentially enhance the anti-tumor effect of PD-L1 antibody through increasing the infiltration of T-lymphocyte in the tumor micro-environment.

Example 5

Fisogatinib modulates immune genes

RNA was extracted and purified from treated cells as recommended (Qiagen, Catalog Number 74104) . Isolated RNA was used for downstream RNA-Seq on an Illumina HiSeq platform with a 2x150 bp configuration by Genewiz. Greater than 350 million raw paired-end reads were generated per lane and FASTQ files were used for data analysis. Briefly, using the STAR v2.3.1q aligner (1) , RNA-seq data from each sample was aligned to version hg19 of the human genome while also providing transcriptome and splice junction annotations from the Gencode project v18 (2) . RNA-seq expression quantification, measured in fragments per kilobase of mRNA per million mapped reads, was calculated for all CCDS transcripts (3) in the Gencode v18 database (2) using Cufflinks v2.2.1 (4) . Results are shown in Figure 5.

Example 6

Assess the combination potential of anti-PD-L1 antibody and fisogatinib

Study Design

Cytotoxic T cells play an essential role in cancer immunotherapy. PBMC-mediated tumor cells killing efficacy (PBMCs: Hep-3B=10: 1) was evaluated in tumor cells in the context of fisogatinib and the anti-PD-L1 antibody.

Materials and Methods

Reagents:

● Hep-3B cell (SIBS, CAT#TCHu106)

● PBS sterile (Hyclone, CAT#SH30256.01) ;

● FBS (Gibco CAT#100991-148) ;

● TRYPSIN 0.25%EDTA (Gibco, CAT#25200-072) ;

● CFSE (eBioscience, CAT#65-0850-84) ;

● Assay medium: RPMI1640+10%FBS;

● Purified NA/LE Mouse Anti-Human CD3 (BD, Cat. No.: 555336) ;

● Purified NA/LE Mouse Anti-Human CD28 (BD, Cat. No.: 555725) ;

● Fixable Viability Stain 700 (BD, Cat. No.: 564997) .

Test articles:

fisogatinib: Stock solution: 10mM;

Ab-1: anti-PD-L1 antibody, which comprises: a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49; Stock solution: 30mg/ml.

PBMCs:

The frozen PBMCs from two differernt donors, coded as 1911150123 from Stemcells, was ordered from the vendor Stemexppress.

Assay Procedure

Protocol A:

On day 1, Hep-3B cells were dissociated and collected (medium: MEM+10%FBS+1%PS+2%NaHCO3+ 1%Sodium pyruvate) and re-suspended in appropriate volume of PBS with 5-20×10 ⁶ cells/mL. 2X CFSE solution (5 μM) was prepared in PBS (pre-warmed to RT) and added into Hep-3B cell solution in an equal volume, followed by mixing immediately. After incubation for 5-10 minutes at 37℃ in the dark, CFSE labeling was completed by adding 4-5 volumes of ice-cold assay media (containing ≥10%serum) . After incubating on ice for 5 minutes, the labeled cells were washed twice with pre-warmed culture media and re-suspended in 5-6 mL of assay medium and adjusted to the appropriate cell density. The cells were cultured in medium and plated in 24-well plate with 1×10 ⁵ cells /well.

On the same day, the frozen PBMC cells were revived and activated, and then transferred into 10 mL of assay medium (RPMI-1640+10%FBS) and centrifuged at 1500rpm for 10 minutes. The PBMC cells were re-suspended in appropriate volume of assay medium containing 1 μg/mL of CD3 and 1 μg/mL of CD28 Ab, to make the cell density at 1.5×10 ⁶ cells/mL and incubated at 37℃ for 24h.

On day 2, for cell killing assay, the activated PBMCs were collected into a 50 mL tube, centrifuged at 1500rpm for 10 minutes, suspended with appropriate volume of assay media and adjusted to appropriate concentration with the same media. Then, the activated PBMCs were added into the Hep-3B cell to make the E: T ratio (E/T ratio is effector/target cell ratio) at 10: 1. The co-cultured Hep-3B and PBMCs were treated following a protocol design as shown in Table 1 (2 independent wells for each treatment) , and cultured at 37℃ for 72 hrs.

Following the treatment, L/D+%of CFSE+ tumor cells were detected and analyzed by flow cytometry using FlowJo on day 5. Results were summarized in Figure 6-A.

Table 1. Treatment group

Note: “iso” or “isotype control” means a negative control antibody from the same species, immunoglobulin class, subclass and light chain as the primary antibody used in a particular scientific application. It is used to differentiate a positive signal or result over background noise or other nonspecific interactions.

Protocol B:

On day 1, Hep-3B cells were labeled by CFSE, cultured and plated into 24-well plate in the same way as described in Protocol A, followed by treating the cells with 20 ng/ml IFN-gamma for 24 hrs. On the same day, PBMC cells were revived and activated as described in Protocol A.

On day 2, for cell killing assay, the activated PBMCs were collected into 50 mL tube, centrifuged at 1500rpm for 10 minutes, suspended with appropriate volume of assay media and adjusted to appropriate concentration with the same media. Then, the activated PBMCs were added into the Hep-3B cell to make the E: T ratio at 10: 1. The co-cultured Hep-3B and PBMCs were treated following a protocol design as shown in Table 2 (2 independent wells for each treatment) , and cultured at 37℃ for 48 hrs.

Following the treatment, L/D ⁺%of CFSE ⁺ tumor cells were detected and analyzed by flow cytometry using FlowJo on day 5. Data was analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. Results were summarized in Figure 6-B. These results demonstrated that fisogatinib and/or Ab-1 (mono or combo) exhibited significantly enhanced PBMCs-mediated tumor killing efficacy in rhIFN-γ pre-treated Hep-3B.

Table 2. Treatment group

Discussion

Briefly, these results demonstrated that fisogatinib and/or Ab-1 (mono or combo) exhibited significantly enhanced PBMCs-mediated tumor killing efficacy in rhIFN-γ pre-treated Hep-3B.

Specifically, as shown in Fig. 6-B, the combination of Ab-1 at 10 μg/ml and fisogatinib at 0.01 or 1 μM exhibited significantly enhanced PBMCs-mediated tumor killing efficacy in rhIFN-γ pretreated Hep-3B compared with using Ab-1 or fisogatinib alone (*p<0.05; ***p<0.001) . However, in Fig. 6-A, the same combination of Ab-1 and fisogatinib did not show synergistically enhanced PBMCs-mediated efficacy in Hep-3B without rhIFN-γ pretreatment, compared with using Ab-1 or fisogatinib alone.

The synergistical efficacy of Ab-1 or fisogatinib in PBMCs-mediated tumor killing was only detected in rhIFN-γ-induced PD-L1 up-regulated Hep-3B cells. Our previous study found that Hep-3B cells were PD-L1 negative on the surface in basal condition and 20 ng/ml rhIFN-γtreatment increased Hep-3B surface PD-L1 expression after 24 hrs. Thus, the present combination therapy is useful for the treatment of a condition of a subject that would benefit from modulating the immune function, especially cancer.

The references are below:

1. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 29, 15-21 (2012)

2. Harrow, J. et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774 (2012) .

3. Pruitt, K.D. et al. The consensus coding sequence (CCDS) project: Identifying a common protein-coding gene set for the human and mouse genomes. Genome Res. 19, 1316–1323 (2009) .

4. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010) .

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

Claims

A method for treating a condition of a subject that would benefit from modulating the immune function comprising administering to the subject a pharmaceutically effective amount of a PD-1 axis binding antagonist and a pharmaceutically effective amount of fisogatinib.
Use of a combination of a PD-1 axis binding antagonist and fisogatinib for the manufacture of a medicament for treating a condition of a subject that would benefit from modulating the immune function.
Use of a PD-1 axis binding antagonist for the manufacture of a medicament for treating a condition of a subject that would benefit from modulating the immune function, which is combined with fisogatinib.
Use of for the manufacture of a medicament for treating a condition of a subject that would benefit from modulating the immune function, which is combined with a PD-1 axis binding antagonist.
A combination of PD-1 axis binding antagonist and fisogatinib for use in a condition of a subject that would benefit from modulating the immune function, comprising administering to the individual a pharmaceutically effective amount of PD-1 axis binding antagonist and a pharmaceutically effective amount of fisogatinib.
A pharmaceutically amount of a combination of PD-1 axis binding antagonist and fisogatinib for use in a condition of a subject that would benefit from modulating the immune function.
A pharmaceutical composition, medicament, agent or kit comprising a PD-1 axis binding antagonist for use in combination with fisogatinib for treating a condition of a subject that would benefit from modulating the immune function.
A pharmaceutical composition, medicament, agent or kit comprising fisogatinib for use in combination with a PD-1 axis binding antagonist for treating a condition of a subject that would benefit from modulating the immune function.
A pharmaceutical composition, medicament, agent or kit, comprising of a PD-1 axis binding antagonist, fisogatinib, and one or more pharmaceutically acceptable carriers.
The method, use, combination, composition, composition, agent, or kit of any one of claims 1-9, wherein the fisogatinib modulates the CCL20 gene after administration alone and before administration of the PD-1 axis binding antagonist, and/or during concurrent administration with the PD-1 axis binding antagonist.
The method, use, combination, composition, composition, agent, or kit of any one of claims 1-10, wherein the fisogatinib promotes influx of T-cells into a tissue that is the subject of treatment, after administration alone and before administration of the PD-1 axis binding antagonist, and/or during concurrent administration with the PD-1 axis binding antagonist.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-11, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist; preferably, the PD-1 axis binding antagonist is a PD-L1 binding antagonist, inhibiting the binding of PD-L1 to PD-l.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-12, wherein the PD-1 axis binding antagonist is an antibody or antigen-binding fragment thereof; preferably, the antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof; more preferably, the antibody is an anti-PD-L1 antibody; most preferably, the antibody is a monoclonal antibody.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-13, wherein the antibody is a humanized antibody or a human antibody or a mammal antibody.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof specifically binds to human PD-L1, preferably with a Kd value of no more than 10 ^-8M; more preferably, the antibody or antigen-binding fragment thereof binds to monkey PD-L1 and/or does not bind to mouse PD-L1; most preferably, the antibody or antigen-binding fragment thereof does not substantially bind to PD-L2.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-15, wherein the antibody or antigen-binding fragment thereof blocks the binding of human or monkey PD-L1 to its receptor; preferably, further providing at least one of the following activities:

a) inducing IL-2 production in CD4 ⁺T cells;

b) inducing IFNγ production in CD4 ⁺T cells;

c) inducing proliferation of CD4 ⁺T cells; and

d) reversing T reg inhibition function.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain CDR sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39, and 41.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-17, wherein the antibody or antigen-binding fragment thereof further comprises a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33 and 35.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of:

a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5;

b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15 and/or SEQ ID NO: 17;

c) a heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; and

d) a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39 and/or SEQ ID NO: 41.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of:

a) a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 11;

b) a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23; and

c) a light chain variable region, SEQ ID NO: 31, SEQ ID NO: 33 and/or SEQ ID NO: 35.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-20, wherein the antibody or antigen-binding fragment thereof comprises:

a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3 and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9 and/or SEQ ID NO: 11;

b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 17; and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23;

c) a heavy chain variable region comprising the SEQ ID NO: 25, SEQ ID NO: 27 and/or SEQ ID NO: 29; and a light chain variable region comprising SEQ ID NO: 31, SEQ ID NO: 33 and/or SEQ ID NO: 35; or

d) a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39 and/or SEQ ID NO: 41; and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-21, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of: SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51 and SEQ ID NO: 55, and homologous sequences with at least 80%, (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of sequence identity.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, and the kit of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 53, and homologous sequences with at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequences of identity.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-23, wherein the antibody or antigen-binding fragment thereof comprises:

a) a heavy chain variable region comprising SEQ ID NO: 43; and a light chain variable region comprising SEQ ID NO: 45;

b) a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49;

c) a heavy chain variable region comprising SEQ ID NO: 51; A chain variable region comprising SEQ ID NO: 53; or

d) a heavy chain variable region comprising SEQ ID NO: 55; and a light chain variable region comprising SEQ ID NO: 49.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-24, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-25, wherein the antibody or antigen-binding fragment thereof is a camelized single chain domain antibody, a diabody, a scFv, a scFv dimer, a BsFv, a dsFv, (dsFv) 2, dsFv-dsFv', Fv fragment, Fab, Fab', F (ab') 2, ds difunctional antibody (dsdiabody) , Nanobody, domain antibody or bivalent domain antibody.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-26, wherein the antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-27, wherein the subject is a human.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-28, wherein the subject has received at least one round of a prior therapy.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-29, wherein the prior therapy is surgical resection, transplantation, local regional therapy, systemic therapy, and/or best supportive care.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-30, wherein the subject has up-regulated PD-L1 expression.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-31, wherein the condition is immune-related diseases or conditions, tumor, cancer or a chronic viral infection.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-32, wherein immune-related diseases or conditions are the PD-L1 associated conditions and disorders selected from autoimmune diseases, such as systemic lupus erythematosus (SLE) , psoriasis, systemic scleroderma, and autoimmune diabetes.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-33, wherein immune-related diseases or conditions are the PD-L1 associated conditions and disorders including infectious disease such as chronic viral infection for example, viral infection of hepatitis B, hepatitis C, herpes vims, Epstein-Barr vims, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, adenovirus, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus, JC virus or BK virus.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-34,

wherein the condition is cancer, specifically, the cancer is a solid tumor or a blood cancer,

such as non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL) , plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the central nervous system (CNS) , such as primary CNS lymphoma, spinal axis tumor, brain stem glioma.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-35, wherein the cancer is selected from the group consisting of: melanoma, lung cancer, kidney cancer, breast cancer, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, including HCC, prostate cancer, endometrial cancer, esophageal cancer, colorectal cancer, glioblastoma, neurogenic tumor, sarcoma, bladder cancer and stomach cancer.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-36, wherein the cancer is selected from the group consisting of: leukemia, lymphoma, hemangioma and multiple myeloma.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-37, wherein the cancer is metastatic, especially metastatic tumors expressing PD-L1.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-38, wherein the cancer is re-current or metastatic, is locally advanced and/or metastatic or relapsed or refractory.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-39,

wherein the PD-1 axis binding antagonist is an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-40, wherein the PD-1 axis binding antagonist is formulated as a liquid medicament, especially as injection, at a dose of about1200 mg; and fisogatinib is formulated as a tablet, especially, at a dose of about 300 mg, about 400 mg, or about 600 mg.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-41, wherein the PD-1 axis binding antagonist and fisogatinib are administered sequentially in either order or simultaneously.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-42, wherein the PD-1 axis binding antagonist is administered by intravenous infusion.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-43, wherein the PD-1 axis binding antagonist is administered once every three weeks during the treatment cycle, at a dose of about 1200 mg.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-44, wherein fisogatinib is administered orally.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-45, fisogatinib is administered once every day during the treatment cycle, at a dose of about 300 mg, about 400 mg, or about 600 mg.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-46, fisogatinib is administered first.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-47, wherein when the PD-1 axis binding antagonist and fisogatinib are both administered on the same day, fisogatinib is administered first.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-48, wherein the PD-1 axis binding antagonist and fisogatinib are administered simultaneously.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-49, which comprises a first container, a second container and a package insert, wherein the first container comprises at least one dose of a medicament comprising an PD-1 axis binding antagonist, the second container comprises at least one dose of a medicament comprising a second agent, wherein the second agent is fisogatinib.
The method, the use, the PD-1 axis binding antagonist and fisogatinib for use, the combination, the composition, the medicament, the agent and the kit of any one of claims 1-50, wherein the instructions state that the medicaments are intended for use in treating a subject having a cancer.
A method of treating a PBMCs-mediated tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of fisogatinib and an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region, wherein the treatment results in improved efficacy as compared to administration of fisogatinib or the anti-PD-L1 antibody alone.
The method of claim 52, wherein the heavy chain variable region comprises SEQ ID NO: 47.
The method of claim 52 or 53, wherein the light chain variable region comprises SEQ ID NO: 49.